Advances in Cell Aging and Gerontology, Volume 5: Programmed Cell Death, Volume I

VOLUME I PROGRAMMED C E L L DEATH

CELLULAR

AND MOLECULAR

MECHANISMS

PREFACE

This first installment of the two volume book on Programmed Cell Death provides a series of concise review articles presenting our current understanding of the cascades of events involved in apoptosis or programmed cell death and signaling pathways that can be engaged to prevent apoptosis. The past five years have seen a virtual explosion in the number of laboratories studying, at the molecular and cellular levels, the mechanisms that lead to cell death of the form called apoptosis which manifests specific morphological, biochemical, and molecular features that distinguish this form of cell death from necrosis. The morphological features of cells undergoing apoptosis include cell shrinkage, surface membrane blebbing, cytoskeletal perturbation, and nuclear chromatin condensation and fragmentation. In addition, organellar structure, (i.e. structure of mitochondria, endoplasmic reticulum, and golgi) are largely preserved in cells undergoing apoptosis. Biochemical features of apoptosis include activation of a class of cysteine proteases called caspases, mitochondrial membrane depolarization, and release of factors, such as cytochrome c, that seem to be critical for nuclear manifestations of apoptosis. In addition, a relatively stereotyped sequence of gene and protein expression occurs involving upregulation of proteins involved in inducing mitochondrial dysfunction and caspase activation, on the one hand, and anti-apoptotic genes that may be involved in suppressing the apoptotic cascade, on the other hand. The first chapter of this book by Jason Mills describes some of the hallmark characteristics of the execution-phase of apoptosis, that is the phase subsequent to initiation of the cell death process but prior to final nuclear changes. The second chapter by Anna Bruce-Keller describes a well-characterized death signaling cascade involving receptors, such as Fas and TNF receptors, that are linked through death domain motifs to the activation of cascades involving caspase activation and certain kinases that ultimately lead to cell death. Carol Troy covers, in some detail, the multiple caspases involved in various paradigms of apoptosis and how caspase cascades may effect the cell death process under various conditions. The next chapter focuses on events occurring in mitochondria during the cell death process that appear to be critical mediators of the final execution-phases of the cell death process. This chapter by Bernard Mignotte and Jean-Luc Vayssiere describes critical roles of membrane permeability transition and release of apoptotic factors. Manicini and colleagues then discuss a quite intriguing concept concerning the role of mitochondria in apoptosis. Namely, the evidence suggesting that mitochondrial proliferation induced by apoptoic signals contributes to mitochondrial dysfunction and cell death. The Bcl-2 family of proteins plays a powerful vii Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. v i i - - v i i i © 2 0 0 1 Elsevier Science. Printed in the Netherlands.

viii

and key role in either promoting or preventing cell death. Qing Guo and colleagues describe the members of this emerging family of death-related proteins and describe what is known about their sites of action within the cell and their roles in various cell death paradigms in non-neuronal cells and neurons. The next chapter by Steve Tammariello, Gary Landreth, and Steven Estus focuses on the involvement of Jun kinases in programmed cell death in neurons. Work done in models of trophic factor withdrawal suggests a critical active role for these kinases in the cell death process. Vivek Rangnekar describes a novel and important death-promoting protein called Par-4, which appears to act very early in the cell death process prior to mitochondrial dysfunction and caspase activation. Par-4 contains both a leucine zipper domain and a death domain and can be induced at the translational level very early stages in the cell death process. It has been known for some time that cytoskeletal alterations occur in cells undergoing apoptosis. Rakesh Srivastava and Dan Longo describe the possible role of cytoskeleton alterations in the cell death process. The next two chapters consider two prominent anti-apoptotic signaling pathways. Mark Mattson describes the evidence supporting an anti-apoptoic role for the transcription factor NF-•B in a variety of cell death paradigms in different mitotic and post-mitotic cell types. NF-~B appears to be linked to genes that suppress oxidative stress and preserve cellular ion homeostasis. Quinn Deveraux and John Reed then cover an intriguing and emerging family of inhibitor of apoptosis proteins, IAP. The IAPs can suppress cell death in a remarkable array of programmed cell death paradigms. In the final chapter, Marcel Leist and Pierluigi Nicotera describes the roles of exicitatory amino acids and nitric oxide in programmed cell death in neurons. Emerging data suggest the overriding importance of activation of glutamate receptors in neuronal apoptosis in many different in vivo and cell culture settings. The links of calcium influx and nitric oxide production to the apoptotic machinery are discussed. The findings presented in this first volume lay the framework for the second volume of Programmed Cell Death which focuses on the involvement of apoptosis or anti-apoptosis in a variety of disease conditions ranging from neurodegenerative disorders to cancer to cardiovascular disease.

M E C H A N I S M S U N D E R L Y I N G THE H A L L M A R K F E A T U R E S OF THE E X E C U T I O N - P H A S E OF APOPTOSIS

JASON C. MILLS

Table of Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolution o f the Execution-Phase Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Brief History o f the Study o f the Apoptotic Time Course . . . . . . . . . . . . . . . . . . . . . . . Asynchrony . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Circumventing A s y n c h r o n y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Defining the T e r m s of the Apoptotic Time-Course . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Importance o f the Individual Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stricter Definitions o f Time Course Phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A Brief Discussion of the C o m m i t m e n t Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Execution-phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuclear Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extranuclear Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary ................................................................

1 2 2 4 8 9 9 9 11 12 12 17 28

Introduction

Natural selection has favored the evolution of a process that allows organisms to dispose efficiently of defective, infected, or surplus cells. This process is apoptosis, the individual cell's consummate act of altruism. It is cell death characterized by: (1) lack of subsequent inflammation (which would damage neighboring, healthy cells); (2) inhibition of replication and spread of potentially dangerous genetic information from exogenous or endogenous sources; and (3) prevention of leakage of potentially injurious, normally sequestered, intracellular substances. To date, much more is understood about how, when, and where, apoptosis is initiated than about what happens once a cell has started the evolutionarily conserved apoptotic process itself. The reasons for this bias toward the study of upstream events in apoptosis are manifold, including simple technical limitations of assays for downstream events. Recently, however, with some technical advances and a general increase in interest in the process of apoptosis, large strides have been made toward understanding downstream events. The downstream, post-initiation phases of apoptosis begin with the commitment phase, when a cell uses highly evolutionarily conserved machinery to commit irreversibly to death and subsequently invoke the multiple pathways that characterize the final stage of apoptosis: the execution-phase. The execution-phase represents the culmination of apoptosis when the committed cell fulfills the evolutionarily driven purpose of apoptosis (namely, altruistic self-elimination). The telltale morphologic patterns that have defined 1 Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. 1 -- 38 © 2001 Elsevier Science. Printed in the Netherlands.

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J.C. Mills

apoptosis (e.g. dynamic membrane blebbing and chromatin condensation) since its initial recognition (Kerr et al., 1972) occur during the execution-phase and show remarkable evolutionary conservation, with the final stages of apoptosis looking about the same no matter the organism, tissue, or apoptotic inducing agent. It has been presumed that the conserved morphologic patterns reflect similar conservation of the underlying execution-phase intracellular pathways. As these execution-phase pathways drive the characteristic, altruistic elimination process that defines apoptosis, they are of considerable interest. The aim of this chapter is to review recent significant advances in understanding the intracellular pathways mediating the execution-phase. As some of the progress (namely much of that having to do with nuclear execution-phase events) has been reviewed elsewhere, this chapter will highlight extranuclear events, which have drawn, up until very recently, less focused attention.

Evolution of the Execution-Phase Concept A Brief History of the Study of the Apoptotic Time Course To understand the execution-phase, it helps to understand the concept of the stages of apoptosis as they have developed so far. To this end, this section represents a brief, broad, by no means comprehensive, history of the evolution of the time course of apoptosis. Initially, apoptosis was defined by its morphologic features. It was distinguished from necrosis as noninflammatory death affecting individual cells showing: chromatin condensation, nuclear envelope convolution, cytoplasmic blebbing or process formation, and cell shrinkage with ultrastructural preservation of organelles (see e.g. Wyllie et al., 1980). The bulk of early studies, for the most part, were concerned with characterizing hallmark features (e.g. Chu-Wang and Oppenheim, 1978; or for reviews: Clarke, 1990; Uchiyama, 1995) and identifying the conditions that lead to apoptosis in various cell types. These early studies established apoptosis as a common, if not fundamental, process, and increased interest in identifying the biochemical mechanisms involved. To investigate such mechanisms, cellular model systems were developed and characterized (see e.g. Wyllie et al., 1984; Martin et al., 1988; Pittman et al., 1993; Mills et al., 1995a). The bulk of apoptotic research switched to the factors responsible for initiating apoptosis within a cell population. If the execution-phase is the goal or end-stage of apoptosis, these studies were of what can be called the "initiation phase" of apoptosis: the first stage of the apoptotic time course, characterized by the cell's recognition and response to an apoptotic signal. General categories of inducers of apoptosis that were studied included growth factor withdrawal (e.g. interleukins or nerve growth factor; NGF), treatment with radiation (UV or X-ray), and pharmacological agents (e.g. chemotherapeutic drugs) (see Figure 1). The initiation phase lends itself easily to study because, for one, induction is a binary process. Either apoptosis ensues, or it is inhibited. Experiments can be designed so that inducing agents or their inhibitors can be applied and, 24 h later, cell death can be assessed. Another advantage of studying this phase is that most of the biochemical pathways involved are shared with already defined cellular processes and, thus, had previously been characterized. The overlap of pathways occurs because

Mechanisms in the Execution-phase

3

the initiation phase is essentially the study of upstream, signal transduction processes that a cell uses to respond to any of a variety of inputs. However, the pathway promiscuity that facilitates study of the initiation phase is no aid in understanding the apoptotic process per se, as the characteristic features of apoptosis occur downstream of initiation. Furthermore, apoptotic initiation phase processes tend to be cell- and usually inducer- specific with limited cross-talk; thus, results are of little general importance outside the specific model system being used. Clearly, to understand the specific process of apoptosis, investigations had to move further downstream from the initiation events. Two relatively early discoveries helped start investigation of downstream events, events that seemed specific to apoptosis and were beyond the initiating signal transduction machinery in the time course. One of these was the watershed discovery and characterization of the bcl-2 family of genes whose products seemed directly involved with the control of apoptosis in perhaps every cell in every species (Vaux et al., 1988; Hockenberry et al., 1990; Korsmeyer, 1992; Vaux et al., 1992). The second was the ongoing characterization of genes involved in C. elegans programmed death, among these the bcl-2 homologue, ced-9, along with ced-3 and ced-4, genes whose importance is in the execution-phase (Ellis et al., 1991; Hengartner and Horvitz, 1994). The finding that Bcl-2 and its related proteins (which, depending on the gene, are either pro- or anti-apoptotic: Adams and Cory, 1998) can initiate or inhibit death in almost every cell suggests that they are downstream of the multiple, promiscuous-initiating pathways. But the fact that, in most cases, initiation or inhibition of apoptosis by the bcl-2 family is still binary suggests that this family is still upstream of or right around the time of commitment of an individual cell to death. In other words, the bcl-2 family is involved with a stage between initiation and execution that has been called the "commitment phase". Study of this phase has been very active during the last several years, with considerable advances made in our understanding of the Bcl-2 family and the role of the mitochondrion (see later this chapter for discussion). Soon after an apoptotic cell commits to death, it undergoes a series of evolutionarily conserved, morphological, and biochemical changes. As mentioned, the stage when these occur has come to be called the execution-phase. The execution-phase is post commitment and, thus, downstream of Bcl-2 and its kin. Investigation into the execution-phase has stemmed primarily from efforts to characterize the mammalian family of ced-3 homologues: the caspases. A simple schematic of the relationship of the three active phases of apoptosis in a given apoptotic cell is identified in Figure 1. Note the initiation phase has many inputs; there are many pathways that will induce apoptosis. The commitment phase is the neck of the apoptotic funnel ', the gateway into the process. It is here modeled as only one common pathway that all cellular apoptotic inducers must invoke if the ultimate goal of apoptosis, the execution-phase, is going to be initiated. Once the execution-phase begins, the processes fan out again, as several relatively independent nuclear and nonnuclear pathways occur. Since the execution-phase is the primary concern of this chapter, these pathways will be discussed at length later. Up until very recently, research into the execution-phase has been limited relative to the other phases of apoptosis. One of the principal reasons for this delay is a technical problem that has long plagued study of execution-phase-specific pathways. This problem is discussed in the next section.

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J.C. Mills

INITIATION

COMMITMENT

EXECUTION ~hromatin ondensation I

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Figure 1. Apoptosis can be divided into three distinct phases. The initiation phase is defined as the stage wherein the signal from any of a variety of inducers of apoptosis is reversibly transduced to the commitment phase. The commitment phase, which seems to involve a single evolutionarily conserved pathway, involves the cell's irreversible commitment to death, ending in the execution phase. The execution phase is irreversible and characterized by the numerous pathways (nuclear and extranuclear) that result in the apoptotic morphology of cell death. Abbreviations used: GF - growth factor; PM - plasma membrane.

Asynchrony To understand why studying the execution-phase is inherently problematic, one must understand that apoptosis is a remarkably asynchronous process. Following a given inducer, onset of the characteristic morphologic features within each cell occurs with marked temporal variability across a population (Oppenheim, 1991; Barres et al., 1992; Raff, 1992; Earnshaw, 1995; Collins et al., 1997). This asynchrony was one of the early defining features of the process in vivo. Wyllie and coworkers (1980), for example, stressed that apoptosis, unlike necrosis, affects individual cells and not populations. Indeed, this feature of apoptosis is one of the key reasons why the role of apoptosis in many human disease processes may have been '. and may continue to be '. underestimated by pathologists (see e.g. Camp and Martin, 1996, and Staunton and Gaffney, 1998, for recent discussions). Despite a presumably clonal population of cells under controlled conditions, apoptosis is asynchronous in the vast majority of in vitro model systems as well (Evan et al., 1992; Earnshaw, 1995; Mills et al., 1997; Messam and Pittman, 1998), although it should be noted that Fas-receptor-mediated apoptosis shows somewhat more synchrony. Asynchrony dictates that cells showing the morphologic features of apoptosis (i.e. those in the execution-phase) represent only a small percentage of the population at any given time. Thus, because biochemical assays generally require large numbers of viable cells, asynchrony hinders biochemical study of the execution-phase (Pittman et al., 1998). To illustrate the point, suppose one wanted to assess the role of specific, key aspects of energetics and metabolism in apoptosis. A typical time course of a population following an apoptotic stimulus is shown in Figure 2A. The curve crudely approximates first-order kinetics (i.e. a consistent proportion of the remaining

Mechanisms in the Execution-phase

5

A. 100

~

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I I Hrs Following Induction of Apoptosis

B. 100

75

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I

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Hrs Following Induction of Apoptosis

Figure 2. Apoptosis is asynchronous, and decrease in ATP levels (as well as many other biochemical parameters) parallels general loss of viability in the population. A) Idealized curve of viability loss following induction of apoptosis. The approximate first-order kinetics of the curve is typical of apoptosis in most model systems. Depending on the system, the x-axis would range from a maximum of 4 h to 4 days. B) Solid line --- plot of ATP levels (idealized from multiple experiments) in a population of apoptotic cells. Note that the curve follows similar kinetics to that in (A). Dashed line -- plot of ATP/ADP ratio in the same cell population. Note there is little change in this parameter (an estimate of energetic viability) across the population, indicating that loss of ATP merely parallels loss of viability across the culture. Thus, intracellular de-energizadon is likely a late-stage event in individual cells involving only a small portion of the viable population at any one time.

population is lost in each block of time). D e p e n d i n g on the cell system, the x-axis can range f r o m a p p r o x i m a t e l y 4 hours to several days, but the shape of the c u r v e is always essentially the same. In the current illustrative e x a m p l e , let us a s s u m e the c u r v e represents N G F - d e p r i v e d neuronal P C 1 2 cells and, thus, the abscissa scale ranges up to 36 h (Pittman et al., 1993; Mills et al., 1995a). A f e w years ago we (the author, with Mr. D a v i d Nelson, Drs. Maria Erecinska and R a n d y Pittman) p e r f o r m e d studies of various energetic parameters on populations of cells, with the hope o f delineating h o w cellular energetics c h a n g e d during early (i.e., a p p r o x i m a t e l y the first 6 h), middle

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J.C. Mills

(approximately 6 to 14 h), and late (14 to 24 h) apoptosis. However, the resulting plot of ATP (and other triphosphate nucleotide) levels in the population almost directly paralleled the viability plot (Figure 2B, and see Mills et al., 1995b). The naive interpretation would be that ATP and energy decrease as cells progress through apoptosis, but this is true only for the population of cells. If the ATP data are replotted as a function of viable cells, a drop to about 80% of control levels occurs around when the first cells in the population begin to die. This level is then maintained more or less until quite late in the time course of death (not shown here; see Mills et al., 1995b). To reinforce the conclusions suggested by these results, we took the ATP-ADP ratio at each timepoint from the entire population of dying cells. This ratio remains essentially constant at around 85 to 90% of control nonapoptotic cultures, so that at 24 h when around half the cells have died, and the cultures are strewn with debris, the remaining cells still have energy levels similar to cells in untreated, healthy control cultures (not shown). The point of this example is that the asynchrony of apoptosis means that only a small percentage of cells will be undergoing characteristic execution-phase events at any time. These execution-phase cells will (no matter when the assay is done in the time course) thus be statistically overrun by unaffected cells, which always constitute the vast majority of the population. Therefore, our experiments demonstrate that changes in cellular energetics occur only late (i.e. possibly during execution-phase) within the time course of apoptosis of individual cells. There will be additional discussion about execution-phase energetics later in the chapter. Our early energetic studies also highlight the importance of specifically isolating execution-phase cells to study apoptosis-specific processes. To characterize the asynchrony observed by other investigators and highlighted by our early energetics studies, the same model system (withdrawal of NGF from neuronal differentiated PC12 cells: Greene and Tischler, 1976) was used to generate long-term time-lapse videotape of fields of apoptotic cells. An example of the type of results obtained using these methods is depicted in Figure 3A. Onset of the morphologic (execution) phase of death, as assessed by membrane blebbing, is highly variable across a population of cells. However, as can be seen in Figure 3B, once the execution-phase begins, it is highly temporally invariant, lasting approximately 45 min in this system (regardless of time following NGF withdrawal or differentiation state of the population at the time of withdrawal; Mills et al., 1997). Similar results were seen with nondifferentiated PCI2 cells (Messam and Pittman, 1998) and have been reported by others in a wide variety of systems (Evan et al., 1992; Earnshaw, 1995; Vidair et al., 1996; Simm et al., 1997). Thus, in most systems, the execution-phase lasts about an hour. One could see how such a short duration relative to the comparably long duration time course of death of the population would tend to skew any biochemical analyses. Graphically speaking, if one were to draw a vertical line through the time course in Figure 3A at any point after about 4 h, one would intersect approximately 85% of the morphologically normal cells. Thus, a harvest at that time to assess energetics or other biochemical parameters (e.g. protein phosphorylation) would be skewed to represent largely the stages of apoptosis upstream of the execution-phase. Only relatively recently have methods to study events specific to the execution-phase been developed and widely applied (and, similarly, only relatively recently have

Mechanisms in the Execution-phase

7

A. Cell# 1 2 3 4 5 6 7 8 9 10 11 12 13 14

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Figure 3. Time-lapse video confirms t h a t o n s e t o f t h e e x e c u t i o n p h a s e is a s y n c h r o n o u s b u t s h o w s t h a t t h e e x e c u t i o n p h a s e , o n c e i n i t i a t e d is of h i g h l y i n v a r i a n t d u r a t i o n . A) Hypothetical data characteristic of time-lapse video microscopy studies of apoptotic cultures. 14 individual ceils in a microscopic field are depicted. Shaded horizontal bars indicate when cells show execution phase morphology (as identified by dynamic membrane blebbing); thin straight lines indicate time spent showing normal morphology. Dashed vertical line indicates time of apoptosis induction. Note that cells can undergo apoptosis prior to induction (indicative of normal background cell loss). Note also that, at any given time, only a small percentage of cells ever show morphologic abnormalities. B) Hypothetical data characteristic of multiple videomicroscopic studies, representing duration of execution phase (as identified by blebbing) relative to time before or after induction of apoptosis in the population (time of induction represented by dashed vertical line). Each point represents a single cell. Note the execution phase lasts approximately 45 rains to an hour in the vast majority of cells regardless of time of onset.

e n o u g h d a t a e m e r g e d to w a r r a n t a r e v i e w o f w h a t is k n o w n specifically a b o u t the e x e c u t i o n - p h a s e ) . S u c h m o d e l s will b e d i s c u s s e d in the n e x t section.

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J.C. Mills

Circumventing Asynchrony The technical problem asynchrony presents to the study of the execution-phase cannot be ignored. Several methods of directing studies specifically to the execution-phase have been developed recently. Some investigators have used differential centrifugation (e.g. Casciola-Rosen et al., 1994) or flow cytometry (e.g. MacFarlane et al., 1997) to isolate actively apoptotic (i.e. execution-phase) cells and study biochemical patterns. These techniques, similar to mechanical methods used to synchronize cells in mitosis, were successfully used in these studies to identify caspase substrates and activation patterns. The limitation of such mechanical isolation methods is that the execution-phase is of such short duration that most cells that have begun the execution-phase will be well into it (or dead) by the time cell sorting is completed. Also, it isn't clear what effect subjecting the (by definition) morphologically altered execution-phase cells to strong mechanical forces like centrifugation would have on many execution-phase processes. Lazebnik et al. (1993, 1994), Solary et al. (1993), and Newmeyer et al. (1994) developed cell-free systems to look at the effect of certain cytoplasmic factors on nuclear features of apoptosis. These systems manipulate cytoplasmic constituents to study the effects on isolated nuclei and have been very helpful in delineating nuclear execution-phase events. From these studies, the mitochondrion has emerged as the crux of the commitment phase. Cell-free systems have also been successfully employed to characterize the nuclear execution-phase. Most of the results detailing such nuclear events to be discussed later in this section were achieved in cell-free systems. Of course, the limitation of cell-free studies is that the nucleus is the endpoint. These systems don't lend themselves to study of cytoplasmic execution-phase events, and plasma membrane-associated events are impossible to study in cell-free systems (at least as they have been used to date). A few other techniques to circumvent asynchrony have been for the biochemical study of nonnuclear execution-phase events. One method is to scan cultures for execution-phase ceils (using nuclear executionphase morphology as a marker) and collect data from this small subset (see e.g. Oberhammer et al., 1994). We used this technique in NGF-deprived neuronal PC12 cells to study phosphorylation of the microtubule-associated protein tau and then used tau phosphorylation state to assess protein phosphatase 2A activity (PP2A; Mills et al., 1998a; see also later this chapter). This technique is limited because only a relatively small subset of biochemical experiments can be performed on individual cells. Another technique for studying nonnuclear execution-phase events was introduced by McCarthy et al. (1997) who found that cultures of fibroblasts overexpressing c-myc could be halted in early execution-phase by inhibitors of caspases (such as z-Val-Ala-Asp-fluoromethylketone, z-VAD-fmk). This technique was further developed to study mediators of apoptotic blebbing (Mills et al., 1998b). Its limitations are the expense of the inhibitors and the fact that, in some cells, z-VAD inhibits blebbing (and presumably other upstream execution-phase events) as well as the nuclear executionphase. Before discussing specific advances in our understanding of nuclear and nonnuclear execution-phase events, it is worth refocusing this discussion on definitions of the phases of death. With the recent mushrooming of research, there has been a concomitant Babelesque mushrooming of terms, and the execution-phase has come to

Mechanisms in the Execution-phase

9

mean different things to different investigators. In the next section, the phases will be as narrowly defined as possible. The definitions should be generally useful, but barring that, will at least be employed consistently to categorize the studies discussed in the remainder of this chapter.

Defining the Terms of the Apoptotic Time-Course The Importance of the Individual Cell To understand the time course of apoptosis, the three phases of initiation, commitment, and execution, one has to remember that these are stages of apoptosis within an individual cell, not within the population. For example, the "commitment point" in apoptosis was initially defined several years ago as the point in a population of dying cells, when 50% of the original population had irreversibly committed to die. That definition is useful for characterizing death kinetics in apoptotic cultures. But to understand, biochemically, what constitutes the initiation, commitment, and executionphases of apoptosis, one must restrict definitions to individual cells. Thus, in Figure 3A, if one follows the timeline of each individual cell, defining the execution-phase becomes relatively straightforward: The commitment and initiation phases are upstream and not represented by outward, morphologic changes; the execution-phase is downstream and characterized by morphologic changes like membrane blebbing. "Commitment point", for the purposes of the time course of apoptosis in an individual cell is the time when that cell commits irreversibly to death. Similarly, the terms "upstream" and "downstream" (or "early" and "late") should refer to progression through the phases of apoptosis in an individual cell. To illustrate a cell that begins blebbing (i.e. enters execution-phase) 6 h following NGF removal (Figures 2A, 3A) is still in a downstream portion of the apoptotic time course, even though 95% of the original population of cells would be alive at that timepoint. On the other hand, a cell that eventually dies is still in the initiation or commitment phase if it has not begun to bleb, even at 48 h, when 75% of the original population has already died. Stricter Definitions of Time-Course Phases So far, the initiation, commitment, and execution-phases have been introduced. In this section, they will be specifically defined (Table 1). The initiation phase comprises multiple pathways that are cell-type and inducing agent specific. NGF withdrawal, for example, induces apoptosis via jun kinase activation (Xia et al., 1995). Ceramidemediated (Cuvillier et al., 1996), PI3-kinase-mediated (Dudek et al., 1997; KauffmanZeh et al., 1997), and stress-activated protein kinase-mediated (Johnson et al., 1996; Verheij et al., 1996; Ichijo et al., 1997) pathways also have all been described. These are initiation events, because the initiation phase is what takes a given cell from recognition of an apoptotic stimulus to the evolutionarily conserved, bottleneck pathway (or gatekeeper) pathway of the commitment phase. So little is known about the onset of the commitment phase that it is difficult to define the transition from initiation, but it can

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be assumed that the commitment phase begins once conserved, predominately deathspecific cellular machinery is engaged. Because the commitment phase is evolutionarily conserved, cytoplasm from any cell that has begun the commitment phase should be able to initiate apoptosis in a cell-free system or in another cell. As certain Bcl-2 family members can induce apoptosis-like death even in yeast and other unicellular eukaryotes, there is evidence for such conservation (see e.g. Ink et al., 1997; Madeo et al., 1997). Note: as we have defined it in this chapter, transition into the commitment phase is not equivalent to commitment to death. Rather, the commitment phase is when the various initiation pathways funnel into evolutionarily conserved machinery that, at some point during this phase, commit a cell to death. The commitment phase, as it is currently understood, hinges on mitochondrial mechanisms, and Bcl-2 family members play the principal role in commitment-phase regulation. Table 1.

Cellular Pathways Location in Cell Duration Cell-type Specificity Reversibility

Initiation P h a s e

Commitment Phase

Numerous(convergent) Plasma membrane, cytoplasm Highly variable (usually long) Cell-typedependent Reversible

One or a small number Numerous(divergent) Mitochondrion Whole cell

Evolution Conservation Variable

??? (probablyshort)

Execution-phase

Invariant,short (~1 hour)

Cell-type independent Cell-typeindependent Switch from reversible Irreversible to irreversibleoccurs during this phase Evolutionarily conserved Evolutionarilyconserved

The execution-phase begins with the pathways leading directly to the hallmark morphologic changes. In many, if not most, cells, it seems to begin with the pathway leading to dynamic membrane blebbing and is well past the actual cellular commitment point (McCarthy et al., 1997; Mills et al., 1997, Brunet et al., 1998; Messam and Pittman, 1998). Note that this is a simple but relatively more restrictive definition than many have adopted, but it serves the purpose of delineating execution from commitment. The execution-phase involves a branching out of several more or less parallel pathways from the bottleneck of commitment. These pathways, which lead to the various hallmark apoptotic changes, are likely much more conserved than those in the initiation phase, because apoptosis (by definition) looks more or less the same regardless of inducer or celltype. However, like the initiation phase, multiple concomitant parallel pathways can be ongoing. It is not binary with regard to death (unlike the commitment phase and the initiation phase). In other words, one can inhibit the morphologic changes individually or as a group and cell death still results. Staurosporine, for example, or inhibitors of myosin light-chain kinase can inhibit blebbing but not death (Mills et al., 1998b). Cytoplasmic and membrane execution

Mechanisms in the Execution-phase

I1

events can occur in cells without a nucleus (Jacobson et al., 1994). Indeed biochemical apoptotic cascades can be activated locally in synaptic compartments. Thus, Mattson et al. (1998a,b) showed that exposure of synaptosomes to staurosporine, or oxidative insults that induce apoptosis in intact neurons, can induce caspase activation, loss of plasma membrane phospholipid asymmetry, mitochondrial membrane depolarization, and release of factors into the cytosol capable of inducing nuclear chromatin condensation and fragmentation. Moreover, Par-4 (prostate apoptosis response-4), a novel apoptosis related protein, can be induced locally in_synaptic compartments. Par-4 is a critical mediator of mitochondrial dysfunction and caspase activation (Guo et al., 1998; Duan et al., 1999). Further, the various apoptotic nuclear events such as DNA fragmentation, chromatin condensation, and lamin disassembly are interrelated but distinct from one another; inhibition of any one pathway does not necessarily affect death (Lazebnik et al., 1993; Oberhammer et al., 1993; Tomei et al., 1993; D.Y. Sun et al., 1994; Hara et al., 1996; Mpoke and Wolfe, 1996; Allera et al., 1997; Hirata et al., 1998). With these definitions, the remainder of the chapter will discuss what constitutes the execution-phase but, first, a brief discussion of recent advances in the study of the commitment phase to aid in further delineating it from execution. A Brief Discussion of the Commitment Phase Commitment-phase research has been prolific and exciting in recent years. To study the commitment phase is to study the mitochondrion (Hirsch et al., 1997; Green and Reed, 1998; Mignotte and Vayssiere, 1998). Mitochondria are the source for two factors known to activate caspases in non-Fas-mediated apoptosis. These two factors are apoptosis-inducing factor (AIF) and cytochrome c. AIF is a protein that has been shown to activate procaspase-3 in vitro and leads to z-VAD-inhibitable initiation of executionphase events (Susin et al., 1996, 1997). Cytochrome c is an inner mitochondrial membrane protein involved in electron transport during oxidative phosphorylation. Disruption of mitochondrial membrane potential leads to release of cytochrome c and/or AIF (Marzo et al., 1998). Cytochrorne c is also released following a variety of apoptotic stimuli I with or without loss of membrane potential (Kluck et al., 1997; Yang et al., 1997) I and microinjected cytochrome c can induce apoptosis in many cell types (Li, F. et al., 1997; Brustugun et al., 1998). Large-scale cytochrome c release is thought by many to mark the commitment point of an individual cell to apoptosis (Green and Reed, 1998). Many Bcl-2 family members have long been known to localize to the mitochondrial outer membrane (Krajewski et al., 1993; Riparbelli et al., 1995) or to reside there under certain conditions (Zha et al., 1996; Wolter, 1997). Anti-apoptotic Bcl-2 family members can inhibit permeability transition, which leads to mitochondrial membrane potential loss (Zamzami et al., 1996; Hirsch et al., 1997). They can also inhibit cytochrome c release (Kluck et al., 1997; Yang et al., 1997). Bax (an apoptosisinducing Bcl 2 bomologue) can directly activate the permeability transition pore (a complex of several trans-mitochondrial membrane proteins that, when activated, forms pores, leading to permeability transition and eventual membrane potential collapse) by binding a key pore protein, the adenine nucleotide translocator (Marzo et al., 1998).

12

J.C. Mills

These various commitment-phase interactions have been linked to the subsequent execution-phase events via AIF and cytochrome c. As discussed above, AIF leads to caspase-3 activation and nuclear execution-phase events. Cytochrome c binds APAF1, the mammalian homologue of the ced-4 C. elegans death gene (Ellis and Horvitz, 1986), in a complex with pro-caspase-9 (P. Li et al., 1997; Zou et al., 1997). The binding of cytochrome c and APAF1 is thought to activate pro-caspase-9, which, in turn, activates caspase-3 and leads to the hallmark events of the execution-phase (P. Li et al., 1997; Wilson, 1998). Bcl-2 family members seem only to be involved with the commitment phase, and have little effect once the execution-phase is in progress (see e.g. Ellerby et al., 1997). Multiple studies of a variety of systems, however, suggest that caspase activation is a postcommitment event (excluding Fas-mediated death, where caspases play a role in the initiation phase). Inhibition of caspases in multiple-cell types delays or alters the morphology of death but does not prevent death from occurring (Xiang et al., 1996; McCarthy et al., 1997; Brunet et al., 1998; Mills et al., 1998b). On the other hand, some non-Fas-dependent apoptotic systems show a slightly more upstream role for a caspase or caspases (Deshmukh et al., 1996; Levkau et al., 1998). Clearly, this issue is not fully resolved (and caspase activation may in fact be different in different cell types), but for the purposes of this chapter, caspase induction will be considered an execution-phase (i.e. postcommitment phase) event.

The Execution-phase Nuclear Events The execution-phase, as discussed, seems to involve multiple nonintersecting pathways that lead from the relative bottleneck of commitment to the many distinctive, presumably critical features that distinguish apoptosis from nonphysiologic death. The data to date suggest that the mitochondrion mediates the commitment phase, but execution-phase transduction of the apoptotic signal must eventually involve pathways to the cytoplasm, cell membrane, and nucleus (Figure 4). So far, the majority of what is known about the execution-phase concerns nuclear events largely because cell-free systems have proved the most valuable for execution-phase studies so far, and these use nuclei as the target of study. Nuclear execution-phase events can be classified readily as: (1) upstream, involving transduction of the signal from the mitochondrion into the nucleus which is mediated predominately by caspases; and (2) downstream, the events or endpoints themselves (DNA laddering, lamin disassembly, chromatin condensation).

Upstream Events: The Caspases Caspases have been discussed and reviewed in numerous recent articles as well as elsewhere in this volume, so the discussion here will be brief. There are at least 13 different caspases (Thornberry and Lazebnik, 1998), and some of these have been shown to have several forms (Faleiro et al., 1997; Martins et al., 1997). Caspases are cysteine

Mechanisms in the Execution-phase

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proteases that exist in the cytoplasm in proenzyme form and have specific (relative to other protease families) substrate requirements: they cleave only after aspartate residues in certain configurations (Porter et al., 1997). Pro-caspases are excellent substrates for activated caspases; thus, it is generally thought that apoptosis may involve a cascade of caspase activation with potential positive feedback amplification. Like all proteases, caspase substrate specificity is easiest studied in vitro, but in vitro systems cannot account for compartmentalization, concentration, natural inhibitors, and competitors that occur in cells. Thus, there is still controversy about the in vivo "significance" of many proteins that have been identified as caspase substrates. Another complication of caspase study is that, although good inhibitors exist to differentiate intracellular caspases from other proteases, there are no well-characterized inhibitors that are specific to individual caspases (Villa et al., 1997). Despite the difficulty in studying normal intracellular caspase activity, the most important upstream execution-phase pathways have been recently established. As discussed in the previous section, cells leave the commitment phase with the release of cytochrome c, which binds APAF-1 and then apparently binds and activates caspase-9 (Figure 4). Activated caspase-9, in turn, activates caspase-3 (P. Li et al., 1997; Zou et al., 1997; Wilson, 1998). In Fas-mediated apoptosis, caspase-8, which is directly linked to the cell surface signaling transduction machinery, seems to play a role like that of caspase-9 (Srinivasula et al., 1996; Medema et al., 1997). What role the mitochondrion and commitment-phase machinery have in the Fas system is unclear, although the downstream, execution-phase events are similar to those in every other system. Inhibition of caspase-8 appears to inhibit Fas apoptosis completely, so caspase-8 is an initiation- or commitment-phase protease. Caspase-3, on the other hand, is downstream of commitment and seems to have the same execution-phase role it does in non-Fas-mediated apoptosis (Hirata et al., 1998). Caspase-3 is a key transducer and effector of execution-phase nuclear events in almost every system. Inhi~bition of caspase-3 in dozens of studies abrogates nuclear apoptosis, and caspase-3 has a broad range of biologically relevant substrates (some of which will be discussed). The other caspase with a well-characterized role in nuclear apoptosis is caspase-6. Caspase-6 seems to be the principal lamin caspase in intact cells (see below and Takahashi et al., 1996). Though in some systems caspase-6 was thought possibly to be upstream of caspase-3, recent studies in intact monocytes (MacFarlane et al., 1997), a cell-free Xenopus system (Farschon et al., 1997), and Fas-stimulated Jurkat cells (Hirata et al., 1998), place caspase-3 as an upstream activator of caspase-6. Corroborating a slightly downstream role for caspase-6, the nuclear lamin breakdown mediated by caspase-6 has generally been shown to be a later step that precedes chromatin condensation but follows initial DNA fragmentation (Oberhammer et al., 1994; Ghibelli et al., 1995; Lazebnik et al., 1995). Accordingly, inhibition of lamin breakdown either by caspase-resistant mutant lamins (Rao et al., 1996), or by specific caspase-6 (but not caspase-3) inhibition (Hirata et al., 1998), leads to DNA fragmentation and death, but not lamin breakdown and chromatin condensation. Summarizing, the upstream regulation of nuclear apoptosis appears to depend on activation of caspase-3, which cleaves a wide variety of substrates itself and also results in activation of caspase-6, which cleaves a few of its own, mainly downstream substrates. The next section

Mechanisms in the Execution-phase

15

discusses what is known about how the cleavage of some of these substrates may result in the nuclear features that have come to define apoptosis. Downstream Nuclear Apoptotic Events DNA fragmentation DNA fragmentation is one of the hallmarks of the execution-phase. The characteristic cleavage of DNA into fragments that are the size of multiples of the intemucleosomal length occurs in the vast majority of apoptotic cells and is very specific for apoptosis. This fragmentation appears to be both an early and late execution-phase event, as many cells show a sequence of fragmentation stages, and fragmentation can be inhibited at various intermediate steps by protease inhibitors or chelators (Weaver et al., 1993; Sun and Cohen, 1994; Lagarkova et al., 1995; Pandey et al., 1997). Generally, it is convenient to distinguish two phases. The first involves the formation of high molecular weight DNA fragments, (2 Mb down to 50 kb), and is thought to occur early in the execution-phase, upstream or concomitant to chromatin condensation (Ghibelli et al., 1995; Hara et al., 1996). The second and final phase involves cleavage of DNA into the characteristic, intemucleosomal-sized fragments (DNA "laddering"); this phase appears to be downstream of chromatin condensation and may, in fact, be among the last things a cell does before the end of the execution-phase (Mpoke and Wolfe, 1996). Numerous proteases and endonucleases have been implicated in DNA fragmentation in both upstream and downstream roles. The topic has been one of intense debate (see Montague and Cidlowski, 1996 for review and see also Pandey et al., 1997). However, there have been recent large strides forward in characterizing the mechanisms underlying this hallmark nuclear execution-phase event. For example, the recent identification of DNA fragmentation factor (DFF), a protein heterodimer of 40 and 45 kDa subunits that can be cleaved by caspase-3 to induce DNA fragmentation (Liu et al., 1997), represents the first direct link between the upstream event of caspase activation and one of the hallmark, downstream nuclear features of apoptosis. Two recent articles substantially strengthen the links between the caspases and internucleosomal fragmentation (Enari et al., 1998; Sakahira et al., 1998). These authors describe a caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD). ICAD, which is a murine homologue of the human DFF 45 protein, appears to act as a chaperone necessary for CAD cytoplasmic stability until caspase-3 activation mediates ICAD cleavage. ICAD cleavage presumably frees CAD to enter the nucleus in the execution-phase, where the authors show specifically that internucleosomal cleavage is dependent on CAD. It isn't clear what role, if any, CAD has in the upstream, large molecular weight fragmentation. Thus, critical caspase-linked, upstream endonucleases may still be undiscovered. Other indirect links between DNA fragmentation and the caspases also exist. DNA-dependent protein kinase (DNA-PK), for example, is cleaved by caspase-3 and thereby inactivated (Song et al., 1996; McConnell et al., 1997). DNA-PK is a DNA repair enzyme whose inactivation would be expected to promote fragmentation by ceasing normal restorative efforts. Poly(ADP-ribose) polymerase (PARP) is one of the earliest and most well-characterized substrates of caspases. PARP is cleaved by caspase-3

16

J.C. Mills

(Takahasi et al., 1996) and/or caspase-7 (Lippke et al., 1996; Hirata et al., 1998). PARP is also involved in DNA repair and is deactivated by caspase cleavage, which, as for DNA-PK, may promote DNA fragmentation by hindering normal attempts to stop DNA damage (for review, see Porter et al., 1997). Chromatin Condensation

Chromatin condensation is the process characterized by the formation of globular and marginated, hemispheric aggregates of chromatin that are easily recognizable by light and electron microscopy. This phenomenon is perhaps the most universal, specific feature of apoptosis. Yet, very little is known about its underlying mechanisms. In addition to the technical hindrance of asynchrony (see above discussion), a factor that might have impeded study is that condensation was long thought to be a byproduct of DNA fragmentation. However, as discussed above, the two processes, if not wholly independent can be largely separated from each other and almost certainly involve many independent mechanisms. Two studies have addressed the issue of condensation itself and shown it to be associated with histone alterations. One group describes a specific deubiquination of histone H2A (Marushige and Marushige, 1995). Condensation of chromatin in metaphase of mitosis also involves a similar (albeit reversible) deubiquination of H2A. The authors state that ubiquination is thought to inhibit higher ordering of chromatin. It remains to be seen how H2A deubiquination might be mediated or how the process might be tied to already characterized executionphase pathways. The other histone study shows that histone H4 becomes deacetylated as condensing chromatin undergoes fundamental changes in nucleosome arrangement (Allera et al., 1997). They report progressive "clumping" of chromatin into dense central bodies with tortuous 11 nM fibers rosetting around each core. Nucleosomes were seen to lose their normal, 30 nM solenoid-ordered radial configuration and adopt tight face-to-face packaging. Again, it is unclear how these changes relate to known execution pathways. The only other significant headway into the problem of chromatin condensation involves a link to lamin disassembly. Lamins, which provide the structural support for the nuclear envelope, are disassembled in apoptosis by a process that requires caspases, most likely caspase-6 (Takahashi et al., 1996; Martelli et al., 1997; Hirata et al., 1998). As lamin disassembly and chromatin condensation are late execution-phase events and chromatin condensation involves substantial margination to the nuclear membrane, it was proposed several years ago that dismantling of the lamin network may be critical for condensation (Lazebnik et al., 1993). Although no direct biochemical evidence links the two processes, several other studies support the theory that lamin disassembly precedes and is required for normal condensation (Rao et al., 1996; Hirata et al., 1998). As caspase-mediated cleavage of lamins is well established, the dependence of chromatin condensation on lamin disassembly represents the closest link from chromatin condensation to the upstream execution-phase pathways.

Mechanisms in the Execution-phase

17

Extranuclear Events

Although there have been quite a few studies in recent years, the investigation of extranuclear execution-phase events has lagged that of nuclear events. Extranuclear events cannot be effectively studied in cell-free systems, so investigators have used a number of model systems for circumventing asynchrony. This variation in cell systems and the current lack of focused approach of the field toward any specific extranuclear apoptotic event complicate somewhat the review of this aspect of the execution-phase. Also, to date, there are no direct links between the commitment phase and actual downstream morphologic processes, as can be shown for caspase-mediated activation of CAD and DNA fragmentation. However, a number of potentially important caspase substrates have been identified that seem to play key roles in many cell types in the execution-phase. There has been some success in the study of membrane blebbing and apoptotic body formation. Namely, a few of the important cytoskeleton-associated proteins have been identified and a putative mechanism identified for cellular blebbing. Certain caspase-dependent pathways have been identified as critical for apoptotic body formation. Study of these cell shrinkage-related processes seems on the verge of important advances, and there are also some promising recent discoveries in other aspects of extranuclear execution-phase. The remainder of this chapter will consist of a relatively comprehensive review of what is known about extranuclear execution-phase events. Upstream Extranuclear Events

The upstream and downstream distinction that was made for nuclear execution-phase events is less useful in the extranuclear setting, largely because the events upstream of extranuclear execution-phase events are much less well-characterized. As will be discussed, there is a role for caspase-3 in several downstream extranuclear events; thus, presumably, activation of caspase-3 (the mechanism of which has already been discussed) is a key event. Interestingly, however, dynamic plasma membrane blebbing, a key feature of extranuclear apoptosis, one of the hallmarks of apoptosis, and one of the earliest signs of the execution-phase, does not require caspases in many model systems (Xiang et al., 1996; McCarthy et al., 1997; Mills et al., 1998b). One study using a Fas-dependent model death implicated caspase-7 in membrane blebbing, because specific inhibition of caspases 3 and 6 had no effect on the process (Hirata et al., 1998). However, because plasma membrane changes were only abrogated by inhibition of all caspases, and Fas-mediated death requires caspase-8 as an initiation or commitment phase event, caspase-7 could not be specifically implicated. Another group reports that caspase-3 is required for blebbing in TNF-mediated (i.e., Fas-dependent) death, as tumor cells homozygous for loss of caspase-3 function only bleb when caspase-3 is reintroduced (Janicke et al., 1998). There have been no studies to date of how cytochrome c might affect membrane blebbing or other extranuclear events independent of caspases, and this avenue of exploration might be promising. Until upstream events are better characterized, the best way to review and analyze the extranuclear executionphase is to examine the downstream processes.

18

J.C. Mills

Downstream Extranuclear Events Cell shrinkage, membrane blebbing, apoptotic body formation One of the hallmark, apoptosis-specific, execution events is plasma membrane blebbing (also termed "zeiosis" or "cytoplasmic boiling"). Necrotic cells also extrude blebs, but these are unidirectional (i.e., they are extruded but not retracted), large, and represent an end-stage change (see e.g. Coakley, 1987; Phelps et al., 1989; Herman et al., 1990). Membrane blebbing also sometimes occurs as a byproduct of mitosis, though, unlike apoptotic blebbing, mitotic blebbing is preceded by microvilli formation, is characterized by smaller blebs that are more varied in size, and shows a different pattern of underlying F-actin organization (Laster and Mackenzie, 1996). Apoptotic blebbing is one of the first signs of the execution-phase and is characterized by dynamic extrusion and retraction of surface protrusions. Apoptotic blebbing can be specifically inhibited without inhibition of nuclear changes or death (Cotter et al., 1992; Endresen et al., 1995; Levee et al., 1996; McCarthy et al., 1997; Mills et al., 1998b). This is to be expected, because, as previously discussed, execution-phase events are post-commitment to death, so inhibition of any execution-phase event does not stop a cell from dying. In addition, because postcommitment events seem to depend on multiple, non-intersecting pathways, inhibition of one pathway does not necessarily inhibit another. For example, as has been mentioned, DNA fragmentation and chromatin condensation, two nuclear phase execution-phase hallmarks, depend on parallel, mostly independent pathways, and neither is critical for a cell to die (Lazebnik et al., 1993; Sun et al., 1994; Rao et al., 1996; Brunet et al., 1998). Before discussing some of the mechanics underlying blebbing, it will help to organize and redefine some of the associated terminology. Blebbing is part of a broader category of extranuclear apoptotic change: reduction in cell volume. Almost all apoptotic cells shrink, the vast majority bleb, and many (though possibly not the majority) form apoptotic bodies. The onset of the execution-phase is marked by centripetal contraction and retraction from the substrate; the cell "rounds up". Next, an execution-phase cell blebs for a while, eventually stops, and then undergoes a final condensation or forms apoptotic bodies. These bodies are the result of the pinching of the endexecution-phase cell into multiple small, membrane-enclosed fragments (Kerr and Harmon, 1991). Blebbing and apoptotic body formation may be related, but they are not identical processes. For example, agents (such as the cytochalasins, inhibitors of actin polymerization, and staurosporine, a general kinase inhibitor) that inhibit blebbing (Tanaka et al., 1994; Endresen et al., 1995; Mills et al., 1998b) also inhibit apoptotic body formation (Cotter et al., 1992; Tanaka et al., 1994; Levee et al., 1996). However, early plasma membrane/cytoplasm changes, such as initial cell body shrinkage and blebbing, seem to be upstream of apoptotic body formation in most cells. One recent study showed that specific caspase-3 and 6 inhibition prevents apoptotic body formation but does not affect the blebbing that occurs earlier in the executionphase (Hirata et al., 1998). These data correlate well with the finding that apoptotic body formation depends on DEVD-inhibitable (i.e. caspase-3-1ike) caspases via a mechanism that involves caspase-mediated cleavage of p21-activated kinase 2 (PAK2) (Rudel and Bokoch, 1997;

Mechanisms in the Execution-phase

19

Lee et al., 1997; reviewed in Bokoch, 1998). Hirata et al. (1998) further define PAK2 cleavage (and apoptotic body formation) as being caspase-3 (and not 6 or 7) dependent. The PAKs are a family of kinases whose activity is modulated by the binding of the small G-proteins Rac and Cdc42 (for review: Sells and Chernoff, 1997). Caspasemediated cleavage results in activation of PAK2, which can then mediate apoptotic morphological changes, the best characterized of which seems to be apoptotic body formation. It is also of interest that PAKs have been found to phosphorylate myosin light chain in vitro (see Bokoch, 1998 for review and references), as conventional non-muscle myosin (myosin II) is regulated by specific phosphorylation of its light chain. As will be discussed later in this section, actin/myosin II interactions have been shown to be important in execution-phase cytoplasmic morphologic changes. The dramatic, dynamic shape changes that characterize apoptotic blebbing suggest that the cytoskeleton plays a prominent role in the process. And, indeed, it has been shown that blebbing, like apoptotic body formation depends on polymerized actin, as actin disassembly promoters, namely, the cytochalasins, prevent it (Endresen et al., 1995; Mills et al., 1998b). Furthermore, alterations in actin binding proteins have been associated with a blebbing morphology. For example, tumor cells lacking actin binding protein (also known as filamin) bleb continuously (Cunningham et al., 1992), and peroxideinduced blebbing correlates with alpha-actinin and talin cleavage (Miyoshi et al., 1996). Similarly, one of the best-characterized substrates of caspase-3-1ike proteases is alphafodrin, which is an actin-binding protein that helps stabilize actin-membrane interactions (Martin et al., 1995; Cryns et al., 1996; Nath et al., 1996; Vanags et al., 1996). And actin itself has been purported to be cleaved by caspases in apoptosis (Mashima et al., 1995; Kayalar et al., 1996; McCarthy et al., 1997), although many other groups report seeing no caspase-mediated actin proteolysis in multiple systems (Levee et al., 1996; Song et al., 1997; Brancolini et al., 1997). Still another group sees actin cleavage mediated by calpains but not caspases (Brown et al., 1997). In any case, neither actin nor fodrin cleavage has been strictly correlated with the onset of execution-phase (when blebbing occurs), and both may be late-stage execution-phase events. Furthermore, polymerized actin seems indisputably necessary for blebbing to occur, and proteolysis would counteract the process. And, finally, blebbing occurs in many systems even in the presence of broad-spectrum caspase inhibition (McCarthy et al., 1997; Mills et al., 1998b). Perhaps actin cleavage is an end-stage means of shutting offblebbing. Another cytoskeletal-associated protein, Gas2, is also caspase-cleaved in apoptosis and has been reported to play a role in apoptosis-associated extranuclear morphologic changes (Brancolini et al., 1995). Gas2, when hyperphosphorylated during GO to G1 transition, correlates with membrane ruffle formation. Overexpression of a caspasetruncated Gas2 leads to cell-shrinkage and process formation. Both the cell body and the processes in the shrinking cell become filled with prominent filamentous (i.e. phalloidin-stainable) actin, suggesting a role for caspase-cleaved Gas2 in apoptotic morphology changes. The authors note, however, that not all cells contain appreciable Gas2, and those that don't still undergo normal apoptosis. Furthermore, the authors do not report that the cytoskeletal reorganization seen during Gas2 overexpression involves blebbing, and other studies of actin in normal apoptosis do not correlate with the actin changes seen in the Gas2 cells (see below and Laster and MacKenzie, 1996;

20

J.C. Mills

Pitzer et al., 1996). Also, as mentioned, blebbing occurs in many cells in the absence of, or upstream of, caspase-3 activation. Thus, the role of Gas2 is likely to be primarily downstream of blebbing, perhaps to further promote shrinkage or to assist in apoptotic body formation. Recently, gelsolin, another protein with actin-interacting properties was identified as a caspase substrate (Kothakota et al., 1997). Gelsolin is a calcium-activated, polyphosphoinositide-inhibited actin regulatory protein that can sever and cap the fastgrowing end of filamentous actin (promoting disassembly of long filaments) but also can serve as a nucleator to promote reassembly (see e.g. Kwiatkowski et al, 1989; Weeds et al., 1991). Thus, gelsolin is often thought of as an actin reorganizer, breaking down long, older filaments in favor of new polymerization sites. Kothakota et al. (1997) have recently shown that gelsolin is specifically cleaved by caspase-3 in vitro and in vivo. The caspase-truncated gelsolin shows greatly increased actin severing in vitro, so that severing predominates over monomer binding (i.e. nucleating). Over-expression of cDNA designed to encode a protein mimicking post-caspase-cleaved gelsolin leads to rapid actin depolymerization and unspecified cytoplasmic morphologic changes. Neutrophils from gelsolin knockout animals (i.e. gelsolin -/- mice) take twice as long to initiate blebbing and DNA fragmentation as controls. HeLa cells, which are normally devoid of gelsolin, initiate apoptosis much more rapidly when they are transfected with full-length gelsolin. Interestingly, Kothakota et al. found no effect on caspase-3 activity by expression of gelsolin. This is in contrast to another recent study (Ohtsu et al., 1997), whose authors report inhibition of apoptosis by gelsolin overexpression in a mechanism that seems to be upstream of caspase-3 activation. In other words, gelsolin overexpression leads to blocking of both apoptosis and caspase-3 activation. The differing results suggest the need for additional, clarifying experiments. Both studies use predominately Fas-mediated apoptosis as a model system; thus, the implications for morphologic changes in non-Fas systems have not yet been explored. The implications of the studies with regard to the mechanisms underlying cell blebbing also await further study, as, although Kothakota et al. report "morphological changes" following overexpression of the activated, truncated gelsolin, they do not specify whether this means normal blebbing (e.g. with the same kinetics and duration). Also, they report that cells devoid of gelsolin (either HeLa cells or neutrophils from knockout animals) still bleb, even though onset is delayed, so gelsolin must not be absolutely necessary for blebbing. Finally, blebbing also occurs in caspase-3-inhibited cells, which further suggests that gelsolin truncation is not critical for the process of blebbing. It would be interesting to determine whether gelsolin expression has any effect on duration of blebbing. If not, one would expect that gelsolin may be involved either directly or indirectly in initiation, rather than maintenance of, blebbing. A direct role in initiation is a tempting hypothesis, because gelsolin is known to mediate switching of cytoplasm from a firmer ("gel") state to a more soluble ("sol") state; thus, in apoptosis, it might reorganize the cytoskeleton so that it is in a "bleb-ready" sol state (Yin and Stossel, 1979; see also: Janson and Taylor, 1993, for discussion of gel-sol switching). Presumably, as most cells continue to bleb for about an hour, some sort of steady state is reached, and gelsolin might not be expected to have any role in the maintenance of this state. The fact that homozygous

Mechanisms in the Execution-phase

21

gelsolin null cells still bleb could mean that another reason why blebbing might occur even in gelsolin's absence is that other actin severing proteins can compensate. Recent studies of neurons from gelsolin knock-out mice suggest an interesting role for this actin-severing protein in modulating neuronal cell death. Previous studies have shown that treatment of cultured hippocampal neurons with cytochalasin D, an actin-disrupting agent, can protect those neurons against excitotoxic and oxidative injury (Furukawa et al., 1995; Furukawa and Mattson, 1995). Data in the latter study suggested that the protective mechanism of actin depolymerization involved stabilization of intracellular calcium levels. Studies of hippocampal neurons cultured from gelsolin knock-out mice have shown that neurons lacking gelsolin exhibit increased vulnerability to excitotoxic cell death (Furukawa et al., 1997). Whole cell patch-clamp analyses of ion currents in neurons containing or lacking gelsolin showed that currents through voltagedependent calcium channels and NMDA type glutamate receptor channels are enhanced in neurons lacking gelsolin. Specifically, the rundown of currents seems to be reduced in neurons lacking gelsolin, which is correlated with enhanced calcium influx through voltage-dependent calcium channels and NMDA receptors (Furukawa et al., 1997). Administration of the seizure inducing excitotoxin, kainic acid, to wild-type and gelsolin knock-out mice demonstrated increased vulnerability of hippocampal neurons to seizureinduced injury in the gelsolin knock-out mice. Collectively, these studies have led to the proposal that gelsolin plays an important role in a feedback pathway in which increased intracellular calcium levels lead to active depolymerization, which in turn suppresses calcium influx through voltage-dependent channels and NMDA receptors. Recently, we also undertook experiments that helped elucidate some of the mechanisms underlying blebbing (Mills et al., 1998b). Z-VAD-fmk, a broad-spectrum caspase inhibitor, was used to arrest apoptosis of serum-deprived PC12 cells. Z-VAD delays death and nuclear apoptosis for several days in this system (similar to those reported in other models of apoptosis by McCarthy et al., 1997) without preventing normal progression into the blebbing phase. Thus, because z-VAD-arrested cells seem to stall in an early, pre-nuclear, execution-phase, much of the asynchrony inherent to apoptosis is avoided, and biochemical determinations of mediators can be performed. Using several kinase inhibitors, a critical role for myosin light chain kinase (MLCK) activation was demonstrated, and, in other experiments, myosin light chain phosphorylation was shown to correlate with a blebbing morphology. As mentioned earlier, conventional, smooth-muscle or non-muscle myosin (myosin II) is activated to contract against actin by phosphorylation of its myosin light chain, as mediated, in part, by myosin light chain kinase (Kohama et al., 1996; Gallagher et al., 1997). Myosin light chain (MLC) phosphorylation can also be stimulated by the small G protein, Rho, which activates Rho-associated kinase (ROK), which, in turn, both phosphorylates MLC directly (Amano et al., 1996) and inactivates the MLC phosphatase (Noda et al., 1995; Kimura et al., 1996). C3 transferase, an enzyme which inactivates Rho (Sekine et al., 1989; Aktories et al., 1990; Paterson et al., 1990) also inhibits blebbing. Cytochalasin D, similar to earlier reports (Endresen et al., 1995) also stops blebbing, and a general inhibitor of motor function of both conventional and non-conventional myosins (2,3-butanedione monoxime, BDM) greatly slows blebbing kinetics (i.e. both extrusion and retraction of blebs). A report from another group demonstrates blebbing in response

22

J.C. Mills

to microinjection of catalytically active MLC (Fishkind et al., 1991). Taken together, the data suggest that blebbing depends on myosin II-actin-mediated contraction. Myosin II in non-muscle cells has been implicated in multiple, critical forcegeneration-dependent functions (for reviews: Grebecki, 1994; Maciver, 1996; Mitchison and Cramer, 1996), but it is a large, filamentous protein, which is not thought to be membrane-associated. It is, on the other hand, thought to interact with the cortical actin ring, which is linked to the plasma membrane, regulating membrane structural stability and motility. Myosin contraction of the cortical actin is thought to produce a centripetal force that compresses the cytoplasm (see e.g. Grebecki, 1994). Although there are some decreases in the levels of detergent-soluble and insoluble, F-actin in the execution-phase (Levee et al., 1996), filamentous, polymerized (and polymerizable) actin is necessary for blebbing (Tanaka et al., 1994; Endresen et al., 1995; Levee et al., 1996; Mills et al., 1997). In addition, execution-phase F-actin has been shown to re-organize, concentrating specifically at the base (but not inside) of blebs (Laster and MacKenzie, 1996; Levee et al., 1996; Pitzer et al., 1996; Brancolini et al., 1997). A recent report indicates that cholecystokinin-mediated blebbing (which resembles apoptotic blebbing) in pancreatic acinar cells also appears to depend on actin reorganization along with myosin II relocation and activation (Torgerson and McNiven, 1998). To explain how a ring of myosin-II-generated actin contraction can cause extrusion of a bleb without being directly associated with the bleb itself, a model of blebbing was proposed. The model postulates that an as yet unidentified upstream execution-phase signal induces MLCK and/or Rho activation, which causes myosin II-mediated cell contraction and shrinkage (Figure 5). In non-apoptotic cells, just such a Rho-mediated contraction has been shown to occur without cell blebbing (Jalink et al., 1994; Tigyi et al., 1996; Gebbink et al., 1997). In apoptotic cells, however, membrane-actin interactions might not be as sturdy (due to weaknesses in membrane-cytoskeletal links) so that when myosin contracts the actin ring centripetally, the plasma membrane may not contract uniformly. Blebs may form in these regions of weakness as cytoplasm is extruded through a focal opening in the cortical cytoskeleton (Figure 5). At least two good candidates for proteins critical for maintaining actin-membranecytoskeletal links that are broken down in the execution-phase have been described. Fodrin, for example, is cleaved by caspases in apoptosis (as was noted above), but it also can be cleaved by calpains (Miyoshi et al., 1996; Nath et al., 1996). Interestingly, preliminary experiments in the PC12 system show that calpain inhibition prevents blebbing in apoptosis in the absence of caspases (Mills et al., 1998b). Another group has recently shown that ezrin/moesin/radixin (a family of closely related proteins that, like fodrin, normally help link the membrane to the actin cytoskeleton) translocate from the plasma membrane to the cytoplasm in an early stage of the execution-phase (Kondo et al., 1997). The translocation in these experiments was prevented by caspase inhibition, but apoptosis was induced in this system by the Fas receptor, and, as discussed above, caspase activation is an initiation (i.e. upstream) event in Fas-mediated apoptosis. Thus, ERM translocation may not directly involve caspases. One question not addressed by the model in Figure 5 is how apoptotic blebs retract. Given that the general inhibitor of myosin motor function, BDM, seems to inhibit both extrusion and retraction of blebs (whereas cytochalasins and MLCK inhibitors

23

Mechanisms in the Execution-phase

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J.C. Mills

seem to inhibit extrusion only), there may be a role for non-conventional myosins in bleb retraction (Mills et al., 1998b). It is also possible that blebs simply recoil from some inherent elastic properties of the membrane. Blebbing is a dynamic process and, therefore, requires energy, but it may only need energy in one direction (in this case, i.e. for extrusion). A final note on blebbing: Although caspase inhibition does not stop blebbing in several non-Fas-mediated systems, it apparently does stop blebbing in normal endothelial cells induced to die by growth factor withdrawal (Levkau et al., 1998), in COS7 cells induced to die by tamoxifen (Nicole Stone and Randy Pittman, personal communication), and in HL-60 human leukemia cells treated with camptothecin (Shimizu and Pommier, 1997). Also, primary cultures of sympathetic neurons shrink over a long time course but don't bleb when treated with z-VAD under an apoptotic stimulus and apparently recover completely when the apoptotic stress is removed (Deshmukh et al., 1996). And dorsal root ganglion neurons in culture shrink but don't die for up to 11 days following an apoptotic stimulus when micro-injected with cDNA encoding crmA (Gagliardine et al., 1994), a viral gene whose product blocks caspases 1 and 8 and also possibly 3 and 6 (Villa et al., 1997). Thus, caspase activation may be upstream of blebbing in many cell types, just as it seems to be upstream of nuclear execution-phase events in almost all cell types. Perhaps in those cell types not dependent on caspases for blebbing and initial non-nuclear execution-phase changes, other proteases (such as calpains) may play the role caspases typically play in other cells (or even in the same cells under conditions when caspases are not being actively inhibited). Or put another way, maybe many cells have no means of compensating for caspase blockade in the initiation of extranuclear execution-phase events. Cytoskeletal events not necessarily related to cell shrinkage and miscellaneous cytoplasmic events

Given the dramatic shape changes in the execution-phase, most investigators have approached the cytoskeleton in apoptosis with the intent to understand blebbing and shrinkage. And, as discussed, actin and myosin seem to play an important role in cellular blebbing. However, other structural and cytoskeletal changes have been described that don't necessarily have a direct role in hallmark execution-phase morphologic features. For example, intermediate filaments, such as cytokeratin and vimentin, have been shown to aggregate and eventually be degraded in the executionphase (van Engeland et al., 1997). And it has long been known that the activation of transglutaminase (an enzyme that catalyzes protein-protein crosslinking) in apoptosis can lead to profound structural changes within cytoplasmic proteins (Fesus, 1993). Of the non-actin cytoskeletal proteins, most is known about microtubules. The microtubular system has been shown to undergo execution-phase reorganization by some groups (Pittman et al., 1994; Ireland and Pittman, 1995; van Engeland et al., 1997) and specific disassembly by others (Bonfoco et al., 1996; Canu et al., 1998; Mills et al., 1998a). Further temporal analysis of microtubules in apoptosis indicate that microtubule disassembly is one of the first execution-phase events in neuronally differentiated PC12 ceils (Mills et al., 1998a). Microtubule assembly in this system appears to be upstream of chromatin condensation. Two other early extranuclear

Mechanisms in the Execution-phase

25

execution-phase events were also noted: protein phosphatase 2A activation (PP2A) and resulting dephosphorylation of the microtubule-stabilizing protein tau. Microtubule disassembly also occurs in apoptotic Chinese hamster ovary (CHO) cells, along with PP2A-mediated activation of exogenously expressed tau (Mills et al., 1998a). Inhibition of microtubule disassembly (with the microtubule stabilizing agent taxol) inhibits PP2A activation and tau dephosphorylation, suggesting that microtubule disassembly occurs first, leading to release of microtubule-bound PP2A, which, in turn, mediates tau dephosphorylation. In cerebellar granule neurons, tau is not only dephosphorylated in the execution-phase but also cleaved in a caspase-dependent fashion (Canu et al., 1998). Tau dephosphorylation may lead to easier degradation, as hyperphosphorylated tau is not as easily proteolyzed and can accumulate in the characteristic paired helical filaments of Alzheimer's disease (Litersky and Johnson, 1992). Experiments from another group indicate that PP2A, in Fas-mediated death in Jurkat cells, can be a substrate for Caspase-3 (Santoro et al., 1998). The Caspase-3 cleavage site is on the regulatory Act subunit and results in apparent dissociation of the regulatory subunit from the PP2A trimer. Furthermore, the authors show a general increase in PP2A activity late in the time course of death of the population of cells. Although the increase was not correlated specifically to the execution-phase, the results suggest a model whereby Caspase-3 activates PP2A during the execution-phase. As execution-phase PP2A activation appears to occur in three vastly different cell types (PC12 neuronal, CHO stromal, and Jurkat lymphoid), it seems to be conserved and likely leads to other important execution-phase changes. It is known, for example, that the ezrin/moesin/radixin family of plasma membrane-actin linking proteins becomes enzymatically dephosphorylated early in apoptosis, which leads to their translocation away from the plasma membrane to the cytoplasm (see earlier section and Kondo et al., 1997). Although inhibition of microtubule disassembly with taxol does not prevent eventual apoptotic body formation (Levee et al., 1996), its effect on blebbing has not been reported. However, it has been shown that taxol can protect cultured hippocampal neurons against excitotoxic and apoptotic cell death (Furukawa and Mattson, 1995). An association is suggested because microtubule assembly by itself can initiate or augment blebbing even in non-apoptotic cells (Keller et al., 1985; Keller and Zimmerman, 1986; Mills et al., 1998a). Thus, microtubule disassembly could be one of the first stages in extranuclear apoptosis and might be a fruitful avenue of study to link commitment phase mitochondrial events to the extranuclear execution-phase. Changes in cell-cell and cell-matrix interaction in execution-phase In an organism, apoptosis is an act of cellular altruism that does not occur in a vacuum. The apoptotic cell communicates its status to neighboring cells. The vast majority of studies of apoptosis (and almost all those reviewed so far in this chapter), on the other hand, have only dealt with what the cell does to itself. One way a cell presumably aids in its own clearance is by shrinking and, in many cases, forming apoptotic bodies. But there is also evidence that execution-phase cells help induce their own phagocytosis by altering cell surface components. For example, one well-characterized change is the expression of phosphatidyl serine (PS) on the surface of dying cells. PS is a

26

J.C. Mills

phospholipid that is normally maintained on the internal plasma membrane leaflet. Apoptotic cells show altered activity of a flippase enzyme, whose activity is needed to maintain asymmetric PS localization, and extracellular exposure of PS has been shown to mediate recognition of apoptotic ceils by phagocytes (Fadok et al., 1992). In at least one system, PS exposure is downstream of blebbing and caspase activation (McCarthy et al., 1997). Several types of apoptotic ceils have also been shown to express cell-surface molecules which bind the macrophage vitronectin receptor (Savill et al., 1990) and/or thrombospondin (CD36) receptor (Ren and Savill, 1995; Ren et al., 1995). The nature of the execution-phase event that underlies these changes in cell surface expression remains to be characterized. Apoptotic cells also show execution-phase changes in cell-cell interactions with neighbors that are not necessarily associated with phagocytosis. As already discussed, one theory of blebbing is that it is simply a marker of increased centripetal contraction in cells that have lost much of their cell-cell and cell-matrix anchors (i.e. show the characteristic apoptotic "rounding up") and are on their way to shrinking and/or forming apoptotic bodies. It has been proposed that this process of shrinking and rounding up could play an important role at least in epithelial cells, which must preserve their sheetlike barrier function. Thus, it is likely epithelia would have mechanisms for preserving the integrity of the monolayer barrier when single cells die, rather than simply having large holes form that are gradually repaired by neighboring cells. It is possible, then, that the shrinkage of apoptosis, as mediated by actinomyosin contraction, actually pulls neighboring cells in to cover the space that would have been formed if the cell had simply died. Thus, the hallmark shrinkage (with its associated blebbing) seen in the execution-phase might represent a dying apoptotic cell exhausting its energy to pull on neighboring cells in an altruistic attempt to preserve the epithelial barrier without forcing neighboring cells to expend their own energy (Peralta-Soler et al., 1996; Mills et al., 1998b). In support of this hypothesis, in tissue culture monolayers of renal epithelial cells, apoptotic cell shrinkage in the execution-phase coincides with stretching of neighboring cells toward the center of the apoptotic cell. This process ensures that only very small (if any) holes form in the monolayer as the result of apoptosis (Peralta-Soler et al., 1996). As expected given the model for execution-phase shrinkage, cytochalasin-mediated filamentous actin disruption prevents this "rosetting" of neighboring cells around the apoptotic cell and leads to large gaps in the monolayer. The apoptotic cells show focal concentration of actin and the cadherin-catenin complex of epithelial cell adhesion proteins at the plasma membrane sites where the apoptotic and neighboring cells interact. Another group of investigators show similar shrinkage and rosette formation in apoptosis in retinal pigment epithelium (Nagai and Kalnins, 1996). These results are interesting in light of data from another group in a non-epithelial system showing caspase-mediated cleavage of 13-catenin in fibroblastic (non-epithelial) cells (Brancolini et al., 1997). 13-catenin (among other functions) links the transmembrane E-cadherin, which interacts on its extracellular surface with E-cadherin from neighboring cells, to the cytoplasmic ct-catenin, which interacts with the actin cytoskeleton. Presumably, cleavage of 13-catenin would disrupt cadherin/cytoskeletal interaction and lead to loss of cell-cell interaction in the execution-phase, a finding that would seem

Mechanisms in the Execution-phase

27

contrary to that seen in epithelial cells. The two systems might, in fact, be different, as fibroblasts do not form barrier functions and don't need to maintain monolayers. However, it should be noted that the authors in the fibroblast study were not specifically examining whether apoptotic shrinkage correlated with drawing in of neighboring cells. Thus, it is possible that, despite cleavage of cadherins, enough cell-cell contact is maintained for the shrinkage occurring in the apoptotic cell to mediate pulling on neighbors. A final example of execution-phase cell communication-related changes is the finding that caspases mediate cleavage of focal adhesion kinase (FAK) in endothelial cells (Levkau et al., 1998). FAK is a tyrosine kinase that plays an important role in integrating signaling from integrin receptors (which link the extracellular matrix with the actin cytoskeleton). Dismantling of FAK could be an important step in the very early rounding up of apoptosis. Cells centripetally contracting their actinomyosin cytoskeleton at the same time they lose matrix attachments would tend to round. Indeed, if epithelial cells cleaved FAK as they began to contract (while maintaining cell-cell interactions), the force of contraction would be applied all the more directly toward neighboring cells (rather than the matrix). Cellular energetics Another group of potentially interesting non-nuclear execution-phase events involves the changes in energy metabolism seen in apoptosis. ATP and cellular energy levels are maintained in cultures of asynchronous apoptotic cells (Mills et al., 1995b; for review, see also Nicotera and Leist, 1997), suggesting any substantial loss of cellular energetics occur in the execution-phase or perhaps the commitment phase. Cell-free models for execution-phase events require ATP (see e.g. Lazebnik et al., 1993; Ellerby et al., 1997), and the dramatic cytoskeletal blebbing and cell shrinkage involves myosin-mediated force generation and, therefore, must be energy-dependent. Furthermore, some groups report a direct requirement of ATP for the execution-phase (Nicotera and Leist, 1997; Tsujimoto, 1997). There may be substantial impairment of oxidative-phosphorylationmediated ATP generation in the execution-phase, though this is still an issue of active debate (Zhivotovsky et al., 1998 for review). Thus, it is possible that a larger portion of the ATP required for execution-phase events might be generated by glycolysis. Studies with apoptotic neuronal cells showed consistent, relatively pronounced increases in rate of lactate production in apoptotic cultures (Mills et al., 1995b), suggesting increased glycolytic flux possibly secondary to oxidative phosphorylation impairment (i.e. the "Pasteur effect"). An inhibitor of glycolysis, 2-deoxyglucose, has been shown to inhibit apoptosis (Thakkar and Potten, 1993). Some studies have shown that nonapoptotic cells might preferentially use glycolytic pathways for possibly focal generation of ATP in processes involving the actin cytoskeleton and the plasma membrane (see e.g. Pagliaro and Taylor, 1992). Thus, one intriguing, though entirely speculative hypothesis, for the data suggesting increased glycolysis in the execution-phase is that blebbing and cell shrinkage rely heavily on glycolysis to generate ATP for the actin-myosin force generation.

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J.C. Mills

Summary As can easily be assessed from the other chapters in this volume (or by any literature search with "apoptosis" as a key word), the study of apoptotic death has mushroomed in the last few years, and many processes and mechanisms specific to apoptosis have been discovered and characterized. The explosion of interest has also naturally led to an impressive proliferation of terms. As it can be helpful to organize all the new findings within a few set categories, the first aim of this chapter was to define more specifically the terms relating to the progression of an individual cell through the time course of death. The time course can be summarized as follows: the initiation phase - (during which the cell responds to numerous types of signals, with cell-type-specific signal transduction pathways, to transduce the signal for apoptosis to the commitment phase); the commitment phase (during which the cell uses a limited number of evolutionarily conserved pathways to integrate the various initiation phase signals into a decision to refrain from apoptosis or commit irreversibly to it, thereby triggering the execution-phase), and the execution-phase l~during which the cell invokes multiple parallel pathways that lead to the hallmark features of apoptosis). The second aim of this chapter was to review recent advances in study of specifically the execution-phase, as this is the stage when all the defining (evolutionarily important) features of apoptosis occur, and there is great potential for fruitful research into identifying the mechanisms responsible for these features. Thus, in this chapter the execution-phase was grouped into nuclear and extranuclear events, and those were further subdivided into upstream and downstream processes. It was noted that the caspase family of proteases comprises the best-characterized upstream regulators in extranuclear and especially nuclear events. Downstream nuclear events were also discussed. These included: DNA fragmentation, which has been well studied and has seen recent great advances with the identification of caspase-inducible endonucleases, and chromatin condensation, which is a process that is still largely uncharacterized. Downstream extranuclear events were discussed, with special attention paid to cell blebbing and apoptotic body formation as part of general cell shrinkage. These morphologic changes have seen recent advances with the identification of caspase-activated kinases necessary for apoptotic body formation and characterization of the possible cellular mechanism underlying apoptotic contraction and blebbing. Other downstream extranuclear events were discussed and included: changes in the cell's interaction with its environment, a field that has seen some progress with the identification of several caspase-mediated changes in membrane-associated proteins; changes in cytoskeleton and cytoskeleton-associated proteins, which have been recently shown to occur in the form of microtubule disassembly and protein phosphatase 2A activation; changes in cellular energetics, which have not been as well studied in the execution-phase itself but the importance of which is hinted at by the dependence of the execution-phase on ATP (as possibly generated preferentially by glycolysis). All the recent advances in the study of the execution-phase discussed in this chapter serve only to highlight how complicated and multifaceted this stage of apoptosis is. Many of the mechanisms underlying the telltale execution-phase morphologic events, especially those mediating extranuclear events, have undoubtedly not even been identified, let alone characterized. Thus, the future seems bright for those interested in discovering how a cell brings about this fascinating, final act of altruism.

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Acknowledgments I am deeply indebted to Dr. Randy Pittman. The studies cited on which I am an author were performed in his laboratory under his supervision. I also would like to thank Jesse Mills, who helped to edit this chapter and provided useful and insightful comments.

References Adams, J.M. & Cory, S. (1998). The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322-1326. Aktories, K., Mohr, C. & Koch, G. (1992). CIostridium botulinum C3 ADP-ribosyltransferase. Curt. Top. Micro. Immunol. 175, 115-131. Allera, C., Lazzarini, G., Patrone, E., Alberti, I., Barboro, P., Sanna, P., Melchiori, A., Parodi, S. & Balbi, C. (1997). The condensation of chromatin in apoptotic thymocytes shows a specific structural change. J. Biol. Chem. 272, 10817-10822. Amano, M., Ito, M., Kimura, K., Fukata, Y., Chihara, K., Nakano, T., Matsuura, Y. & Kaibuchi, K. (1996). Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J. Biol. Chem. 27 l, 20246-20249. Barres, B.A., Hart, I.K., Coles, H.S., Burne, J.F., Voyvodic, J.T., Richardson, W.D. & Raft, M.C. (1992). Cell death and control of cell survival in the oligodendrocyte lineage. Cell 70, 31-46. Bokoch, G.M. (1998). Caspase-mediated activation of PAK2 during apoptosis: proteolytic kinase activation as a general mechanism of apoptotic signal transduction? Cell Death Diff. 5,637-645. Bonfoco, E., Leist, M., Zhivotovsky, B., Orrenius, S., Lipton, S.A. & Nicotera, P. (1996). Cytoskeletal breakdown and apoptosis elicited by NO donors in cerebellar granule cells require NMDA receptor activation. J. Neurochem. 67, 2484-2493. Brancolini, C., Benedetti, M. & Schneider, C. (1995). Microfilament reorganization during apoptosis: the role of Gas2, a possible substrate for ICE-like proteases. EMBO J. 14, 5179-5190. Brancolini, C., Lazarevic, D., Rodrigquez, J. & Schneider, C. (1997). Dismantling cell-cell contacts during apoptosis is coupled to a caspase-dependent proteolytic cleavage of B-catenin. J. Cell Biol. 139, 759-771. Brown, S.B., Bailey, K. & Savill, J. (1997). Actin is cleaved during constitutive apoptosis. Biochem. J. 323,233-237. Brunet, C.L., Gunby, R.H., Benson, R.S.P., Hickman, J.A., Watson, A.J.M. & Brady, G. (1998). Commitment to cell death measured by loss of clonogenicity is separable from the appearance of apoptotic markers. Cell Death Diff. 5, 107-115. Brustugun, O.T., Fladmark, K.E., Doskeland, S.O., Orrenius, S. & Zhivotovsky B. (1998). Apoptosis induced by microinjection of cytochrome e is caspase-dependent and is inhibited by Bcl-2. Cell Death Diff. 5,660-668 Camp, V. & Martin, P. (1996). The role of macrophages in clearing programmed cell death in the developing kidney. Anat. Embryol. 194, 341-348. Canu, N., Dus, L., Barbato, C., Ciotti, M.T., Brancolini, C., Rinaldi, A.M., Novak, M., Cattaneo, A., Bradbury, A. & Calissano, P. (1998). Tau cleavage and dephosphorylation in cerebellar granule neurons undergoing apoptosis. J. Neurosci. 18, 7061-7074. Casciola-Rosen, L.A., Miller, D.K., Anhalt, G.J. & Rosen, A. (1994). Specific cleavage of the 70-kDa protein component of the U1 small nuclear ribonucleoprotein is a characteristic biochemical feature of apoptotic

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cell death. J. Biol. Chem. 269, 30757-30760. Chu-Wang, I.-W. & Oppenheim, R.W. (1978). Cell death of motoneurons in the chick embryo spinal cord: I. A light and electron microscopic study of naturally occurring and induced cell loss during development. J. Comp. Neurol. 177, 59-86. Clarke, P.G.H. (1990). Developmental cell death: morphological diversity and multiple mechanisms. Anat. Embryol. 181,195-213. Coakley, W.T. (1987). Hyperthermia effects on the cytoskeleton and on cell morphology. Symp. Soc. Exp. Biol. 41,187-211. Collins, J.A., Schandl, C.A., Young, K.K., Vesely, J. & Willingham, M.C. (1997). Major DNA fragmentation is a late event in apoptosis. J. Histochem Cytochem. 45,923-934. Cotter, T.G., Lennon, S.V., Glynn, J.M. & Green, D.R. (1992). Microfilament disrupting agents prevent the formation of apoptotic bodies in tumor cells undergoing apoptosis. Cancer Res. 52, 997-1005. Cryns, V.L., Bergeron, L., Zhu, H., Li, H. & Yuan J. (1996). Specific cleavage of alpha-fodrin during Fas- and tumor necrosis factor-induced apoptosis is mediated by an interleukin-lbeta-converting enzyme/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease. J. Biol. Chem. 271, 31277-31282. Cunningham, C.C, Gorlin, J.B., Kwiatkowski, D.J., Hartwig, J.H., Janmey, P.A., Byers, H.R. & Stossel, T.P. (1992). Actin-binding protein requirement for cortical stability and efficient locomotion. Science 255, 325-327. Cuvillier, O., Pirianov, G., Kleuser, B,.Vanek, P.G., Coso, O.A., J.,Gutkind, S. & Spiegel, S. (1996). Suppression of ceramide-mediated programmed cell death by sphingosine-l-phosphate. Nature 381, 800-803. Deshmukh, D., Vasilakos, J., Dechwerth, T.L., Lampe, P.A., Shivers, B.D. & Johnson, E.M., Jr. (1996). Genetic and metabolic status of NGF-deprived sympathetic neurons saved by an inhibitor of ICE family proteases. J. Cell Biol. 135, 1341-1354. Duan, W., Rangnekar, V. & Mattson, M.P. (1999). Par-4 production in synaptic compartments folllowing apoptotic and excitotoxic insults: evidence for a pivotal role in mitochondrial dysfunction and neuronal degeneration. J. Neurochem. In press. Dudek, H., Datta, S.R., Franke, T.F., Birnbaum, M.J., Yao, R., Cooper, G.M., Segal, R.A., Kaplan, D.R. & Greenberg, M.E. (1997). Regulation of neuronal survival by the serine-threonine protein kinase Akt. Science 275,661-665. Earnshaw, W.C. (1995). Nuclear changes in apoptosis. Curr. Opin. Cell Biol. 7, 337-343. Ellerby, H.M., Martin, S., Ellerby, L.M., Nalem, S.S., Rabizadeh, S., Salvesen, G.S., Casiano, C.A., Cashman, N.R., Green, D.R. & Bredesen, D.E. (1997). Establishment of a cell-free system of neuronal apoptosis: comparison of premitochondrial, mitochondrial, and postmitochondrial phases. J. Neurosci. 17, 6165-6178. Ellis, H.M. & Horvitz, H.R. (1986). Genetic control of programmed cell death in the nematode C. elegans. Cell 44, 817-829. Ellis, R.E., Yuan, J. & Horvitz, H.R. (1991). Mechanisms and functions of cell death. Annu. Rev. Cell Biol. 7,663-698. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A. & Nagata, S. (1998). A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43-50. Endresen, P.C., Fandrem, J., Eide, T.J. and Aarbakke, J. (1995). Morphological modification of apoptosis in HL-60 cells: effects of homocystein and cytochalasins on apoptosis initiated by 3-deazaadenosine. Virch. Arch. 426, 257-266. Evan, G.I., Wyllie, A.H., Gilbert, C.S., Littlewood, T.D., Land, H., Brooks, M., Waters, C.M., Penn, L.Z. &

Mechanisms in the Execution-phase

31

Hancock, D.C. (1992). Induction of apoptosis in fibroblasts by c-myc protein. Cell 69, 119-128. Fadok, V.A. Voelker, D.R., Campbell, P.A., Cohen, J.J., Bratton, D.L., & Henson, P.M. (1992). Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. lmmunol. 48, 2207-2216. Faleiro, L., Kobayashi, R., Fearnhead H. & Lazebnik Y. (1997). Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells. EMBO J. 16, 2271-2281. Farschon, D.M., Couture, C., Mustelin, T. & Newmeyer, D.D. (1997). Temporal phases in apoptosis defined by the actions of Src homology 2 domains, ceramide, Bcl-2, interleukin-lB converting enzyme family proteases, and a dense membrane fraction. J. Cell Biol. 137, 1117-1125. Fesus, L. (1993). Biochemical events in naturally occurring forms of cell death. FEBS Letters 328, 1-5. Fishkind, D.J., Cao, L. & Wang, Y. (1991). Microinjection of the catalytic fragment of myosin light chain kinase into dividing cells: effects on mitosis and cytokinesis. J. Cell Biol. 114,967-975. Furukawa, K., Smith-Swintosky, V.L. & Mattson, M.P. (1995). Evidence that actin depolymerization protects hippocampal neurons against excitotoxicity by stabilizing [Ca2+]i. Exp. Neurol. 133, 153-163. Furukawa, K. & Mattson, M.P. (1995). Cytochalasins protect hippocampal neurons against amyloid b-peptide toxicity: evidence that actin depolymerization suppresses Ca 2+ influx. J. Neurochem. 65,1061-1068. Furukawa, K. & Mattson, M.P. (1995b. Taxol stabilizes [Ca2+] and protects hippocampal neurons against excitotoxicity. Brain Res. 689, 141-146. Furukawa, K. Fux, W. Witke, W., Kwiatkowski, D.J. & Mattson, M.P. (1997). The actin-severing protein gelsolin modulates calcium channel and NMDA receptor activities and vulnerability to excitotoxicity in hippocampal neurons. J. Neurosci. 17, 8178-8186. Gagliardini, V., Fernandez, P.-A., Lee, R.K.K., Drexler, H.C., Rotello, R.J., Fishman, M.C. & Yuan, J. (1994). Prevention of vertebrate neuronal death by the crmA gene. Science 263,826-828. Gallagher, P.J., Herring, B.P. & Stull, J.T. (1997). Myosin light chain kinases. J. Muscle Res. Cell Motil. 18, 1-16. Gebbink, M.F.B.G., Kranenburg, O., Poland, M., van Horck, F.P.G., Houssa, B. & Moolenaar, W.H. (1997). Identification of a novel, putative Rho-specific GDP/GTP exchange factor and a RhoA-binding protein: control of neuronal morphology. J. Cell Biol. 137, 1603-1613. Ghibelli, L., Maresca, V., Coppola, S. & Gualandi, G. (1995). Protease inhibitors block apoptosis at intermediate stages: a compared analysis of DNA fragmentation and apoptotic nuclear morphology. FEBS Letters 377, 9-14. Grebecki, A. (1994). Membrane and cytoskeleton flow in motile cells with emphasis on the contribution of free-living amoebae. Int. Rev. Cytol. 148, 37-80. Green, D.R. & Reed, J.C. (1998). Mitochondria and apoptosis. Science 281, 1309-1312. Greene, L.A. & Tischler, A. (1976). Establishment ofa noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73, 2424-2428. Guo, Q., Fu, W., Xie, J., Luo, H., Sells, S.F., Geddes, J.W., Bondada, V., Rangnekar, V. & Mattson, M.P. (1998). Par-4 is a novel mediator of neuronal degeneration associated with the pathogenesis of Alzheimer's disease. Nature Med. 4, 957-962. Hara, S., Halicka, D., Bruno, S., Gong, J., Traganos, F. & Darzynkiewicz, Z. (1996). Effect of protease inhibitors on early events of apoptosis. Exp. Cell Res. 223, 372-384. Hengartner, M.O. & Horvitz, H.R. (1994). Programmed cell death in Caenorhabditis elegans. Curr. Opin. Genetics Development. 4,581-6. Herman, B., Gores, G.J., Nieminen, A.L., Kawanishi, T., Harman, A. & Lemasters, J.J. (1990). Calcium and pH in anoxic and toxic injury. Crit. Rev. Toxicol. 21,127-148.

J.C. Mills

32

Himta, H., Takahashi, A., Kobayashi, S., Yonehara, S., Sawai, H., Okazaki, T., Yamamoto, K. & Sasada, M. (1998). Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis. J. Exp. Med. 187,587-600. Hirsch, T., Matzo, I. & Kroemer, G. (1997). Role of the mitochondrial permeability transition pore in apoptosis. Biosci. Reports 17, 67-75. Hockenberry, D., Nunez, G., Milliman, C., Schreiber, R.D. 8,: Korsmeyer, S.J. (1990). Bcl-2 is an inner mitochondrial protein that blocks programmed cell death. Nature 348,334-336. Ichijo, H., Nishida, E., Irie, K., ten Dijke, P., Saitoh, M., Mofiguchi, T., Takagi, M., Matsumoto, K., Miyazono, K. & Gotoh, Y. (1997). Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275, 90-94. Ink, B., Zornig, M., Baum, B., Hajibagheri, N., James, C., Chittenden, T. & Evan, G. (1997). Human Bak induces cell death in Schizosaccharomyces pombe with morphological changes similar to those with apoptosis in mammalian cells. Mol. Cell. Biol. 17, 2468-2472. Ireland, C.M. & Pittman, S.M. (1995). Tubulin alterations in taxol-induced apoptosis parallel those observed with other drugs. Biochem. Pharmacol. 49, 1491-499. Jacobson, M.D., Burne, F.F. & Raft, M.C. (1994). Programmed cell death and Bcl-2 protection in the absence of a nucleus. EMBO J. 13, 1899-1910. Jalink, K., van Corven, E.J., Hengeveld, T., Morii, N., Narumiya, S. & Moolinaar, W.H. (1994). Inhibition of lysophosphatide- and thrombin- induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho. J. Cell Biol. 126, 801-810. Janicke, R.U., Sprengart, M.L., Wati, M.R. & Porter, A.G. (1998). Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J. Biol. Chem. 273, 9357-9360. Janson, L.W. & Taylor, D.L. (1993). In vitro models of tail contraction and cytoplasmic streaming in amoeboid cells. J. Cell Biol. 123, 345-356. Johnson, N.L., Gardner, A.M., Diener, K.M., Lange-Carter, C.A., Gleavy, J., Jarpe, M.B., Minden, A., Kafin, M., Zon, L.I. & Johnson, G.L.. (1996). Signal transduction pathways regulated by mitogenactivated/extracellular response kinase kinase kinase induce cell death. J Biol. Chem. 271, 3229-3237. Kauffmann-Zeh, A., Rodriguez-Viciana, P., Ulrich, E., Gilbert, C., Coffer, P., Downward, J. & Evan, G. (1997). Suppression of c-Myc-induced apoptosis by Ras signaling through PI(3)K and PKB. Nature 385, 544-548. Kayalar, C., Ord, T., Testa, M.P., Zhong, L.-T. & Bredesen, D.E. (1996). Cleavage of actin by interleukin lfS-converting enzyme to reverse DNaseI inhibition. Proc. Natl. Acad. Sci. USA 93, 2234-2238. Keller, H.U. & Zimmermann, A. (1986). Shape changes and chemotaxis of Walker 256 carcinosarcoma cells in response to colchicine, vinblastine, nocodazole and taxol. Invasion Metastasis 6, 33-43. Keller, H.U., Zimmermann, A., & Cottier, H. (1985). Phorbol myristate acetate (PMA) suppresses polarization and locomotion and alters F-actin content of Walker carcinosarcoma cells. Int. J. Cancer 36, 495-501. Kerr, J. F.R., Wyllie, A.H. & Curie, A.R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239. Kerr, J.F.R. & Harmon, B.V. (1991) Definition and incidence of apoptosis: an historical perspective. In: Apoptosis: the Molecular Basis of Cell Death, Current Communications in Cell & Molecular Biology, vol. 3. Cold Spring Harbor Laboratory Press, New York, pp. 5-29. Kimura, K., Iho, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., Yamamori, B., Feng, J., Nakano, T., Okawa, K., Iwamatsu, A. & Kaibuchi, K. (1996). Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273,245-248. Kluck, R.M., Bossy-Wetzel, E., Green, D.R. & Newmeyer, D.D. (1997). The release of cytochrome c from

Mechanisms in the Execution-phase

33

mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 1132-1136. Kohama, K., Ye, L.H., Hayakawa, K. & Okagaki T. (1996). Myosin light chain kinase: an actin-binding protein that regulates an ATP-dependent interaction with myosin. Trends Pharmacol. Sci. 17,284-287. Kondo, T., Takeuchi, K., Doi, Y., Yonemura, S., Nagata, S., Tsukita, S. & Tsukita, S. (1997). ERM (Ezrin/Radixin/Moesin)-based molecular mechanism of microvillar breakdown at an early stage of apoptosis. J. Cell Biol. 149, 749-758. Korsmeyer, S.J. (1992). Bcl-2 initiates a new category of oncogenes: regulators of cell death. Blood 80, 879-86. Kothakota, S., Azuma, T., Reinhard, C., Klippel, A., Tang, J., Chu, K., McGarry, T.J., Kirschner, M.W., Koths, K., Kwiatkowski, D.J. & Williams, L.T. (1997). Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science 278,294-298. Krajewski, S., Tanaka, S., Takayama, S., Shibler, M.J., Fenton, W. & Reed, J.D. (1993). Investigation of the subcellular distribution of the Bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Cancer Res. 53, 4701-4714. Kwiatkowski, D.J. Janmey, P.A. & Yin, H.L. (1989). Identification of critical functional and regulatory domains in gelsolin. J. Cell Biol. 108, 1717-1726. Lagarkova, M.A., Iarovaia, O.V. & Razin, SV. (1995). Large-scale fragmentation of mammalian DNA in the course of apoptosis proceeds via excision of chromosomal DNA loops and their oligomers. J. Biol. Chem. 270, 20239-20241. Laster, S.M. & Mackenzie, J.M. (1996). Bleb formation and F-actin distribution during mitosis and tumor necrosis factor-induced apoptosis. Micro. Res. Tech. 34, 272-280. Lazebnik, Y.A., Cole, S., Cooke, C.A., Nelson, W.G. & Earnshaw, W.C. (1993). Nuclear events of apoptosis

in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis. J. Cell Biol. 123, 7-22. Lazebnik, Y.A., Kaufmann, S.H., Desnoyers, S., Poirier, G.G. & Earnshaw, W.C. (1994). Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 371,346-347. Lazebnik, Y.A., Takahashi, A., Moir, R.D., Goldman, R.D., Piurier, G.G., Kaufman, S.H. & Earnshaw, W.C. (1995). Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution. Proc. Natl. Acad. Sci. USA 92, 9042-9046. Lee, N., MacDonald, H., Reinhard, C., Halenbeck, R., Roulston, A., Shi, T. & Williams, L.T. (1997). Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis. Proc. Natl. Acad. Sci. USA 94, 13642-13647. Levee, M.G., Dabrowska, M.I., Lelli, J.L. & Hinshaw, D.B. (1996). Actin polymerization and depolymerization during apoptosis in HL-60 cells. Am. J. Physiol. 271 (Cell Physiol. 40) C1981-C1992. Levkau, B., Herren, B., Koyama, H., Ross, R. & Raines, E.W. (1998). Caspase-mediated cleavage of focal adhesion kinase pp125FAK and disassembly of focal adhesions in human endothelial cell apoptosis. J. Exp. Med. 187,579-586. Li, F., Srinivasan, A., Wang, Y., Armstorng, R.C., Tomaselli, K.J. & Fritz, L.C. (1997). Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-xl has activity independent of cytochrome c release. J. Biol. Chem. 272, 30299-30305. Li P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S. & Wang, X. (1997). Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91,479-489. Lippke, J.A., Gu, Y., Sarnecki, C., Caron, P.R. & Su, M.S. (1996). Identification and characterization of CPP32/Mch2 homolog 1, a novel cysteine protease similar to CPP32. J. Biol. Chem. 271, 1825-1828. Litersky, J.M. & Johnson, G.V. (1995). Phosphorylation of tau in situ: inhibition of calcium-dependent

34

J.C. Mills

proteolysis. J. Neurochem. 65,903-911. Liu, X., Zou, H., Slaughter, C. & Wang, X. (1997). DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89, 175-184. MacFarlane, M., Cain, K., Sun, X.-M., Alnemri, E.S. & Cohen, G. M. (1997). Processing/activation of at least four intedeukin-lB converting enzyme-like proteases occurs during the execution-phase of apoptosis in human monocyte tumor cells. J. Cell Biol. 137,469-479. Maciver, S.K. (1996). Myosin II function in non-muscle cells. BioEssays 18, 179-182. Madeo, F., Frohlich, E. & Frohlich, K.-L. (1997). A yeast mutant showing diagnostic markers of early and late apoptosis. J. Cell Biol. 139, 729-734. Martelli, A.M., Bareggi, R., Bortul, R., Grill, V., Narducci, P. & Zweyer, M. (1997). The nuclear matrix and apoptosis. Histochem. Cell Biol. 108, 1-10. Martin, D.P., Schmidt, R.E., DiStefano, P.S., Lowry, O.H., Carter, J.G. & Johnson, E.M. (1988). Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation. J. Cell Biol. 106, 829-844. Martin, S.J., O'Brien, G.A., Nishioka, W.K., McGahon, A.J., Mahboubi, A., Saido, T.C. & Green, D.R. (1995). Proteolysis of fodrin (non-erythroid spectrin) during apoptosis. J. Biol. Chem. 270, 6425-6428. Martins, L.M., Kottke, T., Mesner, P.W., Basi, G.S., Sinha, S., Frigon, N. Jr., Tatar, E., Tung, J.S., Bryant, K., Takahashi, A., Svingen, P.A., Madden, B.J., McCormick, D.J., Earnshaw, W.C. & Kaufmann, S.H. (1997). Activation of multiple interleukin-lbeta converting enzyme homologues in cytosol and nuclei of HL-60 cells during etoposide-induced apoptosis. J. Biol. Chem. 272, 7421-7430. Marushige, Y. & Marushige, K. (1995). Disappearance of ubiquinated histone H2A during chromatin condensation in TGFBl-induced apoptosis. Anticancer Res. 15,267-272. Marzo, I., Brenner, C., Zamzami, N., Jurgensmeier, J.M., Susin, S.A., Vieira, H.L.A., Prevost, M.-C., Xie, Z., Matsuyama, S., Reed, J.C. & Kroemer, G. (1998). Bax and adenine nucleotide translocator cooperate on the mitochondrial control of apoptosis. Science 281, 2027-2031. Mashima, T., Naito, M. Fujita, N., Noguci, K. & Tsuruo, T. (1995). Identification of actin as a substrate of ICE and an ICE-like protease and involvement of an ICE-like protease but not ICE in VP-16-induced U937 apoptosis. Biochem. Biophys. Res. Comm. 217, 1185-1192. Mattson, M.P., Keller, J.N. & Begley, J.G. (1998). Evidence for synaptic apoptosis. Exp. Neurol. 153, 35-48. Mattson, M.P., Partin, J. & Begley, J.G. (1998). Amyloid B-peptide induces apoptosis-related events in synapses and dendrites. Brain Res. 807, 167-176. McCarthy, N.J., Whyte, M.K.B., Gilbert, C.S. & Evan, G.I. (1997). Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak. J. Cell Biol. 136, 215-227. McConnell, K.R., Dynan, W.S. & Hardin, J.A. (1997). The DNA-dependent protein kinase catalytic subunit (p460) is cleaved during Fas-mediated apoptosis in Jurkat cells. J. lmmunol. 158, 2083-2089. Medema, J.P., Scaffidi, C., Kischkel, F.C., Shevchenko, A., Mann, M., Krammer, P.H. & Peter, M.E. (1997). FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J. 16, 2794-2804. Messam, C.A. & Pittman, R.N. (1998). Asynchrony and commitment to die during apoptosis. Exp. Cell Res. 238, 389-398. Mignotte, B. & Vayssiere, J.-L. (1998). Mitochondria and apoptosis. Eur. J. Biochem. 252, 1-15. Mills, J.C., Wang, S.L., Erecinska, M. & Pittman, R.N. (1995a). Use of cultured neurons and neuronal cell lines to study morphological, biochemical, and molecular changes occurring in cell death. Methods Cell Biol. 46, 217-242.

Mechanisms in the Execution-phase

35

Mills, J.C., Nelson, D., Erecinska, M. & Pittman, R.N. (1995b). Metabolic and energetic changes during apoptosis in neural cells. J. Neurochem. 65, 1721-1730. Mills, J.C., Kim, L.H, & Pittman, R.N. (1997). Differentiation to an NGF-dependent state and apoptosis following NGF removal both occur asynchronously in cultures of PC12 cells. Exp. Cell Res. 231, 337-345. Mills, J.C., Lee, V. M.-Y. & Pittman, R.N. (1998a). Activation ofa PP2A-like phosphatase and dephosphorylation of tau protein characterize onset of the execution-phase of apoptosis. J. Cell Sci. 111,625-626. Mills, J.C., Stone, N.L., Erhardt, J. & Pittman, R.N. (1998b). Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation. J. Cell Biol. 140, 627-636. Mitchison, T.J. & Cramer, L.P. (1996). Actin-based cell motility and cell locomotion. Cell 84, 371-379. Miyoshi, H., Umeshita, K., Sakon, M., lmajoh-Ohmi, S., Fujitani, K., Gotoh, M., Oiki, E., Kambayashi, J. & Monden, M. (1996). Calpain activation in plasma membrane bleb formation during tert-butyl hydroperoxide-induced rat hepatocyte injury. Gastroenterol. 110, 1897-1904. Montague, J.W. & Cidlowski, J.A. (1996). Cellular catabolism in apoptotic DNA degradation and endonuclease activation. Experentia 52,957-962. Mpoke, S. & Wolfe, J. (1996). DNA digestion and chromatin condensation during nuclear death in tetrahymena. Exp. Cell Res. 225,357-365. Nagai, H. & Kalnins, V. (1996). Normally occurring loss of single cells and repair of resulting defects in retinal pigment epithelium in situ. Exp. Eye Res. 62, 55-61. Nath, R., Raser, K.J., Stafford, D., Hajimohammadreza, I., Posner, A., Allen, H., Talanian, R.V., Yuen, P., Gilbertsen, R.B. & Wang, K.K. (1996). Non-erythroid alpha-spectrin breakdown by calpain and interleukin 1 beta-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis. Biochemical J. 319, 683-690. Newmeyer, D.D., Farschon, D.M. & Reed, J.C. (1994). Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria. Cell 79, 353-364. Nicotera, P. & Leist, M. (1997). Energy supply and the shape of death in neurons and lymphoid cells. Cell Death Diff. 4, 435-442. Noda, M., Yasuda-Fukazawa, C., Moriishi, K., Kato, T., Okuda, T., Kurokawa, K.& Takuwa, Y. (1995). Involvement of Rho in GTP'/S-induced enhancement of phosphorylation of 20 kDa myosin light chain in vascular smooth muscle cells: inhibition of phosphatase activity. FEBS Letters. 367,246-250. Oberhammer, F., Wilson, J.W., Dive, C., Morris, I.D., Hickman, J.A., Wakeling, A.E., Walker, P.R. & Sikorska, M. (1993). Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 12, 3679-3684. Oberhammer, F.A., Hochegger, K., Froschl, G., Tiefenbacher, R. & Pavelka, M. (1994). Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase. J. Cell Biol. 126, 827-837. Ohtsu, M., Sakai, N., Fujita, H., Kashiwag, M., Gasa, S., Shimizu, S., Eguchi, Y., Tsujimoto, Y., Sakiyama, Y., Kobayashi, K. & Kuzumaki, N. (1997). Inhibition of apoptosis by the actin-regulatory protein gelsolin. EMBO J. 16, 4650-4656. Oppenheim, R.W (1991). Cell death during development of the nervous system. Ann. Rev. Neurosci. 14, 453-501. Pagliaro, L. & Taylor, D.L. (1992). 2-Deoxyglucose and cytochalasin D modulate aldolase mobility in living 3T3 cells. J. Cell Biol.118, 859-863. Pandey, S., Walker, R. & Sikorska, M. (1997). Identification of a novel 97 kDa endonuclease capable of internuclesosmal DNA cleavage. Biochem. 36, 711-720.

36

J.C. Mills

Paterson, H.F., Self, A., Garrett, M.D., Just, I., Aktories, K. & Hall, A. (1990). Microinjection of recombinant p21 Rho induces rapid changes in cell morphology. J. Cell. Biol. 111, 1001-1007. Peralto Soler, A., Mullin, J.M., Knudsen, K.A. & Marano, C.W. (1996). Tissue remodeling during tunor necrosis factor-induced apoptosis in LLC-PK1 renal epithelial cells. Am. J. Physiol. 270, F869-F879. Phelps, P.C., Smith, M.W. & Trump, B.F. (1989). Cytosolic ionized calcium and bleb formation after acute cell injury of cultured rabbit renal tubule cells. Lab Investigation 60, 630-642. Pittman, R.N., Wang, S.L., DiBenedetto, A.J. & Mills, J.C. (1993). A system for characterizing cellular and molecular events in programmed neuronal cell death. J. Neurosci. 13, 3669-3680. Pittman, R.N., Messam, C.A. & Mills, J.C. (1998). Asynchronous death as a characteristic feature of apoptosis. In: Cell Death in Diseases of the Nervous System (Koliatsos, V.E and Rataned., R., eds.) Humana Press, Totowa, N J, 29-43. Pittman, S.M., Strickland, D. & Ireland, C.M. (1994). Polymerization of tubulin in apoptotic ceils is not cell cycle dependent. Exp. Cell Res. 215,263-272. Pitzer, F., Dantes, A., Fuchs, T. Baumeister, W. & Amsterdam, A. (1996). Removal of proteasomes from the nucleaus and their accumulation in apoptotic blebs during programmed cell death. FEBS Letters 394, 47-50. Porter, A.G., Ng, P. & Janicke, R.U. (1997). Death substrates come alive. Bioessays 19, 501-507. Raft, M.C. (1992). Social controls on cell survival and cell death. Nature 356, 397-400. Rao, L., Perez, D. & White, E. (1996). Lamin proteolysis facilitates nuclear events during apoptosis. J. Cell Biol. 135, 1441-1455. Ren, Y. & Savill, J. (1995). Proinflammatory cytokines potentiate thrombospondin-mediated phagocytosis of neutrophils undergoing apoptosis. J. Immunol. 154, 2366-2374. Ren, Y., Silverstein, R.L., Alien, J. & Savill, J. (1995). CD36 gene transfer confers capacity for phagocytosis of cells undergoing apoptosis. J. Exp. Med. 181, 1857-1862. Riparbelli, M.G., Callaini, G., Tripodi, S.A., Cintorino, M., Tosi, P. & Dallai, R. (1995). Localization of the Bcl-2 oncoprotein to the outer mitochondrial membrane by electron microscopy. Exp. Cell Res. 221,363-369. Rudel, T. & Bokoch, G.M. (1997). Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. Science 276, 1571-1574. Sakahira, H., Enari, M. & Nagata, S. (1998). Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391,96-99. Santoro, M.F., Annand, R.R., Robertson, M.M., Peng, Y.W., Brady, M.J., Mankovich, J.A., Hacket, M.C., Ghayur, T., Walter, G., Wong, W.W. & Giegel D.A. (1998). Regulation of protein phosphatase 2A activity by caspase-3 during apoptosis. J. Biol. Chem. 273, 13119-13128. Savill, J., Dransfield, I., Hogg, N. & Haslett C. (1990). Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis. Nature 343, 170-173. Sekine, A., Fujiwara, M. & Narumiya, S. (1989). Asparagine residue in the Rho gene product is the modification site for botulinum ADP-ribosyltransferase. J. Biol. Chem. 264, 8602-8605. Sells, M.A. & Chernoff, J. (1997). Emerging from the PAK - the p21-acitvated protein kinase family. Trends Cell Biol. 7, 162-167. Shimizu, T. & Pommier, Y. (1997). Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases. Leukemia 11, 1238-1244. Simm, A., Bertsch, G., Frank, H., Zimmermann, U. & Hoppe, J. (1997). Cell death of AKR-2B fibroblasts after serum removal: a process between apoptosis and necrosis. J. Cell Sci. 110, 819-828.

Mechanisms in the Execution-phase

37

Solary, E., Bertrand, R., Kohn, K.W. & Pommier, Y. (1993). Differential induction of apoptosis in undifferentiated and differentiated HL-60 cells by DNA topoisomerase 1 and II inhibitors. Blood 81, 1359-1368. Song, Q., Lees-Miller, S.P., Kumar, S., Zhang, Z., Chan, D.W., Smith, G.C., Jackson, S.P., Alnemri, E.S., Litwack, G., Khanna, K.K. & Lavin, M.F. (1996). DNA-dependent protein kinase catalytic subunit: a target for an ICE-like protease in apoptosis. EMBO J. 15, 3238-3246. Song, Q., Wei, T., Lees-Miller, S., Alnemri,E., Watters, D. & Lavin, M.F. (1997). Resistance of actin to cleavage during apoptosis. Proc. Natl. Acad. Sci. USA 94, 157-162. Srinivasula, S.M., Ahmad; M., Fernandes-Alnemri, T., Litwack, G. & Alnemri, E.S. (1996). Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases. Proc. Natl. Acad. Sci. USA 93, 14486-14491. Staunton, M.J. & Gaffney, E.F. (1998). Apoptosis: basic concepts and potential significance in human cancer. Arch. Pathol. Lab. Med. 122, 310-319. Sun, D.Y., Jiang, S., Zheng, L.M., Ojcius, D.M. & Young, J.D. (1994). Separate metabolic pathways leading to DNA fragmentation and apoptotic chromatin condensation. J. Exp. Med. 179, 559-568. Sun, X.M. & Cohen, G.M. (1994). Mg(2+)-dependent cleavage of DNA into kilobase pair fragments is responsible for the initial degradation of DNA in apoptosis. J. Biol. Chem. 269, 14857-14860. Susin, S.A., Zamzami, M., Castedo, T., Hirsch, P., Marchetti, A., Macho, E., Daugas, M., Geuskens, M. & Kroemer, G. (1996). Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J. Exp. Med. 184, 1331-1342. Susin, S.A., Zamzami, N., Castedo, M, Daugas, E., Wang, H.-G., Geley, S., Fassy, F., Reed, J.C. & Kroemer, G. (1997). The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis. J. Exp. Med. 86, 25-37. Takahashi, A., Alnemri, E.S., Lazebnik, Y.A., Fernandes-Alnemri, T., Litwack, G., Moir, R.D., Goldman, R.D., Poirier, G.G., Kaufmann, S.H. & Earnshaw, W.C. (1996). Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis. Proc. Natl. Acad. Sci. USA. 93, 8395-8400. Tanaka, Y., Yoshihara, K., Tsuyuki, M. & Kamiya, T. (1994). Apoptosis induced by adenosine in human leukemia HL-60 ceils. Exp. Cell Res. 213,242-252. Thakkar, N.S. & Potten, C.S. (1993). Inhibition of doxorubicin-induced apoptosis in vivo by 2-deoxy-Dglucose. Cancer Res. 53, 2057-2060. Thornberry, N.A. & Lazebnik, Y. (1998). Caspases: enemies within. Science 281, 1312-1316. Tigyi, G., Fischer, D.J., Sebok, A., Yang, C., Dyer, D.L. & Miledi, R. (1996). Lysophosphatidic acid-induced neurite retraction in PC12 cells: control by phosphoinositide-fa2÷ signaling and Rho. J. Neurochem. 66, 537-548. Tomei, L.D., Shapiro, J.P. & Cope, F.O. (1993). Apoptosis in C3H/10T1/2 mouse embryonic cells: evidence for internucleosomal DNA modification in the absence of double-strand cleavage. Proc. Nat. Acad. Sci. USA 90, 853-857. Torgerson, R.R. & McNiven, M.A. (1998). The actin-myosin cytoskeleton mediates reversible agonist-induced membrane blebbing. J. Cell Sci. 111,2911-2922. Tsujimoto,Y. (1997). Apoptosis and necrosis - intracellular ATP levels as a determinant for cell death modes. Cell Death Diff. 4, 429-434. Uchiyama, Y. (1995). Apoptosis: the history and trends of its studies. Arch. Histol. Cytol. 58, 127-137. Van Engeland, M., Kujipers, H.J.H., Ramaekers, F.C.S., Reutelingsperger, C.P.M. & Schutte, B. (1997). Plasma membrane alterations and cytoskeletal changes in apoptosis. Exp. Cell Res. 236, 421-430.

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Vanags, D.M., Porn-Ares, M.I., Coppola, S., Burgess, D.H. & Orrenius, S. (1996). Protease involvement in fodrin cleavage and phosphatidylserine exposure in apoptosis. J. Biol. Chem. 271, 31075-31085. Vaux, D.L., Cory, S. & Adams, J.M. (1988). Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335,440-~42. Vaux, D.L., Weissman, I.L. & Kim, S.K. (1992). Prevention of programmed cell death in Caenorhabditis elegans by homan bcl-2. Science 258, 1955-1956. Verheij, M., Bose, R., Lin, X.H., Yao, B., Jarvis, W.D., Grant, S., Birrer, M.J., Szabo, E., Zon, L.I., Kyriakis, J.M., Haimovitz-Friedman, A., Fuks, Z. & Kolesnick, R.N. (1996). Requirement for ceramide-initiated SAPK/JNK signaling in stress-induced apoptosis. Nature 380, 75-79. Vidair, C.A., Chen, C.H., Ling, C.C. & Dewey, W.C. (1996). Apoptosis induced by X-irradiation of rec-myc ceils is postmitotic and not predicted by the time after irradiation or behavior of sister cells. Cancer Res. 56, 4116-4118. Villa, P., Kaufmann, S.H. & Earnshaw, W.C. (1997). Caspases and caspase inhibitors. Trends Biochem. Sci. 22, 388-393. Weaver, V.M., Lach, B., Walker, P.R. & Sikorska, M. (1993). Role of proteolysis in apoptosis: involvement of serine proteases in internucleosomal DNA fragmentation in immature thymocytes. Biochem. Cell Biol. 71,488-500. Weeds, A.G., Gooch, J., Hawkins, M., Pope, B. & Way M. (1991). Role of actin-binding proteins in cytoskeletal dynamics. Biochem. Soc. Trans. 19, 1016-1020. Wilson, M.R. (1998). Apoptosis: unmasking the executioner. Cell Death Diff. 5,646-652. Wolter, K.G., Hsu, Y.T., Smith, C.L., Nechushtan, A., Xi, X.G. & Youle, R.J. (1997). Movement of Bax from the cytosol to mitochondria during apoptosis. J. Cell Biol. 139, 1281-1292. Wyllie, A.H., Kerr, J.F.R. & Currie, A.R. (1980). Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251-306. Wyllie, A.H., Morris, R.G., Smith, A.L. & Dunlop, D. (1984). Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol. 142, 67-77. Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J. & Greenberg, M.E. (1995). Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270, 1326-1331. Xiang, J., Chao, D.T. & Korsmeyer, S.J. (1996). Bax-induced cell death may not require interleukin lf3-converting enzyme-like proteases. Proc. Natl. Acad. Sci. USA 93, 14559-14563. Yang, J., Liu, X., Bhalla, K., Kim, C.N., lbrado, A.M., Cai, J., Peng, T.I., Jones, D.P. & Wang, X. (1997). Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129-1132. Yin, H.L. & Stossel, T.P (1979). Control of cytoplasmic actin gel-sol transformation by gelsolin, a calciumdependent regulatory protein. Nature 281,583-586. Zamzami, N., Susin, S.A., Marchetti, P., Hirsch, T., Gomez-Monterrey, I., Castedo, M. & Kroemer, G. (1996). Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183, 1533-1544. Zha, J., Harada, H., Yang, E., Jockel, J. & Korsmeyer, S.J. (1996). Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not Bcl-xL. Cell 87,619-628. Zhivotovsky, B., Wade, D., Gahm, A., Orrenius, S. & Nicotera P. (1994). Formation of 50 kbp chromatin fragments in isolated liver nuclei is mediated by protease and endonuclease activation. FEBS Letters 351,150-154. Zou, H., Henzel, W.J., Liu, X., Lutschg, A. & Wang, X. (1997). Apaf-1, a human protein homologous to C. elegans Ced-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90, 405-413.

DEATH DOMAIN SIGNALING AND ITS ROLE IN THE CENTRAL NERVOUS SYSTEM A N N A D O R A J. B R U C E - K E L L E R

Table of Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Death Domain Signaling Components -- Pathways to Cell Death . . . . . . . . . . . . . . . . . . . Initiation of the Death Signal -- Death Receptor Associated Proteins . . . . . . . . . . . . . . . Execution of the Death Signal -- Caspase Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternative Death Receptor Signaling -- Pathways to Cell Life . . . . . . . . . . . . . . . . . . . . . Inhibitors of Apoptosis -- FLIPs and lAPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NFKB Activation -- Survival Gene Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Death Receptors in the Central Nervous System -- Physiology and Pathophysiology . . . . Fas and the CNS: Tumorigenesis and Chronic Neurodegenerative Disease . . . . . . . . . . . TNF and Neuronal Resistance to Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

39 40 41 44 46 46 48 50 51 52 56

Introduction

Apoptosis or programmed cell death is a controlled death process that eliminates excess cells for the greater good of multicellular organisms. Apoptosis occurs during both development and adulthood, and in addition to physiological roles in development and tissue remodeling, alterations in apoptosis contribute to such pathophysiological disease states such as cancer, Alzheimer's disease and stroke. Higher organisms have developed the means to rapidly and effectively eliminate unwanted cells by apoptosis through the activation of death-inducing receptors. While these receptors can transmit cytotoxic signals from the extracellular space and rapidly induce apoptosis in cells, in many cases these receptors are also involved in unrelated processes such as cell activation or differentiation. Whether death receptor activation ultimately results in life or death for a cell is both tightly regulated and cell-type specific, but is still a poorly understood process. The elucidation, therefore, of the receptor mechanisms underlying these divergent effects could augment scientific understanding o f cell death and survival in general, and also highlight potential clinical therapeutic strategies for human disease states. An important family of receptors with roles in cell death, differentiation, and survival in mammalian cells is the tumor necrosis factor (TNF) receptor superfamily. These death-inducing receptors are structurally similar in that each possesses two to six extracellular domains of imperfect repeats of 40 amino acids containing approximately six Cys residues (Figure 1). Fas (Apol/CD95), was the first family member to 39 Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. © 2 0 0 1 Elsevier Science. Printed in the Netherlands.

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be described exclusively in term of its death-inducing function (Itoh et al., 1991; Oehm et al., 1992). Comparisons of the intracellular domains of Fas with tumor necrosis factor receptor-1 (TNF-R1) revealed that both receptors contain a conserved cytoplasmic sequence of 80 amino acids. These conserved, C-terminal residues have since termed "death domains" and the ability of these receptors to transduce a death signal is strictly dependent on an intact death domain (DD) (Tartaglia et al., 1993a; Itoh and Nagata, 1993). Members of the DD receptor family that have been identified on the basis of sequence, structural, and functional similarity include to date (Figure 1): TNF-R1, Fas, TRAMP (death receptor 3/DR3/APO-3/LARD/wsl-1), TRAIL-R1 (death receptor 4/DR4/APO-2), TRAIL-R2 (death receptor 5/DR5), and DR6 (death receptor 6) (Bodmer et al., 1997; Chinnaiyan et al., 1996; Pan et al., 1997a,b, 1998; Sheridan et al., 1997; Walczak et al., 1997; Chaudhary et al., 1997; Schneider et al., 1997). The TRAIL subfamily also contains the truncated decoy receptors DcR1 (TRAIL-R3/TRID/LIT) and DcR2 (TRAIL-R4), which contain the external TRAIL-binding motif and the membrane anchor domain, but lack the full intracellular DD-containing region (Figure 1) (Pan et al., 1997a; Sheridan et al., 1997; Degli-Esposti et al., 1997; Marsters et al., 1997). The decoy receptors bind TRAIL, and thus limit the biological actions of the full length death receptors, an observation that confirms the importance of the intracellular DD motif in the transduction of apoptotic signals. Cognate ligands for many of the DD-containing receptors have been identified, and include Fas ligand (CD95L), TNFct, lymphotoxin-B (TNFB), and TRAIL (TNF-related apoptosis inducing ligand). Interestingly, the ligands themselves display some structural similarity, including a receptor-recognition motif composed of anti-parallel b-sheets (Schulze-Osthoff et al., 1998). It is generally assumed that biologically active ligands are made up of a trimer of identical proteins that activate their receptors by oligomerization (Banner et al., 1993; Dhein et al., 1992).

Death Domain Signaling Components -- Pathways to Cell Death

In general, apoptosis caused by DD-containing receptors is initiated by oligomerization of ligands and receptors, followed by intracellular protein-protein interactions that elicit the recruitment and physical association of caspases, followed finally by caspase activation and resultant cell death. Subsequent to receptor occupation, DD motifs can both self-associate and bind the DD of other proteins through electrostatic interactions. While DD sequences mediate receptor/adaptor interactions, truncated adapter proteins that lack DD's can still transduce a death when overexpressed to high enough levels. Hence, its seems that the DD acts to stabilize protein-protein interaction and facilitate the necessary local increase in death signaling molecules, a situation that is bypassed by overexpression. Since no post-translation modifications have been shown to be required for apoptotic signaling through DD-containing receptors, the oligomerization of receptors and recruitment of their associated "adapter" proteins seem to be the determining factors in executing the death cascade.

41

Death Domains and the Brian

TRAMP

Figure l.

TNF-R1 (~

Fas (CD95/APO-1) TRAIL-R1 (DR4) TRAIL-R2 DcR1 (DR5) Dr6 ~-~ (TRAIL-R3) DcR2 . (TRAIL-R4)

Schematic illustration of death domain-containing receptors. Members of this receptor family

are characterized by the presence of a conserved, 80 amino acid intracellular sequence known as the death domain, here depicted as a striped, upright rectangle. Additionally, these receptors contain similarities in their extracellular domain, consisting of two to four cystiene-rich repeats, depicted here as open ovals. The decoy receptors DcR1 and DcR2 either lack an intracellular domain, or contain a truncated, inactive death domain, and serve to limit activtion of functional TRAIL receptors.

Initiation of the Death Signal -- Death Receptor Associated Proteins The best characterized DD-containing signal transduction pathway is the Fas system (Figure 2), activated by Fas ligand or specific Fas antibodies. FADD(MORT-1), originally cloned by the two-hybrid method using Fas as a bait (Boldin et al., 1995; Chinnaiyan et al., 1995), interacts with Fas through its DD, and can induce death by itself when overexpressed in cell lines (Chinnaiyan et al., 1995). Together with the death receptor (Fas), these proteins form the death-inducing signaling complex (DISC). FADD can be dissociated into an N-terminal "MORT" domain and the C-terminal DD. The MORT domain contains a motif called the death effector domain (DED), as it can transduce the death signal even in the absence of the C-terminal DD (Schulze-Osthoff et al., 1998). On the other hand, a C-terminal truncated protein with only the DD acts as a dominant negative inhibitor of Fas-induced death, suggesting that the DD is required for proper association of FADD with the intracellular region of Fas, while the DED is coupled to intracellular death machinery. FADD was found to associate with a second adapter molecule called FLICE(MACH/caspase 8), again using the yeast two-hybrid system (Boldin et al., 1996; Muzio et al., 1996). FLICE (for FADD-like ICE) contains two N-terminal DEDs that associate with the DED of FADD, and a C-terminal domain that has the typical structure of a cystein protease like interleukin b-converting enzyme (ICE). FLICE belongs to the cystein proteases of the caspase family, and is generally referred to as caspase 8 (Almenri et al., 1996). Following Fas activation, FADD and caspase 8 are requited to the DISC within seconds, and binding of caspase 8 to FADD causes structural modification that result in autoproteolytic activation of the caspase.

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The active subunits pl0 and p18 are released into the cytoplasm, while portions of the procaspase domain remain bound to the DISC. Presently, it is assumed that active caspase 8 subunits cleave subsequent caspases and additional death substrates leading to execution of apoptosis (Schulze-Osthoff et al., 1998). Thus, caspase 8 provides a direct link from the membrane signal to the proteolytic death execution stage (see following section -- Execution of the Death Signal -- Caspase Activation). Again, the DED domains of caspase 8 are responsible for the association with FADD, and can act as dominant-negative inhibitors of Fas-induced death when overexpressed in the absence of the C-terminal end; and several natural isoforms which act as dominant-negative inhibitors have been described (Boldin et al., 1996). Death signaling through TNF-R1 shares many similarities with the Fas pathway (Figure 2). Upon cross-linking, TNF-R1 rapidly recruits a molecule called TRADD (TNF receptor associated death domain-containing protein), which in turn interacts with FADD. (Schulze-Osthoff et al., 1998). Death signaling through TRADD requires an ICE-family protease activity to induce cell death (Hsu et al., 1995; Miura et al., 1995), likely reflecting the association with caspase 8 through FADD. Similar to FADD, the C-terminus of TRADD contains a DD which enables self-association and interaction with the DD's of FADD and other signaling proteins. However, TRADD does not contain the typical N-terminal DED, and hence death signals are propagated through interactions with other DD adapter proteins. In addition to FADD, TRADD also associates with the DD containing protein RIP (receptor interacting protein) as well as the RING domain adapter protein TRAF2 (TNF receptor associated protein 2) which does not have a DD (Hsu et al., 1995, 1996a,b). Both TRADD and RIP can induce apoptosis, but also cause the activation of NFKB, which is a common component of TNF signaling (Hsu et al., 1995; Park and Baichwal, 1996; Ting et al., 1996). The TRAF2-associated kinase NIK (NFKB-inducing kinase) has recently been shown to be important in TNF-Rl-mediated NFKB activation, in that it facilitates the activation of IKB kinase (Verma and Stevenson, 1997) and subsequent I~B phosphorylation and degradation (see NFKB Activation -- Survival Gene Induction). RIP contains an N-terminal kinase domain and a C-terminal DD, and was originally thought to play a role in Fas signaling (Stanger et al., 1995). However, later studies demonstrated that RIP does not bind directly to Fas, and is recruited to TNF-R1 through TRADD and TRAF2 (Hsu et al., 1996a). TRAF2 leads to the activation of the NFr~B, which elicits anti-apoptotic signaling in cells (see NF~B Activation -- Survival Gene Induction) and hence may protect cells from apoptosis. TNF-R1 activation has also been reported to induce NFKB activity through ceramide generation (Kolesnick and Golde, 1994), by activation of both neutral and acidic sphingomyelineases (SMase's) (Figure 2). Acidic SMase (aSMase) has a pH optimum of 5.5, is activated by diacylglyceral, is located in lysosomes, and is generally thought to transduce death signals. The DD-containing region of TNF-R1 activates aSMase by prior activation of phosphotidylcholine-specific phospholipase C. Increases in lysosomal ceramide can trigger apoptosis, but it has also been shown that TNF-Rl-mediated apoptosis can occur when this pathway is blocked, indicating that aSMase activation is not necessary for death-signaling by TNF (Santana et al., 1996). Neutral SMase (nSMase) has a pH optimum of 7.4, is Mg+ dependent, and is localized intracellularly

43

Death Domains and the Brian

Activated TNF-R1 C~ ~

Activated Fas

\

,

Sequentional ~ " caspase

V/f, O

TRAMP (DR3)

Activationof NF~cB

//

l¥ Cell Survival

/

~ '"/

/

///

/ /

???

/

Programmed Cell Figure 2. Apoptotic signal transduction pathways of death domain-containing receptors. The initial step in Fas-induced cell death is trimerization of the Fas receptor, followed by recruitment of the adapter protein FADD, which interacts with the death domain of Fas through its own death domain (depicted as a striped, upright rectangle). The death effector domain (DED -- depicted as a shaded rectangle) of FADD in turn interacts with the DED of procaspase 8, which together make up the death-inducing signaling complex (DISC). Cleavage and activation of caspase 8 takes place at the DISC, after which caspase 8 presumably initiates a caspase cascade that results in death of the cell. An alternate pathway of Fas-induced death may involve interactions of FADD with Apaf-1 and caspase 9, which bind through their respective caspase recruitment domains (CARD), depicted here as striped rectangles. Caspase activation through CARD interactions is facilitated by mitochondrial damage. Death signaling through the TNF receptor may proceed through interactions of the adapter protein TRADD (TNF receptor associated death domain) with FADD, and subsequent activation of the FADD/caspase 8 pathway. Alternatively, TNF-RI also activates neutral and acidic sphingomyelinases (aSM and nSM), leading to the production of ceramide. Specifically, i n c r e a s e s in ceramide levels in lysosomes following acidic sphingomyelinase activation has been shown to induce apoptosis. TNF-RI can also cause activation of the transcription factor NFKB, either through ceramide production, or activation of NFKB-inducing kinase (N1K), which associates with TRAF2 (TNF receptor associated factor 2). The role of NFKB in TNF-mediated apoptosis is not clear, but evidence indicates that NFKB may be anti-apoptotic. The signal transduction pathways of the TRAMP, TRAIL, and DR6 receptors have not been characterized, but may involve interactions with FADD and NFKB.

n e a r the p l a s m a m e m b r a n e . A m e m b r a n e p r o x i m a l r e g i o n o f the intracellular r e g i o n o f T N F - R 1 ( w h i c h is u p s t r e a m o f the DD, and is h o m o l o g o u s to a s e q u e n c e in the c o r r e s p o n d i n g r e g i o n o f Fas) b i n d s a m o l e c u l e called F A N ( f a c t o r - a s s o c i a t e d neutral S M a s e ) w h i c h in turn i n c r e a s e s n S M a s e activity. T h e s u b s e q u e n t p r o d u c t i o n o f c e r a m i d e

44

A.J. Bruce-KeHer

can trigger multiple transduction pathways, including activation of the translocatable transcription factor NFKB. Neutral SMase signaling is independent of death signaling by TNF-R1 (Adam-Klages et al., 1996), but the role of ceramide production in death receptor signaling (apoptosis and otherwise) is not yet clear. It is known, however, that once activated NFKB translocates to the nucleus, where it can activate target cytoprotective genes (see NFKB aActivation -- Survival Gene Induction). TRAIL (TNF-related apoptosis inducing ligand) binds the two apoptosis signaling receptors TRAIL-R1 and TRAIL-R2, as well as the two decoy receptors DcR1 and DcR2 (Bodmer et al., 1997; Pan et al., 1997a; Sheridan et al., 1997; Marsters et al., 1997; Walczak et al., 1997; Chaudhary et al., 1997; Schneider et al., 1997) (Figure 2). As is the case for Fas and TNF-R 1, TRAIL-induced death signaling is contingent upon caspase activation, as caspase inhibitors can block apoptosis mediated by TRAIL receptors. Caspase 8 has been reported to be activated by TRAIL receptor activation, but a structurally similar caspase, caspase 10 (FLICE-2) seems to be the preferential caspase activated by TRAIL (Pan et al., 1997b). The signaling aspects of the TRAIL receptors are not wello characterized, but initial reports indicated that TRAIL receptors can signal death through a FADD-independent pathway (Marsters et al., 1996; Pan et al., 1997b). However, it has since been shown that overexpression of dominant negative FADD (DN-FADD) can block TRAIL-induced apoptosis (Walczak et al., 1997; Chaudhary et al., 1997; Schneider et al., 1997). This discrepancy amongst reports may be a reflection of the distinct cell lines used in experiments, and the differences in relative concentrations of overexpressed and endogenous genes. For instance, the concentration of endogenous cellular FADD relative to the expression of the transfected DN-FADD construct will have profound quantitative effects on the ability of dominant negative FADD to block apoptosis. Hence, further investigation is required to determine whether FADD is directly or indirectly involved in TRAIL-induced apoptosis. Likewise, the signal transduction and biological function of TRAMP are also largely unknown. TRAMP is structurally related to TNF-R1, and overexpression of TRAMP leads to both NFKB activation and apoptosis (Bodmer et al., 1997; Chinnaiyan et al., 1996). TRAMP is abundantly expressed in thymocytes and lymphocytes, and may play a role in their development (Bodmer et al., 1997; Chinnaiyan et al., 1996), but the putative ligand for TRAMP has not yet been identified. Execution of the Death Signal -- Caspase Activation Activation of members of the caspase family may be the critical final common pathway mediating most (if not all) apoptotic signals (for review see Cohen, 1997; Nicholson and Thornberry, 1997). Caspases are synthesized as dormant precursor proteins that are activated by proteolytic cleavage, and once activated, cleave substrates after a P1 Asp residue. Based on phylogenetic analyses, the more than 10 mammalian caspase family members can be divided in three main groups. The ICE-like protease family includes caspase 1 (ICE), caspase 4 (ICH-2/ICEreIII), caspase 5 (TY/ICE jlII), and caspase 11 (ICH-3). The CED-3 family includes caspase 3 (CPP32/YAMA/apopain), caspase 6 (Mch2), and caspase 7 (Mch3/ICE-LAP3), caspase 8 (FLICE/MACHI/Mch5), caspase 9 (Mch6/ICE-LAP6), and caspase 10 (Mch4/FLICE2). The third family contains only caspase 2 (Nedd2/ICH-1).

Death Domains and the Brian

45

The importance of caspases in DD receptor signaling was first demonstrated in experiments in which TNF-R1- and Fas-mediated cell killing was inhibited by the caspase inhibitors YVAD and DEVD (Enari et al., 1995; Los et al., 1995; Tewari and Dixit, 1995). Measurement of caspase enzymatic activity also confirmed increased caspase activity within minutes following Fas activation (Los et al., 1995). Caspases can be divided into initiators and executors, based on their structure and relative order of induction in the death pathway. Caspase 8 was cloned as part of the DISC (Figure 2), and hence is the initial upstream caspase in DD receptor signaling. Caspase 8 is known to directly cleave (and hence activate) caspase 3, 4, 7, 9, and 10 in vitro (Muzio et al., 1977). Some reports have since demonstrated caspase 6 to be upstream of caspase 3 and 7 (Orth et al., 1996) in mammalian apoptosis, but it has also been shown that caspase 3 can activate caspase 6, 7, and 9 (Srinivascula et al., 1996; Fernandes-Alnemri et al., 1995, 1996). Hence, while it is known that caspase activation following death receptor occupation is sequential, the exact order of participating caspases is still obscure. An increasing number of proteins have been found to be cleaved by caspases in the apoptotic cascade (for review see Cohen, 1997; Nicholson and Thornberry, 1997). Specific substrates include proteins involved in genomic function, including the DNA repair enzyme poly (ADP-ribose) polymerase (PARP), DNA-polymerase kinase, heteronuclear ribonucleoproteins, and the 140 kDa component of the DNA replication complex. Regulators of cell cycle progression are also cleaved, including the p53 regulator MDM-2, retinoblastoma protein, the nuclear mitotic-associated protein NuMA, and the kinases PKC-~5 and MEKK1. Reorganization of cellular morphology during apoptosis is likely mediated by cytoskeletal alterations, and cytoskeletal proteins that are cleaved by caspases include b-catenin, keratin-18, and spectrin. Additionally, the caspase 6-dependent cleavage of laminins may be important in disassembling the nuclear cytoarchitecture during apoptosis. A direct link between caspase activation and DNA fragmentation has also been demonstrated by the cloning of the murine endonuclease CAD (caspase activated DNAse (Enari et al., 1998). Inactive CAD is sequestered in the cytosol by association with the inhibitory subunit ICAD, which is a specific substrate of caspase 3. Upon induction of apoptosis, ICAD is cleaved, and CAD translocated into the nucleus where it can enzymatically degrade DNA. Interestingly, overexpression of ICAD can block the chromatin alterations characteristic of apoptosis, but does not effect other manifestations (Enari et al., 1998). Besides the FADD/caspase 8 pathway, a novel apoptotic pathway initiated by DD-containing receptors has been identified which is controlled by Apaf-1 (apoptotic protease-activating factor-l). Apaf-1 is the mammalian homolog of the C. Elegans death regulator ced-4. It is not clear whether the Apaf-1 pathway is independent of the caspase 8 pathway, as direct binding of Apaf-1 to mammalian caspase 1 and caspase 8 has been observed. The N-terminal domain of Apaf-1 shares some similarity with other caspases, and serves as the so-called caspase recruitment domain (CARD). Through CARD interactions, Apaf-1 can bind capase 9 (Figure 2)(Hoffman et al., 1997; Pan et al., 1998). The CARD mediates the associations (i.e. binding) of caspases that have similar CARD motifs at their N-terminus (Hoffman et al., 1997). The CARD domain also links this pathway to mitochondrail alterations in apoptosis, as binding of ATP and cytochrome c, both released from damaged mitochondria (Liu et al., 1996a),

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induce a conformational change in Apaf-1 to unmask the CARD domain. This event can culminate in the recruitment and activation of caspase 9 (Li et al., 1997), and potentially also caspase 1 and caspase 2, which contain CARD domains. Thus it is possible that this pathway may act independently of FADD and other adaptor proteins, but the link between death receptors, mitochondrial alterations, and Apaf-1 remains to be firmly established.

Alternative Death Receptor Signaling -- Pathways to Cell Life

Although the signaling through death domains is a rapid way to induce cell death, there are alternative signaling pathways of DD containing receptors, and regulatory mechanisms that can modulate or inhibit death signaling. Modulation can affect DD-induced apoptosis at various steps in the pathway, from initiation to execution of the death signal. While the expression of downstream death-domain adaptor protein is generally thought to be constitutive, the presence of "cell life" pathways helps to explain the reported variations in sensitivity to death receptor activation. For example, the presence of Fas on a cell surface is not sufficient to cause Fas-ligand sensitivity. In vitro induction of Fas expression in T cells can precedes Fas-based killing by several days (Klas et al., 1993), and while Fas is expressed on most thymocyte subsets, only double positive cells are sensitive to anti-Fas antibodies (Ogasawara et al., 1995). Hence, apoptotic signaling through DD receptors is subject to multiple intracellular resistance mechanisms that can counteract the apoptotic signal. In addition to blocking apoptosis, these alternate pathways likely mediate such functions as differentiation, activation or proliferation following DD-receptor activation. Inhibitors of Apoptosis -- FLIPs and IAPs Following the discovery of caspase 8 which contains two N-terminal domains related to DED of FADD, homologous viral proteins were discovered that lacked caspase activity. Called v-FLIPs (viral FLICE inhibitory proteins), these proteins bind to the DISC, but prevent caspase 8 recruitment and activation (Bertin et al., 1997; Hu et al., 1997a; Thome et al., 1997). Shortly following v-FLIP identification, a cellular homolog (c-FLIP) was cloned and shown to have important regulatory functions for death signaling in mammalian cells (Irmler et al., 1997). c-FLIP was initially identified by several groups independently, and hence is called c-FLIP, CASPER, I-FLICE, FLAME-l, CASH, CLARP, and MRIT (Shu et al., 1997; Hu et al., 1997b; Srinivasula et al., 1997; Goltsev et al., 1997; Inohara et al., 1997; Han et al., 1997). c-FLIP protein is expressed as two splice variants, including a short form that contains two N-terminal DED motifs through which it associates with other DED proteins (Figure 3). The long form of c-FLIP contains an additional C-terminal region that resembles the proteolytic domain of caspase 8 and caspase 10, but has an amino acid substitution in the active site (Schulze-Osthoff et al., 1998). Hence, c-FLIP is thought to be proteolytically inactive but has been shown to interact with DD adaptor protein including FADD, TRAF2, caspase 8 and caspase 3 (Shu et al., 1997). It has been demonstrated that following transfection into cells, c-FLIP can antagonize

Death Domains and the Brian

47

apoptotic signaling induced by either TNF-R1, Fas, TRAIL, or TRAMP (h'mler et al., 1997; Shu et al., 1997; Srinivasula et al., 1997).

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The initial step of ligand binding to death domain-containing receptors can be blocked by truncated receptors that lack an intracellular death domain (Decoy receptors). The adapter protein c-FLIP (FLICE inhibitory protein) can block interaction of FADD with procaspase 8, and thereby prevent activation of the caspase cascade following Fas or TNF-R1 occupation. Likewise, interactions of TRAF2 (TNF receptor associated protein 2) with IAP (inhibitor of apoptosis protein) family members can block downstream sites caspase activation. Additionally, IAP has been shown to increase NFKB activity. Activation of the transcription factor NF~:B can prevent apoptosis in many cell types, and is induced following TNF-R1 occupation. Several mechanisms exist that cause dissociation of NFKB from the inhibitory subunit I~:B. Increases in neutral sphingomyelinase activity cause increases in ceramide concentrations, which activate NFKB through the action of the ceramide activated kinase (CA-Kinase). Likewise, activation of NFKB-inducing kinase (NIK), following association with TRAF2 (TNF receptor associated factor 2) also activates NFKB. Once dissociated from IKB, NFKB translocates to the nucleus where it can increase expression of protective proteins, thereby preventing apoptotic cell death.

T h e first m e m b e r o f the h u m a n i n h i b i t o r o f a p o p t o s i s p r o t e i n ( l A P ) f a m i l y w a s o r i g i n a l l y i s o l a t e d b a s e d o n its c o n t r i b u t i o n to n e u r o n a l d e g e n e r a t i o n in spinal m u s c u l a r a t r o p h y ( R o y et al., 1995). T h i s n e u r o n a l i n h i b i t o r o f a p o p t o s i s p r o t e i n ( N A I P ) was

48

A.J. Bruce-Keller

shown to be part of a conserved family of proteins originally discovered in baculovirus. In addition to NAIP, four additional human family members have been identified, c-IAP1, c-IAP2, X-lAP, and survivin, and all members have the ability to counteract apoptotic cell death (Rothe et al., 1995; Duckett et al., 1996; Ambrosini et al., 1997). Additionally, c-IAP1 and c-IAP2 have been shown to interact with TNF-R1 through TRAF2 (Rothe et al., 1995; Shu et al., 1996), but the role of c-IAP1/2 recruitment to TNF-R1 is unclear. A common feature of the lAP family is the Cys/His rich domain, repeated in the protein from one to three times, called the baculovirus lAP repeat (BIR). The fact that all family members have this domain suggests that it may be important in inhibiting apoptosis. Additionally, c-IAP1 and c-IAP2 also contain a zinc-finger RING domain at their C-terminus. In vitro studies of the caspase inhibitory properties of these proteins demonstrate that the BIR domains are sufficient for caspase inactivation, but that the proteins that also contain the RING structures are more effective (Deveraux et al., 1997; Cheng et al., 1996). Lastly, it has been demonstrated that c-IAP2 may be associated with activation of NF~B, an effect requiring an intact RING domain of c-IAP2 (Chu et al., 1997). Hence, IAPs could modulate DD-receptor induced apoptosis directly by inhibiting caspase activation, or indirectly, through increases in NFKB-mediated gene activation (see below). NFKB Activation -- Survival Gene Induction NFnB can be rapidly activated by DD-containing receptors by a variety of pathways, both direct and indirect. TNF-R1 causes activation of NFKB directly through TRAF2 and RIP, and indirectly through ceramide generation (Figure 3) (see above section Death domain signaling components -- pathways to cell death). Activation of nSMase causes the release of ceramide and sphingosine from sphingomyelin (Kolesnick and Golde, 1994; Cuvillier et al., 1996), and once formed, ceramide and sphingosine have diverse biological activities that can promote cell survival. Sphingosine and sphingosine-l-phosphate have been recently shown to promote cell survival in Jurkat T cells (Cuvillier et al., 1996), through a mechanism that involves activation of MAP kinase and protein kinase C (Cuvillier et al., 1996). Ceramide release, on the other hand, can also induce translocation of NFKB, via activation of a serine/threionine specific ceramide-activated kinase (CA-kinase), which induces phosphorylation and proteolysis of the NFKB inhibitory factor, IxB (Kolesnick and Golde, 1994). NFKB has also been reported to be activated by Fas (Ravi et al., 1998), and TRAMP (Bodmer et al., 1997; Chinnaiyan et al., 1996), indicating that NFnB may represent a broad-spectrum cellular mechanism to counteract DD-induced apoptotic signals. Additionally, TNF receptors mediate multiple pro-inflammatory pathways in immunocompetent cells (for review see Vassalli, 1992; Bazzoni and Beutler, 1996), and NFnB is an important mediator of inflammatory reactions. NFrB was originally identified in B lymphocytes where it stimulates transcription of the immunoglobulin K light chain (Sen and Baltimore, 1986). NFKB exists in the cytosol as an inducible 3 subunit complex consisting of two active KB subunits and an inhibitory subunit called IrB, and activation occurs when IKB is induced to dissociate from the complex. The active dimer then translocates to the nucleus and binds to 5' regulatory elements of genes responsive to NFKB; consisting of

Death Domains and the Brian

49

a decameric sequence (5'-GGGACTTTCC-3'). As NF~B is a preformed transcription factor with regulated activity, it differs from those in the AP-1 family (for example, whose members are regulated by gene induction). Similar to nuclear hormone receptors, NFKB activation directly transduces an extracellular signal to the nucleus, ensuring a rapid transcriptional response. Until recently, the prevailing view was that NF~zB may be a pro-apoptotic transcription factor, based its association with DD containing receptors. However, while the exact role(s) of NFKB in programmed cell death is not firmly established, recent evidence clearly demonstrates that NFKB activation can prevent apoptosis, both in neurons (Barger et al., 1995; Goodman and Mattson, 1996; Mattson et al., 1997), and in non-neuronal cells (Wu et al., 1996; Beg and Baltimore, 1996; Wang et al., 1996). Furthermore, in experiments in which downstream effectors of TNF-R 1 (FADD, TRAF2 and RIP) were manipulated, data obtained indicated that NFKB activation counteracts FADD-induced death (Liu et al., 1996b). Collectively, these data suggest that TNF-R 1 occupation simultaneously activates a cell death pathway and a cytoprotective pathway involving NFKB. Studies in different laboratories have identified several genes induced by NFKB that likely play important roles in increasing cellular resistance to injury. The antioxidant enzyme MnSOD is induced in several different cell types by TNF receptor stimulation in an NF~B-dependent manner (Eddy et al., 1992; Mattson et al., 1997; Bruce-Keller et al., 1999). In tumor cells resistant to TNF-induced apoptosis, MnSOD activity is increased following TNF treatment, whereas cells vulnerable to TNF-induced apoptosis did not increase their levels of MnSOD (Wong and Goeddel, 1988). MnSOD is a mitochondrially localized antioxidant enzyme that specifically detoxifies superoxide radicals. Superoxide production is increasingly recognized as a critical event in the cell death cascade, as it interacts with nitric oxide resulting in formation of peroxynitrite, a damaging free radical that induces membrane lipid peroxidation (Keller et al., 1998) and causes apoptosis (Beckman and Crow, 1993; Estevez et al., 1995). Peroxynitrite formation in neurons following amyloid B-peptide or iron treatment can be decreased by TNF-R1 stimulation (Mattson et al., 1997), presumably through NFKB-mediated MnSOD activation (Mattson et al., 1997; Furukawa and Mattson, 1998; Bruce-Keller et al., 1999). The existence of calcium dependent, mitochondrially localized nitric oxide synthase isotypes has been reported (Nichol et al., 1995; Buchwalow et al., 1997; Holmqvist and Ekstrom, 1997), supporting a role of peroxynitrite formation in mitochondrial dysfunction. The role of mitochondrial alterations in DD signaling is still disputed, but the role of mitochondrial secretions (ATP and cytochrome c) in Apaf-1 activation (see Execution of the Death Signal -- Caspase Activation) compel further investigation into the role of mitochondrial alterations in the FADD/caspase 8 pathway. For instance, it may be possible that caspase 8 has substrates in the outer mitochondrial membrane, the cleavage of which contributes to mitochondrial swelling and dysfunction, and may be modulated by MnSOD activity. A second gene target of NFI(B that may play a role in its anti-apoptotic action is the calcium-binding protein calbindin D28k. Activation of TNF-R1 can increase expression of calbindin D28k in neurons (Cheng and Mattson, 1994) and in astrocytes (Mattson et al., 1994), and the induction of calbindin can be associated with increased cellular resistance to death induced by agents that elevate intracellular calcium levels

50

A.J. Bruce-Keller

(i.e. excitatory amino acids in neurons and a calcium ionophore in astrocytes). Overexpression of calbindin D28k in pheochromocytoma cells protects those cells against apoptosis induced by calcium ionophore, trophic factor withdrawal, and an apoptosis-enhancing mutation in the presenilin-1 gene linked to Alzheimer's disease (Guo et al., 1998), but the role of calbindin in FADD-induced apoptosis has not been explored. Additional KB responsive genes linked to regulation of cell survival are emerging. In the nervous system, overactivation of glutamate receptors can induce neuronal apoptosis by a mechanism involving calcium overload and free radical production (Ankarcrona et al., 1995; Bonfoco et al., 1995). It was recently reported that NF~:B can modulate neuronal excitability by altering whole-cell currents through voltagedependent calcium channels and ionotropic glutamate receptor channels (Furukawa and Mattson, 1998). Whole-cell perforated patch camp recordings in cultured rat hippocampal neurons showed that long-term treatment (24-48 h), but not acute exposure to TNF results in an increase in Ca 2+ current density and a decrease in currents induced by glutamate. These effects were mimicked by pharmacological agents that specifically activate NFKB, and were prevented by agents that specifically block NFKB, demonstrating the important role of NF~:B activation in these TNF receptor pathways. Calcium imaging studies showed that neurons pretreated with TNF exhibit increased [Ca2+]i following membrane depolarization, but reduced [Ca2+]i responses to glutamate, compared to neurons in untreated control cultures. These findings demonstrate transcription-dependent modulation of voltage-dependent Ca 2÷ channels and glutamate receptors by TNF (Furukawa and Mattson, 1998), and suggest mechanisms whereby NF~:B may regulate neuronal survival, particularly in injury settings where both calcium and NFKB activation are known to be increased.

Death Receptors in the Central Nervous System -- Physiology and Pathophysiology The role of DD-containing receptors in mammalian physiology and pathophysiology is coming to light. It is quite clear, for example, that DD-containing proteins, in particular Fas, is necessary for the down-regulation of the immune system (Depraetere and Golstein, 1997). The elimination of T cells through apoptosis is very important for regulation of the immune response and maintenance of self-tolerance. Mutations of Fas or FasL in mice leads to severe autoimmune pathologies (Watanabe-Fukunaga et al., 1994; Takahashi et al., 1994). Both lpr (for lymphoproliferative) and gld mice (for generalized lymphoproliferative disease) which lack functional Fas and FasL, respectively, show severe autoimmune pathologies that highly resemble systemic lupus erythematosus in humans. Additionally, there is considerable evidence that inappropriate expression of TNF and TNF receptors can play a crucial role in both acute and chronic inflammatory disorders (for review see Vassalli, 1992; Bazzoni and Beutler, 1996). There is, however, considerably less clarity concerning the role of DD receptors in the central nervous system (CNS). TNF receptors are abundantly expressed in the mammalian brain, and as described below, TNF may be an important aspect of neuronal resistance to injury under pathophysiological conditions. In the case of Fas, however, there are conflicting reports concerning even the presence of

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51

Fas in normal mammalian brain, with some reports describing a low but detectable constitutive level of Fas and FasL expression in the CNS (French et al., 1996; Saas et al., 1997), while others describe the brain as completely devoid of Fas (Nishimura et al., 1995; Leithhauser et al., 1993). However, Fas expression known to be increased in post-ischemic brain, and in brain tissue from multiple sclerosis, Parkinson's disease, and Alzheimer's disease patients (see below), suggesting a role for Fas in causing or exacerbating neuronal injury. Additionally, since the brain is an immune-privileged organ, it is possible, as is the case for the eye and the testis, Fas and FasL are involved in maintaining immune privilege of the CNS. Fas and the CNS: Tumorigenesis and Chronic Neurodegenerative Disease While excessive inflammatory reactions in the brain are generally considered harmful (Giulian et al., 1990, 1993, 1995) and isolation from peripheral immune cells may be beneficial to neurons, the same situation could paradoxically facilitate tumor growth in the CNS. Recent studies have shown that many CNS tumor cells express Fas (Leithhauser et al., 1993; Saas et al., 1997; Gratas et al., 1997), through which the cells may counteract the immune system, and evade immune-mediated detection and elimination (Hahne et al., 1996). In support of this scenario, it has been shown that in contrast to normal brain, astrocytoma and gliobastomas are infiltrated to various degrees by peripheral inflammatory cells (Burger and Scheithauer, 1994), raising the possibility that FasL expression on these cells may prevent cytotoxic T cells or NK cells from removing tumorigenic cells. Moreover, monocytes, neutrophils, and activated T cells all express Fas (Lynch et al., 1995; Iwai et al., 1994), and are susceptible to death signals delivered by active FasL. Hence the same Fas-based mechanisms that protect cells from autoimmune destruction may be used by tumors, especially in immune-privileged organs such as the brain, to eliminate activated immune cells that may attempt to attack and remove the tumor cells. On the other hand, tumor cells in some cases express Fas and, hence, could be susceptible to FasL-induced death (in contrast to the low levels of Fas in normal brain tissue). A modified Fas-based anti-tumor therapy is under study, in many clinical settings, for the treatment of malignant brain tumors (Friesen et al., 1997; Fulda et al., 1997). Based on the roles of Fas and FasL in self-tolerance and immune system regulation, it was hypothesized that this pathway may have a role in CNS autoimmune disorders. Both Fas and FasL have been shown to be expressed in glial cells in chronic inflammatory lesions in multiple sclerosis (MS) (Dowling et al., 1996). Microglial cells at sites of CNS lesions express FasL, and these cells have been proposed to be mediators of apoptotic cell death in MS (D'Souza et al., 1996). Further in vitro studies demonstrate that oligodendrocytes, which are the cellular target in MS, are the main cells in the adult CNS that express Fas, while human neurons, in particular, do not have detectable Fas expression (Becher et al., 1998). These data suggest a mechanism whereby inflammatory cells could selective induce cell-type specific (i.e. oligodendrocyte) injury in the CNS. In further support of this scenario, a significant percentage of immunocompetent cells in MS lesions do indeed express FasL (Dowling et al., 1996). The colocalization of TUNEL-positive dying cells with FasL, and the presence of numerous

52

A.J. Bruce-KeUer

Fas-positive corpses within activated macrophages support the theory that Fas expressing oligodendrocytes are indeed a target of FasL attack in MS lesions (Dowling et al., 1996). Based on the role of caspase activation in Fas-mediated cell death and in vitro evidence for caspase mediated oligodendrocyte death (Enari et al., 1996), inhibition of caspases may have some therapeutic value in MS. The role of neuroimmune interactions in age-related neurodegeneration in the CNS is not well understood, but is has been suggested that chronic neurodegenerative disorders of aging such as Alzheimer's (AD) and Parkinson's disease (PD) may be, in part, mediated by altered immune responses (Fiszer et al., 1991; McGreer and McGreer, 1995, 1996). Based on observations that neuronal loss in AD and PD may be apoptotic, and the reported beneficial effects of anti-inflammatory regimens in especially AD, one could hypothesize a role for Fas in neuronal death in these diseases. Indeed, Fas-positive astrocytes have been documented in Alzheimer's brain samples, and data indicate that Fas expression is associated with astrocyte activation (Nishimura et al., 1995). The presence of Fas in astrocytes surrounding senile plaques is also consistent with this view, but it is not clear at this point what signals induce Fas expression on these cells, and there is as of yet no indication that Fas positive astrocytes are subject to apoptosis in AD brain. In PD brain samples, on the other hand, studies have demonstrated a significant increase in soluble Fas in the dopaminergic nigro-striatal pathway of affected individuals (Mogi et al., 1996). Soluble Fas (sFas) was first identified by molecular cloning and nucleotide sequence analysis that detected a Fas mRNA variant capable of encoding a soluble molecule that lacked the transmembrane anchor due to an exon deletion (Cheng et al., 1994). While the physiological function of sFas are unknown, many features of Fas are shared by the soluble molecule, especially in relation to autoimmune abnormalities (Knipping et al., 1995), indicating that an idiopathic alteration in sFas production may somehow be involved in the specific pattern of dopaminergic neurodegeneration in PD. Collectively, these observations suggest that while the majority of CNS neurons and glial cells have low levels of Fas expression, Fas and FasL are increased in specific, discrete regions under injury conditions, and may contribute to local tissue destruction. In further support of this, detectable increases in FasL have been found in cerebrospinal fluid following severe head injury, and in microglial cells following hypoxia/reoxygenation (Ertel et al., 1997; Vogt et al., 1998). However, only a correlative association can be made between neuronal apoptosis and increases in Fas and FasL expression in brain cells under injury conditions, and delineation of the exact consequences of Fas expression in the brain await further studies. TNF and Neuronal Resistance to Injury TNF levels are known to be increased in human brain in cases of AD, PD, or head trauma, but the consequences are still unclear. Unlike Fas, the role of TNF-R1 in neuronal physiology may be unrelated to its death-inducing functions, as accumulating experimental evidence indicates that TNF acts as a neuroprotective agent in the brain. Studies have demonstrated that TNF-R1 receptor signaling affords significant protection against subsequent glutamate toxicity, oxidative insults, and amyloid f5 peptide toxicity in hippocampal neurons in vitro (Cheng and Mattson, 1994; Barger et al., 1995).

Death Domains and the Brian

53

While two membrane associated TNF receptors (TNF-R1 and TNF-R2) have been characterized (Kinouchi et al., 1991; Wolvers et al., 1993; Lewis et.al., 1991), in vitro studies indicate that TNF-R1 is both necessary and sufficient to for TNF-mediated neuroprotection (Figure 4). Furthermore, most in vitro studies employ a combination of human recombinant TNF applied to rodent cells, and as human TNF will activate only TNF-RI in rodent cells, it is clear that TNF-R1 activation in neurons is neuroprotective. The role of TNF-R2 in protective paradigms is unclear, but may be limited to facilitating TNF-R1 occupancy (Tartaglia et al., 1993b). The protective role of endogenous TNF has been demonstrated through the use of transgenic mice deficient in TNF receptors. Transgenic mice lacking both TNF receptors show significantly increased damage excitotoxic and ischemic insults when compared to wild-type controls (Bruce et al., 1996), indicating that the reported increases in TNF following brain injury may stimulate neuroprotective signaling pathways. Subsequent studies using mice lacking either TNF-R 1 or TNF-R2 demonstrated again that TNF-R 1 is responsible for the neuroprotective effects of TNF in the brain (Gary et al., 1998), and data further indicate that the signal transduction pathway elicited by TNF-R1 in neuroprotection likely involves NF~cB and increases in MnSOD activity (Mattson et al., 1997; Bruce et al., 1996). As mentioned previously, the divergent effects of DD-containing receptor activation is a tightly regulated, cell type specific process. Therefore, the neuroprotective effects of TNF in the brain must also be considered from the standpoint of cell-cell (i.e. neuron-glial) interactions. While TNF-R1 signaling is trophic to neurons, it can be toxic to oligodendrocytes, induce astrocyte and microglial proliferation, and also stimulate trophic factor release from these cells (Goossens et al., 1995; Jaattela, 1991; Gadient et al., 1990; Le and Vilcek, 1987). Experiments aimed at determining the signals elicited by TNF-RI in different cell types in the brain again demonstrate that induction of MnSOD is critical for TNF-R1 mediated cytoprotection (Figure 5). Data indicate that while TNF application is not directly toxic to neurons, astrocytes, or oligodendrocytes, TNF-R1 signaling is beneficial only in cell types in which it can increase MnSOD activity (Figure 5) (Bruce-Keller et al., 1999). It is not clear, at this point, what factors are responsible for the divergent TNF signaling pathways in different cell types in the brain, and further experimentation is necessary to determine if these pathways are hard-wired or can be altered. Expression of TNF in the brain is increased following neuronal injury (Minami et al., 1991; Taupin et al., 1993; Tchelingerian et al., 1993; Liu et al., 1994; Kim, 1996; Buttini et al., 1994; Szaflarski et al., 1995), and the profile of TNF expression following injury is biphasic, with both an early (minutes to hours) peak and a sustained (days) plateau in TNF levels (Liu et al., 1994; Kim, 1996). The immediate and delayed pattern of TNF expression in these studies likely reflects different sources of TNF production, as TNF mRNA is rapidly (1-4 h) increased in affected neurons following injury (Minami et al., 1991; Taupin et al., 1993; Tchelingerian et al., 1993; Bruce et al., 1996), while the protracted expression (2-5 days) is detected in microglial and macrophage cells surrounding the damaged tissue (Liu et al., 1994; Kim, 1996). These observations support the following scenario: immediate neuronal alterations in response to injury conditions leads to early production of TNF in neurons, which when secreted aids in the recruitment and proliferating of resident microglia, which also secrete TNF. The resulting cascade of TNF autocrine and paracrine

54

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signaling can provide trophic support to neurons while propagating a brain-resident inflammatory cascade that promotes resolution of injury and functional recovery. This hypothesis is supported by experimental observations, in mice lacking TNF receptors, which show significantly increased neuronal loss but remarkably decreased reactive inflammatory gliosis following excitotoxicity or ischemia (Bruce et al., 1996), Furthermore, additional studies have also confirmed that microglial activation can attenuate excitotoxic or ischemic injury in rodents (Berezovskaya et al., 1995), prevent apoptosis in vitro (Toku et al., 1998), and increase neurite outgrowth and functional recovery following brain injury (Lazarov-Spiegler et al., 1996; Prewitt et al., 1997; Rabchevsky and Streit, 1997).

Concluding Remarks This review summarizes the different death receptors and the intracellular pathways they activate to induce cell life or death. Unfortunately, the cloning and characterization of death receptors and their myriad of control mechanisms proceeds well ahead of the understanding of their roles in human physiology or pathophysiology. Hence, a major goal of death receptor research in the future will be to determine the biological function of the death-receptor signaling, and to delineate what additional intracellular signals dictate signaling for life or death for a cell. Such an understanding is crucial if therapeutic interventions are to be based on death receptor signaling. Pharmacological targeting of death-inducing pathways could be very important for cancer therapy, in which resistance to apoptosis is often associated with chemotherapeutic drug resistance. Additionally, a death-domain based therapy would have promising clinical applications for autoimmune disorders such as multiple sclerosis and systemic lupus erythematosus. On the other hand, the potent neuroprotective properties of TNF suggests that mimicry of these pathways could be of significant therapeutic importance in neurodegenerative disorders, including AD, PD, epilepsy and stroke. The neuroprotective signal transduction pathway utilized by TNF-R1 highlights several sites for potential pharmacological intervention. One mode would be to activate TNF-R1 receptor directly, either through application of TNF itself, or through design of a TNF-Rl-specific agonist. However, potentially harmful side effects of this paradigm could occur upon TNF receptor stimulation of other cell types, including microglia, oligodendrocytes, and endothelial cells. The successful harnessing of events downstream of TNF-R 1 receptor activation would preclude such effects, and would include ceramide application, NFKB activation, or the up-regulation of neuroprotective genes such as MnSOD. The observation that Fas and TNF-R1 receptors, which have such extensive overlap in intracellular mediators, could have such divergent functional roles in the CNS further illustrates the highly pleiotrophic nature of these remarkable receptors, and should drive meaningful research for years to come.

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References Adam-Klages, S., Adam, D., Wiegmann, K., Struve, S., Kolanus, W., Schneider-Mergener, J. & Kroenke, M. (1996). Fan, a novel WD repeat protein, couples the p55 TNF-receptor to neutral sphingomyelinase. Cell 86, 937-947. Alnemri, E.S., Livingston, D.J., Nicholson, D.W., Salvesen, G., Thornberyy, N.A., Wong, W.W. & Yuan, J. (1996). Human IVE/CED-3 protease nomenclature. Cell 87, 171. Ambrosini, G., Adida, C. & Altieri, D. (1997). A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nature Med. 3,917-921. Ankarcrona, M., Dypbukt, J.M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S.A. & Nicotera, P. (1995). Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15,961-973. Banner, D.W., D'Arcy, A., Janes, W., Gentz, R., Schoenfeld, H.J., Broger, C., Loetscher, H. & Lesslauer, W. (1993). Crystal structure of the soluble human p55 TNF receptor-human TNFbeta complex: implications for TNF receptor activation. Cell 73,431-445. Barger, S.W., Horster, D., Furukawa, K., Goodman, Y., Krieglstein, J. & Mattson, M.P. (1995). Tumor necrosis factors alpha and beta protect neurons against amyloid B-peptide toxicity: Evidence for involvement of a kappa B-binding factor and attenuation of peroxide and Ca2+ accumulation. Proc. Nat. Acad. Sci. U.S.A. 92, 9328-9332. Bazzoni, F. & Beutler, B. (1996). The tumor necrosis factor ligand and receptor families. New Engl. J. Med. 334, 1717-1725. Becher, B., D'Souza, S.D., Trout, A.B. & Antel, J.P. (1998). Fas expression on human fetal astrocytes without susceptibility to fas-mediated cytotoxicity. Neuroscience 84, 627-634. Beckman, J.S. & Crow, J.P. (1993). Pathological implications of nitric oxide, superoxide and peroxynitrite formation. Biochem. Soc. Trans. 21,330-334. Beg, A.A. & Baltimore, D. (1996). An essential role for NF-KB in preventing TNF-a-induced cell death. Science 274, 782-784. Berezovskaya, O., Maysinger, D. & Fedoroff, S. (1995). The hemapoietic cytokine, colony stimulating factor 1, is also a growth factor in the CNS: congenetal absence of CSF-1 in mice results in abnormal microglial response and increased neuron vulnerability to injury. Int. J. Dev. Neurosci. 13,285-299. Bertin, J., Armstrong, R.C., Ottilie, S., Martin, D.A., Wany, Y., Banks, S., Wang, G.H., Senkevich, T.G., Alnemri, E.S., Moss, B., Lenardo, M.J., Tomaselli, K.J. & Cohen, J.I. (1997). Death effector domaincontaining herpesvirus and poxvirus proteins inhibit both Fas- and TNFRl-induced apoptosis. Proc. Nat. Acad. Sci. U. S. A. 94, 1172-1176. Bodmer, J.L., Burns, K., Schneider, P., Hofman, K., Steiner, V., Thome, M., Bornand, T., Hahne, M., Schroter, M., Becker, K., Wilson, A., Frence, L.E., Browning, J.L., MacDnoald, H.R. & Tschopp, J. (1997). TRAMP, a novel apoptosis mediating receptor with sequence homology toTNF-R1 and Fas. Immunity 6, 79-88. Boldin, M.P., Goncharov, T.M., Goltsev, Y.V. & Wallach, D. (1996). Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1 and TNF receptor-induced cell death. Cell 85, 803-815. Boldin, M.P., Varfolomeev, E.E., Pancer, Z., Mett, I.L., Camonis, J.H. & Wallach, D. (1995). A novel protein that interacts with the death domains of Fas/APO1 contains a sequence motif related to the death domain. J. Biol. Chem. 270, 7795-7798. Bonfoco, E., Krainc, D., Ankarcrona, M., Nicotera, P. & Lipton, S.A. (1995). Apoptosis and necrosis: two

58

A.J. Bruce-Keller

distinct events induced, respectively, by mild and intense insults with N-methyl-D- aspartate or nitric oxide/superoxide in cortical cell cultures. Proc. Natl. Acad. Sci. U.S.A. 92, 7162-7166. Bruce, A.J., Boling, W.W., Kindy, M.S., Peschon, J., Kraemer, P.J., Carpenter, M.K., Holtzberg, F.W. & Mattson, M.P. (1996). Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors. Nat. Med. 2, 788-794. Bruce-Keller, A.J., Geddes, J.W., Knapp, P.E., McFall, R.W., Keller, J.N., Holtsberg, F.W., Steiner, S.M., Partharasy, S. & Mattson, M.P. (1999). Anti-death properties of TNF against metabolic poisoning: mitochondrial stabilization by MnSOD. J. Neuroimmunol. 93, 53-71. Buchwalow, I.B., Schulze, W., Kostic, M.M., Wallukat, G. & Morwinski, R. (1997). Intracellular localization of inducible nitric oxide synthase in neonatal rat cardiomyocytes in culture. Acta. Histochem. 99, 231-40. Burger, P.C. & Scheithauer, B.W. (1994). Tumors of the central nervous system. (Washington, DC: Armed Forces Institute of Pathology.). Buttini, M., Sauter, A. & Boddeke, H.W. (1994). Induction of interleukin-1 beta mRNA after focal cerebral ischaemia in the rat. Mol. Brain Res. 25, 126-134. Chaudhary, P.M., Eby, M., Jasmin, A., Bookwater, A., Murray, J. & Hood, L. (1997). Death receptor 5, a new member of the TNFR family & DR4 induce FADD-dependent apoptosis and activate the NF-kappa B pathway. Immunity 7, 821-830. Cheng, B. & Mattson, M. (1994). Tumor necrosis factors protect neurons against excitotoxic/metabolic insults and promote maintenance of calcium homeostasis. Neuron 12, 139-153. Cheng, E.H., Levine, B., Boise, L.H., Thompson, C.B. & Hardwick, J.M. (1996). Bax-independent inhibition of apotosis by BcI-XL. Nature 379, 554-556. Cheng, J., Zhou, T., Liu, C., Shapiro, J.P., Brauer, M.J., Keifer, M.C., Barr, P.J. & Mountz, J.D. (1994). Protection from Fas-mediated apoposis by a soluble form of the Fas molecule. Science 263, 1759-1762. Chinnaiyan, A.M., O'Rourke, K., Tewari, M. & Dixit, V.M. (1995). FADD, a novel death domain-contianing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81,505-512. Chinnaiyan, A.M., O'Rourke, K., Yu, G., Lyons, R.H., Gear, M., Duan, D.R., Xing, L., Gebtz, R., Ni, J. & Dixit, V.M. (1996). Signal transduction by DR3, a death domain containing receptor related to TNF-R1 and CD95. Science 274, 990-992. Chu, Z.L., McKinsey, T.A., Liu, L., Gentry, J.J., Malim, M. & Ballard, D.W. (1997). Suppression of tumor necrosis fcator-induced cell death by inhibitor of apoptosis c-lAP is under NF-KB control. Proc. Nat. Acad. Sci. U.S.A. 94, 10657-10667. Cohen, G.M. (1997). Caspases: the executioners of apoptosis. Biochem. J. 326, 1-16. Cuvillier, O., Pirianiv, G., Kleuser, B., Vanek, P.G., Coso, O.A., Gutkind, J.S. & Spiegel, S. (1996). Supression of ceramide-mediated programmed cell death by sphingosine-1-phosphate. Nature 381,800-803. D'Souza, S.D., Bonetti, B., Balasingam, V., Cashman, N.R., Barker, P.A., Troutt, A.B., Raine, C.S. & Antel, J.P. (1996). Multiple sclerosis: Fas signaling in oligodendrocyte cell death. J. Exp. Med. 184, 2361-70. Degli-Esposti, M.A., Smolak, P.J., Walczak, H., Waugh, J., Huang, C.P., DuBose, R.F., Goodwin, R.G., & Smith, C.A. (1997). Cloning and characterization of TRAIL-R3, a novel member of the emerging TRAIL receptor family. J. Exp. Med. 186, 1165-1170. Depraetere, V. & Golstein, P. (1997). Fas and other cell death signaling pathways. Sere. Immunol. 9, 93-107. Deveraux, Q., Takahashi, R., Salvesen, G.S. & Reed, J.C. (1997). X-linked lAP is a direct inhibitor of cell death processes. Nature 388, 300-303. Dhein, J., Daniel, P.T., Trauth, B.C., Oehm, A., Moeller, P. & Krammer, P.H. (1992). Induction of apoptosis by monoclonal antibody anti-APO-1 class switch variants is dependent on cross-linking of APO-1 cell surface receptors. J. Immunol. 149, 3166-3173.

Death Domains and the Brian

59

Dowling, P., Shang, G., Raval, S., Menonna, J., Cook, S. & Husar, W. (1996). Involvement of the CD95 (APO-I/Fas) receptor/ligand system in multiple sclerosis brain. J. Exp. Med. 184, 1513-1518. Duckett, C.S., Nava, V.E., Gedrich, R.W., Clem, R.J., Van Dongen, J.L., Gilfillan, M.C., Shiels, H., Hardwick, J.M. & Thompson, C.B. (1996). A conserved family of cellular genes related to the baculovirus iap gene and encoding apoptosis inhibitors. EMBO J. 15, 2685-2694. Eddy, L., Goeddel, D. & Wong, G. (1992). Tumor necrosis factor-alpha pretreatment is protective in a rat model of myocardial ischemia-reperfusion injury. Biochem. Biophys. Res. Commun. 184, 1056-1059. Enari, M., Hug, H. & Nagata, S. (1995). Involvement of an ICE-like protease in Fas-mediated apoptosis. Nature 375, 78-81. Enari, M., Sakahiera, H., Yokoyama, H., Okawa, K., Iwamatsu, A. & Magata, S. (1998). A caspase activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43-50. Enari, M., Talanian, R., Wong, W. & Nagata, S. (1996). Sequential activation of ICE-like and CPP32-1ike proteases during Fas-mediated apoptosis. Nature 380, 723-726. Ertel, W., Keel, M., Stocker, R., Imhof, H. G., Leist, M., Steckholzer, U., Tanaka, M., Trentz, O. & Nagata, S. (1997). Detectable concentrations of Fas ligand in cerebrospinal fluid after severe head injury. J. Neuroimmunol. 80, 93-96. Estevez, A.G., Radi, R., Barbeito, L., Shin, J.T., Thompson, J.A. & Beckman, J.S. (1995). Peroxynitriteinduced cytotoxicity in PC12 cells: evidence for an apoptotic mechanism differentially modulated by neurotrophic factors. J. Neurochem. 65, 1543-1550. Fernandes-Alnemri, T., Armstrong, R.C., Krebs, J., Srinivasula, S.M., Wang, L., Bullrich, F., Fritz, L.C., Trapani, J.A., Tomaselli, K.J., Litwack, G. & Alnemri, E.S. (1996). In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-Iike domains. Proc. Natl Acad. Sci. U. S. A. 93, 7464-7469. Fernandes-Alnemri, T., Takahashi, A., Armstrong, R.C., Krebs, J., Tomaselli, K.J., Wang, L., Yu, Z., Croce, C.M., Salveson, G., Earnshaw, W.C., Litwack, G. & Alnemri, E.S. (1995). Mch3, a novel human apoptotic cysteine protease related to CPP32. Cancer Res. 55, 6045-6052. Fiszer, U., Piotrowska, K., Kodak, J. & Czlonkowska, A. (1991). The immunological status in Parkinson's disease. Med. Lab. Sci. 48, 196-200. French, L.E., Hahne, M., Viard, I., Radlgruber, G., Zanone, R., Becker, K., Muller, C. & Tschopp, J. (1996). Fas and Fas ligand in embryos and adult mice: ligand expressin in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover. J. Cell. Biol. 133,335-343. Friesen, C., Herr, I., Krammer, P.H. & Debatin, K.M. (1997). Involvement of the CD95 (APO-1/Fas) receptor ligand system in drug-induced apoptosis in leukemia ceils. Nat. Med. 2, 574-577. Fulda, S., Seiverts, H., Friesen, C., Herr, 1. & Debatin, K.M. (1997). The CD95 (APO-I/Fas) system mediates drug-induced apoptosis in neuroblastoma cells. Cancer Res. 57, 3823-3829. Furukawa, K. & Mattson, M.P. (1998). The transcription factor NF-KB mediates increases in calcium currents and decreases in NMDA and AMPA/kainate-induced currents in response to TNFa in hippocampal neurons. J. Neurochem. 70, 1876-1886. Gadient, R.A., Cron, K.C. & Otten, U. (1990). Interleukin-1 13and tumor necrosis factor-alpha synergistically stimulate nerve growth factor (NGF) release from cultured rat astrcytes. Neurosci. Lett. 117, 335-340. Gary, D.S., Bruce-Keller, A.J., Kindy, M.S. & Mattson, M.P. (1998). Ischemic and excitotoxic brain injury is enhanced in mice lacking the p55 tumor necrosis factor receptor. J. Cereb. Blood Flow Metab. 12, 1283-1287. Giulian, D., Haverkamp, L.J., Li, J., Karshin, W.L., Yu, J., Tom, D., Li, X. & Kirkpatrick, J.B. (1995). Senile plaques stimulate microglia to release a neurotoxin found in Alzheimer brain. Neurochem. Int. 27, 119-137.

60

A.J. Bruce-Keller

Giulian, D., Vaca, K. & Corpuz, M. (1993). Brain glia release factors with opposing actions upon neuronal survival. J. Neurosci. 13, 29-37. Giulian, D., Vaca, K. & Noonan, C. (1990). Secretion of neurotoxins by mononuclear phagocytes infected with HIV-1. Science 250, 1593-1596. Goltsev, Y.K., Kovalenko, A.V., Arnold, E., Varfolomeev, E.E., Brodianskii, V.M. & Wallach, D. (1997). CASH, a novel caspase homolog with death effector domains. J. Biol. Chem. 272, 19641-19644. Goodman, Y. & Mattson, M.P. (1996). Ceramide protects hippocampal neurons against excitotoxic and oxidative insults, and amyloid B-peptide toxicity. J. Neurochem. 66, 869-872. Goossens, V., Grooten, J., De Vos, K. & Fiers, W. (1995). Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proc. Natl. Acad. Sci. U.S.A. 92, 8115-8119. Grams, C., Tohma, Y., Van Mier, E.g. Klein, M., Tenan, M., Ishii, N., Tachibana, O., Kleihues, P. & Ohgaki, H. (1997). Fas ligand expression in glioblastoma cell lines and in primary astrocytic brain tumors. Brain Pathol. 7, 863-869. Guo, Q., Robinson, N. & Mattson, M.P. (1998). Secreted APPa counteracts the pro-apoptotic action of mutant presenilin-1 by activation of NF-KB and stabilization of calcium homeostasis. J. Biol. Chem. 273, 12341-12351. Hahne, M., Rimoldi, D., Schroter, M., Romero, P., Schreier, M., French, L.E., Schneider, P., Borland, T., Fontana, A., Lienard, D., Cerottini, J. & Tschopp, J. (1996). Melanoma cell expression of Fas ligand: immplications for tumor immune escape. Science 274, 1363-1366. Han, D.K.M., Chaudry, P.M., Wright, M.E., Friedman, C., Trask, B.J., Reidel, R.T., Baskin, D.G., Schwartz, S.M. & Hood, L. (1997). MRIT, a novel death-effector domain-containing protein interacts with caspases and BclXL and initiates cell death. Proc. Nat. Acad. Sci. U. S. A. 94, 11333-11338. Hoffman, K., Bucher, P. & Tschopp, J. (1997). The CARD domian, a new apoptotic signaling motif. Trends Biochem. Sci. 22, 155-156. Holmqvist, B. & Ekstrom, P. (1997). Subcellular localization of neuronal nitric oxide synthase in the brain of a teleost; an immunoelectron and confocal microscopical study. Brain Res. 745, 67-82. Hsu, H., Huang, J., Shu, H.B., Baichwal, V. & Goeddel, D.V. (1996a). TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex. Inmmunity 4, 387-396. Hsu, H., Shu, H.B., Pan, M.G. & Goeddel, D.V. (1996b). TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal tmnsduction pathways. Cell 84, 299-308. Hsu, H., Xiong, J. & Goeddel, D.V. (1995). The TNF receptor 1-associated protein TRADD signals cell death and NF-~:B activation. Cell 81,495-504. Hu, S., Vincenz, C., Buller, M. & Dixit, V.M. (1997a). A novel family of viral death effector domiancontaining molecules that inhibit both CD95- and TNF-Rl-induced apoptosis. J. Biol. Chem. 272, 9621-9624. Hu, S., Vincenz, C., Ni, J., Gentz, R. & Dixit, V.M. (1997b). I-FLICE, a novel inhibitor of tumor necrosis factor receptor-l- and CD95-induced apoptosis. J. Biol. Chem. 275, 17255-17257. Inohara, N., Koseki, T., Hu, Y., Chen, S. & Nunez, G. (1997). CLARP, a death effector domain-containing protein interacts with caspase 8 and regulates apoptosis. Proc. Nat. Acad. Sci. U. S. A. 94, 1017-1022. Irmler, M., Thome, M., Hahne, M., Schneider, P., Steiner, V., Bodme, J.L., Schroeter, M., Burns, K., Mattmann, C., Hofmann, C., Rimoldi, D., French, L.E. & Tschopp, J. (1997). Inhibition of death receptor signals by cellular FLIP. Nature 388, 190-195. Itoh, N. & Nagata, S. (1993). A novel protein domain required for apoptosis. Mutational analysis of human Fas antigen. J. Biol. Chem. 268, 10932- 10937.

Death Domains and the Brian

61

Itoh, N., Yonehara, S.I.A., Yonehara, M., Mizushima, S., Sameshima, M., Hase, A., Seto, Y. & Nagata, S. (1991). The polypeptide encoded for the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66, 233-243. Iwai, K., Miaywaki, T., Takizawa, T., Konno, A., Ohta, K., Yachie, A., Seki, H. & Taniguchi, N. (1994). Differential expression of bcl-2 and susceptability to anti-Fas-mediated cell death in peripheral blood lymphocytes, monocytes & neutrophils. Blood 84, 1201-1208. Jaattela, M. (1991). Biological activities and mechanisms of action of tumor necrosis factor alpha / cachectin. Lab. Invest. 64, 724-742. Keller, J.N., Kindy, M.S., Holtsberg, F.W., St Clair, D.K., Yen, H.C., Germeyer, A., Steriner, S.M., Bruce-Keller, A.J., Hutchins, J.B. & Mattson, M.P. (1998). Mitochondrial MnSOD prevents neuroal apoptosis and reduces ischemic brian injury: supression of peroxynitrite production, lipid peroxidation & mitochondrial dysfunction. J. Neurosci. 18, 687-697. Kim, J.S. (1996). Cytokines and adhesion molecules in stroke and related diseases. J. Neurol. Sci. 137, 69-78. Kinouchi, K., Brown, G., Pasternak, G. & Donner, D.B. (1991). Identification and characterization of receptors for tumor necrosis factor-alpha in the brain. Biochem. Biophys. Res. Commun. 181, 1532-1538. Klas, C., Debatin, K.M., Jonker, R.R. & Krammer, P.H. (1993). Activation interferes with the APO-I pathway in mature human T cells. Immnuol. 5,625-630. Knipping, E., Debatin, K.M., Strucker, K., Heilig, B., Eder, A. & Krammer, P.H. (1995). Identification of soluble APO-1 in supernatents of human B- and T-cell leukemias. Blood. 85, 1562-1569. Kolesnick, R. & Golde, D.W. (1994). The sphingomyelin pathway in tumor necrosis factor and interleukin-I signaling. Cell 77,325-328. Lazarov-Spiegler, O., Solomon, A.S., Zeev-Brann, A.B., Hirschberg, D., Lavie, V. & Schwartz, M. (1996). Transplantation of activated macrophages overcomes central nervous system regrowth failure. FASEB J. 10, 1296-302. Le, J. & Vilcek, J. (1987). Tumor necrosis factor and interleukin 1: cytokines with multiple overlapping biological activities. Lab. Invest. 56, 234-248. Leithhauser, F., Dhein, J., Mechtersheimer, G., Koretz, K., Bruderlein, S., Henne, C., Schmidt, A., Debatin, K.M., Krammer, P.H. & Moiler, P. (1993). Constituative and induced expression of APO-1, a new member of the NGF/TNF receptor family superfamily, in normal and neoplastc cells. Lab. Invest. 69, 415-429. Lewis, M., Tartaglia, L.A., Lee, A., Bennett, G.L., Rice, G.C., Wong, G.H., Chen, E.Y. & Goeddel, D.V. (1991). Cloning and expression of cDNAs for two distinct murine tumor necrosis factor receptors demonstrate one receptor is species specific. Proc. Nat. Acad. Sci. U. S. A. 88, 2830-2834. Li, P., Hijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S. & Wang, X. (1997). Cytochrome c and dATP dependent formation of Apaf-l/Caspase-9 complex initiates an apoptotic protease cascade. Cell 91,155-156. Liu, T., Clark, R.K., McConnell, P.C., Young, P.R., White, R.F., Barone, F.C. & Feuerstein, G.Z. (1994). Tumor necrosis factor in ischemic neurons. Stroke 25, 1481-1488. Liu, X., Kim, C.N., Yang, J., Jemmerson, R. & Wang, X. (1996a). Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86, 147-157. Liu, Z.-G., Hsu, H., Goeddel, D.V. & Karin, M. (1996b). Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-•B activation prevents cell death. Cell 87,565-576. Los, M., Van de Craen, M., Penning, L.C., Schenk, H., Westendorp, M., Baeuerle, P.A., Drodge, W., Krammer, P.H., Fiers, W. & Shultz-Osthoff, K. (1995). Requirement of an ICE/CED-3 protease for Fas/APO-l-mediated apoptosis. Nature 375, 81-83.

62

A.J. Bruce-Keller

Lynch, D.H., Ramsdell, F. & Alderson, M.R. (1995). Fas and FasL in the homeostatic regulation of the immune system. Immunol. Today 16, 569-574. Marsters, S.A., Pitti, R.M., Donaheu, C.J., Ruppert, S., Bauer, K.D. & Ashkenazi, A. (1996). Activation of apoptosis by Apo-2 ligand is independent of FADD but blocked by CrmA. Curr. Biol. 6, 750-752. Marsters, S.A., Sheridan, J.P., Pitti, R.M., Skubatch, M., Baldwin, D., Yuan, J., Gurney, A., Goddard, A.D., Godowski, P. & Ashkenazi, A. (1997). A novel receptor for Apo21/TRAIL contains a truncated death domain. Curr. Biol. 7, 1003-1006. Mattson, M., Cheng, B., Baldwin, S., Smith-Swintosky, V., Keller, J., Geddes, J., Scheff, S. & Christakos, S. (1994). Brain injury and tumor necrosis factors induce expression of calbidin D-28 in astrocytes: evidence for a cytoprotective response. J. Neurosci. Res. 42, 357-370. Mattson, M.P., Goodman, Y., Luo, H., Fu, W. & Furukawa, K. (1997). Activation of NFKB protects hippocampal neurons against oxidative stress-induced apoptosis: evidence for induction of MnSOD and supression of peroxynitrie production and protein tyrosine nit~:ation. J. Neurosci. Res. 49, 681-697. McGeer, P.L. & McGeer, E.G. (1996). Immune mechanisms in neurodegenerative disorders. Drugs Today 32, 149-158. McGreer, P.L. & McGreer, E.G. (1995). The inflammatory response system of brain: impliications for therapy of Alzheimer and other neurodegenerative diseases. Brain Res. Rev. 21, 195-218. Minami, M., Kuraishi, Y. & Satoh, M. (1991). Effects of Kainic acid on messenger rna levels of IL-1B, IL-6, TNFct and LIF in the rat brain. Biochem. Biophys. Res. Commun. 176, 593-598. Miura, M., Friedlander, R.M. & Yuan, J. (1995). Tumor necrosis factor-induced apoptosis is mediated by a CrmA-sensitive cell death pathway. Proc. Natl. Acad. Sci. U. S. A. 92, 8318-8322. Mogi, M., Harada, M., Kondo, T., Mizuno, Y., Narabayashi, H., Riederer, P. & T.N. (1996). The soluble form of Fas molecule is elevated in Parkinsonian brain tissues. Neurosci Lett. 220, 195-198. Muzio, M., Chinnaiyan, A.M., Kischkel, F.C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J.D., Zhang, M., Gentz, R., Mann, M., Krammer, P.H., Peter, M.E. & Dixit, V. M. (1996). FLICE, a novel FADD-homologous ICE/CED-3 like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85, 817-827. Muzio, M., Salvesen, G.S. & Dixit, V.M. (1977). FLICE induced apoptosis in a cell-free system. Cleavage of caspase zymogens. J. Biol. Chem. 272, 2952-2956. Nichol, K.A., Chan, N., Davey, D.F. & Bennett, M.R. (1995). Location of nitric oxide synthase in the developing avian ciliary ganglion. J. Auton. Nerv. Syst. 51, 91-102. Nicholson, D.N. & Thornberry, N.A. (1997). Caspases: killer proteases. Trends Biochem. Sci. 22, 299-306. Nishimura, T., Akiyama, H., Yonehara, S., Kondo, H., lkeda, K., Kato, M., Iseki, E. & Kosaka, K. (1995). Fas antigen expression in brains of patients with Alzheimer-type dementia. Brain Res. 695, 137-145. Oehm, A., Behrmann, I., Falk, W., Pawlita, M., Maier, G., Klas, C., Li Weber, M., Richards, S., Dhein, J., Trauth, B.C., Pansting, I.K. & Krammer, P.H. (1992). Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor superfamily. Sequence identity with the Fas antigen. J. Biol.Chem. 267, 10709- 10715. Ogasawara, J., Suda, T. & Nagata, S. (1995). Selective apoptosis in CD8+ thymocytes by the anti-Fas antibody. J. Exp. Med. 181,485-491. Orth, K., O'Rourke, K., Salvesen, G.S. & Dixit, V.M. (1996). Molecular ordering of apoptotic mammalian CED-3/ICE-Iike proteases. J. Biol. Chem. 271, 20977-20980. Pan, G., Bauer, J.H., Haridas, V., Wang, S., Lui, D., Yu, G., Vincenz, C., Aggarwal, B.B., Ni, J. & Dixit, V.M. (1998). Identification and functional characterization of DR6, a novel death domain-containing TNF receptor. FEBS Lett. 431,351-356.

Death Domains and the Brian

63

Pan, G., Ni, J., Wei, Y.F., Yu, G., Gentz, R. & Dixit, V.M. (1997a). An antagonist decoy receptor and a death domian-containing receptor for TRAIL. Science 277, 815-818. Pan, G., O'Rourke, K., Chinnaiyan, A.M., Gentz, R., Ebner, R., Ni, J. & Dixit, V.M. (1997b). The receptor for the cytotoxic ligand TRAIL. Science 276, 815-818. Pan, G., O'Rourke, K. & Dixit, V.M. (1998). Caspase 9, BcI-XL, and Apaf-1 form a tertiary complex. J. Biol. Chem. 273, 5841-5845. Park, A. & Baichwal, V.R. (1996). Systematic mutational analysis of the death domain of the tumor necrosis factor receptor 1-associated protein TRADD. J. Biol.Chem. 271, 9858-9862. Prewitt, C.M., Niesmann, I.R., Kane, C.J.M. & Houle, J.D. (1997). Activated macrophage/microglial cells can promote the regeneration of sensory axons into the injured spnial cord. Exp. Neurol. 148,433-443. Rabchevsky, A.G. & Streit, W.J. (1997). Grafting of cultured microglial cells into the lesioned spinal cord of adult rats enhances neurite outgrowth. J. Neurosci. Res. 47, 34-48. Ravi, R., Bedi, A., Fuchs, E.J. & Bedi, A. (1998). CD95 (Fas)-induced caspase mediated proteolysis of NF-•B. Cancer Res. 58,882-886. Rothe, M., Pan, M.-G., Henzel, W.J., Ayres, T.M. & Goeddel, D.V. (1995). The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell 83, 1243 - 1252. Roy, N., Mahadevan, M.S., McLean, M., Shutler, G., Yaraghi, Z., Farahani, R., Baird, S., Besner-Johnston, A., Lefebvre, C., Kang, X., Salih, M., Aubry, H., Tamai, K., Guan, X., Ioannou, P., Cmwford, T.O., de Jong, P.J., Surh, L., Ikeda, J.-E., Korneluk, R.G. & MacKenzie, A. (1995). The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy. Cell 80, 167-178. Saas, P., Walker, P.R., Hahne, M., Quiquerez, A., Schnuriger, V., Perrin, G., French, L., Van Meir, E.G., de Tribolet, N., Tschopp, J. & Dietrich, P. (1997). Fas expression by astrocytoma in vivo: maintaining immune privilege in the brain? J. Clin. Invest. 99, 1173-1178. Santana, P., Pena, L.A., Haimovitz Freidman, A., Martin, S., Green, D., McLoughlin, M., Cordon Cardo, C., Schuchman, E.H., Fuks, Z. & Kolesnick, R. (1996). Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis. Cell 86, 189-199. Schneider, P., Thome, M., Burs, K., Bodmer, J.L., Hofman, K., Kataoka, T., Holler, N. & Tschopp, J.(1997). TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD-dependent apoptosis and activate NF- Kappa B. Immunity 7, 831-836. Schulze-Osthoff, K., Ferrari, D., Los, M., Wesselborg, S. & Peter, M.E. (1998). Apoptosis signaling by death receptors. FEBS Lett. 254, 439-459. Sen, R. & Baltimore, D. (1986). Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46, 705 - 16. Sheridan, J.P., Marsters, S., Pitti, R.M., Gurney, A., Skubatch, M., Badwin, D., Ramakrishnan, L., Gray, C.L., Baker, K., Wood, W.I., Goddard, A.D., Godowski, P. & Ashkenazi, A. (1997). Control of TRIAL-induced apoptosis by a family of signaling of decoy receptors. Science 277, 818-821. Shu, H.B., Halpin, D.R. & Goeddel, D.V. (1997). Casper is a FADD- and caspase-related inducer of apoptosis. Immunity 6, 751-763. Shu, H.B., Takauchi, M. & Goeddel, D.V. (1996). The tumor necrosis factor receptor 2 signal transducers TRAF2 and c-IAPI are componets of tumor necrosis factor receptor 1 signaling complex. Proc. Nat. Acad, Sci. U. S. A. 93, 13973-13978. Srinivascula, S.M., Ahmad, M., Fernandes Alnemri, T., Litwack, G. & Alnemri, E.S. (1996). Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activaters multiple Ced-3/ICE-like cysteine proteases. Proc. Natl. Acad. Sci. U. S. A. 93, 14486-14491.

64

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Srinivasula, S.M., Ahmad, M., Ottilit, S., Bullrich, F,, Banks, S., Wang, Y., Femandes-Alnemri, T., Croce, C.M., Litwack, G., Tomaselli, K.J., Armstrong, R.C. & Alnemri, E.S. (1997). FLAME-l, a novel FADD-Iike apoptotic molecule that regulates Fas/TNFRl-induced apoptosis. J. Biol. Chem. 272, 18542-18545. Stanger, B.Z., Leder, P., Lee, T.H., Kim, E. & Seed, B. (1995). RIP: a novel protein containing a death domain that interacts with Fas/APO-1 (CD95) in yeast and causes cell death. Cell 81,513-523. Szaflarski, J., Burtrum, D. & Silverstein, F.S. (1995). Cerebral hypoxia stimulates cytokine gene expression in perinatal rats. Stroke 26, 1093-1100. Takahashi, T., Tanaka, M., Brannan, C.I., Jenkins, N.A., Copeland, N.G., Suda, T. & Nagata, S. (1994). Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell 76, 969-976. Tartaglia, L.A., Ayes, T.M., Wong, G.H. & Goeddel, D.V. (1993a). A novel domain within the 55kD TNF receptor signals cell death. Cell 74, 845-853. Tartaglia, L.A., Pennica, D. & Goeddel, D.V. (1993b). Ligand passing: the 75-kDa tumor necrosis factor (TNF) receptor recruits TNF for signaling by the 55-kDa TNF receptor. J. Biol. Chem. 268, 18542-18548. Taupin, V., Toulmond, S., Serrano, A., Beuavides, J. & Zavala, F. (1993), Increases in IL-6, IL-1 and TNF levels in rat brain following traumatic lesion. J. Neuroimmunol. 42, 177-186. Tchelingerian, J., Quinonero, J., Booss, J. & Jacque, C. (1993). Localization of TNFct and IL-lct immunoreactivities in striatal neurons after surgical injury to the hippocampus. Neuron 10, 213-224. Tewari, M. & Dixit, V.M. (1995). Fas- and tumor necrosis factor induced apoptosis in inhibited by the poxvirus crmA gene product. J. Biol. Chem. 270, 3255-3260. Thome, M., Schneider, P., Hofmann, K., Fickenscher, H., Meinl, E., Neipel, F., Mattmann, C., Bums, K., Bodmer, J.-L., Schroter, M., Scaffidi, C., Krammer, P.H., Peter, M.E. & Tschopp, J. (1997). Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 386, 517-521. Ting, A.T., Pimentel Muinos, F.X. & Seed, B. (1996). RIP mediates tumor necrosis factor receptor 1 activation of NF-KB but not Fas/APO-l-initiated apoptosis. EMBO J. 15, 6189-6196. Toku, K., Tanaka, J., Yano, H., Desaki, J., Zhang, B., Yang, L., lshihara, K., Sakanaka, M. & Maeda, N. (1998). Microglial cells prevent nitric oxide-induced neuronal apoptosis in vitro. J. Neurosci. Res. 53, Vassalli, P. (1992). The pathophysiology of tumor necrosis factors. Ann. Rev. Immunol. 10, 411-452. Verma, I.M. & Stevenson, J. (1997). IKB kinase: beginning, not the end. Proc, Nat. Acad. Sci. U.S.A. 94, 11758-11760. Vogt, M., Bauer, M.K., Ferrari, D. & Schulze-Osthoff, K. (1998). Oxidative stress and hypoxiaJreoxygenation trigger CD95 (APO-1/Fas) ligand expression in microglial cells. FEBS. Lett. 429, 67-72. Walczak, H., Delgi Esposti, M.A., Johnson, R.S., Smolak, P.J., Waugh, J.Y., Boiani, N., Timour, M.S., Gerhart, M.J., Schooley, K.A., Smith, C.A., Goodwin, R.G. & Rauch, C.T. (1997). TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J. 16, 5386-5397. Wang, C.-Y., Mayo, M.W. & Baldwin Jr., A.S. (1996). TNF- and cancer therapy-induced apoptosis: Potentiation by inhibition of NF-KB. Science 274, 784-787. Watanabe-Fukunaga, R., Brannan, C.I., Copeland, N.G., Jenkins, N.A. & Nagata, S. (1994). Lymphoproliferation disorder in mice explained by deficits in Fas antigen that mediates apoptosis. Nature 356, 314-317. Wolvers, D.A., Marquette, C., Berkenbosch, F. & Haour, F. (1993). Tumor necrosis factor alpha: specific binding sites in the rodent brain and pituitary gland. Eur. Cytokine Netw. 4, 377-381. Wong, G.H. & Goeddel, D.V. (1988). Induction of MnSOD by TNF: possible protective mechanism. Science 242, 941-944.

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Wu, M., Lee, H., Bellas, R.E., Schauer, S.L., Arsura, M., Katz, D., Fitzgerald, M.F., Rothstein, T.L., Sherr, D.H. & Sonenshein, G. E. (1996). EMBO J. 15, 46824690.

Inhibition of NF-kB/Rel induces apoptosis of murine B cells.

DIVERSITY OF CASPASE I N V O L V E M E N T IN N E U R O N A L CELL D E A T H C A R O L M. TROY

Table of Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caspase Family of Proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification of Caspases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caspase Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caspase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assessment of Caspase Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measurement of Caspase Activation in Cells and Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . Models of Neuronal Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Paradigms of Programmed Cell Death in Cultured Neuronal Cells . . . . . . . . . . . . . . . . . . . . Caspase Specificity in Neuronal Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caspases Essential for Normal Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caspases Necessary for Other Types of Neuronal Cell Death . . . . . . . . . . . . . . . . . . . . . . . . Death Receptors and Neuronal Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . All Caspase Activity Does Not Produce Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

67 68 69 70 71 72 73 73 74 76 78 78 83 84 85

Introduction Programmed cell death (or apoptosis) in neurons is an active, genetically regulated suicide mechanism which is essential for normal neuronal development and is a commonly observed feature in neurodegenerative diseases (Bains and Shaw, 1997). The responses of cells to apoptotic stimuli are morphologically and biochemically similar across cell-types and species. Classically, morphologic hallmarks of apoptosis include cell shrinkage, nuclear condensation, clumping of chromatin and formation of apoptotic bodies (Wyllie, 1980; Wyllie et al., 1984). The dying cells disappear without any accompanying inflammation (Kerr et al., 1972). Associated DNA fragmentation can be visualized as a ladder by gel electrophoresis (Wyllie et al., 1984; Batistatou and Greene, 1991). The machinery employed in cell death is evolutionarily conserved. Genetic studies on development in C. Elegans identified three genes which regulated cell death and survival, demonstrating that specific molecular pathways governed programmed cell death (Hengartner et al., 1992; Yuan and Horvitz, 1992; Yuan et al., 1993). Both proapoptotic (ced-3 and ced-4) and antiapoptotic (ced-9) regulators of cell death were identified in the nematode; all have mammalian homologs. Ced-9 is a homolog of the bcl-2 family of proteins (Vaux et al., 1988, 1992), ced-4 is a homolog of Apaf-1 (Zou et al., 1997). The positive regulator ced-3 was found to be a homolog of the 67 Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. 67 © 2001 Elsevier Publishers. Printed in the Netherlands.

-

-

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previously identified mammalian enzyme, interleukin-1 converting enzyme (Cerretti et al., 1992; Thornberry et al., 1992; Yuan et al., 1993), which is now known as caspase-1. Both ced-3 and caspase-1 induce apoptosis after transient expression in mammalian cells (Miura et al., 1993), providing evidence that a conserved family of proteases is a critical factor in cell death. Soon after the discovery of ced-3 other mammalian caspases were identified and also shown to induce death. At the time of preparation of this chapter 14 members of the caspase family have been identified (Ahmad et al., 1998; Hu et al., 1998; Humke et al., 1998). For recent reviews see: Cohen, 1997; Nicholson and Thornberry, 1997; Bergeron and Yuan, 1998; Cryns and Yuan, 1998; Green, 1998; Thornberry and Lazebnik, 1998).

Caspase Family of Proteases Caspases are cysteine aspartases, each contains a conserved QACXG pentapeptide containing the active site cysteine residue (for a review of structural and enzymatic features see Nicholson and Thornberry, 1997). They are constitutively present in most cells and exist in the cytosol as catalytically inactive proenzymes (zymogens) containing a variable length amino terminal prodomain, a large subunit and a small subunit. They can be activated by autocleavage, cleavage by another caspase or cleavage by granzyme B. Cleavage occurs in two steps, shown in Figure 1. First, the chain is cleaved between the large and small subunits and then a second cleavage removes the prodomain from the large subunit. Crystallographic studies show that the subunits assemble into a heterodimer, and two heterodimers assemble to form a tetramer with two active sites (Walker et al., 1994; Wilson et al., 1994; Rotonda et al., 1996). Once activated there can be additional caspase-caspase interactions and cascades of activation. However, the initial zymogen must be activated to begin these events. An oligomerization model has been proposed to explain the onset of activation of caspases with long prodomains. Procaspase-8 has been shown to possess 1-2% of the activity of mature caspase 8; aggregation of procaspase-8 initiates autoactivation (Muzio et al., 1998; Yang et al., 1998). The aggregation of procaspases is mediated by the prodomain which interacts with the activating apparatus. Two different types of interaction domains have been identified: caspase recruitment domains (CARDs), found in caspases -1, -2, -4, -5, and -9 (Ahmad et al., 1997; Duan and Dixit, 1997; Hofmann et al., 1997); and death effector domains (DEDs), found in caspases -8 and -10 (Boldin et al., 1995; Chinnaiyan et al., 1995; Boldin et al., 1996; Muzio et al., 1996; Vincenz and Dixit, 1997). The procaspases also bind to adaptor molecules containing similar domains. For examples, caspase -8 associates with FADD (Fas-associated protein with death domain) through the DED while caspase -9 forms a complex with APAF- 1 through the CARD. Recruitment of the caspases via the adaptor molecules results in formation of an apoptosome that mediates caspase activation. Details of this model are discussed in other chapters in this book as well as in the review by Green (1998).

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SCHEMATIC DIAGRAM OF CASPASE ACTIVATION

Asp Asp ¢ Largesubunit ~ Smallsubunit

Prodomain

I

!!iiiiiii iiiiii iiiiiii l lst CLEAVAGE

I

iiiiiiiiiiiiiiiiiiiiiiiiiiiii l

2nd CLEAVAGE

i...======........m.l===l. iumnnnmn •mmnnguiH| u u u n n m i n N u l m m n H l | H u n n u a n n n . i u n H m u

lnlllmWlllnHm!=~=llllla

+

l ActiveCaspaseh e t e r o d i m e r ~ ,,,,,,,,=,,, ...... ,, ......... ......... i[, H .... ~, ..... Figure 1.

Schematic diagram of caspase activation. A representative pro-caspase is illustrated.

Cleavage occurs in two steps: The first cleavage releases the small subunit and the second cleavage removes the prodomain from the large subunit. The subunits form a heterodimer, and two heterodimers combine to form the active tetramer. The large and small subunits are extensively conserved throughout the family. The prodomain is not conserved among family m e m b e r s .

Classification of Caspases Several classifications of caspases have been used, including subsets based on structure as well as those based on presumed function. The latter divides the caspases into initiators (caspases with long prodomains) and effectors (caspases with short prodomains) with the initiators acting upstream of the effectors (Green, 1998; Thornberry and Lazebnik, 1998). However, in neuronal systems it is not clear whether this classification strictly applies. Structural considerations, which encompass cleavage specificity as well as sequence homologies, allow the separation of caspases into 3 families (Figure 2).

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The Caspase Family

t

Caspase-l," (.ICEI

Caspase- 12 Caspass-11 (Ich-3) - - -

_• m

Caspase-13 (ERICE) Caspase-4 (ICE rel II, TX, ICH2) Caspass-5 (ICE rel III, TY) Caspase-1 (ICE) Caspasa-3* (Yama, CPP32, Apopain) Caspase-7* (ICE-LAP3, Mch3, CMH-1) Caspass-6* (Mch-2) Caspase-8 (FLICE, MACH, Mch5) Caspase-10 (FLICE2, Mch4) Caspass-9 (ICE-LAP6, Mch6)

Caspase-2 (Ich-1, Nedd2)

Caspase-1 subfamily

Caspase-3 subfamily

Caspase-2 subfamily

*short prodomain Figure 2. The caspase family. The three caspase subfamilies, arranged by structural homologies. * indicates caspases with small prodomains, the rest have large prodomains.

The caspase l-like family, which includes caspases -1, -4, -5, -11, -12 -13; the caspase-3-1ike family, composed of caspases -3, -6, -7, -8, -9, -10 and caspase-2, which is the only member of its family. The most recently identified, caspase 14, is unique in that it is not processed in response to several different death signals but does interact physically with several long prodomain caspases (1, 2, 4, 8, and 10) and does induce death (Hu et al., 1998). It is also the only short prodomain member of the caspase-1 subfamily. The other caspases with short prodomains are members of the caspase-3 family, caspases-3, -6 and -7. Caspase Substrates Active caspases cleave their substrates on the carboxy-terminal side of an aspartate residue, the P1 site, illustrated in Figure 3. However, individual caspases differ in their substrate specificities. These differences are determined by the residues immediately amino-terminal to the P1 site, especially the P4 site. The substrate cleavage specificities have been defined using a positional scanning substrate combinatorial library (Thornberry et al., 1997) or synthetic peptides (Talanian et al., 1997). Three subgroups of caspases were defined based on the substrate preference using positional scanning. The first subgroup (caspases-1, -4 and -5) prefers bulky hydrophobic residues in P4 and has the optimal substrate cleavage site WEXD. The second subgroup (caspases-2, -3, -7 and ced-3) prefers an aspartate at P4 and cleaves optimally carboxy terminal to DEXD, with X=V for caspases-3 and -7 and X=H

Diversity of Caspase Involvement

71

Asp Prodomain

Large subunit

Asp ¢ Small subunit

iiiii!iillii !iill iiiii;///YY/',l Q

A

C

X

~

P4 P3 P2 P l Figure 3.

Schematic of pro-caspase. Conserved QACXG active site region and P1-P4 amino acids are

illustrated.

for caspase-2. The third group (caspases-6, -8 and -9) is less specific in P4 preference with an optimal cleavage sequence of (L/V)EXD. These predicted specificities agree well with the known substrates of the caspases. Caspase-1 cleaves pro-IL-lf5 at two sites, FEAD and YVHD, caspase-3 cleaves the DNA repair enzyme PARP at a DEVD site and caspase-6 cleaves lamin A at a VEID site. Many caspase substrates have been identified but it is still unclear how many of these substrates are directly involved in death. Other known substrates include inhibitors of apoptosis (IcAD (Enari et al., 1998), Bcl-2 (Cheng et al., 1997; Xue and Horvitz, 1997; Adams and Cory, 1998)) and cytoskeletal components (nuclear lamina (Lazebnik et al., 1995; Orth et al., 1996; Takahashi et al., 1996), actin (Mashima et al., 1995)) but the relation of cleavage of these substrates to cell death is not yet understood. Caspase Inhibitors Caspases have differential responses to both naturally occurring and synthetic inhibitors, further supporting uniqueness in the cleavage specificity of the individual members of this family. Three naturally occurring inhibitors have been described. Cytokine responsive modifier A (crmA) is a serpin from cowpox virus which is an excellent inhibitor of caspase-l-like proteases, but not particularly effective against caspase-3-1ike family members (Nicholson et al., 1995). p35, from baculovirus, is another viral gene product which prevents apoptosis and is an irreversible inhibitor of caspases-1 through -4 and ced-3 (Bump et al., 1995; Xue and Horvitz, 1995). The IAP (inhibitor of apoptosis) family of gene products are a mammalian group of polypeptides which inhibit caspases (Deveraux et al., 1997, 1998) which are discussed in detail in another chapter in this book. Synthetic pseudosubstrate inhibitors were designed as broad range or specific inhibitors and most are available as reversible (aldehyde) or irreversible (ketone) forms. The broad range inhibitors zVAD and BAF were developed to inhibit as many caspases as possible, although affinities of the different caspases for these inhibitors do vary (Margolin et al., 1997). YVAD and DEVD were designed as specific inhibitors of caspases-1 and -3 respectively, based on knowledge of the substrates of caspase-1 (pro-IL-1B) and caspase-3 (PARP). As the family members identified increased, kinetic studies of substrate and inhibitor specificities showed that DEVD had a broad

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spectrum of action and inhibited most caspases except for caspase-2, at the concentration commonly used (50 /~M). The broad spectrum of DEVD action requires that caution be used in interpreting earlier work attributing apoptosis to caspase-3 on the basis of DEVD inhibition. YVAD was shown to inhibit caspases-1 and -4 but not caspases-3 or -7, so it appears to be more specific for caspase-l-like family members (Margolin et al., 1997). Since the different caspases have different affinities for the inhibitors, dose-response curves with the reversible versions of the inhibitors can provide indications about caspases involved in a particular death paradigm, as will be shown below. A novel strategy for ICE family inhibition is based on the conservation of the active site (QACXG) in all ICE family proteases, from ced-3 to the human homologs (Wang et al., 1994). In a variation on the pseudosubstrate approach to enzyme inhibition, we reasoned that a peptide, IQACRG, comprised of this active site might compete with the enzyme for binding to substrate, and thus inhibit all caspases present in a cell by acting as a pseudoenzyme, regardless of substrate specificity. In vitro, the IQACRG peptide inhibits the cleavage of recombinant pro-IL-1B by recombinant caspase-1 (Troy et al., 1996b), confirming that the peptide blocks caspase-I activity. The peptide is linked by a disulfide bond to a delivery peptide, Penetratinl, which allows rapid translocation through cell membranes (Prochiantz and Theodore, 1995; Theodore et al., 1995; Troy et al., 1996a). Once in the cytosol the inhibitory peptide is released from Penetratin I and can then interfere with the binding of any of the caspases to their substrates. Other broad spectrum inhibitors include zVAD and BAF. All of these are good for general detection of caspase activity, but do not yield specific information about individual caspases. Individual caspases can also be manipulated directly by overexpression, antisense transfection, antisense oligonucleotides and in transgenic and knockout animals. In a redundant family, such as the caspases, it is useful to manipulate the expression of caspases in several different ways to understand their function. The use of these approaches will be discussed below. Assessment of Caspase Activity Several artificial substrates exist which can measure caspase activity. These include the most commonly used DEVD and YVAD, linked either to AMC or pNA, for fluorometric or colorimetric determinations respectively. Fluorometric measurements are more sensitive than colorimetric. Kinetic studies of substrate cleavage by caspases-1, -2, -3, -4, -6 and -7 show that there is considerable overlap in the cleavage specificity of the different caspases (Talanian et al., 1997). The most commonly used substrate, DEVD, is cleaved by all of the caspases tested except caspase-2. Thus DEVD-cleaving activity can be taken as a general measure of caspase activity, excepting caspase-2 activity, but certainly not as a specific measure of caspase-3 activity or even of activity of caspase-3-1ike family members. A 5 amino acid substrate, VDVAD, is cleaved by caspase-2 but is also cleaved by caspases-3 and -7 (Talanian et al., 1997). Differential use of these two substrates could provide data about caspase-2 activity. Another commonly used caspase substrate is YVAD which is cleaved well by caspases-1 and -4, poorly by caspase-3 and not cleaved by caspases-2, -6 and -7 (Talanian et al., 1997;

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Thornberry et al., 1997). Thus, the use of multiple substrates can provide more specific data about individual caspases than the use of only a single substrate. Another measure of caspase-1 activity is the release of interleukin-16. Caspase-1 is the only family member which cleaves pro-IL-113 in cells, although caspase-4 can cleave pro-IL-1B in vitro with 2 orders of magnitude less activity than caspase-1 (Margolin et al., 1997). Cleavage of cellular substrates are also measures of caspase activity. Cleavage of tamin is indicative of caspase-6 activity (Lazebnik et al., 1995; Orth et al., 1996; Takahashi et al., 1996) and PARP, originally proposed as a specific substrate of caspase-3, can also be cleaved by caspases-1, -4 and -7 though less efficiently by caspases-1 and -4 than by caspases-3 and 7 (Margolin et al., 1997). Measurement of Caspase Activation in Cells and Tissues Since caspases exist as inactive zymogens, activation of a particular caspase can be detected by Western blotting when appropriate antibodies are available to detect the active subunits, which range from 17-20 kDa for the large subunit and 10-12 kDa for the small subunit. Usually there is a large amount of the zymogen present relative to the active subunits, even at timepoints where there is detectable caspase activity. It is likely that once there is caspase activation in a cell that it does not survive for long. Therefore, in a population of cells, at any moment only a small proportion will contain activated caspase. Antibodies have been developed which detect only the active p20 fragment of caspase-3, not the proform (Armstrong et al., 1997). These are very useful for studies of cell death in tissues where detection of the active caspase-3 can be combined with TUNEL labeling to detect dying cells (Namura et al., 1998).

Models of Neuronal Cell Death

Much still remains to be determined about the death pathways. A question of great interest is why there are so many caspases. Multiple family members can be present in one cell. Does each have a particular function or is there biologic redundancy? Data from targeted deletions of individual caspases support different functions for specific caspases. Knockouts of caspase-1 (Kuida et al., 1995; Li et al., 1997), -2 (Bergeron et al., 1998) and -11 (Wang et al., 1998) do not have an obvious neuronal phenotype, indicating that these are not critical for early development and/or there is functional redundancy for these caspases. Caspase-3 (Kuida et al., 1996), -8 (Varfolomeev et al., 1998) and -9 (Hakem et al., 1998; Kuida et al., 1998) knockouts have devastating phenotypes, supporting a critical role for these caspases in developmental cell death. The investigation of other individual caspase knockouts are underway and this approach has yielded much data about the roles of specific caspases in developmental neuronal death. However, the effect of knockouts is often influenced by the genetic background of the animals, and it is possible that the redundancy of caspases allows functional substitution at early developmental stages. The availability of "conditional knockouts" will allow a more complete assessment of the question of which caspases are responsible for the aberrant cell death which occurs in models of

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adult neurodegenerative disorders. The ensuing section will present data from model systems of cell death, as well as the specific phenotypes of the null mice and other in vivo data, which address the specific functions of some of the caspases. Caspase activity has been shown to occur during experimental ischemia (Friedlander et al., 1996, 1997b; Namura et al., 1998) and caspases are likely involved in apoptotic neuronal cell death that has been reported in amyotrophic lateral sclerosis (ALS), Alzheimer's and other neurodegenerative diseases. While it is an open question whether apoptosis represents a key step in the disease process, or simply an agonal event eliminating cells which have long before been rendered useless, there is a possibility that caspase inhibition, or blockade of other steps in the apoptotic cascade, might halt the progress of one or more of these diseases. However, a broad spectrum blockade would also pose the risk of perpetuating excessive growth of mutated ceils, which might go on to form tumors. The data suggesting that parallel paths to cell death are taken by neurons, depending on the type of injury they sustain, raises the possibility that therapies could be crafted that are quite specific for a given disease process. Hence, an important question, which remains to be answered, is which caspases are necessary for progression of different neurodegenerative diseases? Among the most promising results is the delay in progression of symptoms in a mouse model of ALS by expression of the dominant negative caspase-1 (Friedlander et al., 1997a), discussed in more detail below. We, and others, have begun to address the question of caspase specificity using models of neuronal cell death. By studying models of apoptotic neuronal death, the progression to death in response to different stimuli can be defined to determine where these pathways converge and diverge. We have employed multiple models for the induction of death in PC12 cells and primary cultures of sympathetic neurons. These models test key theories about cell death in human neurodegeneration. The stimuli we use include oxidative stress, trophic factor deprivation, and DNA damage. Initiating death in the same cell-type with different stimuli allows us to address whether the molecular mechanisms utilized when the cell is exposed to each stimulus are the same or different. Utilizing the same cell-type ensures that the same repertoire of molecules is available. Paradigms of Programmed Cell Death in Cultured Neuronal Cells The three models of cell death which we have studied include: oxidative stress induced by the downregulation of SOD1 (Troy and Shelanski, 1994; Troy et al., 1996a, 1996b); trophic factor withdrawal, in which NGF is withdrawn from cell-types which are dependent on it for survival (Greene, 1978; Rukenstein et al., 1991; Troy et al., 1996b; 1997b; Stefanis et al., 1998); and DNA damage induced by radiation or drugs (Park et al., 1998b). Details of these models follow. Free radicals represent a class of biologically generated species that have been suggested to play a role in many neuronal disorders (Coyle and Puttfarcken, 1993; Brown, 1995; Schapira, 1996). However, the actual mechanism by which they cause neuronal degeneration has not yet been defined. Cu/Zn superoxide dismutase (SOD1) is among the key cellular enzymes by which neurons and other cells detoxify free radicals and protect themselves from damage (McCord and Fridovich, 1969; Fridovich, 1995).

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75

The observation that a subset of cases of familial amyotrophic lateral sclerosis (FALS) are associated with missense mutations of SOD1 provided the first molecular evidence for involvement of this enzyme in neuronal degenerative disorders (Rosen et al., 1993). While the initial report associated FALS mutations with a loss of SOD activity, other disease causing mutations in the enzyme have normal levels of activities. Therefore, the mechanism by which these missense mutations lead to cell death is not clear, though there is evidence to support a role for free radical mediated death (Srinivasan et al., 1996). There is also the possibility that, by analogy with SODI null yeast, copper buffering is disrupted (Culotta et al., 1995). As a model for oxidative stress-mediated cell death, we have employed antisense oligonucleotides to specifically downregulate SOD1. Delivery of the antisense is facilitated by linking the oligonucleotide (ASOD1) to Penetratinl (V-), the delivery peptide discussed above, which allows rapid uptake of the oligonucleotide in serumcontaining medium and increases efficacy by 100 fold over non-vector linked oligonucleotides (Troy et al., 1996a). Within 24 h of downregulation, there is 50-60% apoptotic cell death in PCI2 cells (Troy and Shelanski, 1994; Troy et al., 1996a) which can be prevented by vitamin E and overexpression of bcl-2, but not by insulin (Troy and Shelanski, 1994). In sympathetic neurons downregulation of SOD1 is not sufficient to induce death. However, addition of exogenous nitric oxide concurrent with SOD1 decrease induces death. Nitric oxide by itself is not toxic to the sympathetic neurons or to PCI2 cells (Farinelli et al., 1996; Troy et al., 1996a) and, in fact, protects against trophic factor deprivation (Farinelli et al., 1996). Moreover, the SODl-depleted PC12 cells can be rescued by nitric oxide synthase inhibitors (Troy et al., 1996a). These results, among others, point to the production of peroxynitrite -- which is formed from nitric oxide and superoxide -- as the damaging agent. Manganese superoxide (SOD2) has also been shown to protect from NO-mediated toxicity (Gonzalez-Zulueta et al., 1998; Keller et al., 1998), supporting a role for formation of peroxynitrite in the mechanism of the cell death mediated by an increase in superoxide. Trophic factors represent another influence on neuronal survival. While trophic factors are known to be essential in the developing nervous system (Silos-Santiago et al., 1995), mature neurons also require these substances for their maintenance. There has been much speculation that neurodegenerative disorders may involve disturbances in trophic factor supply or in the ability of cells to respond to them (Williams, 1995). Moreover, even if variations in neurotrophic factors, or the responses to them, are not causally involved in neurodegenerative disorders, there is evidence that these agents can ameliorate or protect neurons from degeneration (Coyle and Puttfarcken, 1993). As a model for trophic-deprivation mediated cell death, removal of serum from naive PC12 cells, or of serum and NGF from NGF-primed PC12 ceils, or NGF from sympathetic neurons, results in 50-85% apoptotic cell death after one day (Greene, 1978). This death can be prevented by multiple agents including NGF (Greene, 1978), insulin (Rukenstein et al., 1991), overexpression of the proto-oncogene bcl-2 (Batistatou et al., 1993) and NO (Farinelli et al., 1996). DNA damage can also lead to apopotosis. Clinically, DNA damage is seen after radiation therapy for tumors as well as in diseases with excessive sensitivity to irradiation,

C.M. Troy

76

such as ataxia-telangiectasia (Enns et al., 1998; Lavin, 1998; Park et al., 1998a). In modeling D N A damage several different agents have been used. These include irradiation, arabinoside C, and camptothecin (Park et al., 1998a, 1998b). The most straightforward is irradiation, either ultraviolet or gamma, leading to direct damage o f the DNA. W e have used neuronal (NGF-primed) PC12 cells and sympathetic neurons to study the path to death after D N A damage.

Caspase Specificity in Neuronal Cell Death Knowledge o f the potential players is important in determining the individual caspase(s) responsible for death. Recent work has begun to identify the caspases present in PC12 cells (J. Angelastro, personal communication). Caspases 1-3, 6-9, I 1 are all present (Angelastro et al., 1998). Caspases- 4 and -5 have been isolated only from humans and m a y not have rodent homologs. Table 1.

Caspase Detection in PC12 cells

CASPASE

mRNA

Cas ~ase-1

Yes Yes Yes No No Yes Yes Yes Yes n.d. Yes

Cas ~ase-2 Cas )ase-3 Cas 9ase-4 Cas ~ase-5 Cas ~ase-6 Cas )ase-7 Cas )ase-8 Cas ~ase-9 Cas )ase-10 Cas ~ase-11

PROTEIN Yes Yes Yes

Not determined Yes Yes Not determined Not determined Not determined

Caspase mRNAs were detected by RT-PCR using probes prepared from rat caspase sequence data, when available, or from mouse or human sequences. Protein was detected by Western blotting of whole cell lysates using available antibodies. Adapted from (Angelastro et al., 1998).

Thus, it appears that most, if not all, the caspases are present in one cell-type. This again raises the question o f whether there is a specific function for each caspase. If not, why is there so much redundancy within a given cell-type? Granted, PC12 cells are a cultured tumor cell line and thus may have more caspases expressed than a normal cell. Now that rodent sequences are known for the different family members normal neurons will be evaluated for presence o f the different caspases.

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77

As an initial approach to determining the caspase requirements in our three death paradigms, several different types of caspase inhibitors were used to generate protection profiles in each of the cell death models using PC12 cells and sympathethic neurons (Stefanis et al., 1996, 1997, 1998; Troy et al., 1996b, 1997a; b; Park et al., 1998b). Y V A D is the most selective inhibitor, V-ICEinh is the least selective. The data support the thesis that different caspases are operative in the different death paradigms and that the death stimulus, not the cell-type, determines the caspase utilized. This is summarized in Table 2. Table 2.

Differentialprotection by caspase inhibitors in three paradigms of cell death YVAD-FMK

ZVAD-FMK DEVD-FMK

downregulation

100% survival

100% survival

100% survival

100% survival

Trophic factor deprivation

No

100%

100%

survival

No protection

100%

protection

survival

survival

No protection

No protection

No protection

100% survival

100% survival

SOD1

DNA damage

BAF

V-ICEinh 100% survival

Cultures of PC12 cells and sympathetic neurons were treated with the various death producing stimuli in the presence of the indicated caspase inhibitors. Survival was quantified as previouslydescribed (adapted from Troy et al., 1996b; Park et al., 1998b; Stefanis et al., 1998).

Despite the effectiveness of the caspase inhibitors in preventing death, there were marked differences in the potency of the inhibitors in the different paradigms. Cells undergoing trophic deprivation-mediated death required higher doses of both the V-ICEInh and zVAD-FMK inhibitors than cells exposed to V-ASOD1. In addition, the inhibitory peptide AcYVAD-CMK protected against SOD1 downregulation but had little effect against trophic factor deprivation or DNA damage in cultures of PC12 cells or sympathetic neurons (Troy et al., 1996b; Park et al., 1998b). DNA damage induced death was prevented only by the broadest spectrum inhibitors, BAF and V-ICEinh. This differential effect of the inhibitors raised the possibility of differences in the substrate(s) utilized in the different paradigms of death or of differences in the caspase(s) involved, or in both. As stated above, the knockout data point to two groups of caspases, those which are essential for normal development and those whose absence has little or no effect on normal development. Data from other systems point to a role for this second group of caspases in neuronal degeneration, which may have pathways that differ from those required for developmental cell death. We will first briefly describe the phenotypes of the mice lacking caspases necessary for normal development and then we will present data, which support roles for caspases-1 and -2 in death paradigms with relevance to neurodegenerative diseases.

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C.M. Troy

Caspases Essential for Normal Development Mice null for caspases-3, -8 or-9 all have abnormal development. Null deletions of caspases-8 and-9 are both embryonic lethal (Varfolomeev et al., 1998; Hakem et al., 1998; Kuida et al., 1998), while the caspase-3 knockout mice die at 1-3 weeks of age (Kuida et al., 1996). In both caspase-3 and -9 knockouts brain, development is profoundly abnormal with a variety of hyperplasias, disorganized cell distribution, and absence of the pyknotic clusters of cells normally seen in CNS, pointing to a failure of morphogenetic death. These caspases are not necessary for all types of cell death. This is illustrated by the sensitivity of thymocytes from animals lacking caspase-3 to induction of apoptosis by anti-Fas antibody, dexamethasone, C2-ceramide, staurosporine and gamma-irradiation, and thymocytes from animals lacking caspase-9 are sensitive to apoptosis induced by UV irradiation or anti-CD95. Caspase processing, particularly of caspase-3, is inhibited in the caspase-9 null ES cells but not in thymocytes or splenocytes, suggesting different apoptotic pathways in mammalian cells. Although caspase-9 has been found to be activated via a complex with Apaf-1, the caspase-9 knockouts do not accurately mimic the Apaf-1 knockouts, indicating that Apaf-1 can act via another caspase than caspase-9, although none of the other known caspases appear to be activated by Apaf-1 (Cecconi et al., 1998; Yoshida et al., 1998). Caspases Necessary for Other Types of Neuronal Cell Death In the caspase- 1, -2, and- 11 null mice, brain development is grossly normal, suggesting that these caspases do not play a critical role in neuronal development (Kuida et al., 1995; Li et al., 1997) Caspase-1 and -11 null mice are deficient in production of interleukin113, as expected, and intedeukin-la, which has yet not been explained. These mice are resistant to lipopolysaccharide-induced endotoxic shock. Caspase-ll physically interacts with caspase-1, suggesting that caspase-11 is a component of the caspase-1 complex and required for the activation of caspase-1. In vitro thymocytes from these caspase-I null mice are resistant to Fas-mediated apoptosis, in contract to caspase-3 mice, and cultured dorsal root gangion cells are resistant to trophic factor deprivation. A dominant-negative mutant caspase-1 (C285G) has also been expressed in mice (Friedlander et al., 1997b). As in the null mouse, neural development is normal, and the mice are deficient in interleukin-ll3 production. Trophic factor deprivation mediated death is inhibited in cultures of dorsal root ganglion cells from these animals. The mice also have a decrease in injury post-ischemia via middle cerebral artery occlusion. It is possible that expression of this construct might inhibit caspases other than caspase-1. However, the phenotype is in remarkable accord with that of the caspase-1 null mouse. Cells from caspase-2 null mice also have differential responses to death stimuli. Their ovaries have an increase in the number of germ ceils and the oocytes were found to be resistant to cell death following exposure to chemotherapeutic drugs. Apoptosis mediated by granzyme B and perforin was defective in the B lymphoblasts. In contrast, cell death of motor neurons during development was accelerated in the mice. In addition, caspase-2-deficient sympathetic neurons underwent apoptosis more quickly

Diversity of Caspase Involvement

79

than wild-type neurons when deprived of NGF. Thus, caspase-2 acts both as a positive and negative cell death effector, depending upon cell lineage and stage of development.

The Case for Caspase-I Although caspase-1 and its relatives caspases-4, -5 and -11 have been proposed to have roles only in inflammation and not in apopotosis (Thornberry and Lazebnik, 1998), there is considerable evidence pointing to a selective role for caspase-1 in mediating neuronal cell death. The caspase-1 knockout and caspase-1 dominant negative mice are each resistant to apoptosis induced by middle cerebral artery occlusion, and cultured DRGs from both mutant animals are resistant to trophic factor deprivation. The lack of a developmental phenotype can be attributed to either lack of a role for caspase-I in development or to compensation by other caspases during development. Action of interleukin-lB (IL-113) at its cell surface receptor is crucial for caspase-1 mediated death. IL-113 itself does not induce death, it must be acting on a cell which has a redox milieu which allows IL-lf3 to be apoptotic. All of the caspase-1 mediated deaths appear to involve excessive free radical production which leads to alterations of the redox setting of the cells. In our death paradigm, the downregulation of SOD1 leads to production of superoxide. Death can be blocked by YVAD treatment or by blocking the action of IL-1B, either with specific blocking antibodies or treatment with the IL-1 receptor antagonist (IL-1Ra), a naturally occurring peptide inhibitor of the IL-1 receptor (Troy et al., 1996b). Similar treatment protects DRG cultures from trophic factor mediated death and reduces the size of ischemic infarcts after MCA occlusion (Friedlander et al., 1996). Hippocampal neurons treated with staurosporine undergo apoptosis which can be blocked by YVAD (Krohn et al., 1998). In this case, there is an increased production of superoxide which is blocked by inhibition of caspase-l-like proteases. The time course of YVAD-cleaving activity parallels the production of superoxide supporting a pivotal role for a caspase-l-like protease. There is also later induction of DEVD-cleaving activity which does not appear necessary for death, as will be discussed below. The requirement for caspase-1 activation in cell death involving the generation of superoxide agrees with our data from PC12 cells and sympathetic neurons (Troy et al., 1996b). The protective effects of blocking IL-1B action led us to investigate the potential role of IL-1B in these systems in trophic factor deprivation. As shown in Figure 4, a blocking antibody to IL-1B protected cells from V-ASOD1 mediated cell death but not from death induced by trophic factor withdrawal (Troy et al., 1996b). Moreover, IL-1RA completely protected against SOD1 downregulation and had only a very modest effect against trophic factor deprivation, and then only at a much higher dose (Troy et al., 1996b). These differential responses to blockade by antibody and by a receptor antagonist raised the question of what changes occurred in IL-1B processing in response to the two stimuli. Measurement of IL-1B secretion in the medium of cells, in the two death paradigms, show that V-ASOD1 elicits an almost three-fold increase in IL-1B secretion which is blocked by V-ICEinh, while trophic factor withdrawal has no effect on IL- 1B secretion.

C.M. Troy

80

Trophic deprivation

V-ASOD1

treatment

T

100-

i

"o

E

7S-

Q =3

Q

SO-

Q ¢ 0

25-

9

8. T NGF

none

T

r

antML.1B

none

antl-lL-18

~r

Figure 4.

Interleukin-lB is differentially involved in death mediated by oxidative stress and by

trophic deprivation. PC12 cells were either treated with V-ASOD1 (50 nM) or trophic factor deprived in the presence or absence of the indicated concentrations of anti-IL-1B. Cell survival was determined at one day. Adapted from (Troy et al., 1996b).

_o G) o

g

15-

o

O

im

10-

e.,

5-

O. -NGF

-NGF

V-ASOD1 V-ASOD1

+V-ICEInh

+V-ICEIn h

w,-

Figure 5. Interleukin-lg levels are increased by by SOD1 downregulation, not by trophic factor withdrawal. PC12 cells were either treated with V-ASOD1 (50nM) or trophic factor deprived in the presence or absence of V-ICEinh. After 20 h treatment, medium was removed and IL-1B measured by ELISA. Adapted from (Troy et al., 1996b).

Diversity of Caspase Involvement

81

Therefore, the lack of response to blocking antibody and receptor block in the trophic deprivation model is not due to an overwhelming release of IL-1B upon trophic factor deprivation. These observations indicated the necessary involvement of IL-1B and its release in apoptosis triggered by SOD1 downregulation. Our findings further indicated that death caused by trophic factor deprivation might involve a different substrate(s) and possibly a different caspase. A second observation of our IL-1B secretion measurements was that NGF treatment provokes the largest effect on IL-IB secretion, a four-fold increase (Troy et al., 1996b). This indicated that IL-1B alone is not toxic to PC12 cells. This was confirmed by the observation that addition of recombinant IL-1B to PC12 cell cultures had no effect on cell survival in the presence of NGF, insulin or serum. Thus, cell death upon SOD 1 downregulation is not due to an increase in IL-1B alone, but rather to an enhanced vulnerability to this cytokine. Consistent with this, addition of IL-1B to cell cultures treated with V-ASOD1 potentiated cell death (Troy et al., 1996b). Several lines of evidence now point to a role for caspase-1 in the pathology associated with the missense mutations of SOD1. As discussed above, missense mutations of SOD1 are found in a subset of FALS cases. There are now more than 50 mutations described. The mechanism by which mutant SOD1 causes motor neuron degeneration is not yet understood. Expression of the human SOD1 mutation G93A in mice causes the development of fatal motor neuron disease (Gurney et al., 1994). Friedlander and colleagues bred the SODG93A mouse with a mouse they had engineered to overexpress a dominant negative mutation of caspase-1, and found that mice expressing both the SODG93A and the dominant negative caspase-1 had a delay in progression of symptoms, although onset of the disease was unchanged (Friedlander et al., 1997a). This indicates a role for caspases in the death induced by the SODG93A mutation. However, the dominant negative may not be completely selective for caspase-1. This possibility is argued against by the phenotypic concordance of the caspase knockout and the caspase-1 dominant negative mice. Caspase-1 has been shown to be activated in neurons expressing mutant SOD1 (Pasinelli et al., 1998). The processing is seen in spinal cords from mice expressing the G37R and G85R mutations, as well as in N2A cells expressing several missense mutations (G37R, G85R, or G41D). The activation of caspase-1 was enhanced by treatment with xanthine/xanthine oxidase, which increases superoxide and hydrogen peroxide production, and triggers cleavage and secretion of IL-1B and apoptosis. Apoptosis was blocked completely by zVADFMK and partially by Ac-YVAD-CHO, suggesting the involvement of caspase-1 and others caspases. It appears that both superoxide and hydrogen peroxide play roles in this death, and block of caspase-1 only affects the superoxide portion of the paradigm. This is consistent with the above data.

Proposed mechanism of caspase-1 mediated neuronal cell death The death mediated by caspase-1 is somewhat different from those induced by the other caspases.

82

C.M. Troy

PROPOSED PATHWAY OF FREE RADICAL MEDIATED DEATH

Increase superoxide

.~._-----~ 0 Activate caspase-1

pro-lL-113

~ tL-113

/ IL-1 receptor

/

activate JNK

activate other caspases

/

Apoptotic cell death

Figure 6. Proposed pathwayof free radical mediatedneuronal cell death.

The data would support an autocrine component of this death, namely the action of IL-1B at the IL-1 receptor. Activation of the receptor may then lead to induction of nitric oxide synthase and activation of c-jun kinase (JNK). Induction of NOS would increase NO which would then be available to combine with superoxide leading to peroxynitrite, which would lead to an increase in IL-1B production, as well as cause damage to DNA and other cell components. JNK activation could lead to activation of other caspases and thus to apoptotic death. Blocking the action of IL-1B at the IL-1 receptor would block all of these effects and thus confer complete protection. Caspase-2 and neuronal cell death Lack of protection against trophic factor deprivation by manipulations of IL-1B effects, as well as differences in the dose-response curves of the various caspase inhibitors in protectiong from trophic factor deprivation and SOD1 downregulation induced deaths, led us to examine which caspase functions in trophic factor deprivation mediated death. There are high levels of caspase-2 (Nedd2) mRNA and protein in PC12 cells (Kumar et al., 1994; Troy et al., 1997b). Caspase-2 exists as a long and short form, with the long form having pro-apoptotic activity and the short form anti-apoptotic activity. We chose to downregulate caspase 2-long in our death paradigms. To block the expression of caspase-2-1ong in neuronal cells, we designed an antisense oligonucleotide (ANedd)

Diversity of Caspase Involvement

83

which corresponds to the last 9 bases in the 5' UTR and the first 12 in the coding region of the caspase-2-1ong transcript (Kumar et al., 1994; Troy et al., 1997b). This sequence is not homologous to caspase-2-short or to any other mRNAs, including those of the other known caspases. For a control, a scrambled oligonucleotide (same base composition, scrambled) was used. The oligonucleotide was linked to the vector peptide Penetratinl (V-) (Troy et al., 1996a) to enhance its cellular uptake. To show that V-ANedd does decrease caspase-2 protein levels we generated an antibody to an N-terminal peptide of caspase-2 and used both immunohistochemistry and Western blotting to show that there is about 70% downregulation of caspase-2 within 24 h of V-ANedd treatment (Troy et al., 1997b). This downregulation was confirmed using a commercial antibody generated to a C-terminal peptide of caspase-2. The scrambled oligonucleotide (V-SNedd) did not downregulate caspase-2. V-ANedd did not affect the protein levels of caspase-3, nor did it alter the DEVD-AFC cleavage seen after trophic factor withdrawal (Stefanis et al., 1997). Treatment with V-ANedd protected PC12 cells from trophic factor deprivation but not from SOD1 downregulation. Protection from trophic deprivation has been confirmed by other investigators using stably expressed antisense constructs for caspase-2 in PC12 cells (Haviv et al., 1998). Western blots of PC12 cells after trophic deprivation show that there is a decrease in pro-caspase-2 and appearance of processed fragments (Stefanis et al., 1997, 1998). Parallel results were obtained with sympathetic neurons. However, as discussed above, sympathetic neurons from a caspase-2 knockout mouse were as susceptible to NGF withdrawal as wild-type neurons (Bergeron et al., 1998), raising the issue of the differences between our work using an antisense oligonucleotide to caspase-2 and the knockout mouse. In the knockout mouse both forms of caspase-2 (long (pro-apoptotic) and short (anti-apoptotic)) are deleted; in our study it is likely that only the long form is downregulated. These apparently contradictory results allow us the opportunity to compare the effects of deleting a gene, as in a knockout animal, with acute downregulation of protein expression in a mature neuron, as with antisense oligonucleotides. Death Receptors and Neuronal Cell Death Most of the data concerning death receptors and cell death is from non-neuronal systems. Very recent data support a role for Fas ligand induction in trophic factor deprivation death (Le-Niculescu et al., 1999). In this study, JNK activation followed by induction of Fas ligand expression and apoptosis were observed after withdrawal of trophic factors from PC12 cells and after KC1 removal from cerebellar granule neurons (CGNs). Apoptosis was blocked by treatment with a Fas-Fc decoy which sequesters Fas ligand. CGNs from gld mice, whose FasL gene encodes a nonfunctional protein, are resistant to KC1 withdrawal. The pathway proposed is stress signals leading to JNK to cJun to FasL to apoptosis. It is unlikely that this path is important during neuronal development as both the gld mice and jnk3 knockout mice have normal neuronal development and behavior. This pathway may be important in various neurodegenerative disorders. The role of caspases and IL-1 in this pathway have not yet been determined. In the primary cultures of CGNs the glial cells, although a minor contaminant, expressed FasL mRNA first after incubation in 5mM KCI. The glial cells are a major source of

84

C.M. Troy

IL-1 and IL-1, while not known to bind to death receptors, is a potent activator of JNK and NFkB and can induce TNF family members (Gupta et al., 1995; DiDonato et al., 1996). This further points to a role for IL-1 in vivo in certain types of neuronal cell injury and death. All Caspase Activity Does Not Produce Death In naive and neuronally differentiated PC12 cells deprived of trophic factors, there is a rapid induction of DEVD-AFC-cleaving, but not YVAD-AFC cleaving, activity. Cleavage of tPARP is also seen in naive and neuronally-differentiated PC12 cells prior to their death following trophic factor deprivation. However, the concentrations of caspase inhibitors which block the DEVD-AFC cleaving activity, as well as tPARP cleavage, do not protect from cell death. 100 -

[]

relative DEVD-AFC cleavage

•

relative survival

75-

50-

25-

0

1

5

10

DEVD-FMK Figure 7.

25

100

,uM

Inhibition of DEVD-cleaving activity does not protect against trophic factor withdrawal. PC12

cells were trophic factor deprived in the presence or absence of the indicated concentrations of DEVD-FMK. After 6 h cells were harvested and DEVD-AFC cleavage was measured. Sister cultures were used for assessment of survival at 20 h (Stefanis et al., 1998).

Protection from cell death required concentrations of the inhibitors (zVAD-FMK or DEVD-FMK) approximately 10-fold higher than the concentrations that inhibited the DEVD-AFC cleaving activity. Our data would, therefore, support the idea that although regulation of DEVD-AFC/tPARP cleaving activity correlates with cell death, it is not the major determinant of apoptosis in trophic factor deprivation. This agrees with the studies of the substrate and inhibitor profiles for caspase-2.

Diversity of Caspase Involvement

85

Similar results have been obtained in another death paradigm, staurosporine induced apoptosis of cultured hippocampal neurons. As discussed above, activation of caspase- 1 and subsequent superoxide production are necessary for cell death in this paradigm. However, there is induction of DEVD-AMC cleaving activity which parallels the increase in superoxide production. But block of the activity by DEVD-CHO prevented the nuclear fragmentation, but not the rise in superoxide production and subsequent death of the cells. These data reinforce the necessity of correlating all parameters of cell death and caspase activation before claiming causalty of a particular caspase.

Summary Much still remains to be determined about the roles of the individual caspases in cell death or in other, as yet, undetermined functions. It is clear, however, that different stimuli do employ different caspases to cause death. It is also clear that caspases may be activated and not lead to cell death. While it is not yet clear what these activated, but non-essential caspases are doing, it is possible that they are involved in cytoplasmic remodeling via cleavage of the cytoskeletal components. CASPASE SPECIFIC[TIES FOR DIFFERENT PARADIGMS OF CELL DEATH

Free radical

1

Caspase I

Trophic factor deprivation

l

Caspese 2

l

Caspase 3

DNA damage

BAF-sensitive caepese

APOPTOTIC CELL DEATH Figure 8. Caspase specificities in three paradigms of neuronal cell death.

In Figure 8 we present a simplistic schematic diagram of our current knowledge about the caspase requirements in several death paradigms. The data we have presented is compatible with either single caspases acting to produce death or with a cascade of caspases, since blocking one element of the cascade will block the entire cascade. Further elucidation of the caspases required for individual death pathways would require careful attention to the answers our tools produce. At present, a combination of knockouts, expression of antisense constructs and antisense oligonucleotides offer the best option of determining caspase specificity. Since caspases are the executors of cell death, this data, together with our ongoing work on the mechanisms of neuronal death, will enable the rational design of drugs for specific neurodegenerative diseases.

C.M. Troy

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Acknowledgements I thank Michael L. Shelanski and Wilma J. Friedman for invaluable critical commentary and editorial assistance. CMT is supported by grants from NINDS and the Muscular Dystrophy Association.

References Adams, J.M. & Cory S. (1998). The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322-1326. Ahmad, M., Srinivasula S.M., Hegde R., Mukattash R., Fernandes-Alnemri T. & Alnemri E.S. (1998). Identification and characterization of murine caspase-14, a new member of the caspase family. Cancer Res. 58, 5201-5205. Ahmad, M., Srinivasula, S.M., Wang L., Talanian R.V., Litwack G., Fernandes-Alnemri T. & Alnemri E.S. (1997). CRADD, a novel human apoptotic adaptor molecule for caspase-2, and FasL/tumor necrosis factor receptor-interacting protein RIP. Cancer Res. 57, 615-619. Angelastro, J.M., Qi H., Stefanis, L., Park, D.S. & Troy, C.M. (1998). Identification and determining the functional roles of caspases in apoptosis and PC12 cells. In ISDN 12tb Biennial Meeting, Vancouver, B.C. Armstrong, R.C., Aja, T.J., Hoang K.D., Gaur S., Bai X., Alnemri E.S., Litwack G., Karanewsky D.S., Fritz L.C. & Tomaselli K.J. (1997). Activation of the CED3/ICE-related protease CPP32 in cerebellar granule neurons undergoing apoptosis but not necrosis. J. Neurosci. 17, 553-562. Bains, J.S. & Shaw C.A. (1997). Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death. Brain. Res. Brain Res Rev. 25,335-358. Batistatou, A. & Greene L.A. (1991). Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity. J. Cell Biol. 115,461-471. Batistatou, A., Merry D.E., Korsmeyer S.J. & Greene L.A. (1993). Bcl-2 affects survival but not neuronal differentiation of PC 12 cells. J. Neurosci. 13, 4422-4428. Bergeron, L., Perez G.I., Macdonald, G., Shi, L., Sun, Y., Jurisicova, A., Varmuza S., Latham K.E., Flaws, J.A., Salter, J.C., Hara, H., Moskowitz, M.A., Li E., Greenberg, A., Tilly, J.L. & Yuan, J. (1998). Defects in regulation of apoptosis in caspase-2-deficient mice. Genes Dev. 12, 1304-1314. Bergeron, L. & Yuan, J. (1998). Sealing one's fate: control of cell death in neurons. Curr. Opin. Neurobiol. 8, 55-63. Boldin, M.P., Goncharov,T.M., Goltsev,Y.V. & Wallach,D. (1996). Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85, 803-815. Boldin, M.P., Varfolomeev,E.E., Pancer, Z., Mett, I.L., Camonis, J.H. & Wallach, D. (1995). A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain. J. Biol. Chem. 270, 7795-7798. Brown, R.H., Jr. (1995). Amyotrophic lateral sclerosis: recent insights from genetics and transgenic mice. Cell 80, 687-692. Bump, N.J., Hackett, M., Hugunin, M., Seshagiri, S., Brady, K., Chen, P., Ferenz, C., Franklin, S., Ghayur, T., Li, P. & et al. (1995). Inhibition of ICE family proteases by baculovirus antiapoptotic protein p35. Science 269, 1885-1888.

Diversity of Caspase Involvement

87

Cecconi, F., Alvarez-Bolado, G., Meyer, B.I., Roth, K.A. & Gruss, P. (1998). Apafl (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94, 727-737. Cerretti, D.P., Kozlosky, C.J., Mosley, B., Nelson, N., Van Ness, K., Greenstreet, T.A., March, C.J., Kronheim, S.R., Druck, T., Cannizzaro, L.A. & et al. (1992). Molecular cloning of the interleukin-1 beta converting enzyme. Science 256, 97-100. Cheng, E.H., Kirsch, D.G., Clem, R.J., Ravi, R., Kastan, M.B., Bedi, A., Ueno, K. & Hardwick, J.M. (1997). Conversion of Bcl-2 to a Bax-like death effector by caspases. Science 278, 1966-1968. Chinnaiyan, A.M., O'Rourke, K., Tewari, M. & Dixit, V.M. (1995). FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81,505-512. Cohen, G. M. (1997). Caspases: the executioners of apoptosis. Biochem. J. 326, 1-16. Coyle, J.T. & Puttfarcken, P. (1993). Oxidative stress, glutamate, and neurodegenerative disorders. Science 262, 689-695. Cryns, V. & Yuan, J. (1998). Proteases to die for. Genes Dev. 12, 1551-1570. Culotta, V.C., Joh, H.D., Lin, S.J. Slekar, K.H. & Strain, J. (1995). A physiological role for Saccharomyces cerevisiae copper/zinc superoxide dismutase in copper buffering. J. Biol. Chem. 270, 29991-29997. Deveraux, Q.L., Roy, N., Stennicke, H.R., Van Arsdale, T., Zhou, Q., Srinivasula, S.M., Alnemri, E.S., Salvesen, G.S. & Reed, J.C. (1998). lAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. Embo J. 17, 2215-2223. Deveraux, Q.L., Takahashi, R., Salvesen, G.S. & Reed, J.C. (1997). X-linked lAP is a direct inhibitor of cell-death proteases. Nature 388, 300-304. DiDonato, J., Mercurio, F., Rosette, C., Wu-Li, J., Suyang, H., Ghosh, S.& Karin, M. (1996). Mapping of the inducible IkappaB phosphorylation sites that signal its ubiquitination and degradation. Mol. Cell. Biol. 16, 1295-1304. Duan, H. & Dixit, V.M. (1997). RAIDD is a new 'death' adaptor molecule. Nature 385, 86-89. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A. & Nagata, S. (1998). A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD [see comments] [published erratum appears in Nature 1998 May 28;393(6683):396]. Nature 391,43-50. Enns, L., Barley, R.D., Paterson, M.C. & Mirzayans, R. (1998). Radiosensitivity in ataxia telangiectasia fibroblasts is not associated with deregulated apoptosis. Radiat Res. 150, 11-16. Farinelli, S.E., Park, D.S. & Greene, L.A. (1996). Nitric oxide delays the death of trophic factor-deprived PC 12 cells and sympathetic neurons by a cGMP-mediated mechanism. J. Neurosci. 16, 2325-2334. Fridovich, 1. (1995). Superoxide radical and superoxide dismutases. Annu. Rev. Biochem. 64, 97-112. Friedlander, R.M., Brown, R.H., Gagliardini, V., Wang, J. & Yuan, J. (1997a). Inhibition of ICE slows ALS in mice [letter] [published erratum appears in Nature 1998 Apr 9;392(6676):560]. Nature 388, 31. Friedlander, R.M., Gagliardini, V., Hara, H., Fink, K.B., Li, W., MacDonald, G., Fishman, M.C., Greenberg, A.H., Moskowitz, M.A. & Yuan, J. (1997b). Expression of a dominant negative mutant of interleukin-1 beta converting enzyme in transgenic mice prevents neuronal cell death induced by trophic factor withdrawal and ischemic brain injury. J. Exp. Med. 185,933-940. Friedlander, R.M., Gagliardini, V., Rotello, R.J. & Yuan, J. (1996). Functional role of interleukin 1 beta (IL-1 beta) in IL-1 beta- converting enzyme-mediated apoptosis. J. Exp. Med. 184, 717-724. Gonzalez-Zulueta,, M., Ensz, L.M., Mukhina, G., Lebovitz, R.M., Zwacka, R.M., Engelhardt, J.F., Oberley, L.W., Dawson, V.L. & Dawson, T.M. (1998). Manganese superoxide dismutase protects nNOS neurons from NMDA and nitric oxide-mediated neurotoxicity. J. Neurosci. 18, 2040-2055. Green, D.R. (1998). Apoptotic pathways: the road to ruin. Cell 94, 695-698. Greene, L.A. (1978). Nerve growth factor prevents the death and stimulates the neuronal differentiation of

88

C.M. Troy

clonal PC12 pheochromocytoma cells in serum-free medium. J. Cell Biol. 78, 747-755. Gupta, S., Campbell, D., Derijard, B. & Davis, R.J. (1995). Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science 267, 389-393. Gurney, M.E., Pu, H., Chiu, A.Y., Dal Canto, M.C., Polchow, C.Y., Alexander, D.D., Caliendo, J., Hentati, A., Kwon, Y.W., Deng, H. X. & et al. (1994). Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation [see comments] [published erratum appears in Science 1995 Jul 14;269(5221): 149]. Science 264, 1772-1775. Hakem, R., Hakem, A., Duncan, G.S., Henderson, J.T., Woo, M., Soengas, M.S., Elia, A., de la Pompa, J.L., Kagi, D., Khoo, W., Potter, J., Yoshida, R., Kaufman, S.A., Lowe, S.W., Penninger, J.M. & Mak, T. W. (1998). Differential requirement for easpase 9 in apoptotic pathways in vivo.Cell 94, 339-352. Haviv, R., Lindenboim, L., Yuan, J. & Stein, R. (1998). Need for caspase-2 in apoptosis of growth-factordeprived PC12 cells [In Process Citation]. J Neurosci Res. 52, 491-497. Hengartner, M.O., Ellis, R.E. & Horvitz, H.R. (1992). Caenorhabditis elegans gene ced-9 protects cells from programmed cell death. Nature 356, 494-499. Hofmann, K., Bucher, P. & Tschopp, J. (1997). The CARD domain: a new apoptotic signalling motif. Trends Biochem Sci. 22, 155-156. Hu, S., Snipas, S.J., Vincenz, C., Salvesen, G. & Dixit, V.M. (1998). Caspase-14 is a novel developmentally regulated protease. J. Biol. Chem. 273, 29648-29653. Humke, E.W., Ni, J. & Dixit, V.M. (1998). ERICE, a novel FLICE-activatable caspase. J. Biol. Chem. 273, 15702-15707. Keller, J.N., Kindy, M.S., Holtsberg, F.W., St. Clair, D.K., Yen, H.-C., Germeyer, A., Steiner, S.M., BruceKeller, A.J., Hutchins, J.B. & Mattson, M.P. (1998). Mitochondrial MnSOD prevents neural apoptosis and reduces ischemic brain injury: Suppression of peroxynitrite production, lipid peroxidation and mitochondrial dysfunction. J. Neurosci. 18:687-697. Kerr, J.F., Wyllie, A.H. & Currie, A.R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239-257. Krohn, A.J., Preis, E. & Prehn, J.H. (1998). Staurosporine-induced apoptosis of cultured rat hippocampal neurons involves caspase-l-like proteases as upstream initiators and increased production of superoxide as a main downstream effector. J. Neurosci. 18, 8186-8197. Kuida, K., Haydar, T.F., Kuan, C.Y., Gu, Y., Taya, C., Karasuyama, H., Su, M. S., Rakic, P. & Flavell, R.A. (1998). Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 94, 325-337. Kuida, K., Lippke, J.A., Ku, G., Harding, M.W., Livingston, D.J., Su, M.S. & Flavell, R.A. (1995). Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme. Science 267, 2000-2003. Kuida, K., Zheng, T.S., Na, S., Kuan, C., Yang, D., Karasuyama, H., Rakic, P. & Flavell, R.A. (1996). Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 384, 368-372. Kumar, S., Kinoshita, M., Noda, M., Copeland, N.G. & Jenkins, N.A. (1994). Induction of apoptosis by the mouse Nedd2 gene, which encodes a protein similar to the product of the Caenorhabditis elegans cell death gene ced-3 and the mammalian IL-1 beta-converting enzyme. Genes Dev. 8, 1613-1626. Lavin, M.F. (1998). Radiosensitivity and oxidative signalling in ataxia telangiectasia: an update [In Process Citation]. Radiother. Oncol. 47, 113-123. I..azebnik, Y.A., Takahashi, A., Moir, R.D., Goldman, R.D., Poirier, G.G., Kaufmann, S.H. & Earnsbaw W.C. (1995). Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution. Proc Natl Acad Sci U S A. 92, 9042-9046.

Diversity of Caspase Involvement

89

Le-Niculescu, H., Bonfoco, E., Kasuya, Y., Claret, F.X., Green, D.R. & Karin, M. (1999). Withdrawal of survival factors results in activation of the JNK pathway in neuronal cells leading to fas ligand induction and cell death [In Process Citation]. Mol. Cell Biol. 19, 751-763. Li, P., Allen H., Banerjee, S. & Seshadri, T. (1997). Characterization of mice deficient in interleukin-1 beta converting enzyme. J. Cell Biochem. 64, 27-32. Margolin, N. & Raybuck S.A., Wilson K.P., Chen W., Fox T., Gu Y. & Livingston D.J. (1997). Substrate and inhibitor specificity of interleukin-1 beta-converting enzyme and related caspases. J. Biol. Chem. 272, 7223-7228. Mashima, T., Naito, M., Fujita, N., Noguchi, K. & Tsuruo, T. (1995). Identification of actin as a substrate of ICE and an ICE-like protease and involvement of an ICE-like protease but not ICE in VP-16-induced U937 apoptosis. Biochem. Biophys. Res. Commun. 217, 1185-1192. McCord, J.M. & Fridovich, I. (1969). Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049-6055. Miura, M., Zhu, H., Rotello, R., Hartwieg, E.A. & Yuan, J. (1993). Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell 75,653-660. Muzio, M., Chinnaiyan, A.M., Kischkel, F.C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J.D., Zhang, M., Gentz, R., Mann, M., Krammer, P.H., Peter, M.E. & Dixit V.M. (1996). FL1CE, a novel FADD-homologous ICE/CED-3-1ike protease, is recruited to the CD95 (Fas/APO-1) death--inducing signaling complex. Cell 85, 817-827. Muzio, M., Stockwell, B.R., Stennicke, H.R., Salvesen, G.S. & Dixit V.M. (1998). An induced proximity model for caspase-8 activation. J. Biol. Chem. 273, 2926-2930. Namura, S., Zhu, J., Fink, K., Endres, M., Srinivasan, A., Tomaselli, K.J., Yuan, J. & Moskowitz, M.A. (1998). Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia. J. Neurosci. 18, 3659-3668. Nicholson, D.W., Ali, A., Thornberry, N.A., Vaillancourt, J.P., Ding, C.K., Gallant, M., Gareau, Y., Griffin, P.R., Labelle, M., Lazebnik, Y.A. & et al. (1995). Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis [see comments]. Nature 376, 37-43. Nicholson, D.W. & Thornberry, N.A. (1997). Caspases: killer proteases. Trends Biochem Sci. 22, 299-306. Orth, K., Chinnaiyan, A.M., Garg, M., Froelich, C.J. & Dixit, V.M. (1996). The CED-3/ICE-Iike protease Mch2 is activated during apoptosis and cleaves the death substrate lamin A. J. Biol. Chem. 271, 16443-16446. Park, D.S., Morris, E.J., Padmanabhan, J., Shelanski, M.L., Geller, H.M. & Greene, L.A. (1998a). Cyclindependent kinases participate in death of neurons evoked by DNA- damaging agents. J. Cell Biol. 143,457-467. Park, D.S., Morris, E.J., Stefanis, L., Troy, C.M., Shelanski, M.L., Geller, H.M. & Greene, L.A. (1998b). Multiple pathways of neuronal death induced by DNA-damaging agents, NGF deprivation, and oxidative stress. J. Neurosci. 18,830-840. Pasinelli, P., Borchelt, D.R., Houseweart, M.K., Cleveland, D.W. & Brown, R.H., Jr. (1998). Caspase-1 is activated in neural cells and tissue with amyotrophic lateral sclerosis-associated mutations in copper-zinc superoxide dismutase. Proc. Natl. Acad. Sci. U S A. 95, 15763-15768. Prochiantz, A. & Theodore, L. (1995). Nuclear/growth factors. Bioessays. 17, 39-44. Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., Donaldson, D., Goto, J., O'Regan, J.P., Deng, H.X. & et al. (1993). Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis [published erratum appears in Nature 1993 Jul

90

C.M.

Troy

22;364(6435):362] [see comments]. Nature 362, 59-62. Rotonda, J., Nicholson, D.W., Fazil, K.M., Gallant, M., Gareau, Y., Labelle, M., Peterson, E.P., Rasper, D.M., Ruel, R., Vaillancourt, J.P., Thornberry, N.A. & Becker, J.W. (1996). The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nat. Struct. Biol. 3,619-625. Rukenstein, A., Rydel, R.E. & Greene, L.A. (1991). Multiple agents rescue PC12 cells from serum-free cell death by translation- and transcription-independent mechanisms. J. Neurosci. 11, 2552-2563. Schapira, A.H. (1996). Oxidative stress and mitochondrial dysfunction in neurodegeneration. Curr. Opin. Neurol. 9, 260-264. Silos-Santiago, I., Greenlund, L.J., Johnson, E.M., Jr. & Snider, W.D. (1995). Molecular genetics of neuronal survival. Curr. Opin. Neurobiol. 5, 42-49. Srinivasan, A., Foster, L.M., Testa, M.P., Ord, T., Keane, R.W., Bredesen, D.E. & Kayalar, C. (1996). Bcl-2 expression in neural cells blocks activation of ICE/CED-3 family proteases during apoptosis. J. Neurosci. 16, 5654-5660. Stefanis, L., Park, D.S., Yan, C.Y., Farinelli, S.E., Troy, C.M., Shelanski, M.L. & Greene, L.A. (1996). Induction of CPP32-1ike activity in PC12 cells by withdrawal of trophic support. Dissociation from apoptosis. J. Biol. Chem. 271, 30663-30671. Stefanis, L., Troy, C.M., Qi, H. & Greene, L.A. (1997). Inhibitors of trypsin-like serine proteases inhibit processing of the caspase Nedd-2 and protect PC12 cells and sympathetic neurons from death evoked by withdrawal of trophic support. J. Neurochem. 69, 1425-1437. Stefanis, L., Troy, C.M., Qi, H., Shelanski, M.L. & Greene, L.A. (1998). Caspase-2 (Nedd-2) processing and death of trophic factor-deprived PC12 cells and sympathetic neurons occur independently of caspase-3 (CPP32)- like activity [In Process Citation]. J. Neurosci. 18, 9204-9215. Takahashi, A., Alnemri, E.S., Lazebnik, Y.A., Fernandes-Alnemri, T., Litwack, G., Moir, R.D., Goldman, R.D., Poirier, G.G., Kaufmann, S.H. & Eamshaw, W.C. (1996). Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis. Proc. Natl. Acad. Sci. U S A. 93, 8395-8400. Talanian, R.V., Quinlan, C., Trautz, S., Hackett, M.C., Mankovich, J.A., Banach, D., Ghayur, T., Brady, K.D. & Wong, W.W. (1997). Substrate specificities of caspase family proteases. J. Biol. Chem. 272, 9677-9682. Theodore, L., Derossi, D., Chassaing, G., Llirbat, B., Kubes, M., Jordan, P., Chneiweiss, H., Godement, P. & Prochiantz, A. (1995). Intraneuronal delivery of protein kinase C pseudosubstrate leads to growth cone collapse. J. Neurosci. 15, 7158-7167. Thornberry, N.A., Bull, H.G., Calaycay, J.R., Chapman, K.T., Howard, A.D., Kostura, M.J., Miller, D.K., Molineaux, S.M., Weidner, J.R., Aunins, J. & et al. (1992). A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature 356, 768-774. Thomberry, N.A. & Lazebnik, Y. (1998). Caspases: enemies within. Science 281, 1312-1316. Thornberry, N.A., Rano, T.A., Peterson, E.P., Rasper, D.M., Timkey, T., Garcia-Calvo, M., Houtzager, V.M., Nordstrom, P.A., Roy, S., Vaillancourt, J.P., Chapman, K.T. & Nicholson, D.W. (1997). A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272, 17907-17911. Troy, C.M., Derossi, D., Prochiantz, A., Greene, L.A. & Shelanski, M.L. (1996a). Downregulation of Cu/Zn superoxide dismutase leads to cell death via the nitric oxide-peroxynitrite pathway. J. Neurosci. 16, 253-261. Troy, C.M. & Shelanski, M.L. (1994). Downregulation of copper/zinc superoxide dismutase causes apoptotic death in PC12 neuronal cells. Proc. Natl. Acad. Sci. U S A. 91, 6384-6387.

Diversity of Caspase Involvement

91

Troy, C.M., Stefanis, L., Greene, L.A. & Shelanski, M.L. (1997a). Mechanisms of neuronal degeneration: a final common pathway? Adv. Neurol. 72, 103-111. Troy, C.M., Stefanis, L., Greene, L.A. & Shelanski, M.L. (1997b). Nedd2 is required for apoptosis after trophic factor withdrawal, but not superoxide dismutase (SOD1) downregulation, in sympathetic neurons and PC12 cells. J. Neurosci. 17,1911-1918. Troy, C.M., Stefanis, L., Prochiantz, A., Greene, L.A. & Shelanski, M.L. (1996b). The contrasting roles of ICE family proteases and interleukin-lbeta in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase downregulation. Proc. Natl. Acad. Sci. U S A. 93, 5635-5640. Varfolomeev, E.E., Schuchmann, M., Luria, V., Chiannilkulchai, N., Beckmann, J.S., Mett, I.L., Rebrikov, D., Brodianski, V.M., Kemper, O.C., Kollet, O., Lapidot, T., Softer, D., Sobe, T., Avraham, K.B., Goncharov, T., Holtmann, H., Lonai, P. & Wallach, D. (1998). Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apol, and DR3 and is lethal prenatally. Immunity. 9, 267-276. Vaux, D.L., Cory, S. & Adams, J.M. (1988). Bcl-2 gene promotes haemopoietic cell survival and cooperates with c- myc to immortalize pre-B cells. Nature 335,440-442. Vaux, D.L., Weissman, I.L. & Kim, S.K. (1992). Prevention of programmed cell death in Caenorhabditis elegans by human bcl-2. Science 258, 1955-1957. Vincenz, C. & Dixit V.M. (1997). Fas-associated death domain protein interleukin-lbeta-converting enzyme 2 (FLICE2), an ICE/Ced-3 homologue, is proximally involved in CD95- and p55-mediated death signaling. J. Biol. Chem. 272, 6578-6583. Walker, N.P., Talanian, R.V., Brady, K.D., Dang, L.C., Bump, N.J., Ferenz, C.R., Franklin, S., Ghayur, T., Hackett, M.C., Hammill, L.D. & et al. (1994). Crystal structure of the cysteine protease interleukin-1 beta- converting enzyme: a (p20/p10)2 homodimer. Cell 78, 343-352. Wang, L., Miura, M., Bergeron, L., Zhu, H. &, Yuan J. (1994). Ich-1, an Ice/ced-3-related gene, encodes both positive and negative regulators of programmed cell death. Cell 78,739-750. Wang, S., Miura, M., Jung, Y.K., Zhu, H., Li, E. & Yuan, J. (1998). Murine caspase-11, an ICE-interacting protease, is essential for the activation of ICE. Cell 92, 501-509. Williams, L.R. (1995). Oxidative stress, age-related neurodegeneration, and the potential for neurotrophic treatment. Cerebrovasc. Brain Metab. Rev. 7, 55-73. Wilson, K.P., Black, J.A., Thomson, J.A., Kim, E.E., Griffith, J.P., Navia, M.A., Murcko, M.A., Chambers, S.P., Aldape, R.A., Raybuck, S.A. & et al. (1994). Structure and mechanism of interleukin-1 beta converting enzyme [see comments]. Nature 370, 270-275. Wyllie, A.H. (1980). Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555-556. Wyllie, A.H., Morris, R.G., Smith, A.L. & Dunlop D. (1984). Chromafin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J. Pathol. 142, 67-77. Xue, D. & Horvitz, H.R. (1995). Inhibition of the Caenorhabditis elegans cell-death protease CED-3 by a CED-3 cleavage site in baculovirus p35 protein. Nature 377,248-251. Xue, D. & Horvitz, H.R. (1997). Caenorhabditis elegans CED-9 protein is a bifunctional cell-death inhibitor. Nature 390, 305-308. Yang, X., Chang, H.Y. & Baltimore, D. (1998). Autoproteolytic activation of pro-caspases by oligomerization. Mol. Cell 1,319-325. Yoshida, H., Kong, Y.Y., Yoshida, R., Elia, A.J., Hakem, A., Hakem, R., Penninger, J.M. & Mak, T.W. (1998). Apafl is required for mitochondrial pathways of apoptosis and brain development. Cell 94, 739-750.

92

C.M. Troy

Yuan, J. & Horvitz, H.R. (1992). The Caenorhabditis elegans cell death gene ced-4 encodes a novel protein and is expressed during the period of extensive programmed cell death. Development. 116, 309-320. Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M. & Horvitz, H.R. (1993). The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell 75, 641-652. Zou, H., Henzel, W.J., Liu, X., Lutschg, A. & Wang, X. (1997). Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3 [see comments]. Cell 90, 405-413.

MITOCHONDRIAL CONTROL OF APOPTOSIS BERNARD M I G N O T T E and JEAN-LUC VAYSSIERE

Table of Contents Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 The Actors of the Execution Stage of the Apoptotic Cascade . . . . . . . . . . . . . . . . . . . . . . . . 93 Bcl-2 Family Proteins Act at Least in Part at the Mitochondrial Level . . . . . . . . . . . . . . . . . 95 Permeability Transition Marks A Point of No-Return of Cells Condemned to Die . . . . . . . . . 97 A Decrease in Mitochondrial Membrane Potential Precedes DNA Fragmentation During Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Mitochondrial Permeability Transition Provokes a AWmDecrease . . . . . . . . . . . . . . . . . . . . 98 Direct Interventions on the Mitochondrial Permeability Transition Modulate Apoptosis... 98 Mitochondria of Cells Undergoing Apoptosis Release Pro-Apoptotic Factors . . . . . . . . . . . . . 99 Mitochondria Undergoing Permeability Transition Liberate AIF Factor . . . . . . . . . . . . . . . . 99 Cytochrome C is Released by Mitochondria During Apoptosis . . . . . . . . . . . . . . . . . . . . . . 101 The Cytochrome C Pathway is Distinct from that of AIF . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Bcl-2 Family Proteins Dock Various Proteins to the Mitochondria . . . . . . . . . . . . . . . . . . . 103 What is the Mechanism Involved in Cytoplasmic Release of Mitochondrial Apoptogenic Factors? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Mitochondrial ROS and Apoptosis Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 ROS as Mediators of PCD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 PCD-Mediating ROS are Produced by Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Mechanisms Of ROS Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Introduction The Actors of the Execution Stage of the Apoptotic Cascade The programmed cell death cascade can be conveniently divided into several phases. During the activation phase, multiple signaling pathways lead from the various deathtriggering signals to the central control of the cell death machinery and activate it. This is followed by the execution stage, in which the activated machinery acts on multiple cellular targets, and, finally, the destruction phase in which the dead or dying cell is broken down. A most important clue to the molecular nature of the death program came initially from genetic studies in C. elegans that led to the identification of a dozen cell death genes (ced) that are responsible for one aspect or another of cell death processes (Ellis et al., 1991). Three of these genes stand out. Two, ced-3 and ced-4 are essential for cell death. The third, ced-9, antagonizes the death activities of ced-3 and ced-4. 93 Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. 93 -- 122 © 2001 Elsevier Science. Printed in the Netherlands.

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Mutational analyses of these genes placed ced-4 between ced-9 and ced-3 in the pathway leading to cell death, suggesting that CED-4 might act as an adaptator, linking the upstream regulator CED-9 to the downstream death effector CED-3 (Shaham and Horvitz, 1996). Biochemical approaches have provided insight into the mechanism by which programmed cell death is regulated in the nematode. CED-4 interacts directly and simultaneously with both CED-3 and CED-9 forming a multimeric protein complex (Chinnaiyan et al., 1997b; Wu et al., 1997a). Furthermore, oligomerized CED-4, acting as a context dependent ATPase, promotes CED-3 activation and its ability to induce apoptosis (Chinnaiyan et al., 1997a). CED-9 binding to CED-4, mutually exclusive with CED-4 oligomerization, prevents it from activating CED-3, thereby blocking cell death (Yang et al., 1998). Remarkably, all three of these C. elegans cell death genes have mammalian counterparts that are likely to play similar, albeit more complex, roles in mammalian cell death (Table 1). CED-3 protein turned out to be a member of a family of cystein proteases, known as caspases (cystein as__laartases). The mammalian caspase family now comprises at least ten known members, most of which have been definitively implicated in PCD (for review see Cryns and Yuan, 1998)). All cleave their substrates after specific aspartic acids and are themselves activated by cleavage at specific aspartic acids (Nicholson and Thornberry, 1997). Caspases mediate PCD by cleaving selected intracellular proteins, including proteins of the nucleus, nuclear lamina, cytoskeleton, endoplasmic reticulum, and cytosol. However, which of these targets is, if any, responsible for the cell blebbing, condensation and fragmentation that characterize PCD remains uncertain. As specific protein or peptide caspase inhibitors can block PCD in all animal and invertebrate cells and in most cell death inducing conditions, it seems likely that caspases form the core of the death program. Table 1.

Programmed cell death in nematodes and mammals is controlled by homologous proteins. Mammalian caspases act either during the activation or the execution-phase of PCD (Nicholson and Thornberry, 1997). Recently, a CED-4 homologue has been identified in human cells (Hofmann et al., 1997; Zou et al., 1997). In mammals some members of the Bcl-2 related proteins are death antagonists while other are death agonists. For a recent review on the structure-function relations of Bcl-2 related proteins see (Kroemer, 1997).

C. elegans

Mammals

CED-3

Activation caspases: caspase-1 (ICE), -4 (ICH-2), -6 (Mch2), -8 (MACH/FLICE)... Execution caspases: caspase-2 (ICH-I), -3 (CPP32), -4 (ICH-2), -7 (ICE-LAP3)...

CED-4 CED-9

Apaf-1 Anti-apoptotic: Bcl-2, Bcl-xe, Bcl-w, Bfl-1, Brag-l, Mcl-1, A1, NR13... Pro-apoptotic: Bax, Bak, Bcl-xs, Bad, Bik, Hrk...

Recent studies have identified and partially characterized Apaf-1 (Apoptotic 12roteaseactivating _factor-I), a mammalian homologue of C. elegans CED-4 (Zou et al., 1997). The N-terminal region of Apaf-1 shares amino acid homology with CED-4 and several caspases with long prodomains, including, caspase-4, -8, -9, and CED-3. This conserved

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region, that has been termed caspase recruitment domain (CARD), mediates the physical association of CARD-containing proteins (Hofmann et al., 1997). Thus, Ced-4 binding to Ced-3 involves their respective amino-terminal protein interaction modules. In such way, Apaf-1 was shown to interact with caspase-9, a mammalian caspase containing a N-terminal long prodomain, and to trigger caspase-9 proteolytic self-activation (Li et al., 1997b). Caspase-9 subsequently proteolyses and activates caspase-3, one of the execution caspases which kill the cell by cleaving key intracellular death targets. Bcl-2 Family Proteins Act at Least in Part at the Mitochondrial Level CED-9 protein is homologous to a family of many members termed the Bcl-2 family in reference to the first discovered mammalian cell death regulator (for review see Adams and Cory, 1998)). Some members, such as Bcl-2 or Bcl-x L, are negative regulators of cell death, able to prevent cells from undergoing apoptosis induced by various stimuli in a wide variety of cell-types (Korsmeyer, 1992; Zhong et al., 1993), whereas others, such as Bax, Bid and Egl-1 promote or accelerate cell death. All members possess at least one of four conserved motifs known as Bcl-2 homology domains (BH1 to BH4). Most pro-survival members contain at least BH1 and BH2, and those most similar to Bcl-2 have all four BH domains. Two pro-apoptotic subfamilies can be define according to their relatedness to Bcl-2. Bax, Bak and Bok, which contain BH1, BH2 and BH3, resemble Bcl-2 fairly closely. In contrast, the other known mammalian and nematode EGL-1 "killers" possess only the central BH3 domain. These "BH3 domain" proteins may represent the physiological antagonists of the pro-survival proteins, because programmed cell death in C. elegans requires Egl-1, which binds to and acts via CED-9. BH3 is essential for the function of the "killers", including Egl-1. The various family members (referred to here as Bct-2s) can heterodimerize and seemingly titrate one another's function, suggesting that their relative concentration may act as a rheostat for the suicide program. Heterodimerization is not required for pro-survival function. For pro-apoptotic activity, heterodimerization is essential in the BH3 domain group, but less so for those of the Bax group, which may have an independent cytotoxic impact. Indeed whether Bax binds to Bcl-2 inside cells has become controversial. Some death agonists may preferentially target subsets of the death repressors. Bok, for example, interacts with Mcl-1 and the Epstein-barr viral protein BHFR1 but not with Bcl-2 or Bcl-x L. Within the BH3 group, Bid is promiscuous, binding to Bax and Bak as well as to the anti-apoptotic proteins, but the others bind only to certain of the death inhibitors. However, the mechanism(s) by which proteins of the Bcl-2 family modulate apoptosis remains elusive and several conflicting theories have been proposed. A widely accepted model postulates that homodimers of Bax promote apoptosis, and that the functional effect of Bcl-2 related proteins is to form competing heterodimers with Bax that cannot promote apoptosis (Oltvai et al., 1993; Sedlak et al., 1995). However, in some systems, Bax binding by Bcl-2 was not sufficient to prevent apoptosis and the overexpression of Bcl-2 or Bcl-x L can repress apoptosis in the absence of Bax (Cheng et al., 1996; Knudson and Korsmeyer, 1997). Thus, while an in vivo competition exists between Bax and Bcl-2, each is able to regulate apoptosis independently.

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CED-9, Bcl-2 (Akao et al., 1994; Chen et al., 1989; de Jong et al., 1994; Hockenbery et al., 1990; Janiak et al., 1994; Krajewski et al., 1993; Nakai et al., 1993; Nguyen et al., 1993), Bcl-x L (Gonzalez-Garcia et al., 1994), Mcl-1 (Wang and Studzinski, 1997; Yang et al., 1995), the BHRF1 Epstein-Barr virus protein (Hickish et al., 1994) and probably other members of the Bcl-2 family are localized to the cytoplasmic surfaces of the nuclear envelope, the endoplasmic reticulum and the outer mitochondrial membrane. However, it must be underlined that only a fraction of Bcl-x L resides on membranes, and that Bax seems to be cytosolic before an apoptotic stimulus, even though both, like most other family members, bear a C-terminal membrane anchor. The membrane association of Bcl-2 is of functional significance as mutant Bcl-2 molecules lacking this membrane anchorage capacity are less effective at preventing apoptosis in some systems (Borner et al., 1994; Nguyen et al., 1994; Zhu et al., 1996). Indeed, it has been reported that, in inhibiting apoptosis of MDCK cells, a mutant Bcl-2 molecule whose anchorage is targeted specifically to the mitochondria is as effective as the wild type protein, whereas mutant Bcl-2 targeted to the ER loses this capacity (Zhu et al., 1996). In contrast, Bcl-2 targeted to the ER in the Rat-1/myc fibroblasts proved to be more active than when targeted to mitochondria. Thus, Bcl-2 mutants with restricted subcellular location reveal distinct pathways for apoptosis depending on celltype. When associated to the endoplasmic reticulum membrane, Bcl-2 could be involved in maintenance of the calcium homeostasis (Distelhorst et al., 1996; He et al., 1997; Lam et al., 1994), while it could modulate protein subcellular trafficking through nuclear pores (Ryan et al., 1994). Recent reports provide spectacular advancements in the understanding of the mechanism of action of antiapoptotic Bcl-2s. It was shown that, in both worm and mammalian cells, the antiapoptotic Bcl-2s act upstream of the "execution caspases" somehow preventing their proteolytic processing into active killers (Golstein, 1997; Shaham and Horvitz, 1996). How these proteins perform this feat remains unknown, although two main mechanisms of action have been proposed to connect Bcl-2s to caspases. In the first one, pro-survival proteins would act by regulating the release of some caspases activators usually sequestered in intracellular compartments. Apoptosis (in vivo and in vitro) involved the preliminary shift to the cytosol of regulatory components, namely cytochrome c or AIF (Apoptosis-Inducing-Factor), previously sequestered in mitochondrial intermembrane space (Kluck et al., 1997a; Susin et al., 1996; Yang et al., 1997). Cytosolic cytochrome c forms an essential part of the vertebrate "apoptosome" which is composed of cytochrome c, Apaf-1 and procaspase-9 (Li et al., 1997b). The result is the activation of caspase-9, which then activates other caspases to orchestrate execution of cell death. AIF is another caspase-activating protein which promotes nuclear apoptosis in vitro probably via the activation of procaspase-3, a major executive caspase. Overexpression of Bcl-2/Bcl-xL in cells or addition of recombinant Bcl-2 to cell-free systems containing mitochondria prevented cytochrome c/AIF exodus from the mitochondria that was triggered normally by a wide variety of apoptogenic stimuli. In contrast, the pro-apoptotic member Bax stimulates mitochondrial cytochrome c release. The ability of Bax, Bcl-2/Bcl-x L to form membranes pores with distinct ionconducting properties may provide a clue to how Bcl-2s can regulate the permeability of the intracellular membranes (Reed, 1997).

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An alternative model proposes that the pro-survival proteins may function downstream of the release of apoptogenic factors by directly inhibiting the ability of CED-4 like proteins to activate caspases. This model arose from the elucidation of the role of the somewhat mysterious CED-4 protein (Hengartner, 1997). Indeed, it was first shown that CED-9 interacts with CED-3 via the bridging protein CED-4 that binds simultaneously to CED-9 and CED-3. CED-9 prevents CED-4 from inducing proteolytic processing and activation of CED-3 (Chinnaiyan et al., 1997b; Wu et al., 1997b). These observations, concerning developmental cell death in C. elegans, were extended to mammalian PCD (Chinnaiyan et al., 1997b). An equivalent ternary complex was found to be present in mammalian cells involving Apaf-1, caspase-9 and Bcl-x L, in which Bcl-x L inhibits Apaf-1-mediated maturation of caspase-9. In this paradigm, pro-apoptotic relatives like Bik may free CED-4/Apaf-I from the death inhibitor. Furthermore, there is some evidence that binding of pro-survival proteins to the apoptosome complex alter its location in cells, pulling it from the cytosol to the intracellular membranes where Bcl-2s often reside (Chinnaiyan et al., 1997b; Wu et al., 1997b). Thus, the mitochondrion seems to constitute a pivotal component of all the mechanisms of activation either as docking sites, via Bcl-2 family proteins, of caspases or as sequestering organelles of caspase activators. This crucial position of mitochondria in PCD control may be reinforced by the results obtained from distinct approaches establishing that mitochondria can contribute to PCD via the production of cell death signaling ROS. This chapter examines the data concerning the mitochondrial features of PCD and the way by which Bcl-2 family proteins participate in the modulation of these mitochondrial events (recent reviews on similar subjects:Mignotte and Vayssi~re, 1998; Zamzami et al., 1998).

Permeability Transition Marks A Point of No-Return of Cells Condemned to Die A Decrease in Mitochondrial Membrane Potential Precedes DNA Fragmentation During Apoptosis Several changes in mitochondrial biogenesis and function are associated with the commitment to apoptosis. A fall of the membrane potential (AqJm) occurs before the fragmentation of the DNA in oligonucleosomal fragments (Kroemer et al., 1995; Petit et al., 1995; Vayssi~re et al., 1994; Zamzami et al., 1995a; Zamzami et al., 1995b). This drop of Attt m is responsible for a defect of maturation of mitochondrial proteins synthesized in the cytoplasm (Mignotte et al., 1990), cessation of mitochondrial translation and an uncoupling of oxidative phosphorylation (Vayssi~re et al., 1994). The drop of AqJm is detectable whatever the apoptosis induction signal, physiological (absence of growth factor, glucocorticoids, TNF) or non-physiological (irradiation, chemotherapy). These data showed, on the one hand, that the nuclear fragmentation is a late event as compared to the drop of the Aq/m and, on the other hand, that this drop marks the point of no-return of a cell condemned to die.

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Mitochondrial Permeability Transition Provokes a AY/m Decrease What is the mechanism involved in the Au.-/mdisruption? The permeability transition (trF) is a phenomenon that is characterized by the opening of large conductance channels known as the mitochondrial PT pores and by its sensitivity to very low concentration of cyclosporine A. These pores, permeable to compounds of molecular mass up to 1500 Da are formed under specific conditions. The opening of these PT pores in the inner membrane allows for an equilibration of ions within the matrix and intermembrane space of mitochondria, thus dissipating the H+ gradient across the inner membrane and uncoupling the respiratory chain. These events lead to the decrease of the AI¥m and the arrest of ATP synthesis (Bernardi et al., 1992; Petronilli et al., 1994). Perhaps more importantly, PT pore opening results in a volume dysregulation of mitochondria due to the hyperosmolarity of the matrix, which cause the matrix space to expand. Because the inner membrane is a larger surface area than the outer membrane, this matrix volume expansion can eventually provoke outer membrane rupture, releasing intermembrane space molecules into the cytosol. Furthermore, permeability transition has properties of self-amplification: the drop of the m ~ m that is linked to depletion of non-oxidized glutathione (Macho et al., 1997), and that result from the opening of the PT pores, would increase the permeability transition in a retrograde manner. We have, therefore, proposed that the opening of the PT pore may constitute an irreversible state of apoptosis and could account for the apparent synchronization in the drop of ALI/m that takes place simultaneously in all the mitochondria of a same cell (Kroemer et al., 1995). The molecular composition of these PT pores remains elusive. The peripheral benzodiazepin receptor, that has recently been implicated in the protection against ROS (Carayon et al., 1996), and the translocase of adenine nucleotides (ANT) are probable components of the PT pore. Indeed, protoporphyrine IX and PK11195, that are ligands of the benzodiazepin receptor, induce a drop of Aq/mand consequently apoptosis (Hirsch et al., 1998; Zamzami et al., 1996a). On the contrary, N-methyl Val-4-cyclosporin A (a derivative of cyclosporine that is not immunosuppressor) and bongkrekic acid that bind to the matrix side of the ANT, prevent the drop of the mitochondrial potential. Altogether, these results sustain the hypothesis that the opening of PT pores is involved in the disruption of AI'I/m observed during apoptosis. Direct Interventions on the Mitochondrial Permeability Transition Modulate Apoptosis Direct alterations of mitochondria can induce apoptosis (Hartley et al., 1994; Wolvetang et al., 1994). The links between mitochondrial perturbations and nuclear alterations have been studied by means of acellular systems where purified nuclei and purified mitochondria are confronted (Newmeyer et al., 1994). Such a system allows study of reciprocal and direct effects of one organelle on another and to characterize at the biochemical level the factors involved. These experiments have shown that when mitochondria are treated with substances capable to induce PT pores opening, they provoke nuclear apoptosis (condensation of the chromatin and fragmentation of the DNA) (Zamzami et al., 1996b). A correlation between induction of the trF and nuclear apoptosis has been observed by using a

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variety of known inductors of the PT such as atractyloside, pro-oxidants, calcium, protonophores and substances that provoke linkage of thiol groups such as diamide. These substances, that have no direct effect on nuclei in absence of mitochondria, confer pro-apoptotic properties upon mitochondria. The pro-apoptotic character (induction of nuclear apoptosis) of the mitochondria treated with atractyloside is altered by inhibitors of the PT such as bongkrekic acid, cyclosporine A and substances like monochlorobimane that block the cross-linking of the thiols. Cyclosporine A can be replaced by its non-immunosuppressor analogue, N-methyl Val-4-cyclosporine A, which shows that its inhibitory effect on PT and nuclear apoptosis is independent from its calcineurine activity. These results suggest the implication of the PT pores opening in the regulation of apoptosis induced via the mitochondria. Nuclei and mitochondria have been purified from hybridomas of T cells transfected by bcl-2 to study how Bcl-2 suppresses apoptosis in in vitro experiments (Zamzami et al., 1996b). Upon treatment with atractyloside, in contrast to mitochondria purified from control cells, mitochondria purified from cells transfected by bcl-2 do not provoke nuclear apoptosis. On the contrary, nuclei purified from cells transfected by bcl-2 show a condensation of the chromatin and a fragmentation of the DNA when they are confronted to control mitochondria treated with atractyloside. Furthermore, bcl-2 inhibits the induction of permeability transition by agents such as atractyloside, oxidants and protonophores. These results show that, even if Bcl-2 intervenes also during latter apoptotic events (Gurnal et al., 1997; Marton et al., 1997) and o n A q J m loss induced by other mechanisms (Shimizu et al., 1998), at least a part of its activity is exerted by acting on the mitochondrial permeability transition (Decaudin et al., 1997). Moreover, the structure of a protein of the Bcl-2 family (Bcl-XL) has been established (Muchmore et al., 1996). It recalls that of bacterial toxins, especially the diphtheria toxin, that form a pH-sensitive transmembrane channel. Furthermore, the pro-apoptotic Bax protein can form channels (Antonsson et al., 1997), as reported also for the anti-apoptotic proteins Bcl-x e (Minn et al., 1997) and Bcl-2 (Schendel et al., 1997). However, the intrinsic properties of Bax and those of Bcl-x e and Bcl-2 reveal differences. The channel forming activity of Bcl-x L and Bcl-2 is observed at highly acidic pH while Bax forms channels at a wide range of pH including at pH=7, those found in cells. Furthermore, Bcl-2 can block the pore-forming activity of Bax. Indeed, recent results suggest that Bax might produce cell death by inducing PT (Pastorino et al., 1998). Bcl-2 might counteract this effect of Bax on mitochondrial membranes (Gross et al., 1998).

Mitochondria of Cells Undergoing Apoptosis Release Pro-Apoptotic Factors Mitochondria Undergoing Permeability Transition Liberate AIF Factor The experiments described above showed that mitochondria from apoptotic cells can effectively control nuclear apoptosis and suggest the involvement of mitochondria derived products in the apoptotic cascade. Acellular experiments have shown that mitochondria contain a pre-formed approximately 50-kD protein, which is released upon PT pores opening and causes isolated nuclei to undergo apoptotic changes,

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such as chromatin condensation and intemucleosomal DNA fragmentation (Figure 1). This apoptosis-inducing factor (AIF) derived from the mitochondria is a protease (or a protease-activating protein), which is blocked by the general caspase inhibitor Z - V A D and is capable of activating purified procaspase-3 (Susin et al., 1996). As expected, when preventing mitochondrial permeability transition, Bcl-2 overexpressed in the outer mitochondrial membrane also impedes the release of A I F from isolated mitochondria. In contrast, Bcl-2 does not affect the formation of AIF, which is contained in comparable quantities in control mitochondria and in mitochondria from Bcl-2-hyperexpressing cells. Furthermore, the presence of Bcl-2 in the nuclear membrane does not interfere with the action of AIF on the nucleus, nor does Bcl-2 overexpression protect cells against AIF. It thus appears that Bcl-2 prevents apoptosis by favoring the retention of an apoptogenic protein in mitochondria (Susin et al., 1996).

.... i

J

Execution caspases activation

Apoptosis

Simplified model of events occurring during apoptosis. Numerous signals can lead to apoptosis. The induction pathways seem to converge to events involving mitochondria. Proteins of the Bcl-2 family act on mitochondrial membrane permeability and regulate the release of pro-apoptotic factors from the intermembrane space to the cytosol.. These factors, directly or indirectly activate execution caspases responsible for the apoptotic phenotype.

Figure 1.

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Cytochrome C is Released by Mitochondria During Apoptosis Another mitochondrial killer component has been demonstrated through the role of cytochrome c in PCD. Indeed, translocation of cytochrome c from mitochondria to cytosol has been shown to be a crucial step in the activation of the PCD machinery in various death models, including Fas-, UV-, staurosporine or etoposide-treated mammalian cells, in a cell-free system using Xenopus egg extracts or dATP-primed cytosols of growing cells (Kluck et al., 1997a; Krippner et al., 1996; Liu et al., 1996; Yang et al., 1997). Furthermore, direct microinjection of cytochrome c into the cytosol can induce apoptosis in various cell-types (Li et al., 1997a; Zhivotovsky et al., 1998). Cytochrome c is an essential component of the mitochondrial respiratory chain: it accepts an electron from cytochrome c reductase and passes it on to cytochrome c oxidase. It is a soluble protein that is located in the intermembrane space and is loosely attached to the surface of the inner mitochondrial membrane. Cytochrome c is translated on cytoplasmic ribosomes as apocytochrome c and follows a unique pathway into mitochondria that does not require the signal sequence, electrochemical potential, and general protein translocation machinery (Mayer et al., 1995). The apoprotein, on entry into the intermembrane space, gains an heine group, to become the fully folded holocytochrome c. This globular, positively charged protein can no longer pass through the outer mitochondrial membrane and is thought to become electrostatically attached to the inner membrane. Upon its release into the cytoplasm during the initiation of apoptosis, cytochrome c promotes the assembly of a multiprotein complex, the apoptosome, that induces proteolytic processing and activation of executive caspases (Li et al., 1997b). Biochemical studies have shown that, besides cytochrome c, caspase-9, Apaf-1 and (d)ATP constituted the activating components of this complex. Apaf-1 and caspase-9 interact via their CARD domains, an association that requires dATP and cytochrome c. Binding of dATP and cytochrome c to Apaf-1 likely alters it's conformation and renders its CARD domain more available to caspase-9. Once bound to caspase-9, Apaf-1 triggers caspase-9 proteolytic self-activation; caspase-9 subsequently proteolyses and activates caspase-3. Many data suggested that the pro-survival Bcl-2 proteins prevent cell death acting upstream of the release of cytochrome c. Overexpresssion of Bcl-2 or Bcl-x L in cells blocks the release of cytochrome c and aborts the apoptotic response triggered by a wide variety of killing signals (Bossy-Wetzel et al., 1998; Kharbanda et al., 1997; Van der Heiden et al., 1997; Yang et al., 1997). On the contrary, caspases inhibitors have no effect on this process. Similarly, addition of recombinant Bcl-2 to Xenopus egg extracts containing mitochondria slows down the release of cytochrome c from these mitochondria and inhibits nuclear apoptosis observed in this cell-free system (Kluck et al., 1997a). Moreover, co-immunoprecipitation studies have shown that Bcl-x L can bind cytochrome c and may thereby act to sequester it in the mitochondria (Kharbanda et al., 1997). Bcl-x s, a pro-apoptotic derivative of Bcl-x L, was found to prevent the formation of the Bcl-XL-Cytochrome c complex. In contrast, overexpression of the pro-apoptotic Bcl-2 family member Bax in cells stimulates both the release of cytochrome c and apoptosis. By using isolated mitochondria and recombinant Bax,

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it was shown that Bax can directly induce cytochrome c release from mitochondria. Similarly, recent reports show that Bid, a BH3 domain-containing pro-apoptotic Bcl-2 family member, induces cytochrome c release and apoptosis after its caspase8-dependent cleavage and subsequent translocation onto mitochondria (Li et al., 1998; Luo et al., 1998). Even though, altogether, these results provide evidence that Bcl-2 family members can modulate the cell death process by directly controlling the availability of cytochrome c in the cytosol (Figure 1), it must underlined that pro-survival actors, as Bcl-2/ Bcl-x L, may also act downstream of cytochrome c to prevent caspase activation under certain circumstances (see below) (Rosse et al., 1998; Zhivotovsky et al., 1998). Although efflux of cytochrome c from mitochondria appears to be a crucial step in the killing cascade initiated by a wide range of apoptogenic stimuli, it must be underlined that this event is not an universal requirement for death signal transduction. Indeed, on the one hand, apoptosis can occur in the absence of detectable cytochrome c release (Chauhan et al., 1997; Tang et al., 1998) and, on the other hand, under certain circumstances, cytochrome c release is not sufficient to promote cell death (Li et al., 1997a). Moreover, it was reported that efflux of cytochrome c could constitute only a later event of the cell death, in other words, a side effect of the terminal dismantelment of the cell (Adachi et al., 1998; Krippner et al., 1996). However, in this Fas-mediated apoptosis model, an inactivation of cytochrome c, correlated to the inhibition of mitochondrial respiration at the cytochrome c level, constitutes an very early causal event in the apoptotic program. It was suggested that inactivation of cytochrome c is associated to its release from the outer surface of the inner mitochondrial membrane where normally it functions as a shuttle connecting respiratory chain energy transducers (Skulachev, 1998). More intriguing, the authors suggest that efflux of cytochrome c which had been associated with early events of apoptosis could be, in some instances, the result of a methodological artifact (Adachi et al., 1998). Indeed, most studies are based on subcellular fractionations, i.e., after cell disruption, centrifugation allows to separate soluble cytosolic elements in the supernatant from heavy membranes, including mitochondria, included in the pellet. In this approach, the in vivo cytochrome c redistribution is inferred from its appearance into the cytosolic fraction during the in vitro procedure. In fact, this method could be more properly considered as a measure of cytochrome c extractability from mitochondria rather than as a exact view of its in situ localization. For instance, some minor alterations of the outer mitochondrial membrane could be compatible with a mitochondrial localization of cytochrome c in the cell and its outflow from mitochondria as fractionation provoked breakdown of the fragilized outer membrane. However, these considerations refute neither the early occurrence of cytochrome c release in various apoptotic models as observed by in situ immunolocalization nor its crucial role in the apoptotic program, ascertained by direct microinjection of cytochrome c in the cytosol. They must be correlated to the evidenced existence of alternative pathways, i.e. cytochrome c independent, in the transduction of apoptogenic stimuli (Chauhan et al., 1997; Li et al., 1997a; Tang et al., 1998).

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The Cytochrome C Pathway is Distinct from that of AIF The above described AIF could be a key regulator of such a cytochrome c independent pathway inasmuch as these two mitochondrial components seem involved in distinct signaling route. Indeed, while AIF release occurs after the PT-associated mitochondrial depolarization, several reports indicates that cytochrome c release is dissociable from PT (Bossy-Wetzel et al., 1998; JiJrgensmeier et al., 1998; Kluck et al., 1997a; Li et al., 1998; Van der Heiden et al., 1997; Yang et al., 1997). Furthermore, cytosolic cytochrome c requires additional cytosolic factors to promote apoptotic changes, via the activation of caspase-3, whereas AIF, once released from mitochondria, directly induces nuclear apoptosis without cytosol. Beyond their differences, these two "execution caspase" activating pathways illustrate the sophistication and the apparent molecular redundancy which characterize the mammalian cell death. They may correspond to alternative and independent links between death-triggering stimuli and the execution machinery or, in contrast, they may work together to induce complete PCD (Golstein, 1997). Bcl-2 Family Proteins Dock Various Proteins to the Mitochondria Even though, increasing reports provide evidence that Bcl-2 family members can modulate the cell death process by directly controlling the availability of cytochrome c in the cytosol, it must be underlined that pro-survival actors, such as Bcl-2/Bcl-xL, may also act downstream of cytochrome c to prevent caspase activation under certain circumstances (Rosse et al., 1998; Zhivotovsky et al., 1998). Moreover, effects of Bcl-2s cannot be reduced merely to models that envision Bcl-2s as regulators of killing caspase activation, inasmuch as Bcl-2 can also block caspase-independent cell deaths such as oxidant and hypoxia-induced necrosis, or Bax-induced yeast killing (Jtirgensmeier et al., 1997; Manon et al., 1997). The ability of Bcl-2 to bind (at least in two hybrid or co-immunoprecipitation experiments) and to dock several cellular proteins that do not belong to the Bcl-2 related proteins family, can provide a clue to understand how the Bcl-2 family governs PCD. First, Bcl-2 has been found to interact with Nip l, Nip2, and Nip3, the function of which is unknown (Boyd et al., 1994), the GTPase R-ras p23 (Fernandez and Bischoff, 1993), Raf-1 (Ali et al., 1997; Wang et al., 1994), BAG-1 (Takayama et al., 1995), the cellular prion protein (PrP) (Kurschner and Morgan, 1995), the p53 binding-protein p53-BP2 (Naumovski and Cleary, 1996), the protein phosphatase calcineurin (Shibasaki et al., 1997) and the mitochondrial membrane protein carnitine palmitoyltransferase I (Paumen et al., 1997). At least some of these interactions could reflect the ability of Bcl-2 to relocalize cellular proteins to mitochondrial membranes. Bcl-2 binds to BAG-1 (Takayama et al., 1995) that can also interact with Raf-1 (Wang et al., 1996b). Active Raf-1 fused with targeting sequences from an outer mitochondrial membrane protein protect cells from apoptosis and phosphorylate BAD, a proapoptotic Bcl-2 homologue (Wang et al., 1996a). Furthermore, plasma membranetargeted Raf-1 did not protect from apoptosis and resulted in phosphorylation of ERK-I and ERK-2 while Raf-1 improved Bcl-2-mediated resistance to apoptosis.

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Bcl-2 can therefore target Raf-1 to mitochondrial membranes, allowing this kinase to phosphorylate BAD. However, the link between Raf-1 and the mitochondrial changes occurring during apoptosis are not yet known and bcl-2 does not always require c-raf-1 kinase activity and an associated mitogen-activated protein kinase signaling pathway for its survival function (Olivier et al., 1997). Second, in accord with C. elegans genetics, biochemical evidence shows that the pro-survival Bcl-2 family proteins may function by directly inhibiting the activation of killing caspases. It was initially shown that binding of CED-9 to CED-4 prevents CED-4 from inducing proteolytic processing and activation of CED-3 (Chinnaiyan et al., 1997b; Wu et al., 1997b). The CED-9 protein, like the mammalian Bcl-2 related proteins, is localized to intracellular membranes and the perinuclear region, whereas CED-4 was distributed in the cytosol. Expression of CED-9, but not a mutant lacking the carboxy-terminal hydrophobic domain, targeted CED-4 from the cytosol to intracellular membranes in mammalian cells (Wu et al., 1997b). A similar mechanism exists in mammalian cells as first suggested by the ability of Bcl-x L to interact with and inhibit the function of CED-4 (Chinnaiyan et al., 1997b). A ternary complex involving Apaf-1, caspase-9 and Bcl-x L, in which Bcl-x L inhibits Apaf-l-mediated maturation of caspase-9, was found to be present in mammalian cells (Hu et al., 1998; Pan et al., 1998). Figure 2 illustrates that pro-apoptotic relatives like Bik, Bak and Bax can promote cell death by disrupting interaction between Bcl-x L and Apaf-1 (Chinnaiyan et al., 1997b; Pan et al., 1998). Such data explain how pro-survival actors, as Bcl-2/Bcl-x L, may also act downstream of cytochrome c to prevent caspase activation under certain circumstances. For instance, overexpression of Bcl-2 and Bcl-x L inhibits apoptosis induced by direct injection of cytochrome c in cytosol (Li et al., 1997a; Zhivotovsky et al., 1998). Similarly, Bcl-2 delays Bax-induced caspase activation and cell death even when cytochrome c is already in the cytosol (Rosse et al., 1998). However, it must underlined that today there is no evidence concerning a physical interaction of Bcl-2 with apoptosome complex. Lastly, there is some evidence that binding of pro-survival proteins to the Apaf-1/caspase-9 complex alters its location in ceils, dragging it from the cytosol to the intracellular membranes where Bcl-2s often reside (Chinnaiyan et al., 1997b; Wu et al., 1997b). In murine thymocytes, Bcl-2 is exclusively membrane-bound, whereas Bcl-x L is present in both soluble and membranebound forms and Bax is present predominantly in the cytosol. Induction of apoptosis by dexamethasone or gamma-irradiation shifts the subcellular locations of Bax and Bcl-x L from soluble to membrane-bound forms. Inhibition of apoptosis with cycloheximide inhibits the movement of Bax and Bcl-x L from the cytosol into intracellular membranes (Hsu et al., 1997). Since Bax and Bik can disrupt the association between CED-9 (or Bcl-xL) and CED-4 (Chinnaiyan et al., 1997b), it is tempting to speculate that Bcl-x L and possibly other members of the Bcl-2 protein family inhibit apoptosis by maintaining the procaspases/Apaf-1 complexes associated to mitochondrial membranes, and that Bax and Bik by dissociating the complexes permit the activation of procaspases.

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"

~caspase-9J1

Survival

Caspase-3 activation

Cell death

Figure 2.

Bcl-2 family proteins regulate the intracellular localization and the activity of caspases

containing complexes. In C. elegans, CED-9 might maintain CED-4/CED-3 complexes at the level of intracellular membranes by its simultaneous binding to mitochondrial membrane and CED-4. Similarly, in mammals, Bcl-xL might anchor Apaf-1/caspase complexes and keep them in an inactive state. The dissociation of the Bcl-XL/Apaf-1interaction by Bax or Bak (or other proapoptotic Bcl-2 family protein) leads to the activation of caspases.

What is the Mechanism Involved in Cytoplasmic Release of Mitochondrial Apoptogenic Factors? The mechanism by which AIF and cytochrome c are released from mitochondria is largely unknown. Although apoptosis can occur in the absence of detectable cytochrome c release (Chauhan et al., 1997; Tang et al., 1998), efflux of cytochrome c from mitochondria appears to be a critical coordinating step in the killing program towards which converge the multiple signaling pathways and beyond which are initiated the entire panel of apoptotic features, pro-caspase 3 expressing cells being then irreversibly committed to die (Li et al., 1997a). With this perspective, the possible mechanisms involved in activation of this central control may be envisaged from data concerning mediators of the death signal transduction cascades. For instance, the release of cytochrome c might result from oxidative imbalance, an upstream event in the apoptotic transduction cascade (see below). This phenomena might lead to the alteration of some redox sensitive crucial regulatory elements of the outer mitochondrial membrane permeability, e.g. by a shift of the redox state of some sulfhydryl groups to a more inactivating oxidized state.

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Another possibility comes from the ability of caspases to promote the release of intermembrane proteins, including cytochrome c and AIF, through the outer mitochondrial membrane. These observations suggested that mitochondria carry a caspase substrate that, when cleaved, promotes cytochrome c release (Mignotte and Vayssi~re, 1998). Recent reports concerning Fas and TNF-ct receptor signaling could provide insight into how caspase induces cytochrome c efflux. Previously, it had been established that activation of caspases, predominantly caspase-8, constitutes an early step in these apoptotic pathways (Nagata, 1997), and that inhibition of these proteases prevents both the release of cytochrome c from mitochondria and the execution of the cell death program (Schulze-Osthoff et al., 1998; Van der Heiden et al., 1997). Recently, it has been shown that caspase-8, activated by cell surface death receptors such as TNF and Fas, cleaves Bid, a death agonist of the Bcl-2 family, which in turn transduces the apoptotic signal from the cell surface to mitochondria (Li et al., 1998; Luo et al., 1998). Thus, while full-length Bid is localized in cytosol, the C-terminal part caspase-8-truncated BID translocates to mitochondria and then induces cytochrome c release and the downstream caspase-dependent apoptotic program. These results point to a model where mitochondria might act as apoptotic amplifiers, fostering a positive feedback loop between cytochrome c-efflux and caspase activation. Any event that primes the loop will initiate the vicious "circle of death", leading to large-scale caspase activation and apoptotic cell death. However, this model does not prevail in all systems as, in numerous PCD, caspase inhibitors have no effect on the loss of cytochrome c (Bossy-Wetzel et al., 1998; Kluck et al., 1997a,b). Alternatively, opening of PT pores, which is a common event of apoptosis, might be involved in AIF and cytochrome c outflow (Skulachev, 1998). However, PT pores, which are thought to connect the mitochondrial matrix to the cytosol, within the contact sites between inner and outer mitochondrial membranes (Beutner et al., 1996) are only permeable to small compounds (molecular mass <1500 Da) and, therefore, are probably not the structure directly responsible for the efflux of these intermembrane space apoptogenic proteins. Nevertheless, opening of the PT pore could be a first step of a cascade of events causing an increase in matrix volume and subsequently a mechanic disruption of the outer membrane. Recent results show that this scenario is possible at least in vitro (Petit et al., 1998; Scarlett and Murphy, 1997; Van der Heiden et al., 1997). Last, the pore-forming properties of Bax and other related proteins might suggest that they directly modulate the permeability of the outer mitochondrial membrane. Indeed, other studies have shown that Bax can induce cytochrome c release from mitochondria and caspase processing and activation (JUrgensmeier et al., 1998). These events are sensitive to Bcl-x L but caspase inhibitors have no effect on release of cytochrome c from mitochondria (Gross et al., 1998; Xiang et al., 1996) although they prevent the subsequent activation of caspases in cytosolic extracts. Unlike Ca ++, Bax did not induce PT and swelling of mitochondria in vitro in this system. These findings imply that Bax could use an alternative mechanism for triggering release of cytochrome c from mitochondria,, maybe through to the action of specific pores in the outer membrane.

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Mitochondriai Ros and Apoptosis Signaling ROS as Mediators of PCD The first evidence suggesting the involvement of mitochondria in cell death arose from the study of the TNF-ct-induced cytotoxicity (Lancaster et al., 1989; SchulzeOsthoff et al., 1992). Indeed, an alteration of the mitochondrial function was associated with the early phases of the cell death and was defined as a crucial step of the process. The observed inhibition of the mitochondrial respiratory chain was assumed to result in the overproduction of Reactive Oxygen Species (ROS) which would act as mediators of the death signaling pathway (Schulze-Osthoff et al., 1993). ROS, such as superoxide anion, hydrogen, organic peroxides and radicals, are generated by all aerobic cells as byproducts of a number of metabolite reactions and in response to various stimuli (Fridovich, 1978). Mitochondria are believed to be a major site of ROS production: superoxide radical is produced by a single electron transfer to molecular oxygen at the level of the respiratory chain, mainly at the ubiquinone site in complex III. However, endoplasmic reticulum and nuclear membranes also contain e-transport chains that can lose e- and generate superoxide radical. Some fatty acid metabolites, such as those derived from arachidonic acid by the lipoxygenase pathway, are also ROS. However, ROS play a role in physiological systems: they were shown to be responsible for the inducible expression of genes associated with inflammatory and immune responses. Current evidence indicates that different stimuli use ROS as signaling messengers to activate transcription factors, such as AP-1 and NF-KB, and induce gene expression (Pinkus et al., 1996). The ability of oxidative stress, which is an excessive production of ROS, to provoke necrotic cell death as a result of massive cellular damage associated with lipid peroxidation and alterations of proteins and nucleic acids, is well documented for a long time (Halliwell and Gutteridge, 1989). The highly reactive hydroxyl radical (" OH), by-product of superoxide anion or hydrogen peroxide, is assumed to be directly responsible for most of the oxidative damages leading to the non-physiological necrosis (Halliwell and Gutteridge, 1990). To prevent oxidative damage, mammalian cells have developed a complex antioxidant defense system that includes nonenzymatic antioxidants (e.g. glutathione, thioredoxine) as well as enzymatic activities (e.g. catalase, superoxide dismutase (SOD)) (Sies, 1991). In this point of view, aerobic cells appear as being under a continual "oxidative siege", their survival depending on a balance between ROS and antioxidants. On the other hand, the possible implication of ROS as signaling molecules in more physiological deaths such as PCD is an emerging concept. Thus, since the initial observation outlining the contribution of ROS to the TNF-ct-induced cytotoxicity, there is mounting evidence that these compounds may be central in the cell death transduction pathways. Indeed, several observations suggest that ROS might mediate PCD: (1) the addition of ROS or the depletion of endogenous antioxidants can promote cell death (Gu6nal et al.; 1997; Kane et al., 1993; Lennon et al., 1991; Ratan et al., 1994; Sato et al., 1995), (2) PCD can sometimes be delayed or inhibited by antioxidants (Greenlund et al., 1995; Mayer and Noble, 1994; Mehlen et al., 1996; Sandstrom and Buttke, 1993; Wong et al., 1989) and (3) increases

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in intracellular ROS are sometimes associated with PCD (Martin and Cotter, 1991; Quillet-Mary et al., 1997; Uckun et al., 1992). Moreover, it was shown that Bcl-2 may act in an antioxidant pathway to block a putative ROS-mediated step in the cascade of events required for PCD (see below). So, in addition to their role in TNF-ct-induced killing, the contribution of ROS to the activation of the execution machinery was extended to PCD triggered by a wide range of influences including UV light, ionizing irradiation, anthracyclines, ceramides, glucocorticoids or survival-factor withdrawal (Jacobson, 1996). Moreover, some data raise the possibility that ROS are also required for the execution of the death program (Kroemer et al., 1995). However, they must be cautiously considered inasmuch as, in the majority of these systems, it is difficult to ascertain that the observed ROS accumulation corresponds to a causal effect and is not a side effect of the other changes accompanying the killing process (Cai and Jones, 1998). Moreover, in these cases, ROS increase most often arises during the later stage of the death program, i.e., during the destruction phase when the cell is broken down, and may be associated with a necrotic type terminal degradation of the cell. Exogenous sources of ROS such as hydrogen peroxide can induce PCD or necrosis depending upon the dose added (Gurnal et al., 1997). So a burst in ROS, in response to a dramatic perturbation of the physiology of the dying cell, could convert the late PCD steps into necrotic death. Therefore, it appears that at any moment the level of intracellular ROS can determine the fate of the cell: low levels of ROS can induce PCD while accumulation of high levels promotes necrosis or can lead PCD-committed cells toward necrotic-like destruction. PCD-Mediating ROS are Produced by Mitochondria The nature of the ROS involved in PCD is a conflicting question that will allow us to return to the central subject of this review, i.e., the role of mitochondria in PCD. Indeed, two opposite models have emerged concerning the source of signaling ROS, in relation with the variety of metabolic reactions and intracellular _sites which can generate ROS (see above) (Jacobson, 1996). While most investigators believe that oxidants are produced by electron chain transport, some data seem to moderate this point of view. Fatty acid metabolites, such as those produced from arachidonic acid by the lipoxygenase pathway, may be better mediators of PCD (O'Donnell et al., 1995). On the one hand, it is argued that these molecules harbor a more specific reactivity than superoxide anion and its by-products, this biological specificity being assumed necessary for a signaling role in PCD transduction pathways. On the other hand, it was shown that exogenous fatty acid metabolites can promote PCD and that, in some cases, their increased production was associated with cell death. It must be underlined that such a situation is limited to systems where the death signal result is mediated by surface receptors. Nevertheless, these considerations do not refute the compelling evidence of the involvement of electron transport chain-produced ROS in cell death signaling. It appears mote reasonable to consider that, depending upon the cell death stimulus and the cell model, these two types of ROS can mediate PCD or even both contribute to the activation of the execution machinery as suggested by studies of TNF-ct-induced PCD.

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Where are localized the electron transport chains that produce cell death signaling ROS such as hydrogen peroxide? The question must address distinct intracellular compartments as reticulum endoplasmic, nuclear layer and especially mitochondria. The more convincing responses have arisen from indirect studies measuring the consequences of an alteration of the electron transport chain on the PCD process. In this way, it was established that both ROS accumulation and PCD process require the presence of a functional mitochondrial respiratory chain in most ROS-dependent cell death systems (Higuchi et al., 1997; Quillet-Mary et al., 1997; Schulze-Osthoff et al., 1993; Sidoti-de Fraisse et al., 1998). Indeed, it was shown that an upstream inhibition, with chemical compounds acting on complex I (Quillet-Mary et al., 1997; Schulze-Osthoff et al., 1992), or an elimination of the electron transfer chain (Higuchi et al., 1997; Schulze-Osthoff et al., 1993; Sidoti-de Fraisse et al., 1998), by depletion of the mtDNA, prevent ROS accumulation and consequently protect cells against PCD. Another indirect argument is provided by the scavenger role of mitochondrial glutathione in the regulation of ROS-mediated PCD (Goossens et al., 1995). The ubiquinone site in complex III appears as the major site of mitochondrial ROS production as this site catalyzes the conversion of molecular oxygen to superoxide anion which can lead to the formation of other potent ROS such as hydrogen peroxide and hydrogen radicals. Such a model is supported by the observed potentiation of cell death processes in ROS-dependent PCD when electron flow was inhibited distal to the ubiquinone pool. Mechanisms of ROS Signaling The involvement of mitochondrial ROS in some cell death transduction pathways next leads to the fundamental questions concerning, on the one hand, the causal event of the increased ROS generation and, on the other hand, the molecular mechanisms underlying the ROS signaling. Two viewpoints must first be considered to address the question of the origin of ROS accumulation, which can indeed result from an increased production or from a reduced scavenging by the cellular detoxifying systems. Much of the available data converge to the hypothesis that ROS increases are the consequence of an impairment of the mitochondrial respiratory chain (Cai and Jones, 1998; Degli Esposti and McLennan, 1998; France-Lanord et al., 1997; Gudz et al., 1997; Quillet-Mary et al., 1997; Schulze-Osthoff et al., 1992). In agreement with the above considerations, the observed alterations are distal to the ubiquinone site of the complex III, but the origin of these electron flow disturbances are not clear. The only strong evidence comes from the study of ceramide-induced PCD, in which an increased HzO 2 production was linked to mitochondrial Ca 2+ homeostasis perturbation as inhibition of the mitochondrial Ca 2+ uptake was shown to abolish both ROS accumulation and cell death. However, the recently observed shift of cytochrome c from mitochondria to cytosol in the early phases of many PCD (see above) could provide a clue to resolve this question (Kluck et al., 1997a; Liu et al., 1996; Yang et al., 1997). Indeed, the release of cytochrome c must lead to a breakdown of the mitochondrial electron flow downstream of the ubiquinone site which, in turn, would result in an increased generation of ROS. Such a model is supported by the described correlation

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between loss of cytochrome c activity and respiratory failure in a Fas-induced PCD model (Krippner et al., 1996) and by the study of mitochondria isolated from apoptotic cells which shows a superoxide production due to a switch from the normal 4-electron reduction of 0 2 to a i-electron reduction when cytochrome c is released from mitochondria. (Cai and Jones, 1998). Beside the question of the process of mitochondrial ROS accumulation, arises the problem concerning the targets of these compounds, or more precisely, how can they mediate PCD? Two models can be proposed to approach this conflicting and not well documented subject. The first model assumes that ROS themselves are signaling molecules which activate some crucial components of the PCD machinery. Conversely, the alternative proposition suggests that ROS can act indirectly by modifying the cellular redox potential, which would regulate some key regulatory proteins involved in PCD. Several lines of evidence agree with an explanation based on indirectly mediated action. First, unlike fatty acid metabolites which harbor specific reactivity and are known to mediate particular signals from surface receptors, mitochondrial ROS are characterized by a lack of biological specificity or even an extreme reactivity, as for the hydroxyl radical: these are all features contrary to the requirements of a specific signaling role (Jacobson, 1996). In this way, a direct influence of ROS on PCD process would be correlated to a general damaging effect on cellular structures resulting in necrotic cell death, or perhaps to a more limited action on mitochondria, their site of production, which in turn could activate some mitochondria-dependent downstream cascades leading to PCD. Secondly, despite the compelling evidence of the role of mitochondrial ROS in PCD signaling pathways, it has been assumed that they do not represent a general mediator of cell death, as suggested by the ability of some PCD to occur in very low oxygen environments (Jacobson and Raff, 1995; Shimizu et al., 1995). However, ROS might be produced in such conditions (Degli Esposti and McLennan, 1998). Another alternative explanation would be to consider that the major effect of an increased ROS production is the subsequent imbalance of intracellular redox status, i.e., an enhancement of the cellular oxidative tonus, and that in fact the oxidative stress is the central common effector of PCD. Hence, ROS accumulation would only be one way which leads in some PCD to an oxidative status. Anti-Fas/APO-1 antibody or IL-3 withdrawal-induced PCD represent good illustrations of this model (Bojes et al., 1997; van den Dobbelsteen et al., 1996). Indeed, no ROS accumulation can be measured in these two systems and anaerobic cultured cells deprived of IL-3 still undergo PCD (Schulze-Osthoff et al., 1994; Shimizu et al., 1995). However, an oxidative stress can be shown in these models as a depletion of glutathione (GSH), a non-enzymatic cellular antioxidant, as a result of a rapid and specific efflux of glutathione, an event that takes place at the very beginning of the apoptotic process (Bojes et al., 1997; van den Dobbelsteen et al., 1996). Moreover, it has been shown that Bcl-2 can protect cells from PCD by shifting the cellular redox potential to a more reduced state (see below). However, the observation that oxidation of thiols other than glutathione can mediate induction of PCD suggests that the intracellular thiol redox status would be the real key factor of the cell death signaling pathways (Kane et al., 1993; Marchetti et al., 1997; Mirkovic et al., 1997; Sato et al., 1995). In this model, the redox state of glutathione or other cellular antioxidants such as thioredoxine, would be in equilibrium with that of

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thiols resident in some redox sensitive crucial components of the execution machinery (Kroemer et al., 1997). Where does ROS fit this thiol hypothesis? In this putative model, an increased production of mitochondrial ROS would result, either by a direct modification of the thiols or indirectly via a depletion of the intracellular antioxidant pool, in a shift of the redox state of the sensor SH groups to a more oxidized state. The nature of the ROS and the level of the intracellular antioxidant defenses would determine in which way regulatory components are activated to commit cells to PCD. In an hypothetical model (Figure 3), mitochondrial ROS modify the membrane permeability leading to the release of pro-apoptotic factors and/or activate directly executing caspases.

TNF-~ Activating Caspase Ceramides ~

~

~ .k

M TOCHOND

~ /

Reactive Oxygen Species

, _ ~

7

Reactive Oxygen Species

Pro-apoptotic factors Mitochondrial ROS contribute to apoptosis signaling. In some cases, as during TNF-ctor ceramide-induced apoptosis, a ROS production is involved in apoptosis signaling. ROS accumulate as a result of a dysfunction of the mitochondrial respiratory chain and contribute to the activation of execution caspases.

Figure 3.

Several lines of evidence support the idea that Bcl-2 acts in an antioxidant pathway to suppress apoptosis. Yeast mutants lacking superoxide dismutase were partially rescued by expression of Bcl-2 (Kane et al., 1993; Longo et al., 1997). Following an apoptotic signal, overexpression of Bcl-2 suppressed lipid peroxidation completely (Hockenbery et al., 1993). Bcl-2 deficient mice turn gray with the second hair follicle cycle, implicating possibly a defect in redox-regulated melanin synthesis (Veis et al., 1993). Bcl-2 can protect neural cells from delayed death resulting from chemical hypoxia and reenergization, and may do so by an antioxidant mechanism (Myers et al., 1995) (reviewed in (Korsmeyer et al., 1995). The way by which Bcl-2 protects from ROS remains unclear. In some systems, Bcl-2 appears to influence the generation of oxygen free radicals (Kane et al., 1993),

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while in other cases it does not affect ROS production but does prevent oxidative damage to cellular constituents (Hockenbery et al., 1993; Tyurina et al., 1997). Alternatively, since superoxide is produced by mitochondria from apoptotic cells due to a switch from the normal 4-electron reduction of 02 to a 1-electron reduction, the block of cytochrome c release could provide a mechanism for the apparent antioxidant function of Bcl-2 (Cai and Jones, 1998). Several authors have studied the effect of Bcl-2 family proteins on cellular redox potential. Activities of antioxidant enzymes and levels of glutathione and pyridine nucleotides have been measured in pheochromocytoma PC12 and the hypothalamic GnRH cell line GT1-7 cells transfected with bcl-2 (Ellerby et al., 1996). Both cell lines overexpressing bcl-2 had elevated total glutathione levels when compared with control transfectants. The ratios of oxidized glutathione to total glutathione in PC12 and GT1-7 cells overexpressing bcl-2 were significantly reduced. In addition, the NAD+/NADH ratio of bcl-2-expressing PC12 and GT1-7 cells was two- to threefold less than that of control cell lines while they had approximately the same level of catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase activities as control ceils. These results indicate that the overexpression of bcl-2 shifts the cellular redox potential to a more reduced state, without consistently affecting the major cellular antioxidant enzymes. Furthermore, depleting cellular thiols reversed the resistance to radiation in Bcl-2 expressing lymphoma cell lines (Mirkovic et al., 1997). The ability of bax and bcl-x L to affect GSH was assessed in interleukin (IL)-3dependent murine prolymphocytic FL5.12 cells (Bojes et al., 1997). Overall levels of GSH increased in bcl-x c transfectants while, in cells overexpressing bax, GSH was reduced by approximately 36%. There were no consistent differences between these cell lines in the activities of superoxide dismutase, catalase, glutathione peroxidase or glutathione reductase. Following IL-3 withdrawal-induced apoptosis, control cells and bax transfectants exhibit a rapid loss of intracellular GSH that seemed to occur due to a translocation out of the cell. Cells overexpressing bcl-x L did not lose significant amounts of GSH upon withdrawal of IL-3, and no apoptosis was evident. These results suggest a possible role for GSH in the mechanism by which Bcl-x L prevents cell death. Thus, both Bcl-2 and Bcl-x L can protect cells from apoptosis by shifting the cellular redox potential to a more reduced state. Assuming that mitochondrial thiols constitute a critical sensor of the cellular redox potential during apoptosis (Marchetti et al., 1997), these effects could be at the mitochondrial level.

Conclusions In conclusion, mitochondria are involved in the decision of cells to survive or not at several levels. At a first level, mitochondria can contribute to apoptosis signaling, as shown in TNF-ct- or ceramide-induced cell death during which increased mitochondrial ROS production appears as an early event of the induction phase. At a second level, mitochondria are involved in the control of the activation of the cell death

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machinery by docking at their surface, via Bcl-2 family proteins, execution caspases or by sequestering, in the intermembrane space, caspase activators such as AIF or cytochrome c. This recent accumulation of data has also generated a number of new questions. Future works must address the connection between survival signals and mitochondrial functions. The study of Bad regulation, a proapoptotic Bcl-2 member which is supposed to counteract Bcl-2 has provided an example of how survival factors counteract PCD. Indeed, Bad is inactivated by phosphorylation by Akt (Datta et al., 1997; del Peso et al., 1997) and Raf-1 (Wang et al., 1996a), two kinases involved in survival signals transduction. It has been suggested that, in the absence of phosphorylation, Bad induces cell death possibly via the formation of heterodimers with Bcl-x L (or Bcl-2 depending of the cell-type) and the concomitant generation of Bax homodimers. Assuming that all mammalian cells constitutively express all the protein components required to execute the death program (Jacobson et al., 1994), these results suggest that Bax (or an other similar pro-apoptotic member of the Bcl-2 family) are ubiquitously expressed and that survival requires their continuous inhibition. Taking into account the mitochondrial membrane localization of these proteins and their pore forming properties, it can be proposed that this kind of regulation operates at the mitochondrial level to control membrane permeability and efflux of AIF and cytochrome c. The mechanism allowing for the release of AIF and cytochrome c needs further investigations. Opening of the PT pore could be just a first step of a cascade of events causing an increase in the permeability of the outer mitochondrial membrane. At present, it remains elusive whether this permeability increase is due to the action of specific pores in the outer membrane and/or to its mechanic disruption secondary to an increase in matrix volume. Another exciting question to resolve is the occurrence of an apoptosis-like phenotype associated with a specific mutation in S. cerevisiae (Madeo et al., 1997). This yeast species seems to not contain caspases or Bcl-2s gene products and its cytochrome c has been shown to be ineffective to induce nuclear apoptosis in an acellular assay (Kluck et al., 1997b). These data suggest the existence of yet unknown PCD pathways in which the place and the role of mitochondria remain to be determined.

Summary Programmed cell death (PCD) serves as a major mechanism for the precise regulation of cell numbers, and as a defense mechanism to remove unwanted and potentially dangerous cells. Despite the striking heterogeneity of cell death induction pathways, the execution of the death program is often associated with characteristic morphological and biochemical changes termed apoptosis. In both worm and mammalian cells, the antiapoptotic members of the Bcl-2 family act upstream of the ~<execution caspases>~ somehow preventing their proteolytic processing into active killers. Two main pathways connect Bcl-2 family proteins to caspases. In the first one, antiapoptotic Bcl-2 family proteins maintain cell survival by dragging execution caspases to intracellular membranes (probably the mitochondrial membrane) and by

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preventing their activation. The mammalian protein Apaf-l, the mammalian equivalent of CED-4, could be the physical link between Bcl-2 family proteins and caspases. In the second one, Bcl-2 would act by regulating the release from mitochondria of caspases activators: cytochrome c and/or AIF (Apoptosis-Inducing-Factor). This crucial position of mitochondria in PCD control is reinforced by the observation that mitochondria contribute to PCD signaling via the production of reactive oxygen species, as shown in TNF-ct- or ceramide-induced cell death during which increased mitochondrial ROS production appears as an early event of the induction phase. In this chapter, we examined the data concerning the mitochondrial features of PCD.

References Adachi, S., Gottlieb, R.A. & Babior, B.M. (1998). Lack of Release of Cytochrome c from Mitochondria into Cytosol Early in the Course of Fas-mediated Apoptosis of Jurkat Cells. J. Biol. Chem. 273, 19892-19894. Adams, J.M. & Cory, S. (1998). The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322-1326. Akao, Y., Otsuki, Y., Kataoka, S., Ito, Y. & Tsujimoto, Y. (1994). Multiple subcellular localization of Bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes. Cancer Res. 54, 2468-2471. Ali, S.T., Coggins, J.R. & Jacobs, H.T. (1997). The study of cell death proteins in the outer mitochondrial membrane chemical cross-linking. Biochem. J. 325, 321-324. Antonsson, B., Conti, F., Ciavatta, A., Montessuit, S., Lewis, S., Martinou, I., Bernasconi, L., Bernard, A., Mermod, J.J., Mazzei, G., Maundrell, K., Gambale, F., Sadoul, R. & Martinou, J.C. (1997). Inhibition of Bax Channel-Forming Activity by Bcl-2. Science 277, 370-372. Bemardi, P., Vassanelli, S., Veronese, P., Colonna, R., Szabo, I. & Zoratti, M. (1992). Modulation of the mitochondrial permeability transition pore. Effect of protons and divalent cations. J. Biol. Chem. 267, 2934-9. Beutner, G., Ruck, A., Riede, B., Welte, W. & Brdiczka, D. (1996). Complexes between kinases, mitochondrial porin and adenylate translocator in rat brain resemble the permeability transition pore. FEBS Lett. 396, 189-195. Bojes, H.K., Datta, K., Xu, J., Chin, A., Simonian, P., Nunez, G. & Kehrer, J.P. (1997). Bcl-xL overexpression attenuates glutathione depletion in FL5.12 cells following interleukin-3 withdrawal. Biochem. J. 325, 315-319. Borner, C., Martinou, I., Mattmann, C., Irmler, M., Schaerer, E., Martinou, J.C. & Tschopp, J. (1994). The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis. J. Cell Biol. 126, 1059-1068. Bossy-Wetzel, E., Newmeyer, D.D. & Green, D.R. (1998). Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 17, 37-49. Boyd, J.M., Malstrom, S., Subramanian, T., Venkatesh, L.K., Schaeper, U., Elangovan, B., D'Sa, E.C. & Chinnadurai, G. (1994). Adenovirus E1B 19 kDa and Bcl-2 proteins interact with a common set of cellular proteins. Cell 79, 341-351. Cai, J. & Jones, D.P. (1998). Superoxide in apoptosis. Mitochondrial generation triggered by cytochrome c loss. J. Biol. Chem. 273, 11401-11404. Carayon, P., Portier, M., Dussossoy, D., Bord, A., Petitpretre, G., Canat, X., Le Fur, G. & Casellas, P. (1996). Involvement of peripheral benzodiazepine receptors in the protection of hematopoietic ceils against

Mitochondrial Control of Apoptosis

1 15

oxygen radical damage. Blood 87, 3170-3178. Chauhan, D., Pandey, P., Ogata, A., Teoh, G., Krett, N., Halgren, R., Rosen, S., Kufe, D., Kharbanda, S. & Anderson, K. (1997). Cytochrome c-dependent and -independent induction of apoptosis in multiple myeloma cells. J. Biol. Chem. 272. 29995-29997. Chen, L.Z., Nourse, J. & Cleary, M.L. (1989). The bcl-2 candidate proto-oncogene product is a 24-kilodalton integral-membrane protein highly expressed in lymphoid cell lines and lymphomas carrying the t(14;18) translocation. Mol. Cell. Biol. 9, 701-10. Cheng, E.H., Levine, B., Boise, L.H., Thompson, C.B. & Hardwick, J.M. (1996). Bax-independent inhibition of apoptosis by Bcl-xL. Nature 379, 554-556. Chinnaiyan, A.M., Chaudhary, D., O'Rourke, K., Koonin, E.V. & Dixit, V.M. (1997a). Role of CED-4 in the activation of CED-3. Nature 388,728-729. Chinnaiyan, A.M., O'Rourke, K., Lane, B.R. & Dixit, V.M. (1997b). Interaction of CED-4 with CED-3 and CED-9: a molecular framework for cell death. Science 275, 1122-1126. Cryns, V. & Yuan, J. (1998). Proteases to die for. Genes and Dev. 12, 1551-1570. Datta, S.R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y. & Greenberg, M.E. (1997). Akt phosphorylation of BAD couples survival signals to the cell- intrinsic death machinery. Cell 91, 231-241. de Jong, D., Prins, F.A., Mason, D.Y., Reed, J.C., van Ommen, G.B. & Kluin, P.M. (1994). Subcellular localization of the bcl-2 protein in malignant and normal lymphoid cells. Cancer Res 54, 256-60. Decaudin, D., Geley, S., Hirsch, T., Castedo, M., Marchetti, P., Macho, A., Kofler, R. & Kroemer, G. (1997). Bcl-2 and Bcl-XL antagonize the mitochondrial dysfunction preceding nuclear apoptosis induced by chemotherapeutic agents. Cancer Res. 57, 62-7. Degli Esposti, M. & McLennan, H. (1998). Mitochondria and cells produce reactive oxygen species in virtual anaerobiosis: relevance to ceramide-induced apoptosis. FEBS Lett. 430, 338-342. del Peso, L., Gonzalez Garcia, M., Page, C., Herrera, R. & Nunez, G. (1997). Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 278, 687-689. Distelhorst, C.W., Lam, M. & McCormick, T.S. (1996). Bcl-2 inhibits hydrogen peroxide-induced ER Ca2÷ pool depletion. Oncogene 12, 2051-2055. Ellerby, L.M., Ellerby, H.M., Park, S.M., Holleran, A.L., Murphy, A.N., Fiskum, G., Kane, D.J., Testa, M.P., Kayalar, C. & Bredesen, D.E. (1996). Shift of the cellular oxidation-reduction potential in neural cells expressing Bcl-2. J. Neurochem. 67, 1259-1267. Ellis, R.E., Yuan, J.Y. & Horvitz, H.R. (1991). Mechanisms and functions of cell death. Annu. Rev. Cell Biol. 7,663-698. Fernandez, S.M. & Bischoff, J.R. (1993). Bcl-2 associates with the ras-related protein R-ras p23. Nature 366, 274-275. France-Lanord, V., Brugg, B., Michel, P.P., Agid, Y. & Ruberg, M. (1997). Mitochondrial free radical signal in ceramide-dependent apoptosis: a putative mechanism for neuronal death in Parkinson's disease. J Neurochem 69. 1612-21. Fridovich, I. (1978). The biology of oxygen radicals. Science 201,875-880. Golstein, P. (1997). Controlling Cell Death. Science 275, 1081-1082. Gonzalez-Garcia, M., Perez-Ballestero, R., Ding, L., Duan, L., Boise, L.H., Thompson, C.B. & Nunez, G. (1994). BcI-XL is the major bcl-x mRNA form expressed during murine development and its product localizes to mitochondria. Development 120, 3033-3042. Goossens, V., Grooten, J., De V.K. & Fiers, W. (1995). Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proc. Natl. Acad. Sci. USA 92, 8115-8119.

116

B. Mignotte and J.-L. Vayssiere

Greenlund, L.J.S., Deckwerth, T.L. & Johnson Jr, E.M. (1995). Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death. Neuron 14, 303-315. Gross, A., Jockel, J., Wei, M.C. & Korsmeyer, S.J. (1998). Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J. 17, 3878-3885. Gudz, T.I., Tsemg, K.Y. & Hoppel, C.L. (1997). Direct inhibition of mitochondrial respiratory cahin complex III by cell-permeable ceramide. J. Biol. Chem. 272, 24154-24158. Gu6nal, I., Sidoti-de Fraisse, C., Gaumer, S. & Mignotte, B. (1997). Bcl-2 and Hsp27 act at different levels to suppress programmed cell death. Oncogene 15, 347-360. Halliwell, B. & Gutteridge, J.M. (1990). Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol. 1-85. Halliwell, B. & Gutteridge, J.M.C. (1989). Free radicals in Biology and Medicine. Oxford University Press, Inc., 2nd Edition. Hartley, A., Stone, J.M., Heron, C., Cooper, J.M. & Schapira, A.H. (1994). Complex I inhibitors induce dose-dependent apoptosis in PC12 cells: relevance to Parkinson's disease. J. Neurochem. 63, 1987-90. He, H., Lam, M., McCormick, T. S. & Distelhorst, C. W. (1997). Maintenance of Calcium Homeostasis in the Endoplasmic Reticulum by Bcl-2. J. Cell. Biol. 138, 1219-1228. Hengartner, M.O. (1997). Apoptosis. CED-4 is a stranger no more. Nature 388, 714-715. Hickish, T., Robertson, D., Clarke, P., Hill, M., di Stefano, F., Clarke, C. & Cunningham, D. (1994). Ultrastructural localization of BHRFI: an Epstein-Ban" virus gene product which has homology with bcl-2. Cancer Res. 54, 2808-11. Higuchi, M., Aggarwal, B.B. & Yeh, E.T. (1997). Activation of CPP32-1ike protease in tumor necrosis factor-induced apoptosis is dependent on mitochondrial function. J. Clin. Invest. 99, 1751-1758. Hirsch, T., Decaudin, D., Susin, S.A., Marchetti, P., Larochette, N., Resche-Rigon, M. & Kroemer, G. (1998). PK11195, a ligand of the mitochondrial benzodiazepine receptor, facilitates the induction of apoptosis and reverses bcl-2-mediated cytoprotection. Exp. Cell Res. 241,426-434. Hockenbery, D., Nunez, G., Milliman, C., Schreiber, R.D. & Korsmeyer, S.J. (1990). Bcl-2 is an inner mitochondrial protein that blocks programmed cell death. Nature 348, 334-336. Hockenbery, D.M., Oltvai, Z.N., Yin, X.M., Milliman, C.L. & Korsmeyer, S.J. (1993). Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75, 241-251. Hofmann, K., Bucher, P. & Tschopp, J. (1997). The CARD domain: a new apoptotic signalling motif. Trensd Biochem. Sci. 22, 155-156. Hsu, Y.T., Wolter, K.G. & Youle, R.J. (1997). Cytosol-to-membrane redistribution of Bax and BcI-X(L) during apoptosis. Proc. Natl. Acad. Sci. USA 94, 3668-3672. Hu, Y., Benedict, M.A., Wu, D., Inohara, N. & Nunez, G. (1998). Bcl-xL interacts with Apaf-1 and inhibits Apaf-l-dependent caspase-9 activation. J. Biol. Chem. 273, 4386-4391. Jacobson, M.D. (1996). Reactive oxygen species and programmed cell death. Trends Biochem. Sci. 21, 83-86. Jacobson, M.D., Burne, J.F. & Raft, M.C. (1994). Programmed Cell Death and Bcl-2 Protection in the Absence of a Nucleus, EMBO J. 13, 1899-1910. Jacobson, M.D. & Raft, M.C. (1995). Programmed cell death and Bcl-2 protection in very low oxygen. Nature 374, 814-816. Janiak, F., Leber, B. & Andrews, D.W. (1994). Assembly of Bcl-2 into microsomal and outer mitochondrial membranes. J. Biol. Chem. 269, 9842-9849. Jtirgensmeier, J., Krajewski, S., Armstrong, R.C., Wilson, G.M., Olterdorf, T., Fritz, L.C., Reed, J.C. & Ottilie, S. (1997). Bax- and Bak-induced cell death in the fission yeast Schizosaccharomyces pombe.

Mitochondrial Control of Apoptosis

117

Mol. Biol. Cell 8, 325-339. JiJrgensmeier, J.M., Xie, Z., Deveraux, Q., Ellerby, L., Bredesen, D. & Reed, J.C. (1998). Bax directly induces release of cytochrome c from isolated mitochondria. Proc. Natl. Acad. Sci. (USA) 95, 4997-5002. Kane, D.J., Sarafian, T.A., Anton, R., Hahn, H., Gralla, E.B., Valentine, J.S., Ord, T. & Bredesen, D.E. (1993). Bcl-2 inhibition of neural death - decreased generation of reactive oxygen species. Science 262, 1274-1277. Kharbanda, S., Pandey, P., Schofield, L., lsraels, S., Roncinske, R., Yoshida, K., Bharti, A., Yuan, Z.M., Saxena, S., Weichselbaum, R., Nalin, C. & Kufe, D. (1997). Role for bcl-XL as an inhibitor of cytosolic cytochrome C accumulation in DNA damage-induced apoptosis. Proc. Natl. Acad. Sci. USA 94, 6939-6942. Kluck, R.M., Bossy-Wetzel, E., Green, D.R. & Newmeyer, D.D. (1997a). The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 1132-1136. Kluck, R.M., Martin, S.J., Hoffman, B., Zhou, J.S., Green, D.R. & Newmeyer, D.D. (1997b). Cytochrome c activation of CPP32-1ike proteolysis plays a critical role in a Xenopus cell-free apoptosis system. EMBO J. 16, 4639-4649. Knudson, C.M. & Korsmeyer, S.J. (1997). Bcl-2 and Bax function independently to regulate cell death. Nat. Genet. 16, 358-363. Korsmeyer, S.J. (1992). Bcl-2 initiates a new category of oncogenes: regulators of cell death. Blood 80, 879-86. Korsmeyer, S.J., Yin, X.M., Oltvai, Z.N., Veis, N.D. & Linette, G.P. (1995). Reactive oxygen species and the regulation of cell death by the Bcl-2 gene family. Biochim. Biophys. Acta 1271, 63-66. Krajewski, S., Tanaka, S., Takayama, S., Schibler, M.J., Fenton, W. & Reed, J.C. (1993). Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Cancer Res. 53, 4701-4714. Krippner, A., Matsuno-Yagi, A., Gootlieb, R.A. & Babior, B.M. (1996). Loss of Function of Cytochrome c in Jurkat Cells Undergoing Fas-mediated Apoptosis. J. Biol. Chem. 271, 21629-21636. Kroemer, G.. (1997). The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nature Med. 3,614-620. Kroemer, G., Petit, P.X., Zamzami, N., Vayssibre, J.L. & Mignotte, B. (1995). The biochemistry of programmed cell death. FASEB J. 9, 1277-1287. Kroemer, G., Zamzami, N. & Susin, S.A. (1997). Mitochondrial control of apoptosis. Immunol. Today 18, 44-51. Kurschner, C. & Morgan, J.I. (1995). The cellular prion protein (PrP) selectively binds to Bcl-2 in the yeast two-hybrid system. Mol. Brain Res 30, 165-168. Lam, M., Dubyak, G., Chen, L., Nunez, G., Miesfeld, R.L. & Distelhorst, C.W. (1994). Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca 2. fluxes. Proc. Natl. Acad. Sci. USA 91, 6569-6573. Lancaster, J.R., Laster, S.M. & Gooding, L.R. (1989). Inhibition of target cell mitochondrial electron transfer by tumor necrosis factor. FEBS Lett. 248, 169-174. Lennon, S.V., Martin, S.J. & Cotter, T.G. (1991). Dose-dependent induction of apoptosis in human tumour cell lines by widely diverging stimuli. Cell Prolif. 24, 203-14. Li, F., Srinivasan, A., Wang, Y., Armstrong, R.C., Tomaselli, K.J. & Fritz, L.C. (1997a). Cell-specific Induction of Apoptosis by Microinjection of Cytochrome c. Bcl-xl has activity independent of cytochrome c release. J. Biol. Chem. 272, 30299-30305. Li, H., Zhu, H. & yuan, J. (1998) Cleavage of BID by Caspase 8 Mediates the Mitochondrial Damage in th Fas Pathway of Apoptosis, Cell 94,

118

B. Mignotte and J.-L. Vayssiere

Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S. & Wang, X. (1997b). Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade. Cell 91,479-489. Liu, X., Kim, C.N., Yang, J., Jemmerson, R. & Wang, X. (1996). Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86, 147-157. Longo, V.D., Ellerby, L.M., Bredesen, D.E., Valentine, J.S. & Gralla, E.B. (1997). Human Bcl-2 reverses survival defects in yeast lacking superoxide dismutase and delays death of wild-type yeast. J. Cell Biol. 137, 1581-8. Luo, X., Budihardjo, I., Zou, H., Slaughter, C. & Wang, X. (1998). Bid, a Bcl-2 Interacting Protein, Mediates Cytochrome c release from Mitochondria in Response to Activation of Cell surface Death receptors. Cell 94, 481-490. Macho, A., Hirsch, T., Marzo, I., Marchetti, P., Dallaporta, B., Susin, S.A., Zamzami, N. & Kroemer, G. (1997). Glutathione depletion is an early and calcium elevation is a late event of thymocyte apoptosis. J. Immunol. 158, 4612-4619. Madeo, F., Frohlich, E. & Frohlich, K. U. (1997). A yeast mutant showing diagnostic markers of early and late apoptosis. J. Cell Biol. 139, 729-734. Manon, S., Chaudhuri, B. & Guerin, M. (1997). Release of cytochrome c and decrease of cytochrome c oxidase in Bax- expressing yeast ceils, and prevention of these effects by coexpression of Bcl-xL. FEBS Lett. 415, 29-32. Marchetti, P., Decaudin, D., Macho, A., Zamzami, N., Hirsch, T., Susin, S. A. & Kroemer, G. (1997). Redox regulation of apoptosis: impact of thiol oxidation status on mitochondrial function. Eur. J. Immunol. 27, 289-96. Martin, S.J. & Cotter, T.G. (1991). Ultraviolet B irradiation of human leukaemia HL-60 cells in vitro induces apoptosis. Int. J. Radiat. Biol. 59, 1001. Marton, A., Mihalik, R., Bratincsak, A., Adleff, V., Petak, I., Vegh, M., Bauer, P. I. & Krajcsi, P. (1997). Apoptotic cell death induced by inhibitors of energy conservation--Bcl- 2 inhibits apoptosis downstream of a fall of ATP level. Eur. J. Biochem. 250, 467-475. Mayer, A., Neupert, W. & Lill, R. (1995). Translocation of Apocytochrome c accross the Outer membrane of Mitochondria. J. Biol. Chem. 270, 12390-12397. Mayer, M. & Noble, M. (1994). N-acetyl-L-cysteine is a pluripotent protector against cell death and enhancer of trophic factor-mediated cell survival in vitro. Proc. Nathl. Acad. Sci. USA 91, 7496-500. Mehlen, P., Schulze-Osthoff, K. & Arrigo, A.P. (1996). Small stress proteins as novel regulators of apoptosis. J. Biol. Chem. 271, 16510-16514. Mignotte, B., Larcher, J.C., Zheng, D.Q., Esnault, C., Coulaud, D. & Feunteun, J. (1990). SV40 induced cellular immortalization: phenotypic changes associated with the loss of proliferative capacity in a conditionally immortalized cell line. Oncogene 5, 1529-1533. Mignotte, B. & Vayssi~re, J.L. (1998). Mitochondria and apoptosis. Eur. J. Biochem. 252, 1-15. Minn, A.J., Velez, P., Schendel, S.L., Liang, H., Muchmore, S.W., Fesik, S.W., Fill, M. & Thompson, C.B. (1997). Bcl-xL forms an ion channel in synthetic lipid membranes. Nature 385,353-357. Mirkovic, N., Voehringer, D.W., Story, M.D., McConkey, D.J., McDonnell, T.J. & Meyn, R.E. (1997). Resistance to radiation-induced apoptosis in Bcl-2-expressing ceils is reversed by depleting cellular thiols. Oncogene 15, 1461-1470. Muchmore, S.W., Sattler, M., Liang, H., Meadows, R.P., Harlan, J.E., Yoon, H.S., Nettesheim, D., Chang, B.S., Thompson, C.B., Wong, S.L., Ng, S.C. & Fesik, S. W. (1996). X-ray and NMR structure of human BcI-XL, an inhibitor of programmed cell death. Nature 381,335-341.

Mitochondrial Control of Apoptosis

119

Myers, K.M., Fiskum, G., Liu, Y., Simmens, S.J., Bredesen, D.E. & Murphy, A.N. (1995). Bcl-2 protects neural cells from cyanide/aglycemia-induced lipid oxidation, mitochondrial injury, and loss of viability. J. Neurochem. 65, 2432-2440. Nagata, S. (1997). Apoptosis by Death Factor. Cell 88, 355-365. Nakai, M., Takeda, A., Cleary, M.L & Endo, T. (1993). The bcl-2 protein is inserted into the outer membrane but not into the inner membrane of rat liver mitochondria in vitro. Biochem. Biophys. Res. Commun. 196, 233-239. Naumovski, L. & Cleary, M. L. (1996). The p53-binding protein 53BP2 also interacts with Bcl-2 and impedes cell cycle progression at G2/M. Mol Cell Biol 16, 3884-92. Newmeyer, D.D., Farschon, D.M. & Reed, J.C. (1994). Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria. Cell 79, 353-364. Nguyen, M., Branton, P.E., Walton, P.A., Oltvai, Z.N., Korsmeyer, S.J. & Shore, G.C. (1994). Role of membrane anchor domain of Bcl-2 in suppression of apoptosis caused by E1B-defective adenovirus. J. Biol. Chem. 269, 16521-16524. Nguyen, M., Millar, D.G., Yong, V.W., Korsmeyer, S.J. & Shore, G.C. (1993). Targeting of bcl-2 to the mitochondrial outer membrane by a COOH-Terminal signal anchor sequence. J. Biol. Chem. 268, 25265-25268. Nicholson, D.W. & Thornberry, N.A. (1997). Caspases: killer proteases. Trends Biochem. Sci. 22, 299-306. O'Donnell, V.B., Spycher, S. & Azzi, A. (1995). Involvement of oxidants and oxidant-generating enzyme(s) in tumour- necrosis-factor-alpha-mediated apoptosis: role for lipoxygenase pathway but not mitochondrial respiratory chain. Biochem J 310, 133-141. Olivier, R., Otter, I., Monney, L., Wartmann, M. & Borner, C. (1997). Bcl-2 does not require Raf kinase activity for its death-protective function. Biochem. J. 324, 75-83. Oltvai, Z.N., Milliman, C.L & Korsmeyer, S.J. (1993). Bcl-2 heterodimerizes in vivo with a conserved homolog, bax, that accelerates programmed cell death. Cell 74, 609-619. Pan, G., O'Rourke, K. & Dixit, V.M. (1998). Caspase-9, Bcl-xL, and Apaf-1 Form a Ternary Complex. J. Biol. Chem. 273, 5841-5845. Pastorino, J.G., Chen, S.T., Tafani, M., Snyder, J.W. & Farber, J.L. (1998). The overexpression of Bax produces cell death upon induction of the mitochondrial permeability transition. J. Biol. Chem. 273, 7770-5. Paumen, M.B., Ishida, Y., Han, H., Muramatsu, M., Eguchi, Y., Tsujimoto, Y. & Honjo, T. (1997). Direct interaction of the mitochondrial membrane protein carnitine palmitoyltransferase I with Bcl-2. Biochem. Biophys. Res. Commun. 231,523-525. Petit, P.X., Goubern, M., Diolez, P., Susin, S.A., Zamzami, N. & Kroemer, G. (1998). Disruption of the outer mitochondrial membrane as a result of large amplitude swelling: the impact of irreversible permeability transition. FEBS Lett. 426, 111-116. Petit, P.X., Lecoeur, H., Zorn, E., Dauguet, C., Mignotte, B. & Gougeon, M.L. (1995). Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocytes apoptosis. J. Cell Biol. 130, 157-167. Petronilli, V., Nicolli, A., Costantini, P., Colonna, R. & Bernardi, P. (1994). Regulation of the permeability transition pore, a voltage-dependent mitochondrial channel inhibited by cyclosporin A. Biochem. Biophys. Acta 1187, 255-259. Pinkus, R., Weiner, L.M. & Daniel, V. (1996). Role of Oxidants and Antioxidants in the Induction of AP-1, NF-kB, and Glutathione S-Transferase Gene Expression. J. Biol. Chem. 271, 13422-13429. Quillet-Mary, A., Jaffrezou, J.P., Mansat, V., Bordier, C., Naval, J. & Laurent, G. (1997). Implication

120

B. Mignotte and J.-L. Vayssiere

of mitochondrial hydrogen peroxide generation in ceramide- induced apoptosis. J. Biol. Chem. 272, 21388-95. Ratan, R.R., Murphy, T.H. & Baraban, J.M. (1994). Oxidative stress induces apoptosis in embryonic cortical neurons. J. Neurochem. 62, 376-9. Reed, J.C. (1997). Double identity for proteins of the Bcl~2 family. Nature 387,773-776. Rosse, T., Olivier, R., Monney, L., Rager, M., Conus, S., Fellay, I., Jansen, B. & Borner, C. (1998). Bcl-2 prolongs cell survival after Bax-induced release of cytochrome c. Nature 391,496-499. Ryan, J.J., Prochownik, E., Gottlieb, C.A., Apel, l.J., Merino, R., Nunez, G. & Clarke, M.F. (1994). c-myc and bcl-2 modulate p53 function by altering p53 subcellular trafficking during the cell cycle. Proc. Natl. Acad. Sci. USA 91, 5878-5882. Sandstrom, P.A. & Buttke, T.M. (1993). Autocrine production of extracellular catalase prevents apoptosis of the human CEM T-cell line in serum-free medium. Proc. Natl. Acad. Sci. USA 90, 4708-12. Sato, N., Iwata, S., Nakamura, K., Hod, T., Mori, K. & Yodoi, J. (1995). Thiol-Mediated Redox Regulation of Apoptosis. Possible roles of cellular thiols other than glutathione in T cell apoptosis. J. Immunol. 154, 3194-3203. Scarlett, J.L. & Murphy, M.P. (1997). Release of apoptogenic proteins from the mitochondrial intermembrane space during the mitochondrial permeability transition. FEBS Lett. 418,282-286. Schendel, S.L., Xie, Z., Montal, M.O., Matsuyama, S., Montal, M. & Reed, J.C. (1997). Channel formation by antiapoptotic protein Bcl-2. Proc. Natl. Acad. Sci. USA 94, 5113-8. Schulze-Osthoff, K., Bakker, A.C., Vanhaesebroeck, B., Beyaert, R., Jacob, W.A. & Fiers, W. (1992). Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation. J. Biol. Chem. 267, 5317-5323. Schulze-Osthoff, K., Beyaert, R., Vandevoorde, V., Haegeman, G. & Fiers, W. (1993). Depletion of the mitochondrial electron transport abrogates the cytotoxic and Gene-lnductive effects of TNF. EMBO J. 12, 3095-3104. Schulze-Osthoff, K., Ferrari, D., Los, M., Wesselborg, S. & Peter, M. E. (1998). Apoptosis signaling by death receptors. Eur. J. Biochem. 254, 439-459. Schulze-Osthoff, K., Krammer, P. H. & Droge, W. (1994). Divergent signalling via APO-1/Fas and the TNF receptor, two homologous molecules involved in physiological cell death. EMBO J. 13, 4587-4596. Sedlak, T.W., Oltvai, Z.N., Yang, E., Wang, K., Boise, L.H., Thompson, C.B. & Korsmeyer, S.J. (1995). Multiple Bcl-2 family members demonstrate selective dimerizations with Bax. Proc. Natl. Acad. Sci. USA 92, 7834-7838. Shaham, S. & Horvitz, H.R. (1996). Developing Caenorhabditis elegans neurons may contain both cell-death protective and killer activities. Genes Dev. 10, 578-591. Shibasaki, F., Kondo, E., Akagi, T. & McKeon, F. (1997). Suppression of signalling through transcription factor NF-AT by interactions between calcineurin and Bcl-2. Nature 386, 728-731. Shimizu, S., Eguchi, Y., Kamiike, W., Funahashi, Y., Mignon, A., Lacronique, V., Matsuda, H. & Tsujimoto, Y. (1998). Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating proton flux. Proc. Natl. Acad. Sci. (USA) 95, 1455-1459. Shimizu, S., Eguchi, Y., Kosaka, H., Kamiike, W., Matsuda, H. & Tsujimoto, Y. (1995). Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-XL. Nature 374, 811-813. Sidoti-de Fraisse, C., Rincheval, V., Risler, Y., Mignotte, B. & Vayssi~re, J. L. (1998). TNF-a activates at least two apoptotic signaling cascades, Oncogene 17, 1639-1652. Sies, H. (1991). Oxidative stress: from basic research to clinical application. Am. J. Med. 91, 31S-38S. Skulachev, V. P. (1998). Cytochrome c in the apoptotic and antioxidant cascades. FEBS Lett. 423,275-280.

Mitochondrial Control of Apoptosis

121

Susin, S.A., Zamzani, N., Castedo, M., Hirsh, T., Marchetti, P., Macho, A., Daugas, E., Geuskens, M. & Kroemer, G. (1996). Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J. Exp. Med. 184, 1-11. Takayama, S., Sato, T., Krajewski, S., Kochel, K., Irie, S., Millan, J.A. & Reed, J.C. (1995). Cloning and functional analysis of BAG-l: a novel Bcl-2-binding protein with anti-cell death activity. Cell 80, 279-284. Tang, D.G., Li, L., Zhu, Z. & Joshi, B. (1998). Apoptosis in the absence of cytochrome c accumulation in the cytosol. [In Process Citation], Biochem. Biophys. Res. Commun. 242, 380-384. Tyurina, Y.Y., Tyurin, V.A., Carta, G., Quinn, P.J., Schor, N.F. & Kagan, V.E. (1997). Direct evidence for antioxidant effect of Bcl-2 in PC12 rat pheochromocytoma cells. Arch. Biochem. Biophys. 344, 413-423. Uckun, F.M., Tuelahlgren, L., Song, C.W., Waddick, K., Myers, D.E., Kirihara, J., Ledbetter, J.A. & Schieven, G.L. (1992). Ionizing radiation stimulates unidentified Tyrosine-Specific protein kinases in human Lymphocyte-B precursors, triggering apoptosis and clonogenic cell death. Proc. Natl. Acad. Sci. USA 89, 9005-9009. van den Dobbelsteen, D.J,, Nobel, C.S.I., Schlegel, J., Cotgreave, I.A., Orrenius, S. & Slater, A.F.G. (1996). Rapid and Specific Efflux of Reduced Glutathione during Apoptosis Induced by Anti-Fas/APO-I Antibody. J. Biol. Chem. 271, 15420-15427. Van der Heiden, M.G., Chandel, N.S., Williamson, E.K., Schumacker, P.T. & Thompson, C.B. (1997). Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell 91,627-637. Vayssi~re, J.L., Petit, P.X., Risler, Y. & Mignotte, B. (1994). Commitment to apoptosis is associated with changes in mitochondrial biogenesis and activity in cell lines conditionally immortalized with Simian Virus 40. Proc. Natl. Acad. Sci. USA 91, 11752-11756. Veis, D.J., Sorenson, C.M., Shutter, J.R. & Korsmeyer, S.J. (1993). Bcl-2-Deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 75,229-240. Wang, H., Rapp, U.R. & Reed, J.C. (1996a). Bcl-2 Targets the Protein Kinase Raf-1 to Mitochondria. Cell 87,629-638. Wang, H.G., Miyashita, T., Takayama, S., Sato, T., Torigoe, T., Krajewski, S., Tanaka, S., Hovey, L. r., Troppmair, J., Rapp, U.R. & Reed, J.C. (1994). Apoptosis regulation by interaction of Bcl-2 protein and Raf-1 kinase. Oncogene 9, 2751-2756. Wang, H.G., Takayama, S., Rapp, U.R. & Reed, J.C. (1996b). Bcl-2 interacting protein, BAG-I, binds to and activates the kinase Raf- 1. Proc. Natl. Acad. Sci. USA 93, 7063-7068. Wang, X. & Studzinski, G.P. (1997). Antiapoptotic action of 1,25-dihydroxyvitamin D3 is associated with increased iitochondrial MCL-1 and RAF-1 proteins and reduced release of cytochrome c. Exp. Cell. Res. 235,210-7. Wolvetang, E.J., Johnson, K.L., Krauer, K., Ralph, S.J. & Linnane, A.W. (1994). Mitochondrial respiratory chain inhibitors induce apoptosis. FEBS Lett. 339, 40-44. Wong, G. H. W., Elwell, J. H., Oberley, L. W. & Goeddel, D. V. (1989). Manganous superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor, Cell 58,923-931. Wu, D., Wallen, H.D., Inohara, N. & Nunez, G. (1997a). Interaction and regulation of the Caenorhabditis elegans death protease CED-3 by CED-4 and CED-9. J. Biol. Chem. 272, 21449-21454. Wu, D., Wallen, H.D. & Nunez, G. (1997b). Interaction and regulation of subcellular localization of CED-4 by CED-9. Science 275, 1126-1129. Xiang, J., Chao, D. T. & S.J., K. (1996). BAX-induced cell death may not require interleukin l[~-converting enzyme-like proteases. Proc. Natl. Acad. Sci. USA 93, 14559-14563.

122

B. Mignotte and J.-L. Vayssiere

Yang, J., Liu, X., Bhalla, K., Kim, C.N., Ibrado, A.M., Cai, J., Peng, T.I., Jones, D.P. & Wang, X. (1997). Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129-1132. Yang, T., Kozopas, K.M. & Craig, R.W. (1995). The intracellular distribution and pattern of expression of Mcl-1 overlap with, but are not identical to, those of Bcl-2. J. Cell Biol. 128, 1173-1184. Yang, X., Chang, H.Y. & Baltimore, D. (1998). Essential role of CED-4 oligomerization in CED-3 activation and apoptosis [see comments]. Science 281, 1355-7. Zamzami, N., Brenner, C., Marzo, I., Susin, S.A. & Kroemer, G. (1998). Subcellular and submitochondrial mode of action of Bcl-2-1ike oncoproteins. Oncogene 16, 2265-2282. Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Macho, A., Petit, P.X., Mignotte, B. & Kroemer, G. (1995a). Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J. Exp. Med. 182, 367-377. Zamzami, N., Marchetti, P., Castedo, M., Hirsch, T., Susin, S.A., Masse, B. & Kroemer, G. (1996a). Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis, FEBS Lett. 384, 53-57. Zamzami, N., Marchetti, P., Castedo, M., Zanin, C., Vayssi~re, J.L., Petit, P. X. & Kroemer, G. (1995b). Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181, 1661-1672. Zamzami, N., Susin, S.A., Marchetti, P., Hirsch, T., Gomez-Monterrey, I., Castedo, M. & Kroemer, G. (1996b). Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183, 1533-1544. Zhivotovsky, B., Orrenius, S., Brugstugun, O. T. & Doskeland, S. O. (1998). Injected cytochrome c induces apoptosis. Nature 391,449-450. Zhong, L.T., Sarafian, T., Kane, D.J., Charles, A.C., Mah, S.P., Edwards, R.H. & Bredesen, D.E. (1993). Bcl-2 inhibits death of central neural cells induced by multiple agents. Proc. Natl. Acad. Sci. USA 90, 4533-4537. Zhu, W., Cowie, A., Wasfy, G.W., Penn, L.Z., Leber, B. & Andrews, D.W. (1996). Bcl-2 mutants with restricted subcellular location reveal spatially distinct pathways for apoptosis in different cell-types. EMBO J. 15, 4130-4141. Zou, H., Henzel, W.J., Liu, X., Lutschg, A. & Wang, X. (1997). Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3, Cell 90, 405-413.

PROLIFERATIVE MITOCHONDRIAL DYSFUNCTION AND APOPTOSIS

MARIANGELA MANCINI, SOPHIE CAMILLERI-BRC)ET, BENJAMIN O. ANDERSON and DAVID M. HOCKENBERY

Table of Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Initial Description of a Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial Proliferation in Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Relationship With Apoptosis Models Featuring PTP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of Bcl-2 and Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Examples of Mitochondrial Proliferation in Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Mitochondrial Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction

With the acceptance of the central role of mitochondria in apoptosis, in the last 1-2 years, there has been a new focus on understanding how mitochondria are recruited into apoptotic pathways and what are the important changes in mitochondria function, structure or integrity that matter in apoptosis (Henkart and Grinstein, 1996; Susin et al., 1998). Several key proteins, including representatives of the major categories of apoptotic regulators/effectors, are found in association with mitochondria (Bcl-2, Bax, Caspase-3, cytochrome c, AIF) (Mancini et al., 1998; Samali et al., 1998). Our knowledge of their mitochondrial functions during apoptosis is still incomplete and there is virtually no information on potential functions during non-apoptotic cell pathways for most of these factors. Major gaps in our understanding remain concerning how cellular pathways involved in the triggering of apoptosis (receptor-mediated signal transduction, cell cycle, checkpoints) eventually lead to mitochondrial effects. In light of the multiple reports of functional alterations in mitochondria in apoptosis, there have been surprisingly few descriptions of mitochondrial ultrastructure in these studies. We have characterized mitochondrial function and structure at various time points following apoptotic signaling. The pattern of changes that we observed are suggestive of defects in mitochondrial biogenesis rather than membrane or osmotic damage. Mitochondrial misassembly may be a common mechanism during apoptosis and provides a conceptual basis for understanding the interactions between cell signaling and mitochondrial dysfunction.

123 Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar, © 2 0 0 1 Elsevier Science. Printed in the Netherlands.

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Initial Description of a Model

The colorectal carcinoma Colo-205 cell line undergoes morphologic differentiation when treated with herbimycin A, a destabilizer of Hsp-90-associated proteins (Mancini et al., 1997). Progressive flattening and adherence of cells over 24-48 h is accompanied by development of an extensive microvillus structure, cell-cell junctions and cytoplasmic polarization. Over the next 24-48 h, cells become refractile, round up and detach during a wave of apoptosis. This sequence recapitulates the orderly progression of colonocytes from stem cell through terminal differentiation, exit from cell cycle and extrusion by apoptosis. One of the striking features of the differentiated cells is the numerous mitochondria noted on transmission electron microscopy (EM) (Mancini et al., 1997). The number of mitochondrial cross-sections per differentiated cell is several fold higher than is found in untreated cells (see Figure 1). The number of mitochondria/cell continues to increase with time after herbimycin treatment, including those cells with nuclear changes of early apoptosis. Mitochondria undergoing cleavage are readily apparent, consistent with mitochondrial proliferation. There is a prominent supranuclear localization of mitochondria in the polarized epithelial cells. Mitochondrial proliferation in herbimycintreated Colo-205 cells was also detected using flow cytometry with fluorescent dyes that localize to mitochondria. Cell-labeling with dyes that bind to mitochondria independently of membrane potential increased following herbimycin treatment. Mean fluorescence values for nonyl acridine orange (NAO), which binds the mitochondrial lipid cardiolipin, increased 1.5 fold at 24 h and by a factor of 1.8 after 96 h of treatment (Maftah et al., 1989). The FL1 fluorescence of the lipophilic cationic dye JC-1, indicating potential-insensitive binding of the monomeric probe, increased 1.5 fold at 24 h and 3.0 fold by 96 h (Reers et al., 1995; Smiley, 1991). These results provide confirmation of progressive mitochondrial proliferation in herbimycin-treated Colo-205 cells. In contrast, no increase in functional mitochondria was revealed using potentialsensitive mitochondrial dyes. Rhodamine 123 staining and the FL2 fluorescence of JC-1, arising from J-aggregate formation in polarized mitochondria, did not vary significantly at any time following herbimycin treatment (see Figure 2). One interpretation of this surprising result is that the newly assembled mitochondria in herbimycin-treated Colo-205 cells, have depressed membrane potential de novo and do not make a significant contribution to total cellular mitochondrial function. An examination of the mitochondrial staining pattern by fluorescent microscopy supports this hypothesis. Colo-205 cells, like other cells, have a heterogenous pattern of staining with Aapm-sensitive dyes. Superimposed images of FL1 and FL2 fluorescence in JC-l-labeled Colo-205 cells revealed that mitochondria with bright orange-red (FL2) fluorescence were intermixed with those showing only green (FL1) fluorescence. Following herbimycin treatment, the number of mitochondrial images/cell increased, represented almost entirely by greenstaining mitochondria. A similar number of orange-red mitochondria were present in treated and control cells, demonstrating that mitochondria function was not uniformly depressed in herbimycin-treated cells.

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Figure 1. Electron microscopy analysis of mitochondrial proliferation in apoptosis, a) Colo-205, untreated. b) Colo-205, treated for 48 h with herbimycin A 300 ng/ml. Bar represents 0.2 ,um.

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Coincident with changes in mitochondrial number, an increase in the cellular content of reactive oxygen species was detected in herbimycin-treated Colo-205 cells (Mancini et al., 1997). Mean fluorescence values for oxidized dihydroethidium (DHE) and dichlorodihydrofluorescein (DCFH), indicators of cellular superoxide and peroxides, respectively, increased 24-96 h after treatment. Mitochondria might be the source as well as a target of ROS in these cells. Electron micrographs demonstrated both an increased number and ultrastructural changes of mitochondria in herbimycin-treated cells. These mitochondria have an pyknotic appearance with homogenously dense matrix, closely opposed inner and outer membranes and small cross-sectional area (see Figure lb). In order to assess the contribution of these altered mitochondria to apoptosis in Colo-205 cells, we prepared lysates from herbimycin-treated cells at 24 hours, before the onset of apoptosis. The addition of post-nuclear supernatants from these lysates to rat liver nuclei caused typical apoptotic nuclear changes within 1 h (Mancini et al., 1997). The apoptotic activity of Colo-205 extracts was removed by centrifugation at 10,000 x g to deplete mitochondria.

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Figure 3. Wide-field deconvolution microscopy of cells stained with JC-1. a) CHO, untreated, b) CHO, treated for 18 h with aphidicolin 5 ~tg/ml.

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M. Mancini, S. Camilleri-Brret, B.O. Anderson and D.M. Hockenbery

The diminished functional status of newly formed mitochondria may indicate some defect in their assembly. We have recently begun to characterize the composition of mitochondria in herbimycin-treated Colo-205 cells. In light of the increased NAO staining result, we analyzed mitochondrial lipids by thin-layer chromatography. Cardiolipin, an essential and exclusive mitochondrial lipid, increased by 40-50% in treated cells. Lipids in the nuclear membrane were not affected, demonstrating that there was not a general increase in phospholipid synthesis in herbimycin-treated ceils. We have begun to examine the expression of several mitochondrial proteins by Western blot analysis. We observed an 1.8 fold increase in expression of cytochrome oxidase II (COX II), which is coded by the mitochondrial genome, but no changes in levels of the nuclear-coded subunits COX IV, Va, Vb, or Via.

Mitochondrial Proliferation in Apoptosis Many types of apoptosis have been associated with aberrant regulation of growth or cell cycle pathways. These seemed good candidates to examine for evidence of a proliferation-associated mitochondrial dysfunction, possibly linked to cell growth and division pathways. Treatment of CHO cells with several cytostatic agents results in the loss of clonogenic survival and apoptosis. Schimke observed that high levels of protein synthesis and cell growth are maintained under these conditions, a process he termed nuclear-cytoplasmic cell cycle dissociation (Kung et al., 1993; Urbani et al., 1995). In contrast, cell lines with downregulation of protein synthesis and arrested cell growth after treatment with the same agents retained high viability after the drugs were removed. We examined the responses of CHO cells to a panel of cytostatic and chemotherapeutic agents at doses that led to apoptosis and diminished clonogenic survival (Camilleri-Broet et al., 1998). Cells were treated for an interval of 18 h after which they were harvested for flow cytometry and for assay of clonogenic survival in the absence of drug. Those agents (taxol, ara-C, cisplatin, hydroxyurea, adriamycin, VP-16, diepoxybutane and aphidicolin) causing cell death also resulted in increased mitochondrial mass at 18 h (see Figure 3 and 4). CHO cells that were arrested in G1 with mimosine or early S phase with thymidine excess did not exhibit either mitochondrial proliferation or loss of viability. The drugs that led to mitochondrial proliferation in CHO cells have diverse mechanisms of action and cause cells to arrest at different points within the cell cycle, indicating some generality to this response. Atypical mitochondria with a pyknotic appearance, resembling those in herbimycin A-treated Colo-205 cells, are noted in CHO cells treated with aphidicolin. A decrease in the ratio of JC-1 red/green fluorescence, signifying Al.Pm /mitochondrial mass, is also observed in CHO cells which exhibit drug-induced mitochondrial proliferation.

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Relationship With Apoptosis Models Featuring PTP

Several models of apoptosis have been described in which mitochondrial depolarization, attributable to opening of permeability transition pores, appears to be the initial mitochondrial event (Kroemer et al., 1997). We analyzed one of these models, glucocorticoid-induced apoptosis of the T cell hybridoma 2B4, in order to see there is any overlap between this phenotype and the mitochondrial dysfunction associated with proliferation (Camilleri-Br6et et al., 1998). As previously shown, mitochondrial depolarization is apparent in treated cells by 18 h (decreased JC-1 Red). Analyses of mitochondrial mass with fluorescent probes gave inconsistent results. Using NAO and Mitotracker green fluorescence as indicators, no changes are detected. There is a significant increase in JC-1 Green in the treated population, however, this may reflect shifts in JC-1 monomer and J-aggregate pools associated with the decrease in JC-1 Red staining. Examination of mitochondrial structure by EM showed that by 18 h of treatment, severe mitochondrial swelling and membrane disruption is evident. We evaluated subpopulations of these cells after cell sorting based on AaPM. Cells with reduced JC-1 Red fluorescence contained grossly swollen mitochondria with incomplete outer membranes (see Figure 5). Surprisingly, greater mitochondrial swelling was found in 2B4 cells having high (similar to untreated cells) levels of JC-1 Red staining

130

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M. Mancini, S. Camilleri-BrOet, B.O. Anderson and D.M. Hockenbery

Electron microscopy analysis of cell and mitochonddal morphology in cells with decreased

mitochondrial membrane potential, a) 2B4, untreated, b) 2B4, treated for 18 h with dexamethasone 0.1 OM. JC-1 Red h~subpopulation c) 2B4, treated with dexamethasone as in b). JC-1 Red ~° subpopulation.

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after 18 h of treatment. This appearance was not an artifact of EM processing, as 2B4 cells transfected with Bcl-2 did not show mitochondrial swelling after glucocorticoid treatment. The mitochondrial abnormalities evident in the JC-1 Red hi cells indicate that depolarization, and presumably permeability transition pore (PTP) activation, are secondary changes in this apoptosis model. We next examined earlier time points to identify the initial effects of treatment on mitochondrial ultrastructure. Mitochondrial swelling was observed within 8 h of glucocorticoid treatment. However, prior to mitochondrial swelling, a pyknotic morphology is seen in cells treated for only 4 h (see Figure 6). These mitochondria appear similar to those observed in Colo-205 and CHO cells undergoing apoptosis. 2B4-Bcl-2 cells maintained a non-condensed morphology at this time point, showing that mitochondrial changes occurring earlier than PTP activation are suppressed by Bcl-2. Thus, the early mitochondrial events leading to mitochondrial depolarization or production of dysfunctional mitochondria in apoptosis may be similar.

Effects of Bcl-2 and Antioxidants As noted above, the ultrastructural changes in mitochondria are suppressed by Bcl-2 expression. These effects can also be seen in flow cytometry assays of mitochondrial mass and function. As an example, we studied the human breast carcinoma cell line SkBr3, which undergoes apoptotic death when treated with herbimycin A. Mitochondrial mass was increased at 18 h after drug addition, but mitochondrial function remained unchanged. Reactive oxygen species were also increased following treatment, as assessed by oxidant-sensitive fluorescent dyes. Stable transfectants expressing Bcl-2 were established and determined to be resistant to herbimycin-induced apoptosis. In these cells, Bcl-2 had a strong inhibitory effect on ROS, as previously described (Hockenbery et al., 1993). Bcl-2 also completely blocked the proliferative mitochondrial response to herbimycin A, consistent with the previously mentioned effects on ultrastructure (see Figure 6). We also examined the effect of antioxidants on mitochondrial responses in apoptosis. We previously interpreted the anti-apoptotic properties of antioxidants as neutralizing the increased oxidants formed by dysfunctional mitochondria (Hockenbery, 1993). This mechanism should occur distal to the initial mitochondrial derangement in apoptosis. We found that Colo-205 cells treated with N-acetytcysteine or vitamin E were protected from herbimycin A-induced apoptosis. Analysis of the mitochondrial response in antioxidant-treated cells demonstrated that, similar to Bcl-2, the proliferative response of mitochondria was inhibited. This result indicates that a redox-sensitive pathway lies upstream of the initial mitochondrial response. A proximal effect of antioxidants to prevent mitochondrial dysfunction could explain the dramatic protective effects of antioxidants in some models. Conversely, the examples of apoptosis that do not respond to antioxidant treatment may employ distinct proximal pathways that are not redox-sensitive.

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M. Mancini, S. Camilleri-Br6et, B.O. Anderson and D.M. Hockenbery

Figure 6. Electron microscopy analysis of early changes of mitochondrial morphology in apoptosis, a) 2B4, untreated, b) 2B4, treated for 4 h with dexamethasone 0.1 IxM. c) 2B4-Bcl-2, treated for 4 h with dexamethasone 0.1 IzM.

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Other Examples of Mitochondrial Proliferation in Apoptosis One of the first descriptions of mitochondrial proliferation in programmed cell death was made in chick brainstem auditory nuclei following experimental deafferentation. One quarter of second-order auditory neurons in the ipsilateral nucleus magnocellularis die in 3-4 days following unilateral removal of the cochleus from 10 day old chicks. The number of mitochondrial profiles were greatly increased in neurons in the nucleus magnocellularis on the side of cochlear removal. Stereologic measurements demonstrated that mitochondrial area increased 86% at 6 h and 236% at 12 h compared to unoperated animals (Hyde, 1994a,b). Mitochondrial oxidative phosphorylation activities increased rapidly following deafferentation as shown by histochemical staining. Cytochrome oxidase and succinate dehydrogenase activities increased between 6 and 24 h after cochleus removal (Durham and Rubel, 1985; Hyde and Durham, 1990). Malate dehydrogenase underwent ipsilateral induction at 4 h (Durham et al., 1993). At later time points (3-14 days after deafferentation), enzyme activities are decreased, corresponding to mitochondrial vacuolization observed on EM. Systemic administration of chloramphenicol, an inhibitor of mitochondrial translation, accentuated the cell death response to deafferentation (Hyde, 1994a,b; Garden et al., 1994). Contrary to expectations, in a subset of neurons treated with chloramphenicol even greater mitochondrial proliferation resulted (Hartlage-Rubsamen and Rubel, 1996). Reipert et al. (1996) observed prominent mitochondrial proliferation following etoposide treatment of the pluripotent murine hematopoietic stem cell line, FDCP-mix. The timing of mitochondrial proliferation was observed to precede nuclear condensation and DNA cleavage. Multiparameter flow cytometry using propidium iodide and nonylacridine orange confirmed the increase in mitochondrial mass. Importantly, these studies demonstrated that mitochondrial proliferation occurred in all cell cycle stages and could not be explained solely by an etoposide-triggered G2 accumulation. Ultrastructural changes were not identified in these studies. Autophagy of mitochondria was a prominent finding, consistent with organelle damage or dysfunction. In frankly apoptotic cells, mitochondrial mass, as measured by NAO staining, declined. Interleukin-3 withdrawal resulted in decreased mitochondrial mass and diminished the proliferative response in etoposide-treated cells, suggesting a role of growth factors in drug-induced mitochondrial proliferation. In one study, cardiac myocytes in sinus node tissue (P cells) resected from patients with the long QT syndrome had an greatly increased mitochondrial content together with classic features of apoptosis (James et al., 1993). Notable ultrastructural changes included small cross-sectional area and pleiomorphic, occasionally vermiform, contours. The association of abnormal mitochondrial conformation with tumor necrosis factorinduced and spontaneous apoptosis was noted by Jia et al. (1997). These authors described an ultracondensed appearance of mitochondria in apoptotic cells characterized as small mitochondria with widened cristae and an highly electron-opaque matrix. Similar changes were observed during spontaneous apoptosis of CCRF-CEM cells in culture. Inhibition of TNFet-induced apoptosis with 3-methyladenine, an inhibitor of autophagy, did not effect TNFct-triggered mitochondrial changes. Thus, the actions of TNFct receptor signaling on mitochondria are not dependent on apoptosis and may

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represent a primary site of TNFct action. An ultracondensed mitochondrial morphology is distinct from the orthodox and condensed conformations found in non-apoptotic cells representing resting and activated states, respectively. Progression from orthodox to condensed and ultracondensed mitochondrial morphologies occurred in CCRF-CEM exposed to TNFct and suggested that mitochondrial activation was an underlying mechanism. These authors identified mitochondrial swelling only as a late event following TNFct treatment and did not observe it in cells with apoptotic morphologies. Ultracondensed mitochondria appeared to be structurally intact, although biochemical function was not assessed in this study.

Regulation of Mitochondrial Biogenesis Several hundred individual proteins in mammalian mitochondria are encoded on nuclear chromosomes and translated in the cytoplasm as precursor proteins. The majority of evidence indicates that mitochondrial import of cytoplasmic proteins occurs post-translationally, although examples of co-translational import have been found (Verner, 1993). The circular mitochondrial genome encodes 13 proteins, all of which are subunits of oxidative phosphorylation complexes I, III, IV and F0/FI ATPase. 22 tRNAs and the genes for 12S and 16S rRNA for the separate mitochondrial translation system are also present in mitochondrial DNA. The assembly of mitochondria, and in particular multisubunit enzymes, involves coordinated gene expression at both nuclear and mitochondrial gene loci. Two transcription factors, NRF-1 (nuclear respiratory factor) and NRF-2, control expression of multiple chromosomal genes as one mechanism to ensure co-regulation of mitochondrial components (Scarpulla, 1997). NRF-I is a nuclear phosphoprotein that binds as a homodimer to a consensus DNA sequence (Gugneja and Scarpulla, 1997). NRF-1 stimulates transcription of respiratory proteins, components of mitochondrial DNA replication and transcription activities, and biosynthetic enzymes for heme synthesis. The NRF-2 transcription factor is a multisubunit activator that bind to ets-like recognition sites. Three subunits are shared with the murine GA-binding protein. Both NRF-1 and NRF-2 contain unique activation domains defined by tandemly arranged clusters of hydrophobic residues (Gugneja et al., 1996). The expression of mitochondrial structural genes is coordinated by transcription of H and L strands in mitochondrial DNA as single transcription units, producing 2 polycistronic mRNAs including all 13 genes. Promoter elements for each strand in the mitochondrial D-loop region are under the control of a nuclear transcription factor, MTF-A, which is in turn regulated by NRF-1 and NRF-2 (Virbasius and Scarpulla, 1994). Mitochondrial proliferation occurs in several physiologic contexts (see Table 1). Brown fat adipocytes have a high content of mitochondria important for the tissue function of thermogenesis. Tissue-specific expression of uncoupling protein results in low rates of oxidative phosphorylation despite high substrate turnover, with loss of chemical bond energy as heat. In the normal sequence of brown fat differentiation from interstitial cells to preadipocytes and adipocytes, it has been estimated that the number of mitochondria per cell increases by a factor of five, while relative volume increases

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14-fold. Mitochondrial proliferation is completed at the pre-adipocyte stage prior to uncoupling protein expression (Goglia et al., 1992). Table 1.

Mitochondrial Proliferation in Physiological and Pathological Settings

PHYSIOLOGICAL

PATHOLOGICAL

Brown adipocyte differentiation Myogenic differentiation Stimulation of muscle contractile activity Thyroid hormone

Apoptosis Androgenic hormones Antiviral nucleoside analogs Peripheral benzodiazepine receptor ligands Peroxisome proliferators Mitochondrial encephalomyopathies Cholestasis Oncocytic tumors

Cardiac and striated muscle adapt to exercise with increases in mitochondrial content (Moyes et al., 1998). Coordinated expression of both nuclear and mitochondrial genomes results from heightened contractile activity of sufficient duration. Steady state levels of an essential RNA component of the mitochondrial RNA-processing endonuclease (MRP-RNA) increase 14-fold within 14 days of skeletal muscle stimulation (Ordway et al., 1993). In paced cardiac tissue, transcription of the cytochrome c gene is under the control of immediate early genes c-fos and c-jun, as well as NRF-1 (Xia et al., 1998). Electrical pacing, by activation of c-jun N-terminal kinase activity, also contributes to transcriptional regulation via CRE sites in this model. Cellular differentiation is frequently associated with mitochondrial proliferation (Heerdt et al., 1996). Myogenic differentiation in the C2C12 cell line is associated with increased mitochondrial mass. Inhibition of mitochondrial biogenesis using the mitochondrial translation inhibitor tetracycline impaired myotube formation (Hamai et al., 1997). Several muscle-specific genes (creatine kinase and troponin-I) were suppessed in chloramphenicol-treated C2C12 cells, while others are normally induced (myoD and myogenin), arguing against non-specific toxicity. Several non-peptide hormones have effects on mitochondrial replication. Thyroid hormone has well known effects to increase cellular respiration and mitochondrial proliferation. Several nuclear and mitochondrial genes are induced by thyroid hormone including mt glycerol-3-phosphate dehydrogenase (Gong et al., 1998; Pillar and Seitz, 1997). Uncoupling effects leading to thermogenesis have been described and may involve direct, non-genomic functions of thyroid hormone in addition to transcriptional regulation of uncoupling protein expression in brown adipose tissue (Luvisetto, 1997; Rabelo et al., 1995). Conversely, hypothyroid states are associated with downregulation of several mitochondrial proteins (Izquierdo et al., 1995). Rats treated with the androgenic hormone dehydroepiandrosterone develop foci of proliferating hepatocytes and eventually adenomas and hepatocellular carcinomas. These lesions are composed of amphophilic cells with marked proliferation of mitochondria (Metzger et al., 1995).

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Several drugs in clinical use have direct effects on mitochondrial functions manifested as a mitochondrial proliferation phenotype. Antiviral nucleoside analogs including azidothymidine (AZT) and 2',3'-dideoxycytidine inhibit mitochondrial DNA replication (Lewiset al., 1991; Lewis et ak,, 1992). Increased mitochondriogenesis results as part of a delayed mitochondrial DNA depletion syndrome, but mitochondrial proliferation can also be seen after short exposures, prior to reduction of mitochondrial DNA (Hobbs et al., 1995). Ultrastructural changes similar to mitochondrial pyknosis are the earliest pathologic finding in AZT-induced myopathy (Cupler et al., 1995). The mitochondrial effects of this category of drugs are responsible for the fulminant hepatic failures and deaths observed with the antiviral agent FIUR in hepatitis C-infected patients (Colacino, 1996). The peripheral benzodiazepine receptor is located at the outer mitochondrial membrane and may function as part of the permeability transition pore. The PBR-specific ligands, PKlI195 or Ro5-4864, stimulates mitochondrial proliferation in pituitary tumor GH3 cells (Black et al., 1994). Upregulation of PBR-binding of specific ligands occurs within 24 hr following portal-caval anastomosis in rat models of portosystemic encephalopathy (PSE) (Leong et al., 1994). Mitochondrial proliferation is a pronounced feature of astrocytes in PSE. Another class of drugs with mitochondrial effects are the peroxisome proliferators. Although not as well studied, certain drugs in this class (e.g. clofibrate) result in mitochondrial as well as peroxisome proliferation (Brass, 1992). Among diseases characterized by mitochondrial proliferation, the classic example would be mitochondrial encephalomyopathies. Affected muscle fibers are recognized by light microscopy as ragged red fibrs. This appearance is caused by the accumulation of abnormal mitochondria in subsarcolemmal and intermyofibrillar zones. Mitochondria in "ragged red fibers" are enlarged, contain condensed or vacuolated matrix, and most characteristically para-crystalline inclusions composed of protein, predominantly creatine kinase (Stadhouders et al., 1994). Fast twitch, type I fibres are predominantly affected in these diseases. Histochemical studies demonstrate the association of mitochondrial enzyme deficiency with mitochondrial accumulation and myocyte atrophy. The patchy distribution of the cellular phenotype reflects the heteroplasmic maternal inheritance of mitochondrial DNA mutations, at least in part. The underlying genetic defects are large deletions in mitochondrial DNA or point mutations in tRNA genes and result in complex syndromes with phenotypes reflecting site-specific penetrance of the mitochondrial defect. The cellular changes in non-muscle cell-types have not been studied to the same extent, but mitochondrial proliferation has been described in endothelial cells, perhaps contributing to stroke-like episodes in MELAS, and is probably ubiquitous (Campos et al., 1997; Mita et al., 1995; Kaufmann et al., 1996). Mitochondrial proliferation has been described in both hepatocytes and bile duct epithelial cells in secondary cholestatic diseases (Tobe, 1982; Krahenbuhl et al., 1992). Bile duct ligation in rodents activates mitochondrial synthesis in biliary epithelium and is associated with enhanced expression of nuclear genes encoding mitochondrial proteins (Forestier et al., 1997). Finally, oncocytic tumors that contain high numbers of mitochondria have been described for virtually every neoplastic site (Tallini, 1998). The occurrence of oncocytic

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hyperplasias and metaplasias supports a tumor progression model. Mitochondria in liver and parathyroid oncocytic foci have been documented to have respiratory defects, suggesting a mechanism for mitochondrial proliferation akin to mitochondrial myopathies (Muller-Hocker, 1998). Oncocytic tumors may be benign or malignant, with the proportion depending on the tissue of origin.

Summary While there is little doubt remaining that mitochondrial dysfunction is an important step in apoptosis resulting in the release of important cofactors for cytoplasmic effectors of apoptosis, the mechanism of mitochondrial damage is more contentious. Particularly problematic are the multiplicity of signals and pathways that must have connections with mitochondrial integrity, including cell cycle, growth, DNA and macromolecular damage, growth factors and extracellular matrix. It seems likely that such a mechanism will be fairly complex and include some redundant steps that are linked to distinct signaling pathways. Our analysis of mitochondrial responses in apoptosis indicates that loss of mitochondrial transmembrane potential, either due to permeability transition pore formation or disruption of membrane integrity, is not a universal pathway in apoptosis. When mitochondrial depolarization is a prominent finding in apoptosis, it is a relatively delayed response and an insensitive indicator of mitochondrial damage. The earliest and most common mitochondrial responses in apoptosis involve changes in ultrastructural morphology and biosynthetic activity. These early events can be detected using fluorescent dyes that bind mitochondrial constituents independently of membrane potential. Mitochondrial proliferation in the context of apoptosis is closely linked to the accumulation of hypofunctional mitochondria and, rather than a derangement of existing mitochondria, suggests that bioassembly of new mitochondria is defective during apoptosis. This process may be linked to unbalanced signaling pathways or uncoupled cell growth states that function as apoptotic triggers.

References Black, K.L., Shiraishi, T., Ikezak, K., Tabuchi, K. & Becker, D.P. (1994). Peripheral benzodiazepine stimulates secretion of growth hormone and mitochondrial proliferation in pituitary tumour GH3 cells. Neuro. Res. 16, 74-80. Brass, E.P. (1992). Translation rates of isolated liver mitochondria under conditions of hepatic mitochondrial proliferation. Biochem.J. 288, 175-180. Camilleri-Broet, S., Vanderwerff, H., Caldwell, E. & Hockenbery, D. (1998). Distinct alterations in mitochondrial mass and function characterize different models of apoptosis. Exp. Cell Res. 239, 277-292. Campos, Y., Martin, M.A., Rubio, J.C., Gutierrez del Olmo, M.C., Cabello, A. & Arenas, J. (1997). Bilateral striatal necrosis and MELAS associated with a new T3308C mutation in the mitochondiral ND1 gene. Biochem. Biophys. Res. Comm. 238,323-325.

138

M. Mancini, S. Camilleri-BrOet, B.O. Anderson and D.M. Hockenbery

Colacino, J.M. (1996). Mechanisms for the anti-hepatitis B virus activity and mitochondrial toxicity of fialufidine (FIAU). Antiviral. Res. 29, 125-139. Cupler, E.J., Danon, M.J., Jay, C., Hench, K., Ropka, M. & Dalakas, M.C. (1995). Early features of zidovudine-associated myopathy: histopathological findings and clinical correlations. Acta Neuropathol. 90, 1-6. Durham, D.& Rubel, E.W. (1985). Afferent influences on brain stem auditory nuclei of the chicken: changes in succinate dehydrogenase activity following cochlea removal. J. Comp. Neurol. 231,446-456. Durham, D., Matschinsky, F.M. & Rubel, E.W. (1993). Altered malate dehydrogenase activity in nucleus magnocellularis of the chicken following cochlea removal. Hear Res. 70, 151-159. Forestier, M., Soliez, M., Isbeki, F., Talos, C., Reichen, J. & Krahenbuhl, S. (1997). Hepatic mitochondrial proliferation in rats with secondary biliary cirrhosis: time course and mechanisms. Hepatology 26, 386-391. Garden, G.A., Canady, K.S., Lurie, D.I., Bothwell, M. & Rubel, E.W. (1994). A biphasic change in ribosomal conformation during transneuronal degeneration is altered by inhibition of mitochondrial, but not cytoplasmic protein synthesis. J. Neurosci. 14, 1994-2008. Goglia, F., Geloen, A., Lanni, A., Minaire, Y. & Bukowiecki, L.J. (1992). Morphometric-stereologic analysis of brown adipocyte differentiation in adult mice. Am. J. Physiol. 262, CI018-1023. Gong, D.W., Bi, S., Weintraub, B.D. & Reitman, M. (1998). Rat mitochondrial glycer-3-phosphate dehydrogenase gene: multiple promoters, high levels in brown adipose tissue, and tissue-specific regulation by thyroid hormone. DNA Cell Biol. 17, 301-309. Gugneja, S., Virbasius, C.M. &Scarpulla, R.C. (1996). Nuclear respiratory factors i and 2 utilize similar glutamine-containing clusters of hydrophobic residues to activate transcription. Mol. Cell. Biol. 16, 5708-5716. Gugneja, S. & Scarpulla, R.C. (1997). Serine phosphorylation within a concise amino-terminal domain in nuclear respiratory factor 1 enhances DNA binding. J. Biol. Chem. 272, 18732-18739. Hamai, N., Nakamura, M. & Asano, A. (1997). Inhibition of mitochondrial protein synthesis impairs C2C12 myoblast differentiation. Cell Struct.Funct. 22, 421-431. Hartlage-Rubsamen, M. & Rubel, E.W. (1996). Influence of mitochondrial protein synthesis inhibition on deafferentation-induced ultrastructural changes in nucleus magnocellularis of developing chicks. J. Comp. Neurol. 371,448-460. Heerdt, B.G., Houston, M.A., Rediske, J.J. & Augenlicht, L.H. (1996). Steady-state levels of mitochondrial messenger RNA species characterize a predominant pathway culminating in apoptosis and shedding of HT29 human colonic carcinoma cells. Cell Growth Differ. 7, 101-106. Henkart, P.A. & Grinstein, S. (1996). Apoptosis: Mitochondria resurrected? J. Exp. Med. 183, 1293-1295. Hobbs, G.A., Keilbaugh, S.A., Rief, P.M. & Simpson, M.V. (1995). Cellular targets of 3'-azido-3'deoxythymidine: an early (non-delayed) effect on oxidative phosphorylation. Biochem. Pharmacol. 50, 381-390. Hockenbery, D.M., Oltvai, Z.N., Yin, X.-M., Milliman, C.L. & Korsmeyer, S.J. (1993). Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75,241-251. Hyde, G.E. & Durham, D. (1990). Cytochrome oxidase response to cochlea removal in chicken auditory brainstem neurons. J. Comp. Neurol. 297, 329-339. Hyde, G.E. & Durham, D. (1994a). Rapid increase in mitochondria volume in nucleus magnocellularis neurons following cochlea removal. J. Comp. Neurol. 339, 27-48. Hyde, G.E. & Durham, D. (1994b). Increased deafferentiation-induced cell death in chick brainstem auditory neurons following blockade of mitochondrial protein synthesis with chloramphenicol. J. Neurosci.

Proliferative Mitochondrial Dysfunction and Apoptosis

139

14, 291-300. Izquierdo, J.M., Jimenez, E. & Cuezva, J.M. (1995). Hypothyroidism affects the expression of the beta-F1ATPase gene and limits mitochondrial proliferation in rat liver at all stages of development. Ear. J. Biochem. 232, 344-350. James, T.N., Terasaki, F., Pavlovich, E.R. & Vikhert, A.M. (1993). Apoptosis and pleomorphic micromitochondriosis in the sinus nodes surgically excised from five patients with the long QT syndrome. J Lab Clin Med 122, 309-323. Jia, L., Dourmashkin, R.R., Newland, A.C. & Kelsey, S.M. (1997). Mitochondrial ultracondensation, but not swelling, is involved in TNF alpha-induced apoptosis in human T-lymphoblastic leukaemic cells. Leuk. Res. 21,973-983. Kaufmann, P., Koga, Y., Shanske, S., Hirano, M., DiMauro, S., King, M.P., Schon, E.A. (1996). Mitochondrial DNA and RNA processing in MELAS. Ann. Neurol. 40, 172-180. Krahenbuhl, S., Krahenbuhl-Glauser, S., Stucki, J., Gehr, P. & Reichen, J. (1992). Stereological and functional analysis of liver mitochondria from rats with secondary biliary cirrhosis: impaired mitochondrial metabolism and increased mitochondrial content per hepatocyte. Hepatology 15, 1167-1172. Kroemer, G., Zamzani, N. & Susin, S.A. (1997). Mitochondrial control of apoptosis. Immunol.Today 18, 44-51. Kung, A.L., Sherwood, S.W. & Schimke, R.T. (1993). Differences in the regulation of protein synthesis, cyclin B accumulation, and cellular growth in response to the inhibition of DNA synthesis in Chinese hamster ovary and HeLa $3 cells. J. Biol. Chem. 268, 23072-23080. Leong, D.K., Therrien, G., Swain, M.S. & Butterworth, R.F. (1994). Densities of bindingsites for the "peripheral-type" benzodiazepine receptor ligand 3H-PK11195 are increased in brain 24 hours following portacaval anastomosis. Metab. Brain Dis. 9, 267-273. Lewis, W., Papoian, T., Gonzalez, B., Louie, H., Kelly, D.P., Payne, R.M. & Grody, W.W. (1991). Mitochondrial ultrastructural and molecular changes induced by zidovudine in rat hearts. Lab. Invest. 65, 228-236. Lewis, L.D., Hamzeh, F.M. & Lietman, P.S. (1992). Ultrastructural changes associated with reduced mitochondrial DNA and impaired mitochondrial function in the presence of 2'3'-dideoxycytidine. Antimicrob. Agents. Chemother. 36, 2061-2065. Luvisetto, S. (1997). Hyperthyroidism and mitochondrial uncoupling. Biosci. Rep. 17, 17-21. Maftah, A., Petit, J.-M., Ratinaud, M.-H. & Julien, R. (1989). 10-N Nonyl-acridine orange; a fluorescent probe which stains mitochondria independently of their energetic state. Biochem. Biophys. Res. Commun. 164, 185-190. Mancini, M., Nicholson, D.W., Roy, S., Thornberry, N.A., Peterson, E.P., Casciola-Rosen, L.A. & Rosen, A. (1998). The caspase-3 precursor has a cytosolic and mitochondrial distribution: implications for apoptotic signaling. J. Cell Biol. 140, 1485-1495. Mancini, M., Anderson, B.O., Sedghinasab, M., Paty, P.B., Caldwell, E. & Hockenbery, D. (1997). Mitochondrial proliferation and paradoxical membrane depolarization during herbimycin A-induced terminal differentiation and apoptosis in a human colon carcinoma cell line. J. Cell Biol. 138,449-469. Metzger, C., Mayer, D., Hoffmann, H., Bocker, T., Hobe, G., Benner, A. & Bannasch, P. (1995). Sequential appearance and ultrastructure of amphophilic cell foci, adenomas, and carcinomas in the liver of male and female rats treated with dehydroepiandrosterone. Toxol. Pathol. 23,591-605. Mita, S., Tokunaga, M., Kumamoto, T., Uchino, M., Nonaka, I. & Ando, M. (1995). Mitochondrial DNA mutation and muscle pathology in mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes. Muscle Nerve 3, S113-118. Moyes, C.D., Battersby, B.J. & Leary, S.C. (1998). Regulation of muscle mitochondrial design. J. Exp.

140

M. Mancini, S. Camilleri-Bri~et, B.O. Anderson and D.M. Hockenbery

Biol. 201,299-307. Muller-Hocker, J. (1998). Oncocytic metaplasia/neoplasia - morphology, biochemistry, and molecular genetics. Pathologe 19, 104-114. Ordway, G.A., Li, K., Hand, G.A. & Williams, R.S. (1993). RNA subunit of mitochondrial RNA-processing enzyme is induced by contractile activity in striated muscle. Am. J. Phys. 265, C1511-C1516. Pillar, T.M. & Seitz, H.J. (1997). Thyroid hormone and gene expression in the regulation of mitochondrial respiratory function. Eur. J. Endocrinol. 136, 231-239. Rabelo, R., Schifman, A., Rubio, A., Sheng, X. & Silva, J.E. (1995). Delineation of thyroid hormoneresponsive sequences within a critical enhancer in the rat uncoupling protein gene. Endocrinology 136, 1003-1013. Reers, M., Smiley, S.T., Mottola-Hartshom, C., Chen, A., Lin, M. &Chen, L.B. (1995). Mitochondrial membrane potential monitored by JC-1 dye. Methods Enzymol. 260, 406-417. Reipert, S., Berry, J., Hughes, M.F., Hickman, J.A. & Allen, T.D. (1995). Changes of mitochondrial mass in the hemopoietic stem cell line FDCP-Mix after treatment with etoposide: a correlative study by multiparameter flow cytometry and confocal and electron microscopy. Exp Cell Res. 221,281-288. Samali, A., Zhivotovsky, B., Jones, D.P.& Orrenius, S. (1998). Detection of pro-caspase-3 in cytosol and mitochondria of various tissues. FEBS Lett. 431,167-169. Scarpulla, R.C. (1997). Nuclear control of respiratory chain expression in mammalian cells. J Bioenerg. Biomembr. 29, 109-119. Smiley, S.T. (1991). Intracellular heterogeneity in mitochondrial membrane potential revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci USA 88, 3671-3675. Stadhouders, A.M., Jap, P.H.K., Winkler, H.-P. & Eppenberger, H.M., Wallimann, T. (1994). Mitochondrial creatine kinase: a major constituent of pathological inclusions seen in mitochondrial myopathies. Proc. Natl. Acad. Sci. U.S.A. 91, 5089-5093. Susin, S.A., Zamzani, N. & Kroemer, G. (1998). Mitochondria as regulators of apoptosis: doubt no more. Biochim. Biophys. Acta 1366, 151-165. Tallini, G. (1998). Oncocytic tumours. Virchows Archi. 433, 5-12. Tobe, K. (1982). Electron microscopy of liver lesions in primary biliary cirrhosis. I. Intrahepatic bile duct oncocytes. Acta Pathol Jpn. 32, 57-70. Urbani, L., Sherwood, S. & Schimke, R.T. (1995). Dissociation of nuclear and cytoplasmic cell cycle progression by drugs employed in cell synchronization. Exp. Cell Res. 219, 159-168. Verner, K. (1993). Co-translational protein import into mitochondria: an alternative view. Trends Biochem. Sci. 18, 366-371. Virbasius, J.V. & Scarpulla, R.C. (1994). Activation of the human mitochondrial transcription factor A gene by nuclear respiratory factors: A potential regulatory link between nuclear and mitochondrial gene expression in organelle biogenesis. Proc. Natl. Acad. Sci.U.S.A. 91, 1309-1313. Xia, Y., Buja, L.M. & Mcmillin, J.B. (1998). Activation of the cytochrome c gene by electrical stimulation in neonatal rat cardiac myocytes - role of NRF-1 and c-jan. J. Biol. Chem. 273, 12593-12598.

THE BCL-2 FAMILY OF PROTEINS AND THEIR ACTIONS WITHIN THE MOLECULAR MACHINERY OF CELL DEATH Q I N G GUO, SIC L. C H A N and I N N A K R U M A N

Table of Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Apoptosis: A Significant Form of Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Apoptosis and Necrosis: Differences and Similarities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Four Phases of Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multiple Apoptotic Signal Transduction Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiological and Pathological Significance of Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . The Pro- and Anti-Apoptotic Genes in C. elegans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bcl-2 Family of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure of Bcl-2 Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure-Activity Relationship among Bcl-2 Subfamilies . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Mitochondrial Function by Bcl-2 Family Proteins . . . . . . . . . . . . . . . . . . . . . . Regulation of Apoptosis by Bcl-2 Family Proteins in the Nervous System . . . . . . . . . . . . . . Neuronal Apoptosis during Embryonic Development and in Neurodegenerative Conditions. Expression and Distribution of Bcl-2 in the Nervous System . . . . . . . . . . . . . . . . . . . . . . . . Maintenance of Neuronal Population by Bcl-2 in Normal Physiological Conditions . . . . . In Vitro and In Vivo Evidence for the Neuroprotective Effect of Bcl-2 Following Insults . Evidence for Bcl-x as a Neuronal Survival Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pro-apoptotic Actions of Bax in the Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

141 142 142 145 146 146 146 149 151 153 158 164 164 168 168 169 177 177 178

Introduction

Apoptotic cell death occurs during normal development and in pathological (genetically or environmentally based) settings, in many different invertebrate and vertebrate organ systems, and plays an indispensable role in the development and maintenance of organism homeostasis (Kerr et al., 1972; Wyllie et al., 1984; Ellis et al., 1991; Ameisen et al., 1996; Vaux and Strasser, 1996). Genetic analysis in the nematode C. e l e g a n s uncovered the existence of a death program that is highly conserved throughout evolution and laid the foundation for the identification o f the core components o f the molecular apoptotic machinery (Driscoll, 1992). During nematode development, 131 of the 1090 cells formed in the hermaphrodite are programmed to die. Three genes - - c e d - 3 , c e d - 4 , and c e d - 9 - - have been shown to be essential for all 131 cell deaths (Driscoll, 1992). c e d - 3 gene product is required for cell death to occur 141 Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. 141 -- 195 © 2001 Elsevier Science. Printed in the Netherlands.

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and shows homology to some of the cysteine proteases of the caspase family in mammalian cells (Yuan et al., 1993; Chinnaiyan et al., 1997a,,b). The product of the ced-9 gene functions, in most instances, as a cell survival gene and shares homology with the product of the bcl-2 proto-oncogene in mammalian cells (Hengartner and Horvitz, 1994). Bcl-2 is a 26 kD membrane-associated protein that was originally discovered from the breakpoint of the t(14; 18) chromosomal translocation in follicular lymphoma (Bakhshi et al., 1985; Cleary et al., 1986). Using a variety of techniques, including the analysis of Bcl-2 related proteins by immunoprecipitation and yeast two-hybrid screening, a family of at least fourteen Bcl-2 proteins in mammalian cells has emerged. It rapidly became evident that members of this family contain three highly conserved structural domains called the Bcl-2 homology (BH)I, BH2 and BH3 domains that are responsible for the ability of these proteins to form hetero- or homodimers in dynamic equilibrium. The mammalian Bcl-2 family consists of both pro-apoptotic and anti-apoptotic members. Accumulating evidence suggests that Bcl-2 family members play an important role in the regulation of apoptotic cell death in the nervous system. In this chapter, we will discuss the different forms of cell death, and particularly, the differences and similarities between apoptotic and necrotic cell death. We will also discuss the different apoptotic pathways (including caspase-dependent and caspase-independent pathways) and the actions of the members of the Bcl-2 family with the molecular machinery of cell death. Pro- and anti- apoptotic genes in C. elegans, classification and structure of the Bcl-2 family of proteins will be discussed in detail. Mitochondria are one of the major players in the cell death process in mammalian cells, and Bcl-2 family proteins have been implicated in the regulation of mitochondrial pathophysiology. Regulation of mitochondrial function by Bcl-2 family proteins and activation of caspases in apoptosis will also be discussed. Finally, we will provide experimental evidence from this lab and others showing the specific roles of the Bcl-2 family proteins in neuronal apoptosis that occurs during normal development and in neurological disease and injury conditions.

Apoptosis: A Significant Form of Cell Death Apoptosis and Necrosis: Differences and Similarities Cell death in multicellular organisms may occur in multiple forms. Traditional necrotic cell death occurs in response to highly toxic compounds, severe cold or heat stress, or traumatic injury that promotes random degradation of DNA, rapid membrane damage and extracellular release of the cell content, resulting in an inflammatory response of the organism (Wyllie et al., 1980; Majno and Joris, 1995; Searle et al., 1982). However, to achieve and maintain homeostasis, cells in multicellular organisms selfdestruct in a different form when they are no longer needed or if they are damaged. This is accomplished by activation of genetically regulated cell suicide machinery that requires the active participation of the cell in a process known as apoptosis, a form of programmed cell death (PCD) (Wyllie, 1987a,b). Therefore, in contrast to necrosis, apoptosis is considered to be an active cell death mechanism often requiring novel

The Bcl-2 Protein Family and Cell Death

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transcription and translation of specific genes, which leads to distinct morphological alterations of the cell, such as DNA condensation, cell shrinkage, and membrane blebbing, with no signs of disruption of membrane integrity or spillage of its intracellular milieu into the extracellular space. Cellular endonuclease is often activated during apoptotic cascades, cleaving chromosomal DNA into nucleosome-length fragments that are detectable as 180-200 base-pair (bp)ladders on agarose gels. The membrane of an apoptotic cell actively blebs but remains intact, ultimately vasculating the cell apart into membrane-bound apoptotic bodies that contain cytoplasmic and/or nuclear material. The apoptotic cell debris is removed by a non-inflammatory process (Arends and Wyllie, 1991). Apoptosis was first described more than 20 years ago by Kerr, Wyllie, and Currie from University of Edinburgh, describing apoptosis in the peripheral liver cells after ligation of the portal vein (Kerr, 1971; Kerr et al., 1972). In its ancient context, apoptosis was derived from two Greek roots: apo (away) and ptosis (to fall, also a medical term referring to the dropping of eyelids) that was used to describe petals falling from flowers or leaves from trees. Kerr and colleagues described several epithelial cell-types that underwent orderly death during development. These included cells that died owing to withdrawal of survival signals and cells that had been exposed to toxins. However, it took nearly 20 years for scientists to recognize the fundamental importance of apoptosis. Today, there has been an explosion of discoveries in resolving the structure and function of genes, signal molecules, and signal transduction pathways regulating apoptosis. A number of cellular gene products have been identified that regulate and modulate the cell death pathway (Wyllie, 1981, 1987a,b, 1995; Wyllie et al., 1980,1992; Majno and Joris, 1995; Steller, 1995; Vaux et al., 1994; Stewart, 1994; Ellis et al., 1991; Arends et al., 1990; Schwartzman and Cidlowski, 1993). It has been recognized that apoptosis is conserved throughout evolution from nematode to man, although the process in mammals is considerably more complex involving multiple isoforms of components of cell death machinery (Tomei and Cope, 1991; Osborne and Schwartz, 1994; Majno and Joris, 1995; Steller, 1995; Kroemer, 1997; Kroemer et al., 1995). As stated, apoptotic death can be distinguished from necrotic cell death in many aspects. In apoptosis, cells shrink and dissociate from surrounding cells, their organelles retain definition for a long time, and the nucleus displays a distinctive pattern of heterochromatization and eventual fragmentation. On the other hand, necrotic cells swell, their mitochondria dilate, other organelles dissolve, and plasma membranes rupture, whereas the nuclear changes are relatively unremarkable The fundamental differences between apoptosis and necrosis are summarized in Table 1. However, apoptosis and necrosis are not two completely different and unrelated form of cell death. For example, apoptotic cells may undergo secondary necrosis (Itoh et al., 1995; Sasaki et al., 1996; Shimizu et al., 1996a,b,c). DNA ladders indicative of internucleosomal endoproteolysis can be observed in necrotic cells (Fuduka et al., 1993; Shen et al., 1992). Agents supposed to be classic causes of necrosis have now been shown to cause apoptosis as well, suggesting the same toxin can induce apoptosis or necrosis depending on the dose and duration of treatment used (Kroemer, 1995; Shimizu et al., 1996a,b,c; Mills et al., 1996; Beeri et al., 1995; Tanaka et al., 1994; Vaux et al., 1996). Bcl-2, the vertebrate homologue of ced-9 and the pre-eminent inhibitor of programmed cell death

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and/or apoptosis, also ameliorates necrosis (Martino et al., 1994; Kane et al., 1993; Shimizu et al., 1995; 1996a.b). Mitochondrial permeability transition is involved in both apoptosis and necrosis (Kroemer et al., 1997). Oxidized low-density lipoproteins have been reported to cause necrosis and apoptosis (Escargueil-Blanc et al., 1994). Maintenance of high ATP levels favors apoptosis over necrosis, whereas inhibition of caspase activation transforms apoptosis into necrosis (Leist et al., 1997; Eguchi et al., 1997; Xiang et al., 1996; McCarthy 1997; Hirsch et al., 1997). It is important to note that not all cell death can be readily fitted into an apoptosis or necrosis category (Majno and Joris, 1995; Escargueil-Blanc et al., 1994; Robertson and Thomason, 1982; Grasl-Kraupp et al., 1995; Vaux et al., 1996; Fukuda et al., 1993). Several forms of dying cells fail to fit neatly into the specific morphology supposed to be seen in necrotic or apoptotic cells. Since apoptotic cells shrink, and non-apoptotic cells swell, it has been suggested that oncosis (for swelling) be substituted for necrosis, which is only a subset of oncotic cell deaths (Ellis et al., 1991 ; Columbano, 1995; Clarke, 1990). Some investigators have divided the cell deaths that occur in development into three main types: Type 1 (apoptosis, as described above), Type 2 (autophagic cell death in which abundant autophagic vacuoles can be found), Type 3A (nonlysosomal disintegration in which there is a general disintegration of cytoplasm) and Type 3B (cytoplasmic type in which there is significant dilation of ER, nuclear envelope, Golgi and sometimes mitochondria, forming " empty" spaces in the cytoplasm). It is important to remember, however, that different morphologic appearances of cell death may not necessarily reflect fundamentally different biochemical mechanisms. Four Phases of Apoptosis Apoptotic death represents a cell's transduction of a signal (extrinsic or intrinsic), with subsequent changes in gene expression or the activation/inactivation of proteins already present in the cell. This results in the initiation of the cell death pathway, which if unchecked, leads to the rapid demise of the cell. Diverse stimuli can initiate the apoptotic process, and these signals are mediated through diverse signaling pathways. Among these are cell surface receptors, transmembrane domains, intracellular proteins involved in propagation of death signals (death domains), second messengers including inositol triphosphate and ceramides, Ca 2+ fluxes, reactive oxygen species, cell cycle regulating factors (cyclins and coupled cdc kinases), and proteins that act as either suppressors (e.g. Bcl2, iap) or activators (e.g. Bax) of cell death (Mills et al., 1996; Beeri et al., 1995; Hopcia et al., 1996; Squier et al., 1994). The activation of caspases is now recognized as a key biochemical marker of at least some forms of apoptosis (Miller et al., 1997; Williams and Henkart, 1994; Enafi et al., 1996; Alnemri et al., 1996; Henkart, 1996; Nichoson et al., 1995). The apoptotic process can be roughly divided into four phases: 1) Stimulus phase: The stimulation that provokes the apoptotic response in a cell-type- and signaldependent manner. The stimulation can be an external signal delivered through the surface receptors or signals originate inside the cell from the action of drug, toxin, or radiation. 2) Detection, activation and signaling phase: this phase encompasses a great variety of signal transduction pathways that mediate signals from outside the cell,

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as well as others that originate inside the cell. 3) Effector phase: In this phase, signal transduction pathways send this message to the cell death machinery, as well as their positive and negative regulators are activated. 4) Postmortem phase: In this phase, cell's chromatin condenses and its DNA is degraded. Under in vivo conditions, dying cells are recognized and engulfed by neighboring ceils. Multiple Apoptotic Signal Transduction Pathways One central question in the field of apoptosis is whether an irreversible commitment to death leads from a common biochemical pathway to the morphological features of apoptosis. Considerable evidence supports the view that there is more than one basic genetic program for death and more than one final pathway (McConkey, 1996; McCarthy et al., 1997). For example, it has been demonstrated that the internucleosomal DNA lysis is only a frequently associated but nonessential event of apoptosis. Apoptotic cell death can occur through caspase-independent and caspase-dependent pathways (McConkey, 1996; McCarthy et al., 1997; Sarin et al., 1997; Anel et al., 1997; Lotem and Sachs, 1996). Some cell death pathways require macromolecular synthesis leading to lethal consequences but do not require caspase activation or in which caspase activation is a secondary event. In contrast, some other cell death pathways require caspases as the central agents of death but do not require macromolecular synthesis. The multiple pathways of apoptosis, which include the caspase-dependent and caspaseindependent pathways, are summarized in Figure 1. Physiological and Pathological Significance of Apoptosis Physiologically, apoptosis is important for maintaining homeostasis during embryonic development and plays an important role in normal aging. Apoptosis is triggered during developmental transitions in situations that lead to sculpting of structures, deleting unneeded structures, controlling cell numbers and eliminating superfluous or potentially harmful cells in multicellular organisms (Wyllie et al., 1980; Duke et al., 1996; Fukuda, 1997). In the vertebrate nervous system, approximately 50% of neurons die by apoptosis during maturation. The dysregulation of apoptosis is the basis of many human diseases (Dixon et al., 1997; Gougeon and Montagnier, 1993; Mountz et al., 1994; Fisher, 1994). An abnormal resistance to apoptosis induction correlates with malformations, autoimmune diseases or cancer due to the persistence of self-specific immunocytes or mutated cells, respectively. In contrast, enhanced apoptotic decay of cells participates in acute pathologies (infection by toxin-producing microorganisms, ischemia-reperfusion damage, or infarction) as well as in chronic diseases (neurodegenerative and neuromuscular diseases, AIDS).

The Pro- and Anti-Apoptotic Genes in C. elegans The nematode, C. elegans has been particularly valuable in the studies of the genetic control of apoptotic cell death. During nematode development, 131 of the 1090 cells

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triggered by a variety of apoptotic insults, including trophic factor withdrawal and irradiation, etc., which result in disruption of calcium homeostasis, increased production of free radicals, release of cytochrome C and mitochondrial dysfunction. Propagation of apoptotic signals can lead to activation of caspase-dependent (which requires activation of effector caspases) and caspase-independent (which involves activation of non-caspase proteases and induction of specific gene expressions) pathways of apoptosis. During execution of apoptotic cell death, cell death substrates (such as PARP) are cleaved, nuclear DNA becomes fragmented, and apoptotic bodies are formed. Caspase and other protease inhibitors, the pro- and anti-apoptotic members of the Bcl-2 family, and macromolecular synthesis inhibitors may modulate apoptotic cell death by different mechanisms and through different apoptotic pathways.

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formed in the hermaphrodite are programmed to die (Driscoll, 1992). Genetic analysis of C. elegans has determined a number of genes that are responsible for apoptosis that occurs during development (Driscoll, 1992). Fourteen genes have been identified that function at various steps in this process, among which egl-1, ces-1 and ces-2 are responsible for specification of cell death, ced-3, ced-4, ced-8 and ced-11 are associated with apoptotic cell death, ced-9 is associated with suppression of cell death, ced-1, ced-2, ced-6, ced-5, ced-7 and ced-lO are associated with phagocytosis of dead cells, and nuc-1 is required for phagocytes to degrade the DNA of the dying cell (Driscoll, 1992). Three of these genes -- ced-3, ced-4, and ced-9 -- have been shown to be essential for all 131 cell deaths (Alnemri et al., 1996; Chinnaiyan et al., 1997a,b; Wu et al., 1997; Yuan et al., 1993; Vaux et al., 1997; Zou et al., 1997; Hengartner, 1997; Hengartner and Horvitz, 1994). The ced-3 gene is required for cell death to occur; loss-of-function mutations in either gene eliminate all cell deaths (Xue et al., 1996; Yuan et al., 1990; 1993; Chinnaiyan et al., 1996; Miura et al., 1993). Similarities between the ced-3 gene product and the interleukin-lfS-converting enzyme (ICE) suggest that a cellular protease is responsible for apoptosis. Furthermore, the ced-3 gene product can induce apoptosis in transfected mammalian cells. The ced-4 gene encodes a highly hydrophilic 63-kDa novel protein (Chinnaiyan et al., 1997a,b). CED-4 can act as an adapter that allows interaction between CED-3 and CED-9. In addition, ectopic expression of the ced-4 gene in S. pombe leads to rapid chromatin condensation and apoptotic cell death. The CED-9 protein was isolated as a binding-partner of CED-4 in an interactive genetic screen (Hengartner et al., 1992; Hengartner and Horvitz, 1994). Normally, CED-4 is localized in the cytosol, but when CED-9 is expressed in mammalian cells, it targets CED-4 from the cytosol to intracellular membranes, suggesting that CED-9 plays an important role in the subcellular localization of CED-4. Loss-of-function mutations in ced-9 prevent the corresponding protein from associating with CED-4 and result in additional deaths, whereas gain-of-function mutations allow the survival of the doomed cells. The ced-3 gene product that can be directly inhibited by the product encoded by the ced-9 gene. Conversely, the ced-9 gene product can inhibit the pro-apoptotic function of the ced-3 gene, and it was shown that ced-9 is a C. elegans homologue of the mammalian bcl-2 gene. These results indicate that ced-9 functions as a cell survival gene (Alnemri et al, 1996; Chinnaiyan et al., 1997a,b; Wu et al., 1997; Yuan et al., 1993; Vaux et al., 1997; Zou et al., 1997; Hengartner, 1997; Hengartner and Horvitz, 1994). However, the recent observation that ced-4 exists in both death-promoting and deathprotecting forms, and that both forms are inhibited by ced-9, suggests that ced-9 may function in a complex way to either promote or inhibit death under different physiologic conditions.

Bcl-2 Family of Proteins The mammalian Bcl-2 family falls into two classes depending upon whether they induce or repress apoptosis (Table 2). Homologs of the Bcl-2 members have been found in frogs (Cruz-Reyes and Tata, 1995), birds (Cazals-Hatem et al., 1992; Gillet et al., 1995),

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and DNA viruses (Henderson et al., 1993; Nava et al., 1997; Sarid et al., 1997; Cheng et al., 1997). The latter includes death-inhibitory Bcl-2 family members, such as the adenovirus EIB19K protein (White et al., 1992), Epstein-Barr virus BHRF-1 (Henderson et al. 1993), African swine fever virus LMWF-HL (Neilan et al., 1993), and Kaposi-sarcoma associated herpes virus KSbcl-2 (Cheng et al., 1997). The purpose of these viral Bcl-2 homologs may be to counter the propensity of infected host cells to undergo apoptosis while infection is being established. Numerous experiments have established that Bcl-2 is capable of blocking a wide range of apoptotic stimuli in a variety of different cell-types, which suggests that Bcl-2 may block a final common pathway to apoptosis. Two non-mutually exclusive models for the action of Bcl-2 have been proposed. Heterodimerization between pro-apoptotic and anti-apoptotic Bcl-2 family members has been suggested to be an important property for Bcl-2 proteins to regulate cell survival. For many, but not all apoptotic signals, the balance between these competing activities may ultimately determine the cell fate. Analysis of various bcl-2 and bcl-xl mutants shows a correlation between the inability to heterodimerize and the failure to protect, suggesting that Bcl-2 and its closest homologues bind to and sequester their pro-apoptotic relatives, preventing them from conveying a death signal (Sedlak et al., 1995; Yin et al., 1994). Recently, bcl-2 mutants have been identified that do not bind Bax and Bak yet still block apoptosis (Cheng et al., 1996) suggesting an alternative model which Bcl-2 may regulate the caspase cascade (see below). The latter model was supported by genetic analysis, which suggest that ced-9 acts through ced-4 to keep ced-3 inactivated (Chinnaiyan, 1997a,b; Spector, 1997; Imler, 1997; Hu et al., 1998), and by the recent observations that Bcl-xl, which can directly interact with ced-4, associates with caspase-3 and caspase-9 in mamalians cells (Hu et al., 1998; Pan et al., 1998). The specificity for the induction or repression of apoptosis in any given cell may depend upon the interaction of the members that are expressed in that cell at that time. The existence of multiple Bcl-2 family members in mammals is likely due to the need to regulate cell death in a temporal and tissue-specific manner or to fine-tune the response to multiple survival or death signals. Consistent with this, it has been found that that bcl-2, bcl-xl, bcl-w, mcl-1 and A-1 exhibit differences in tissue expression, developmental expression and inducibility in response to extrinsic stimuli (Boise et al., 1995; Krajewski et al., 1994a,b; 1995; Lin et al., 1993; 1996; Yang et al., 1996). For instance, A-1 is the only known Bcl-2 family member that is inducible by inflammatory cytokines such as TNFct and IL-lft, suggesting it may play a protective role during inflammation. Studies in bcl-2 and bcl-xl null mice which have distinct properties demonstrated that the latter is critical for neuronal survival during embryonic development (Motoyama et al., 1995), whereas the former is required for the maintenance of some neuronal populations after the period of physiological death (Nakayama et al., 1994, Veis et al., 1993). This section will describe the structure of the Bcl-2 protein and the structure-activity relationships among the members of the Bcl-2 family, as well as the pathway in which each member acts to either repress or promote apoptosis.

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Structure of Bcl-2 Proteins The five most closely related mammalian Bcl-2 homologues, Bcl-2, Bcl-xl, Bcl-w, Bax contain the BH1, BH2 and BH3 domains but only the first three, which are anti-apoptotic, also bear the BH4 domain. As shown in Figure 2a, the BH1 domain spans amino acid residues 136-155 of the Bcl-xL protein, the BH2 spans residues 187-202, BH3 spans residues 93-107, and BH4 spans the N-terminal residues 10-30. The region spanning BH1 and BH2 domains is important for pore formation in the artificial membranes and Bcl-2 proteins could function as ion channels in subcellular organelle membrane. Though the small BH3 domain of the pro-apoptotic proteins appears to be essential for heterodimerization with anti-apoptotic Bcl-2 partners, deletions within the BH3 domain also abolish their cytotoxic activity (Chittenden et al., 1995; Ink et al., 1997). Mutations in several highly conserved residues in the BH3 domain have been shown to abolish both heterodimerization and apoptotic efficiency (Wang et al., 1996; Sattler et al., 1997; Zha et al.,. 1997; Boyd et al., 1995, Inohara et al., 1997, 1998). The importance of the BH3 domain for facilitating apoptosis has been highlighted by the discovery of a group of "BH3-only" pro-apoptotic members which can kill cells directly when overexpressed (Wang et al., 1996; Han et al., 1996; Inohara et al., 1997; see below The BH3 subfamily). The conserved N-terminal BH4 domain, which is restricted to some but not all anti-apoptotic members, is essential for the pro-survival function. Deletion of BH4 rendered Bcl-xL inactive though its ability to bind to pro-apoptotic members was not affected. Huang et al., (1998) demonstrated that Bcl-xl lacking BH4 failed to associate with the C. elegans ced-4 protein, a molecule that acts as a catalyst to stimulate CED-3 processing (Seshagiri, 1997; Chinnaiyan, 1997a,b). Zou et al., (1997) identified Apaf-1, a mamalian protein with similarity CED-4. Apaf-1, along with ATP, cytochrome C and caspase 9 is required for caspase 3 activation (Li, 1997). These findings have raised the possiblity that the BH4 domain allows the pro-survival Bcl-2 members to sequester CED-like molecules, thereby preventing activation of caspases (Huang, 1998; Chinnaiyan et al., 1997a,b, James et al., 1997; Wu et al., 1997). This is the first evidence that the Bcl-2 family of proteins directly regulate caspase activation via CED-4 like molecules. The BH4 domain of Bcl-2 is also required for association with Raf-1 kinase and the phosphatase calcineurin, which is essential for anti-apoptotic activity (Wang et al., 1996). Cleavage of this domain by activated caspases in cells undergoing apoptosis yields truncated Bcl-2 molecules resembling the pro-apoptotic members of the Bax subfamily (Jurgensmeier, 1998; Clem et al., 1998). The three dimensional structure of Bcl-xl has been elucidated by a combination of NMR and X-ray crystallography techniques. It is predicted that other closely related Bcl-2 proteins have a similar tertiary structure with seven c~ helices. As illustrated in Figure 2b, Bcl-xl is a predominatly c~ helical protein that consist of two central hydrophobic helices surrounded by five amphipathic helices. The central hydrophobic helices correspond to parts of the BH1 and BH2 domains whereas BH3 and BH4 contribute mostly to the five amphipathic helices. Computer modeling showed that the first 3 BH domains are in close proximity and form an elongated hydrophobic

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cleft along which the amphipathic helix formed by the BH3 domains of pro-apoptotic members can bind (Sattler et al., 1997).

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Many Bcl-2 members encode a stretch of hydrophobic residues in the extreme carboxyl termini that are required for insertion into membranes. Though the outer mitochondrial membrane is typically emphasized, significant amounts of Bcl-2 proteins are also integrated into the membranes of the endoplasmic reticulum and the nuclear envelope (Lithgow et al., 1994, Givol et al., 1994). Deletion of this membrane-anchoring domain reduces the cytoprotective function of Bcl-2 and Bcl-xl, suggesting that insertion into membranes is closely associated with the ability of these Bcl-2 family members to regulate apoptosis (Tanaka et al., 1993). Though the major cytoprotective function of Bcl-2 and Bcl-xL is related to their effects on mitochondria, recent studies showed that ER-targeted Bcl-2 can block apoptosis induced by serum deprivation. In addition, some members of the Bcl-2 family may be located preferentially, if not exclusively, in non-mitochondrial sites. The association of some other Bcl-2 members with intracellular membranes appears to be inducible rather than constitutive. The mechanism responsible for this inducible association of these members with membranes remains unknown. Interestingly, some Bcl-2 members with no membrane-anchoring domain have been

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demonstrated to associate with intracellular membranes via dimerization with other members of the family that are integral membrane proteins (Tanaka S. et al., 1993). The arrangement of the hydrophobic and amphipathic helices in the Bcl-xL protein mimics the membrane translocation domains of some bacterial toxins, in particular the colicins A and diphtheria toxins (Muchmore et al., 1996). Like these toxins, Bcl-2, Bcl-xL, and Bax are all capable of forming ion channels in planar lipid bilayers and synthetic lipid membranes that have distinct properties which include conductance, voltage dependence, and ion selectivity (Minn et al., 1997; Antonsson et al., 1997; Schendel et al., 1997; Schlesinger et al., 1997). Although these studies did not address whether the ability to form ion channels with different properties is related to the ability of these proteins to differentially regulate cell survival, they suggest that these proteins might directly or indirectly function to control membrane permeability. In support of this hypothesis, evidence exists for the regulation of intracellular Ca 2+ levels and release of apoptotic factors from mitochondria by Bcl-2. The latter is the most-studied mechanism by which Bcl-2 might exert its suppressive effect on cell death by preventing the release by mitochondria of cytochrome c and AIF (apoptosis inducing factor) in response to apoptotic signals (Kroemer et al., 1997, Kluck et al., 1997, Yang et al., 1997). Structure-Activity Relationships Among Bcl-2 Subfamilies Using a variety of techniques, including the analysis of Bcl-2 related proteins by immunoprecipitation and yeast two-hybrid screening, a family of at least seventeen Bcl-2 proteins in mammalian cells has emerged (see Table 2), including three that have recently been identified. It rapidly became evident that members of this family contain several recurring structural motifs, commonly denoted Bcl-2 homology (BH)1, BH2, BH3 and BH4 domains important for intra-family protein-protein interaction. The Bcl-2 family members can be classified into three subfamilies based on their structure (BH domain arrangement) and function. Bcl-2 Subfamily

The anti-apoptotic effects of Bcl-2 and Bcl-x have been extensively analysed. The Bcl-2 gene encodes two proteins (26 and 22 kDa) that differ in their C-termini as a result of alternative mRNA splicing (Tsujimoto and Crote, 1986; Tanaka et al., 1993, Chinnadurai et al., 1986). The smaller form (Bcl-2B) lacks the transmembrane domain. The Bcl-x gene encodes three different variants, each with a distinct function: the long form (Bcl-xL) exhibits anti-apoptotic activity, whereas Bcl-x-short (Bcl-xS) and Bcl-xB are pro-apoptotic (Boise et al., 1993; Shiraiwa et al., 1996). Bcl-xS lacks the BH1 and BH2 domains due to alternative splicing, however, it retains the BH3 domain and is the only pro-apoptotic member with a BH4 domain. Mice deficient in Bcl-x die at day 13 of gestation and display massive cell death in hematopoietic tissue as well as neuronal tissue of the brain, spinal cord, and dorsal root ganglia (Motoyama et al., 1995). The similarities in structure, intracellular distribution and function of members of this subfamily suggest that they protect against apoptotic cell death by a common mechanism. Evidence suggests that they act upstream of effector caspases to inhibit their activation.

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Recent studies indicated that Bcl-2 can also inhibit E1A-induced processing of initiator procaspase-8 and prevent NO-induced apoptotic cell death of PC-12 cells via a caspaseindependent mechanism (Nguyen et al., 1998). In many cells that undergo apoptosis, levels of Bcl-2 family proteins do not appear to change. It is likely that heterodimer formation between Bcl-2 family proteins is regulated by post-transcriptional modification. Several studies demonstrated that phosphorylation of Bcl-2 by PKC and possibly other Bcl-2 kinases is required for its anti-apoptotic function. Site-specific mutational analysis of Bcl-2 revealed that an evolutionary conserved serine residue (ser 70) is required for phosphorylation and maximal suppression of apoptosis following stress of IL-3 or NGF withdrawal and chemotherapy (Ito et al., 1997). Treatment of with bryostatin-l, a potent activator of PKC, has been shown to result in increased mitochondrial PKCct localization, increased Bcl-2 phosphorylation, and enhanced resistance to drug-induced apoptosis (Ruvolo et al., 1998). Bcl-2 protein has been demonstrated to also interact with a variety of cellular proteins that have no BH domains. These include Raf-1 kinase (Blagosklonny et al., 1996; Wang et al., 1996), calcineurin (Shibasaki et al., 1997), p28Bap31(Ng et al., 1997), and BI-I (Xu and Reed, 1998). The latter two are ER membrane proteins that participate in apoptosis regulation through yet undefined mechanism. Bcl-2 has also been shown to modulate the activity of transcription factor NF-KB and these observations may link Bcl-2 to NF~cB signaling pathway for rescue from apoptosis under certain conditions (de Moissac et al., 1998). Interestingly, several studies suggest that Bcl-2 has anti-oxidant properties and can prevent the generation of ROS and lipid peroxides under pro-oxidant conditions (Hockenberry et al., 1993; Bruce-Keller et al., 1998). BaxSubfami~ It appears that cells utilize different pro-apoptotic Bcl-2 homologs and strategies to block the function of pro-survival Bcl-2 family members. Bax (Bcl-2 associated x protein) was one of the first pro-apoptotic Bcl-2 members to be identified as a Bcl-2 binding protein in immunoprecipitation experiments (Oltvai et al., 1993). The 21 kD Bax protein contains all three BH domains, as well as the membrane-anchoring domain, and is 45% homologous to Bcl-2. Unlike the Bcl-2 and Bcl-xL proteins, Bax is mainly a cytosolic protein in the inactive monomeric form, but translocates during the propagation of a death signal to the mitochondria where it is a homodimerized integral membrane protein (Hsu et al., 1997; Wolter et al., 1997; Gross et al., 1998). However, the regulatory mechanism underlying Bax translocation is not clear. Translocation to mitochondria may be an important mechanism for the pro-apoptotic members and the propagation of death signals intracellularly (for review see Zamzami et al., 1998; Green and Reed, 1998; Reed et al., 1998). The Bax gene encodes a number of splicing variants (Bax-ct, Bax-B, Bax-y, Bax-6 and Bax-o0) with unknown functions (Oltvai et al., 1993; Zhou et al., 1998). Interestingly, Bax-o overexpression in some cells can be anti-apoptotic and it is not know whether cells undergoing degeneration modify their expression of Bax-ct and Bax-to (Zhou et al., 1998). Bax is upregulated at the transcriptional level by genetoxic stress

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via p53, and levels of Bax expression increase in certain tissues after apoptotic insults (Miyashita et al., 1994). Mice that are deficient for Bax display significant levels of lymphoid hyperplasia (Knudson et al., 1995). Though the tissue distribution of the Bax protein is more widespread than Bcl-2 (Krajewski et al., 1994a,b), Bax expression does not always corresponds to tissue marked by a high turnover rate or apoptotic cell death. Genetic experiments have shown that Bax can also induce cell death in the absence of Bcl-2 or Bcl-xL (Simonian et al., 1996) and seemingly in the absence of caspase activation (Xiang et al., 1996; Miller et al., 1997). Similar results were obtained in transformed yeast cells which lack known Bcl-2 proteins and caspases (Ligr et al., 1998; Tao et al., 1998). In addition, mutations in the BH1, BH2 or BH3 domains of Bax do not affect its ability to promote apoptosis. These studies demonstrate that Bax is the direct effector that kill ceils independently of caspase activation and support the notion that pro-apoptotic members can initiate a new activity that triggers apoptosis. This may involve several mechanisms including alterations in mitochondrial membrane potential and generation of reactive oxygen species. Like Bax, Mtd (Matador) induces cell death by a caspase-independent mechanism. Mtd is 22% identical to Bcl-2 and contains all four conserved BH domains in addition to the hydrophobic membrane anchor region. Its expression in embryonic and adult tissues is highly restricted. In transient assays, Mtd induces cell death in primary neurons and tumor cells that is not inhibited by the synthetic caspase inhibitor z-VAD-fmk nor blocked by Bcl-2/Bcl-xl overexpression (Inohara et al., 1998). Bak was isolated by interaction cloning with the viral bcl-2 homolog E1B 19K (Farrow et al., 1995) and by degenerate PCR cloning using primers directed to the conserved BH1 and BH2 domains of the Bcl-xl protein (Farrow et al., 1995; Chittenden et al., 1995). This 26 kDa pro-apoptotic member contains BH1, BH2 and BH3 domains and has been shown to interact with the anti-apoptotic proteins Bcl-2, Bcl-xL, and E1B 19K (Chittenden et al., 1995; Farrow et al., 1995, Chinnadurai and Lutz, 1995). Though Bak mRNA has been detected in sensory and sympathetic neurons as well as in cells of the CNS, Bak protein has not yet been detected in the latter. Bak is functionally similar to Bax. Bok (Bcl-2-related ovarian killer) was isolated by screening an ovarian fusion cDNA library (Hsu et al., 1997). Its tissue expression is limited to the ovary, uterus, and testis. Two splice variants of Bok have been identified (Bok-L and Bok-S). Bok-L shares the first three BH3 domains and preferentially dimerizes with Mcl-1, Bfl-1, and the viral BHRF-1. On the other hand, Bok-S lacks parts of the BH3 and BH1 domains but still induces cell killing without heterodimerization with anti-apoptotic Bcl-2 proteins, suggesting that this pro-apoptotic protein may form a mitochondrial channel to regulate apoptosis (Hsu et al., 1998). Bok S-mediated apoptosis may be critical in situations when unwanted cells need to eliminated rapidly, despite the presence of anti-apoptotic Bcl-2 proteins in the same cell. Cell-death induced by either Bok variant was antagonized by co-transfection with p35, a baculovirus-derived caspase inhibitor. Diva (Death-_inducer binding to vBcl-2 and Apaf-1) was recently identified by screening the GenBank database for cDNAs encoding proteins with homology to NR- 13, a Bcl-2 related protein in quail (Inohara et al., 1998). In situ hybridization studies

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revealed that Diva mRNA is expressed in brain, liver and heart of mouse embryonic tissues, but is restricted to the reproductive tissues in adult mice. Diva contains all four BH domains as well as the putative transmembrane region and is 41 and 44% homologous to Bcl-2 and Bcl-xL, respectively. Like Bax, Mtd and Bok-S, Diva-induced cell death in primary sensory neurons and tumor cells is independent of the BH3 domain. Diva promotes apoptosis through direct binding to Apaf-1 and therefore prevents Bcl-xL from sequestering Apaf-1 (Inohara et al., 1998). BH3 Subfamily This subfamily of pro-apoptotic proteins shows no amino acid homology to other Bcl-2 family members except for the BH3 amphiphatic ct helical domain (see Table 2). This subfamily includes Bad (Yang et al., 1995), Bik (Boyd et a1.,1995), Bid (Wang et al.,1996) Bim (O'Conner et al., 1998) and Hrk (Inohara et a1.,1997). Since members in this subfamily lack the BH1 and BH2 domains important for pore formation, it is proposed that they are functionally inactive in term of direct killing. However, the presence of the common BH3 domain suggests that they may induce apoptosis by acting as a death ligand to neutralize members of the pro-survival Bcl-2 subfamily. The BH3 region has been proposed to be critical in conferring the pro-apoptotic properties as polypeptides containing this domain are sufficient to induce apoptosis in transfected cells or cell free system (Chittenden et al 1995; Cosulick et al., 1997). In addition, when the BH3 domain of Bax was added to Bcl-2 by mutagenesis, the resulting hybrid molecule promoted apoptosis (Hunter and Parslow 1996). The isolation of Egl-1 in C. elegans which acts upstream of ced-9 to regulate all the developmental deaths proved that BH3 domain-only proteins are evolutionary conserved components of a central death pathway. The BH3-only proteins alone can induce cytochrome c release and the activation of caspases in a cell-free system (Cosulich et al., 1997) and provoke apoptosis when overexpressed (Boyd et al., 1995; Han et al., 1996; Wang et al., 1996; Imaizumi et al., 1997), suggesting that they may play a regulatory role in mediating apoptotic mitochondrial damage. However, some of the BH3-only version members -- Bad and Bid -- do not have a transmembrane anchor and it is not know how they act to induce mitochondrial damage. Bad was originally shown to have weakly homologous BHI and BH2 domains (Yang et al., 1995), but recent works showed that Bad is also a BH3-only protein (Kelekar et al., 1997; Zha et al., 1997). Recent data suggest that the localization of Bad and its pro-apoptotic function may be under the regulation of cell survival signals. Bad (Bcl-xl/bcl-2-_associated death promoter homologue) translocation from cytosol to mitochondria is regulated by protein phosphorylation by the serine-threonine kinase Akt that is stimulated by products of the PI 3-kinase in respond to receptor-mediated cell survival signals, such as IGF-1 (insulin-like growth factor 1), GM-CSF and IL-3 (Franke et al., 1995;Alessi et al., 1996; del Peso, 1997; Songyang, 1997). Phosphorylation of Bad on ser 136 is required, as IGF-1 is unable to suppress Bad-mediated death in cerebellar neurons expressing Bad mutated at ser 136 or dominant-negative mutants of Akt (Songyang, 1997). The phosphorylated form of Bad is subsequently sequestered in the cytosol bound to

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the widely distributed 14-3-3 proteins, molecules that recognize phosphoserine residues. In the absence of IL-3, Bad is dephosphorylated and changes partners from 14-3-3 to Bcl-xL. Binding to Bcl-xL not only redistributes Bad to mitochondrial membrane, but also inactivates the cell survival function of Bcl-xL by displacing prebound Bax/Bak. (Zha et al., 1996). This would result in an overall increase in free Bax/Bak leading to cell death. Expression of Bcl-2 can support survival upon IL-3 withdrawal (Nunez et al., 1990; Vaux et ah, 1988). Wang et al., (1996) showed that Bcl-2 can target Raf-1 to the mitochondria and initiates phosphorylation of Bad that resulted in the dissociation of Bad from Bcl-xl/Bcl-2. These findings suggest that phosphorylation of Bad promotes an anti-apoptotic effect by disassembly of heterodimer with Bcl-xl or Bcl-2. On the other hand, Bid translocation to mitochondria is regulated by caspases. Bid has been shown to contain three putative caspase-8 cleavage sites (Gross et al., 1999). Cleavage by caspase-8 of cytosolic full length Bid allows the truncated Bid to translocate to the mitochondria where it induces release of cytochrome c (Li et al., 1998). Immunodepletion of truncated Bid prevents cytochrome c release. It has been proposed that the truncated Bid which contains the BH3 domain can regulate channels formed by other Bcl-2 members or polymerize itself to form a pore for the selective passage of cytochrome c. (Gross et al., 1999, Li et al., 1998). In addition, truncated Bid could also displace Bcl-xl from Apaf-1, making it available to complex with cytochrome c and trigger ATP-dependent activation of downstream caspases (Li et al., 1998). Cleavage of two other Bcl-2 members, Bcl-2 and Bcl-xL, by activated caspases (Cheng et al., 1997; Clem et al., 1998) and non-ICE cysteine proteinase (Yamamoto AM et al, 1998) during apoptosis has also been reported, which convert them from anti-apoptotic to pro-apoptotic molecules. Thus, caspase cleavage of the Bcl-2 members may represent a feed forward loop to ensure cell death. Several other BH3-only members such as Bik, Blk, Bim, BOD, BOK and Hrk have a hydrophobic C-terminal membrane anchor that is preceded by the BH3 domain. The exact role of these members in apoptosis regulation requires further studies. The murine Blk (Bik -like killer), which shows 43% homology with the human Bik, (Hedge et al., 1998) was isolated from a mouse cDNA library by PCR using random primers. Blk expression is more restricted than human Bik; Blk mRNA is only detectable in the testes, kidney, liver, and heart but not in the brain, spleen, and skeletal muscles. On the other hand, Bik shows widespread tissue expression but its distribution in the nervous system is not known (Boyd et al., 1995). Both Bik and Blk promote apoptosis by interacting and antagonize the protective effects of Bcl-2, Bcl-x, BHRF-1, and E1B 19k or through competition and sequestration of cellular factors such as Apaf-1. Consistent with this notion, Blk-induced apoptosis can be inhibited by the dominant negative caspase-9 mutant (Hedge et al., 1998). Bim was isolated by screening a murine cDNA expression library with a Bcl-2 probe from a lymphoma cell line. Bim has three isoforms (Bim EL, Bim L and Bim S) that vary in their size and cytotoxicity; the shortest, BimS, is the most potent (O'Conner et al., 1998). When overexpressed, Bim proved to be highly cytotoxic for diverse cell-types. Bim-induced cytotoxicity can be neutralized by co-expression of Bcl-2, Bcl-xL or Bcl-c~, all of which bind to Bim in vivo. Like Bim, BOD (Bcl-2related ovarian death agonist) also has three variants (BOD-L, BOD-M and BOD-S).

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BOD was isolated from an ovarian fusion cDNA library by two-hybrid screen for Mcl-1 binding proteins. The BOD gene is conserved in mammals during evolution and its expression was detected in diverse tissues. Since coimmunoprecipitation studies showed that BOD has a wide heterodimerization pattern (Hsu, 1998), BOD could serve as an apoptosis regulator in diverse cell lineages by interacting with diverse anti-apoptotic Bcl-2 proteins including Bcl-2, Bcl-xL, Bcl-w, Bfl-1, Mcl-1, and the viral BHRF- 1.

Regulation of Mitochondrial Function by Bel-2 Family Proteins Mitochondria are unique among cell organelles in their involvement in the concerted consumption of oxygen, production of oxygen radicals and mobilization of intracellular calcium. Mitochondria are now considered major players in the apoptotic process of mammalian cells (Susin et al., 1998; Reed et al., 1998; Green and Reed, 1998). Kinetic studies indicate that mitochondria undergo major changes in membrane integrity before classical signs of apoptosis become manifest. Pharmacological data indicate that certain drugs which stabilize mitochondrial membranes can prevent apoptosis. However, until recently, the role of mitochondria in cell death was typically considered to be passive. It has now become clear that mitochondria play a critical role in mediating apoptotic signal transduction. Bcl-2 family proteins have been implicated in the regulation of two important aspects of mitochondrial pathophysiology: mitochondrial permeability transition (PT) pore opening and release of apoptogenic proteins from mitochondria into the cytosol (for review see Susin et al., 1998; Green and Reed, 1998; Cai et al., 1998). Though the structure and biochemical composition of the PT pore remain poorly defined, its constituents are thought to include both inner membrane proteins such as porin (voltage dependent anion channel; VDAC), which operate in concert (presumably at inner and outer membrane contact sites) to create a channel with approximately 1.5 kDa diameter (Ichas and Mazat, 1998). Thus, the outer membrane should be freely permeable to ions such as Ca 2÷ and most metabolites. Bcl-2 and Bcl-x L may communicate functionally or physically with inner membrane proteins responsible for ion transport, such as components of the PT pore (Susin et al., 1998; Ichas and Mazat, 1998). Since pH regulation in mitochondria is governed by inner membrane transporters, it implies a mechanism for interaction between Bcl-2 family proteins in the outer membrane and H ÷ channels in the inner membrane. Bcl-2 prevents signs of PT in cells (Zamzami et al., 1995a; b), as well as in isolated mitochondria (Zamzami et al., 1996; Susin et al., 1996; Marchinetti et al., 1996). Overexpression of Bcl-2/Bcl-xL has been shown to prevent apoptosis induced by variety of death stimuli (Merry and Korstmeyer, 1997). Also, it prevents the mitochondrial release of apoptogenic proteins (Zamzami et al., 1996; Kluck et al., 1997), the uncoupling of the respiratory chain (Zamzami et al., 1995b), the oxidation of the inner membrane constituent cardiolipin (Zamzami et al., 1995b) and the release of calcium from the mitochondrial matrix (Baffy et al., 1993). The exact mechanisms whereby Bcl-2 prevents PT remains elusive. However, it appears that this effect is a direct one, because the opening of PT pore complexes reconstituted into liposomes is inhibited by Bcl-2 and Bcl-x L (Zamzami

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et al., 1998; Marzo et al., 1998). Bax has been shown to induce both apoptosis and PT (Zamzami et al., 1996; Xiang et al., 1996). Purification of PT pore results in approximately 50-fold enrichment of Bax, implying that Bax may be closely associated with proteins which constitute components of the PT pore (Marzo et al., 1998). Moreover, the partially purified PT pore complex retained many of its expected functional characteristics when reconstituted in liposomes, including suppression of pore opening by recombinant Bcl-2 protein (Zamzami et al., 1998). Considerable clarification of the role of mitochondria in apoptosis was obtained by Liu et al. in 1996 when they found that cytochrome c is an important participant of apoptotic cascade. Cytochrome c is a nuclear DNA encoded protein, a part of the mitochondrial electron transport chain. Numerous studies with intact cells and in vitro cell free systems have provided strong support for the role of cytochrome c in apoptosis. In different cells, a variety of apoptotic stimuli could induce the release of cytochrome c before the caspase-3 activation and nuclear apoptosis (Yang et al., 1997; Kluck et al., 1997; Kharbanda et al., 1997; Vander Heiden et al., 1997). A second protein has been described which can trigger apoptosis when released from mitochondria into cytosol: apoptosis inducing factor (AIF), a putative protease of 50k Da (Susin et al., 1996). AIF appears to directly activate certain members of caspase family resulting in proteolytic processing of their proproteins and production of the mature enzymes (Susin et al., 1996). AIF was shown to cleave in vitro pro-caspase-3, and possibly itself is a member of caspase family (Susin et al., 1997). Mitochondria of some cells have been shown to contain procaspase-3 (Mancini et al., 1998) that is liberated into the cytosol during apoptosis, although it remains unclear whether it becomes activated before release. Caspases (interleukin-1B-converting enzymes; ICE-like proteases), the chief effectors of apoptosis are characterized by specificity for aspartic acid. They are synthesized as inactive proenzymes and lie in a latent state, becoming activated under conditions that culminate in apoptosis (Thurnberry and Lazebnik, 1998). Caspases can be divided by phylogenetic analysis and substrate specificity into three subfamilies. Initiator caspases (caspase-1,-2,-8, and 10) are activated independently of mitochondria. Second level of effector caspases (most prominently caspase-3, -6, and -7) act downstrean of the mitochondria checkpoint or are directly activated by first level caspases (Peter et al., 1997; Thornberry and Lazebnik, 1998). Caspase-9 appears to be the key mediator of the mitochondria-initiated caspase cascade (Kuida et al., 1998; Hakem et al., 1998). Caspase-1 appears to be activated by caspase-11, since caspase-11 knockout mice reveal a defect in activating caspase-1 (Wang et al., 1998). The participation of caspase-1 in apoptosis is controversial. Overexpression of caspase-1 results in programmed cell death (Miura et al., 1993), while transient expression of antisense caspase-1 cDNA blocks cytotoxicity induced by cross-linking of Fas (Los et al., 1993). Caspase-1 deficient mice, however, develop normally (Nicholson and Thornberry, 1997), but thymocytes from caspase-1 knockout mice showed a subtle resistance to apoptosis induced by CD95 ligand (Kuida et al., 1995). Caspase-8 has been demonstrated is activated by cross-linking of the Fas receptor by engagement of the Fas-ligand or agonistic antibodies resulting in the formation of so-called death-inducing signal complex (DISC), which includes adaptor protein FADD/MORT-1 and caspase-8 (Kishkel et al., 1995; Li et al., 1998).

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Caspases contribute to apoptosis through direct disassembly of cell structures. They cut off contacts with surrounding cells, reorganize the cytoskeleton, shut down DNA replication, and disrupt the nuclear structure (Thornberry and Lazebnic, 1998). Caspases also cleave many other proteins, such as proteins that protect cells from apoptosis. CED-9, Bcl-2 and Bcl-x L can be cleaved by caspases (Cheng et al., 1997; Xue and Horvitz, 1997; Clem et al., 1998). The proapototic member of Bcl-2 family Bid has been shown to be a substrate for caspase-8 in the Fas apoptotic signaling pathway (Li et al., 1998). Cytosolic phospholipase A2, an enzyme that plays an important role in the induction of inflammatory processes (Murakami et al., 1997; Leslie, 1997), has been demonstrated to be a substrate for several caspases involved in apoptosis in different cell-types and after different stimuli, such as caspase-3 (Atsumi et al., 1998), caspase-1, and caspase-8 (Luschen et al., 1998). A large body of genetic and biochemical evidence supports a cascade model for effector caspase activation (Figure 3). Cytochrome c activates caspases through its effects on protein called Apaf-1 (the mammalian CED-4 homolog; Zou et al., 1997). Apaf-1 lies in a latent state in the cytosol. Upon binding cytochrome c (Apaf-2), Apaf-1 becomes competent at binding the procaspase-9 (Apaf-3) presumably because of cytochrome c-induced conformational changes in the Apaf-1 protein (Li et al., 1997). Bcl-2 could interact with cytochrome c, either in a direct fashion, as this has been suggested for Bcl-x L (Kharbanda et al., 1997) or in indirect fashion, via Apaf-1, which binds to both Bcl-2 and cytochrome c (Zhou et al., 1997). In accord with Caenorhabditis elegans genetics (Hengartner and Horvitz, 1994), biochemical evidence suggests that the prosurvival proteins may function by directly inhibiting the ability of CED-4-1ike molecules to activate caspases. CED-9 and Bcl-x L can bind to CED-4, which also binds CED-3 and stimulates its activation (Chinnaiyan et al., 1997a,b; Spector et al., 1997). The BH4 region of Bcl-x L is required for pro-survival activity and interaction with CED-4, and might serve as a direct binding site for CED-4 (Huang et al., 1998). Bcl-x L can bind also to the CED-4 like portion of Apaf-1, whereas procaspase-9 binds to its NH2-terminal caspase recruitment domain (CARD; Pan et al., 1998; Hu et al., 1998). Bcl-x L may inhibit the association of Apaf-1 with procaspase-9 and thereby prevent caspase-9 activation. Pro-apoptotic members of Bcl-2 family like Bik may free CED4/Apaf-1 from the death inhibitor (Hu et al., 1998). Since Bcl-2 is located on the outer membrane of mitochondria and orients towards the cytosol, it is able to recruit cytosol proteins with which it interacts to the mitochondrial surface. For example, protein kinase Raf-1 and the phosphatase calcineurin, both of which can be co-immunoprecipitated with Bcl-2 are targeted from the cytosol to the organellar sites where Bcl-2 resides when Bcl-2 is over-expressed in cells (Wang et al., 1996; Shibasaki et al., 1997). A regulator of Hsp70/Hsc70 family of molecular chaperones, BAG-1 can also be targeted to the surface of mitochondria through interaction with Bcl-2 (Reed et al., 1998). Pro- and anti-apoptotic family members can heterodimerize and seemingly titrate one another's function, suggesting that their relative concentration may act as a rheostat for the suicide program (Oltvai et al., 1993). Heterodimerization is not required for prosurvival function (Cheng et al., 1996; Kelekar et al., 1997). Some death agonists may preferentially target subsets of the death repressors. Boc, for example, interacts with McI-1 and Epstein-Barr viral protein BHRFI but not with Bcl-2, Bcl-x L, or Bcl-w

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Ltgana • IFas-L, TNF) •

I

Death recepto! (Fas, TNF-R1J

Mitochondrion Death substrates (PARP, lamin A, spectrin, etc.) Figure 3. Modelfor effector caspase activation. Caspase-8 is activated by cross-linking of the Fas-receptor by engagement of the Fas-ligand or agonistic antibodies regulating in the formation of so called deathinducing signal complex (DISC), which includes adaptor protein FADD/MORT 1 caspase-8. BID is cleaved by caspase-8 and translocates to mitochondria where induces cytochrome c release. Cytochrome c activates caspases through Apaf-1 upon binding cytochrome c. Apaf-1 becomes competent at binding the procaspase-9. Bcl-2 could interact with cytochrome c directly or indirectly, via Apaf-1, which binds to both Bcl-2 and cytochrome c.

(Adams and Cory, 1998). Within the BH3 group, Bid is promiscuous, binding to Bax and Bak as well as to the anti-apoptotic proteins, but the others bind only to certain of the death inhibitors (Wang et al., 1996; O ' C o n n o r et al., 1998). For pro-apoptotic activity heterodimerization is essential in the BH3 domain group (Chittenden et al., 1995; Adams and Cory, 1998), but less so for those of the Bax group, which can have an independent cytotoxic impact. Most pro-apoptotic proteins o f Bcl-2 family of C. elegans regulate all the developmental cell death and map upstream CED-9 (Conradt and Horvitz, 1998) that BH3 domain only molecules are evolutinary conserved components o f central death pathway. The Bax group may also kill by damaging organelles. Although yeast Saccaromyces cerevisiae and Schizosaccharomyces pombe apparently lack Bcl-2-1ike proteins, CED-4, and caspases, both are killed by Bax and Bak (Zha et al., 1996; Jurgenmeier et al., 1997). The structure of Bcl-x L resembles

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the membrane insertion domains of bacterial toxins, suggesting that members having BH1 and BH2 domains function by forming pores in organelles such as mitochondria (Muchmore et al., 1996; Sattler et al., 1997). The anti-apoptotic proteins Bcl-2 and Bcl-x L have been suggested to tend to form small channels preferring cations, whereas the pro-apoptotic protein Bax tends to form larger channels preferring anions (Reed et al., 1998). Under some in vitro conditions, Bcl-2 has been demonstrated to be able to prevent channel formation by Bax (Antonsson et al., 1997). Channel forming Bcl-2 family proteins, such as Bax, probably form large channels in the outer membrane of mitochondria that liberate cytochrome c. Precedence exists for protein transport by other pore-forming proteins, such as diphtheria toxin, that are predicted to share structural similarity with Bax (Donovan et al., 1982). Indeed, recombinant Bax protein can induce specific release of cytochrome c from isolated mitochondria without attendant mitochondrial swelling (Jurgensmeier et al., 1998). Bcl-2, Bcl-x L, and Bax do form channels in bilayers in vitro, and those created by Bax and Bcl-2 have distinct characteristics (Minn et al., 1997; Lam et al., 1998). Even in the presence of caspase inhibitor, overexpression of Bax-like proteins, or their enforced dimerization, kills mammalian cells (Xiang et al., 1996; Gross et al., 1998). Bax and Bax-like proteins might mediate caspase-independent death via channel-forming activity. Bcl-2 was first reported to be a mitochondrial membrane protein (Hockenbery et al., 1990). Detailed knowledge about locations of other family proteins within mitochondrial membranes is relatively scant, but many Bcl-2 family proteins reside in the mitochondrial outer membrane (Reed et al., 1998). The C. elegans Bcl-2 homologue CED-9 is expressed from a bicistronic mRNA that encodes both CED-9 and cytochrome b (Hengartner and Horvitz, 1994). This suggests a functional connection between Bcl-2 family proteins and mitochondria and implies that CED-9 may have originated from the genome of promitochondrial symbionts, transferred along with other mitochondrial genes to the nuclear genome. A vide variety of mitochondrial events have been reported to be modulated by Bcl-2 and its homologs. These include some that directly affect mitochondria such as oligomycin which inhibits complex V of respiratory chain; cyanide, which inhibits complex IV; and BSO, which inhibits glutathione synthesis (Vander Heiden et al., 1997; Zamzami et al., 1996; Green and Reed, 1998). Bcl-2 and Bcl-x L suppress release of sequestered matrix Ca 2÷ induced by uncouplers of respiration (Baffy et al., 1993). In isolated mitochondria, Bcl-2 and Bcl-x L enhance proton extrusion from mitochondria and increases mitochondrial Ca 2÷ buffering capacity (Susin et al., 1996; Shimuzu et al., 1998; Murphy et al., 1996; Kruman et al., 1998). The mechanism whereby Bcl-2 influences mitochondrial calcium handling remains to be established. Possibilities include blocking the pore-forming action of proapoptotic proteins such as Bax, an agonist of the Bcl-2 family (Antonsson et al., 1997), formation of ion channels by Bcl-2 itself (Minn et al., 1997), and suppression of lipid peroxidation in mitochondrial membranes (Bruce-Keller et al., 1998). As mentioned earlier, the association of some Bcl-2 family proteins with mitochondrial membranes appears to be inducible rather than constitutive. For example, typically about half the Bax protein found in the cell resides in the cytosol until an apoptotic signal is delivered (Hsu et al., 1997; Wolter et al., 1997). Monomeric Bax translocates from the cytosol to the mitochondria where it is a homodimerized, integral membrane protein (Gross et al., 1998).

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Induced Bax expression (Xiang et al., 1996) or the enforced dimerization of Bax (Gross et al., 1998) results in downstream program of mitochondrial dysfunction and caspase activation. The other pro-apoptotic members of Bcl-2 family Bid and Bad also translocate from cytosol to mitochondria during propagation of death signal (Zha et al., 1996; Li et al., 1998). Perhaps all pro-apoptotic Bcl-2 family members will prove to have inactive forms which undergo conformational changes as part of their activation. Bcl-2 and Bcl-x L are localized to the nuclear envelope. Immunoelectronmicroscopic studies suggest potential association with nuclear pore complexes (Monaghan et al., 1992; Krajevski et al., 1993). Overexpression of Bcl-2 can prevent translocation of apoptosisinducing protein p53 from cytosol into nucleus (Ryan et al., 1994; Beham et al., 1997), suggesting that Bcl-2 may snag selected proteins to transit through nuclear pore complexes. The survival proteins seem to maintain organelle integrity and may register damage to these compartments and affect their behavior, perhaps by modifying the flux of small molecules of proteins. Membrane attachment of Bcl-2 is presumably due to a hydrophobic amino acid sequence present at the COOH terminus. Although the COOH-terminal hydrophobic domain of Bcl-2 is important in membrane docking, its deletion does not abrogate Bcl-2 survival function (Nguyen et al., 1994; Bomer et al., 1994). Mitochondria are the major source of reactive oxygen species (ROS) production in cells. ROS production and lipid peroxidation are increased during apoptosis induced by myriad stimuli (see McConkey and Orrenius, 1996 for review). Generation of ROS may be a relatively late event, occuring after cells have embarked on a process of caspase activation as a result of cellular damage (Mattson et al., 1992; Kruman et al., 1997; Mark et al., 1997). Considerable circumstantial evidence suggests that mitochondrial signaling of apoptosis may not be limited to the role of release of apoptogenic proteins such as cytochrome c and that ROS may provide an alternative signaling pathway. Several studies show that Fas activation (Albrecht et al., 1994), NGF deprivation (Greenlund et al., 1995a,b; Dugan et al., 1997), TNFct (Hennet et al., 1993) or staurosporine (Kruman et al., 1998) treatment of cells results in stimulated mitochondrial generation of ROS. ROS can result also from overexpression of the proapoptotic anti-oncogene p53 (Polyak et al., 1997) or from treatment of cells with ceramide (Quillet-Mary, 1997). ROS can be generated under conditions of virtual anaerobiosis (Degli et al., 1998), and thus their role in apoptosis cannot be excluded solely on this basis. Because mitochondria have a central role in cellular redox regulation, these findings indicate that mitochondria may provide a redox signal to activate the key processes of apoptosis. Bcl-2 can prevent the accumulation of mitochondrial ROS (Hockenbery et al., 1993; Kruman et al., 1998). It has been suggested that Bcl-2 might protect cells against oxidant injury by altering intracellular Ca 2+ homeostasis (Ichimiya et al., 1998). One mechanism is prevention of ROS-induced mitochondrial Ca 2+ cycling, a process which results in a collapse of mitochondrial membrane potential. Thus, Bcl-2 prevents disturbances of the cellular Ca z+ homeostasis and ROS production at the mitochondrial level.

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Regulation of Apoptosis by Bel-2 Family Proteins in the Nervous System Neuronal Apoptosis during Embryonic Development and in Neurodegenerative Conditions Neuronal apoptosis occurs both during development and in disease and injury conditions. Many physiological cell deaths in the nervous system are apoptotic (Oppenheim, 1991; Clarke, 1990; Server, 1991; Linden, 1994; Lo et al., 1995; Jacobson et al., 1997). In the developing nervous system, apoptosis is largely controlled by a limiting supply of target-derived growth factors, but is controlled by afferent stimulation as well. Apoptosis at this time plays important roles in matching neuronal populations to target size and represents a tightly regulated set of cellular responses to both extrinsic and intrinsic signals that serve to mold and refine neuronal structures. Neuronal apoptosis also occurs under a variety of neurodegenerative conditions and following injury (Borden, 1998; Shivers et al., 1998; Dragunow et al., 1997; Hetts et al., 1998; Hortnagl et al., 1997; Licinio, 1997; Gorman et al., 1996; Vasilakos et al., 1996; Gerlach et al., 1996). It is important to note that other forms of cell death may also be involved in neuronal cell death during embryonic development, since the morphological characteristics of dying neurons during development do not always meet the strict criteria of apoptosis. Apoptosis has been shown in animal models to be involved in neuronal degeneration associated with Alzheimer's disease (Guo et al., 1996, 1997, 1998, 1999; Luquin et al., 1997; Davis, 1996; Siman et al., 1996; Kusiak et al., 1996; Cotman et al., 1995), Down's Syndrome (Buscigolio et al., 1995), prion-induced neuronal degeneration (Kitamoto et al., 1996), amyotrophic lateral sclerosis (Troost et al., 1995), Parkinson's disease (Marsden et al., 1998; Hirsch et al., 1998;), Huntington's disease (Zeitlin et al., 1995), ischemic brain injury (Johnson et al., 1995), excitotoxic neuronal injury and epilepsy (Macaya et al., 1994), HIV-1 associated neurodegeneration (Masliah et al., 1996) traumatic brain injury (Martin et al., 1998). Mutations in presenilin-1 (PS-1) gene on chromosome 14 are causally linked to many cases of early-onset inherited form of Alzheimer's disease (Mattson and Guo., 1997a;; Mattson et al., 1997b,c; Guo et al., 1996; 1997; 1998a;b,c; 1999a, 1999b,c; Keller et al., 1998; Furukawa et al., 1998; Pedersen et al., 1997). To address the mechanisms by which mutations in PS-1 cause neuronal degeneration in AD, we generated and characterized mutant PS-1 M146V knock-in mice (Guo et al., 1999a,b). Primary hippocampal neurons from PS-I mutant knock-in mice, which express the human PS-1 M146V mutation at normal physiological levels, exhibit increased vulnerability to amyloid 13 peptide induced apoptosis (Guo et al., 1999b). The endangering action of the mutant PS-1 was associated with increased superoxide production, mitochondrial dysfunction and caspase activation (Figure 4). In transfected PC12 cells, overexpression of mutant PS-1 sensitizes neurons to apoptosis by a mechanism involving perturbation of endoplasmic reticulum calcium signaling and calcium overload (Guo et al., 1996, 1997). Calcium imaging studies showed that elevations of intracellular calcium concentration induced by agonists that induce calcium release from ER are enhanced in PC12 cells that express mutant PS-I (Guo et al., 1996, 1997). Moreover, primary hippocampal neurons from PS-I mutant knockin mice exhibit

The Bcl-2 Protein Family and Cell Death

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M146V mutation of presenilin-1 sensitizes hippocampal neurons from mutant presenilin-1

knock-in mice to apoptotic cell death induced by amyloid fS-peptide by a mechanism involving increased accumulation of superoxide and production of mitochondrial reactive oxygen species (ROS). (A) Phase contrast micrographs of primary hippocampal neuronal cultures showing amyloid B-peptide (20 IxM for 48h) induces significantly more cell death in neurons expressing mutant presenilin-1. (B) Confocal microscopic propidium iodide fluorescence images showing increased nuclear chromatin condensation and fragmentation in neurons from mutant presenilin-1 knock-in mice. (C and D). Confocal DHR fluorescence (a measure of mitochondrial ROS) and HE fluorscence (a measure of cellular superoxide levels) images showing increased levels of mitochondrial ROS and superoxide in neurons from mutant presenilin-1 knock-in mice. (Modified from Guo et al., 1999b)

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enhanced and prolonged calcium responses to glutamate that can be abolished by treatment with dantrolene (Guo et al., 1999a). These findings suggest a primary adverse effect of PS-1 mutations on endoplasmic reticulum calcium homeostasis. Treatment of neural cells expressing mutant PS-1 with sAPPct stabilizes intracellular calcium homeostasis and activates the neuroprotective transcription factor NF-KB, thereby protects the cells against apoptosis induced by AB. (Guo et al., 1998c). In addition, neural cells overexpressing Bcl-2 are resistant to the pro-apoptotic actions of the mutant PS-1 (see below). Recently a link was made between PS-1 mutations and expression of a novel gene involved in apoptosis. Par-4 is a leucine zipper domain-containing protein recently isolated by differential screening for genes upregulated in prostate cancer cells undergoing apoptosis (Guo et al. 1998a). Par-4 is expressed to various extents in different neuronal populations in the nervous system with levels being particularly high in hippocampal neurons. Levels of Par-4 mRNA and protein are greatly increased in vulnerable regions of AD brain and in association with neurofibrillary tangle-beating neurons (Figure 5). Further in vivo and in vitro studies demonstrated that the induction of Par-4 in AD brain tissues acts at an early stage in the apoptotic process before caspase activation and mitochondrial membrane depolarization and contribute to apoptotic neuronal degeneration (Guo et al., 1998a). Bcl-2 and caspase inhibitors block the pro-apoptotic action of Par-4. Expression and Distribution of Bcl-2 in the Nervous System Bcl-2 expression is developmentally regulated and may modulate physiologic cell death in the nervous system (Merry et al., 1994). The distribution of Bcl-2 in the nervous system has been studied at both the RNA and protein levels (Merry et al., 1994; Abe-Dohmae et al., 1993; Martinou et al., 1994; Castren et al., 1994; Ferrer et al., 1994; Gonzalez-Garcia et al., 1995). In the central nervous system, Bcl-2 protein is widely expressed during CNS development, but undergoes a marked down regulation during maturation and is present only at low concentrations in adult CNS. Neuronal Bcl-2 mRNA is maximal during embryogenesis and becomes downregulated and much more restricted postnatally. In squirrel monkeys (Saimiri sciureus) brain, Bcl-2 can serve as a marker of both proliferating and differentiating neurons during embryogenesis. In human spinal cord, between 5 and 10 weeks of gestation, Bcl-2 immunoreactivity was identified in primitive neuroepithelial cells of the ventricular zone. Individual cells of the mantle zone were stained including clusters of early anterior horn cells. Between 10 and 14 weeks of gestation, Bcl-2 staining was observed in cells lining the central canal, neurons of the dorsal horn (especially laminae I and II), and in anterior horn cells. Bcl-2 immunoreactivity became markedly reduced between 15 and 25 weeks of gestation, persisting only in ependymal cells. In contrast, strong Bcl-x staining was observed in most neurons throughout development and into adulthood. The period of apparent Bcl-2 downregulation overlaps with a peak in physiologic motoneuron death and the establishment of functional neuromuscular synapses in the human spinal cord. During mouse development, Bcl-2 protein is found in dividing cells of the ventricular zones, and in differentiating neurons, most notably in the developing cortical plate. Postnatally, Bcl-2 protein declines in most regions of the brain, except in those areas that

The Bcl-2 Protein Family and Cell Death

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Double label immunohistochemical analysis of hippocampal sections from an Alzheimer's disease (AD) patient and a neurologically normal control patient stained with antibodies against Par-4 and PHF-1 (an antibody that recognizes hyperphosphorylated tau in neurofibrillary tangles). Note Par-4 levels are significantly increased in AD brain and that approximately 40% of PHF-1 immunoreactive neurons (brown, white arrowhead) are also immunoreactive with the Par-4 antibody (black, white arrow). Dystrophic neurites associated with neuritic plaques (black arrow) showed PHF-1 immunoreactivity but little or no Par-4 immunoreactivity. (B). Densometric analysis of Par-4 amounts in three brain regions from control and AD patients. Values are the mean and SD of determinations made in samples from six control and six AD patients. ***P<0.001 and **P<0.01 compared with corresponding control value; *P<0.05 compared with the corresponding control value and P<0.01 compared with values from the hippocampus and inferior parietal cortex from AD patients. ANOVA with Scheffe's post-hoc tests. (Modified from Guo et al., 1998a)

168

Q. Guo, S.L. Chan and L Kruman

continue with postnatal neurogenesis, such as the cerebellar cortex, the hippocampus, and the olfactory bulb. After neurogenesis is complete, however, Bcl-2 protein levels continue to decline throughout the brain. However, bcl-2 mRNA and protein is still readily detectable in neurons of many areas of the adult brain. In contrast to the expression patterns of Bcl-2 protein seen in the brain, the expression of Bcl-2 protein in neurons of the peripheral nervous system remains high throughout life (Merry et al., 1994; Miyashita et al., 1994). Maintenance of Neuronal Populations by Bcl-2 in Normal Physiological Conditions Expression patterns of Bcl-2 during nervous system development are consistent with the observation that Bcl-2 functions as a neuronal survival factor. Transgenic mice that overexpress Bcl-2 in neurons showed an excess of neurons in selected regions of the brain and spinal cord, indicating that Bcl-2 is capable of preventing some developmental deaths (Martinou et al., 1994; Farlie et al., 1995; Dubois-Dauphin et al., 1994; Alberi et al., 1996; Bonfanti et al., 1996; Chen et al., 1996). However, newborn Bcl-2-/- knockout mice are viable and show no overt signs of neurodegenerative abnormalities and no decrease in neuron number following the period of naturally occurring cell death (Veis et al., 1993; Nakayama et al., 1994; Michaelidis et al., 1996). This is likely the result of functional compensation by other factors that promote neuronal survival during embryonic development. For example, Bcl-xL is also expressed in the nervous system during development and may protect neurons in the absence of Bcl-2. However, despite normal nervous system development, the majority of the Bcl-2-/knockout mice die at a few weeks of age. They develop a subsequent loss of motor, sensory, and sympathetic neurons, suggesting that Bcl-2 is critical for the postnatal maintenance of neuronal populations (Michaelidis et al., 1996). Besides, Bcl-2-/mice gradually develop polycystic kidney disease with marked dilatation of proximal and distal tubules and collecting ducts, resulting in renal failure (Veis et al., 1993). In addition, neonatal sympathetic neurons isolated from bcl-2 knockout mice died faster following NGF withdrawal than neurons isolated from their wild-type littermates (Greenlund et al., 1995a,b). In Vitro and In Vivo Evidence for the Neuroprotective Effect of Bcl-2 Following Insults

There has been compelling evidence showing that Bcl-2 can prevent death induced by diverse stimuli in neuronal cell lines and in primary neuronal cultures (Mah et al., 1993; Batistatou et al., 1993; Zhong et al., 1993; Kane et al., 1995). For example, sympathetic neurons deprived of NGF could be rescued by Bcl-2 (Garcia et al., 1992). Bcl-2 can also protect chick sensory neurons deprived of NGF, brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT-3). Bcl-2 overexpression can prevent motor neuron death induced by facial nerve axotomy and sciatic nerve axotomy (Dubois-Dauphin et al., 1994; Farlie et al., 1995). In transgenic mice, overexpression of Bcl-2 rescued retinal ganglion cells from developmental and axotomy-induced death (Bonfanti et al., 1996). In addition, Bcl-2 can protect neurons in vivo from death induced during focal ischemia. In vivo Bcl-2 overexpression in the rat spinal cord enhanced motor neuron survival.

The Bcl-2 Protein Family and Cell Death

169

Upregulation of neuronal Bcl-2 expression during ischemia, kainate-induced excitotoxicity, and axotomy-induced neuronal degeneration has been thought to be a neuroprotective response. Data from our lab support the view that Bcl-2 protects neurons following apoptotic insults and prevents neuronal degeneration associated with neurodegenerative disorders, such as Alzheimer's disease (Guo et al., 1997; Bruce-Keller et al., 1998; Kruman and Mattson, 1998, 1999; Kruman et al., 1997; Furukawa et al., 1997). For example, NGF withdrawal-induced apoptosis of differentiated PC12 cells expressing mutant presenilin-I (L286V) was significantly enhanced as compared with cells expressing wild-type presenilin-1. Co-expression of Bcl-2 in these cells completely blocked the pro-apoptotic actions of the mutant presenilin-1 (Figure 6). Bcl-2 does not reduce the peroxide accumulation promoted by mutant PS-1 following trophic factor withdrawal (Figure 7), but it largely blocks the enhanced calcium release from ER in cells overexpressing mutant presenilin-1 following exposure to thapsigargin, an inhibitor of the ER calcium ATPase (Figure 8). These results suggest that Bcl-2 counteracts the pro-apoptotic actions of the Alzheimer's mutant presenilin-1 by stabilizing calcium regulation. Bcl-2 expression is involved in protecting neurons against apoptosis induced by oxidative insults. In primary hippocampal cultures, neuroprotective concentrations (10-100 nM) of the protein synthesis inhibitor cycloheximide (CHX) protect neurons against oxidative stress induced cell death not by inhibiting protein synthesis but by inducing bcl-2, c-fos and c-jun mRNA expressions (Figure 9). Thus, CHX induced a concentration-dependent increase in levels of bcl-2 mRNA and protein. Bcl-2 antisense oligodeoxynucleotides abrogate the neuroprotective effect of CHX (Figure 10). Another mechanism by which Bcl-2 protects neurons against oxidative insults is to increase intracellular levels of glutathione and reducing the production of 4-hydroxynonenal, an aldehydic product of membrane lipid peroxidation (Kruman et al., 1997) Lipid peroxidation plays an important role in apoptosis, and Bcl-2 is localized to mitochondria, as well as plasma membrane, where it can act locally to suppress oxidative damage induced by AB and hydrogen peroxide (H202). Electron paramagnetic resonance (EPR) spectroscopy was used to determine the loss of EPR-detectable paramagnetism of nitroxyl stearate (NS) spin labels 5-NS and 12-NS and to investigate the local effect of Bcl-2 on membrane lipid peroxidation in transfected PC12 cells. Bcl-2 overexpression significantly prevented the loss of 5-NS and 12-NS signal amplitude in isolated plasma and mitochondrial membranes induced by AB and hydrogen peroxide (Figure 11). Overexpression of Bcl-2 in transfected PC12 cells suppressed staurosporine-induced apoptosis by preventing sustained increase in intracellular calcium levels. Thus, staurosporine induces rapid and delayed elevations of intracellular calcium levels in PC 12 cells. Both Bcl-2 and the caspase inhibitor zVAD-fmk suppressed the sustained elevation of calcium levels, and protect the cultured cells against apoptosis induced by staurosporine (Figure 12). Bcl-2 can also protect cells against apoptosis induced by agents that act primarily by elevating intracellular calcium levels, including exposure to calcium ionophore. These results suggest that one of the mechanisms by which Bcl-2 protects neurons against apoptosis is by stabilizing calcium homeostasis.

170

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(L286V) in transfected PC12 cells following withdrawal of nerve growth factor (NGF). Inducible expression of the wild-type or mutant PS-1 transgene in PC12 cells was achieved using the Tet-Off mammalian expression system (Guo, et al., 1997). Cultures of differentiated PC12 cells were incubated for 48 h in serumfree medium containing or lacking NGF, and the percentage of cells exhibiting nuclear condensation and fragmentation was quantified. Values are the mean and SEM of determinations made in four separate cultures. (A) and (B) show the percent of apoptotic cells following NGF withdrawal in cells expressing wild type and mutant PS-1 respectively. Following withdrawal of NGF, the number of apoptotic cells in cell lines expressing mutant PS-I (PS1L286V TP C1 and C9) was significantly greater than those in cell lines expressing the wild type PS-1 (PS1Tetoff C3 and C7). Co-overexpression of Bcl-2 (bc12-PS1L286V TP C1 and C9, bcl-2-PS1TPC11 and C13) protected the cells against the proapoptotic actions of the mutant PS-1. *P < 0.01 compared with corresponding values for the cell lines co-expressing Bcl-2 following NGF withdrawal. ANOVA with Scheffe's post-hoc tests. (Modified from Guo et al., 1997).

The Bcl-2 Protein Family and Cell Death

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PC12 cells expressing wild or mutant PS-I. Inducible expression of the wild-type or mutant PS-1 transgene in PC12 cells was achieved using the Tet-Off mammalian expression system (Guo, et al., 1997). Cultures of differentiated PC12 cells were incubated for 3 hr in serum-free medium containing or lacking NGF, and levels of cellular peroxides were quantified by confocal laser scanning microscope image analysis of DCF fluorescence. Values are the mean and SEM of determinations made in four separate cultures. (A) and (B) show the relative levels of DCF fluorescence in each cell following NGF withdrawal in cells expressing wild type and mutant PS-1, respectively. Mutant PS-1 (PS1L286V TP C1 and C9) significantly increases cellular peroxides levels compared to wild-type PS-1 (PS1Tetoff C3 and C7). Co-overexpression of Bcl-2 does not prevent NGF withdrawal-induced accumulation of peroxides in PC12 cells overexpressing wild type or mutant PS-1. *P < 0.01 compared with corresponding values for each line expressing with type PS-1. ANOVA with Scheffe's post-hoc tests. (Modified from Guo et al., 1997).

172

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transfected PC12 cells expressing mutant PS-1. Inducible expression of the wild-type or mutant PS-1 transgene in PC12 cells was achieved using the Tet-Off mammalian expression system (Guo et al., 1997). Cultures of differentiated PC12 cells were incubated in serum-free medium and basal [Ca2+]i levels before exposure to 1 [xM Tpg and the peak [Ca2+]i following exposure to 1 [xM Tpg was quantified. Values are the mean and SEM of determinations made in four separate cultures. (A) and (B) show intracellular calcium levels (nM) in ceils expressing wild type and mutant PS-1 respectively before and after exposure to Tpg. Following exposure to Tpg, the increase in intracellular calcium levels in cell lines expressing mutant PS-1 (PS1L286V TP C1 and C9) was significantly greater than those in cell lines expressing the wild type PS-1 (PS 1Tetoff C3 and C7). Co-overexpression of Bcl-2 (bcI2-PS1L286V TP C1 and C9, bel-2-PS1TPCll and C13) significantly attenuates the calcium response following exposure to Tpg in cells expressing either wild type or mutant PS-1. *P < 0.01 compared with corresponding values for the cell lines co-expressing Bcl-2 following exposure to Tpg. ANOVA with Scheffe's post-hoe tests. (Modified from Guo et al., 1997)

The Bcl-2 Protein Family and Cell Death

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174

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Figure 10. Induction of bcl-2 expression is necessary for the neuroprotective effect of CHX: bcl-2 antisense oligonucleotide blocks the neuroprotective actions of CHX. Primary rat hippocampal neuronal cultures were pretreated for 12 h 10 or 50 IxM bcl-2 antisense oligonucleotide (bcl-2 AS) or control missense oligonucleotide (MS). Cultures were then exposed to vehicle (saline) or 10 IxM (A) or 50 ~VI (B) glutamate (Glut). Neuronal survival was quantified. Values were the mean and SEM of three separate experiments and are expressed as percentage of the initial number of neurons. *P<0.05; **P<0.01. ANOVA with Seheffe's post-hoc tests. (Modified from Furukawa et al., 1997)

The Bcl-2 Protein Family and Cell Death

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176

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apoptosis in PC12 cells by suppressing increase in intracellular calcium levels. Cultures of control PC12 (vector transfected) cells and PC12 cells overexpressing Bcl-2 were exposed to vehicle or 1 ~tM STSP. Additional control cells were exposoed to 100 ~tM zVAD-fmk plus 1 IxM STSP. Four hours following the treatment, the intracellular calcium levels were measured using fura-2 fluorescent imaging. Values are the mean and SEM of determinations made in at least 3 separate cultures. The value for PC12 cells exposed to STSP was significantly greater than each of the other values. (P < 0.01; ANOVA with Scheffe's post-hoc tests). Note that overexpression of Bcl-2 completely blocked the calcium response to STSP. (Modified from Kruman et al., 1998).

It is important to note, however, that Bcl-2 does not prevent neuronal cell death induced by all agents (Allsopp et al., 1993; Vanhaesebroeck et al., 1993; Takayama et al., 1995). It is possibly because there exist other cell death pathways that are controlled by other cell survival promoting proteins, including other member proteins of the Bcl-2 family and otherBcl-2- unrelated proteins. For example, Bcl-2 cannot rescue ciliary neurons deprived of ciliary neurotrophic factor (CNTF), nor can it prevent the death of BDNF-dependent neurons that have been exposed to CNTF (Allsopp et al., 1993). Bcl-2 was ineffective in protecting motor neurons from death in the wobblermutant mice. Bcl-2 was inefficient in protecting certain hematopoietic ceils from death induced by Fas antigen (Vanhaesebroeck et al., 1993; Takayama et al., 1995). Moreover, overexpression of Bcl-2 in normal retinal photoreceptors actually caused these cells to die, suggesting that in this particular system the complex protein interactions that determine photoreceptor survival were likely perturbed by Bcl-2 overexpression (Chen et al., 1996).

The Bcl-2 Protein Family and Cell Death

177

Evidence for Bcl-x as a Neuronal Survival Factor Expression and Distribution of Bcl-x in the Nervous System At the amino acid level, Bcl-x shares 56% homology with Bcl-2. However, unlike Bcl-2, Bcl-x is not only expressed early in neurogenesis, but also expressed postnatally, reaching a peak in the adult (Gonzalez-Garcia et al., 1995; Frankowski et al., 1995) Under basal conditions, Bcl-x mRNA and protein are expressed almost exclusively in neurons, and are largely present in soma. In the adult, high levels of RNA and protein are detected throughout the brain. Bcl-xL is the major form of Bcl-x expressed in the murine nervous system. Neuroprotective Roles of Bcl-X in Physiological and Pathological Conditions There is considerable evidence supporting the idea that Bcl-x is a potent survival factor for neurons during development and in adult brain (Gonzalez-Garcia et al., 1995; Frankowski et al., 1995). For example, Bcl-xL protects sympathetic neurons from cell death induced by NGF withdrawal. Transgenic mice lacking Bcl-x die around embryonic day 13 because of the massive death of immature but differentiating neurons that have not yet made synaptic connections (Motoyama et al., 1995). Extensive cell death can be observed in these mice throughout the brain and spinal cord in regions of postmitotic, differentiating neurons, in which Bcl-X is normally highly expressed. These results indicate that Bcl-x is essential for brain development and is required for neuronal survival during differentiation and maturation. This is in sharp contrast to Bcl-2 -/-mice, which showed no signs of neuronal degeneration during embryonic development. Pro-apoptotic Actions of Bax in the Nervous System Bax is widely expressed in most neurons in both central and peripheral nervous systems, although levels of expression among different populations of neurons may vary (Krajewski et al., 1994a,b; Oltvai et al., 1993). Bax has been shown to be a pro-apoptotic factor in the nervous system and is essential for trophic factor withdrawal induced neuronal degeneration (Deckwerth et al., 1996). For example, Bax-deficient sympathetic neurons are independent of NGF for survival, and motor neurons lacking Bax survive disconnection from their targets by axotomy. Bax is also required for neuronal cell death in response to some forms of cytotoxic injury. Bax plays a key role for p53 activation in response to excitotoxic and genotoxic injury. Interestingly, mice deficient in both Bcl-xL and Bax (bcl-x-/-/bax-/-) demonstrate greatly reduced levels of apoptosis in the CNS both in vivo and in vitro compared with the CNS of Bcl-xL-deficient mice, indicating Bax critically interacts with Bcl-xL to regulate survival of immature neurons. Since Bax-/-mice appear healthy and show no overt changes in the nervous system, Bax does not seem to be an essential factor for development of the nervous system,

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although sympathetic and motor neuronal death during development is reduced in Bax-/-mice. It is interesting to note, however, that germ cells of the male Bax-/-mice exhibit a marked increase in cell death, causing a complete cessation of mature sperm cell production and an expansion of the premeiotic 2N cell population (Knudson et al., 1995).

Summary It is quite clear that understanding the actions of the Bcl-2 family of proteins within the cell death machinery is rapidly and continuously growing, but it is far from complete, and the challenge of converting that understanding into new therapeutic modalities has only begun to be approached. How do we define the multiple forms of cell death? What are the differences and similarities between apoptosis and the classical primary necrosis? What are the multiple pathways of apoptosis? Why is apoptosis important physiologically and pathologically? What are the Bcl-2 family proteins and how their structural characteristics dictate their actions within the cell death machinery? Why is regulation of mitochondrial function by Bcl-2 family proteins important in apoptotic cell death? What are the roles of the Bcl-2 family proteins in the physiological and pathological cell death in the nervous system? Those are the questions this chapter considers. Further studies of the Bcl-2 family of proteins and mechanisms of apoptotic cell death will likely reveal a complex and central role for these proteins in mediating signals for cell death and survival. Insights into the functional roles of the protective, as well as the destructive genes of the Bcl-2 family and their molecular mechanisms in various organ systems may provide promising avenues for novel therapeutic approaches to control cell death that occurs in a variety of disease and injury conditions.

References Abe-Dohmae, S., Harada, N., Yamada, K. & Tanaka, R. (1993). bcl-2 gene is highly expressed during neurogenesis in the central nervous system. Biochem. Biophys. Res. Commun. 191(3), 915-21 Adams, J.M. & Cory, S. (1998). The Bcl-2 protein family: arbiters of cell survival. Science. 281, 1322-1326. Akao, Y., Otsuki, Y., Kataoka, S., Ito, Y. & Tsujimoto, Y. (1994). Multiple subcellular localization of bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes. Cancer Res. 54, 2468-2471. Alberi, S., Raggenbass, M., de Bilbao, F. & Dubois-Dauphin, M. (1996). Axotomized neonatal motoneurons overexpressing the bcl-2 proto-oncogene retain functional electrophysiological properties. Proc. Natl. Acad. Sci. USA 93, 3978-83 Albrecht, J., Tschopp, J. & Jongeneel (1994). Bcl-2 protects from oxidative damage and apoptotic cell death without interfering with activation of NF-kappa B by TNF. FEBS Lett 351, 45-48. Alessi, D.R., Caudwell, F.B., Andjelkovic, M., Hemmings, B.A. & Cohen, P. (1996). Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 $6 kinase. FEBS Lett 399, 333-338, Allsopp, T.E., Wyatt, S., Paterson, H.F. & Davies, A.M. (1993). The proto-oncogene bcl-2 can selectively

The Bcl-2 Protein Family and Cell Death

179

rescue neurotrophic factor-dependent neurons from apoptosis. Cell 73,295-307 Alnemri, E.S., Livingston, D.J., Nicholson, D.W., Salvesen, G.S., Thomberry, N.A., Wong, W. W. & Yuan J. (1996). Human ICE/CED-3 protease nomenclature. Cell 87,171. Ameisen, J.C. (1996). The origin of programmed cell death. Science 272, 1278-1279 Anel, A., Gamen, S., Alava, M.A., Schmitt-Verhulst, A.M., Pineiro, A. & Naval, J. (1997). Inhibition of CPP32-1ike proteases prevents granzyme B- and Fas-, but not granzyme A-based cytotoxicity exerted by CTL clones. J. Immunol. 158, 1999-2006 Antonsson, B., Conti, F., Ciavatta, A.M., Montessuit, S., Lewis, S., Martinou, I., Bernasconi, L., Bernard, A., Mermod, J.-J., Mazzei, G, Maundrell, K., Gambale, F., Sadoul, R., & Martinou, J.-C. (1997). Inhibition of Bax channel forming activity by Bcl-2. Science. 277, 370-372. Arends, M.J., Morris, R.G., Wyllie, A.H. (1990). Apoptosis. The role of the endonuclease. Am. J. Pathol. 136, 593~)08 Arends, M.J.& Wyllie, A.H. (1991). Apoptosis: mechanisms and roles in pathology. Int. Rev. Exp. Pathol. 32, 223-254 Atsumi, G.I., Tajima, M., Hadano, A., Nakatani, Y., Murakami, M. & Kudo, I. (1998). Fas-induced arachidonic acid release is mediated by Ca2*-independent phospholipase A2 but not cytosolic phospholipase A2, which undergoes proteolytic inactivation. J. Biol. Chem. 273, 13870-13877. Baffy, G., Miyashita, T., Williamson, J.R. & Reed, J.C. (1993). Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production. J. Biol. Chem. 268, 6511-6519. Bakhshi, A, Jensen, J.P., Goldman, P., Wright, J.J., McBride, O.W., Epstein, A.L. & Korsmeyer, S.J. (1985). Cloning the chromosomal breakpoint of t(14;18) human lymphomas: clustering around JH on chromosome 14 and near a transcriptional unit on 18. Cell 3:899-906. Batistatou, A., Merry, D.E., Korsmeyer, S.J. & Greene, L.A. (1993). Bcl-2 affects survival but not neuronal differentiation of PC12 cells. J. Neurosci. 13,4422-28. Beeri, R., Symon, Z., Brezis, M., Ben-Sasson, S.A., Baehr, P.H., Rosen, S. & Zager, R.A. (1995). Rapid DNA fragmentation from hypoxia along the thick ascending limb of rat kidneys. Kidney Int. 47,1806-1810. Beham, A., Marin, M.C., Fernandez, A., Hermann, J., Brisbay, S., Tari, A.M., Lopez-Berestein, G., Lozano, G., Sarkiss, M. & McDonnell, T.J. (1997). Bcl-2 inhibits p53 nuclear import following DNA damage. Oncogene. 15, 2767-2762. Blagosklonny, M.V., Schulte, T., Nguyen, P., Trepel, J., Neckers, L.M. (1996). Taxol-induced apoptosis and phosphorylation of Bcl-2 protein involves c-Raf-1 and represents a novel c-Raf-1 signal transduction pathway. Cancer Res. 56:1851-1854. Boise, L.H., Gonzalez-Garcia, M., Postema, C.E., Ding, L., Lindsten, T., Turka, L.A., Mao, X., Nunez, G. & Thompson, C.B. (1993). Bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 74:597-608 Boise, L.H., Gottschalk, A.R., Quintans, J., Thompson, C.B. (1995). Bcl-2 and Bcl-2-related proteins in apoptosis regulation.Curr. Top. Microbiol. Immunol. 200:107-121. Bonfanti, L., Strettoi, E., Chierzi, S., Cenni, M.C., Liu, X-H., Martinou, J.C., Maffei, L. & Rabacchi, S.A. (1996). Protection of retinal ganglion cells from natural and axotomy-induced cell death in neonatal transgenic mice overexpressing bcl-2. J. Neurosci. 16, 4186-4194. Borden, K.L. (1998). Structure/function in neuroprotection and apoptosis. Ann. Neurol. 44(3 Suppl 1),$65-71. Borner, C., Martinou, I., Mattman, C., Irmler, M., Schaerer, E., Martinou, J.C. & Tschopp, J. (1994). The

180

Q. Guo, S.L. Chan and 1. Kruman

protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis. Cell. Biol. 126, 1059-1068. Boyd, J.M., Gallo, G.J., Elangovan, B., Houghton, A.B., Malstrom, S., Avery, B.J., Ebb, R.G., Subramanian, T., Chittenden, T. & Lutz, R.J. (1995). Bik, a novel death-inducing protein shares a distinct sequence motif with Bcl-2 family proteins and interacts with viral and cellular survival-promoting proteins. Oncogene 11, 1921-1928. Bruce-Keller, A.J., Begley, J.G., Fu, W., Butterfield, D.A., Bredesen, D.A., Hutchins, J.B., Hensley, K. & Mattson, M.P. (1998). Bcl-2 protects isolated plasma and mitochondrial membranes against lipid peroxidation induced by hydrogen peroxide and amyloid fS-peptide.J. Neurochem. 70, 31-39. Busciglio, J. & Yankner, B.A. (1995). Apoptosis and increased generation of reactive oxygen species in Down's syndrome neurons in vitro. Nature 378,776-779. Cai, J., Yang, J. & Johnes, D.P. (1998). Mitochondrial control of apoptosis: the role of cytochtome c. Biochim. Biophys. Acta. 1366, 139-149. Castren, E., Ohga, Y., Berzaghi, M.P., Tzimagiorgis, G., Thoenen, H. & Lindholm, D. (1994). bcl-2 messenger RNA is localized in neurons of the developing and adult rat brain. Neuroscience 61(1), 165-177 Cazals-Hatem, D.L., Louie, D.C., Tanaka, S. & Reed, J. (1992). Molecular cloning and DNA sequence analysis of cDNA encoding chicken homologue of the Bcl-2 oncoprotein. Biochim. Biophys. Acta 1132, 109-113. Chen, J., Flannery, J.G., La Vail, M.M., Steinberg, R.H., Xu, J. & Simon, M.I. (1996). bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations. Proc. Natl. Acad. Sci. U.S.A. 93,7042-7047. Cheng, E.H., Kirsch, D.G., Clem, R.J., Ravi, R., Kastan, M.B., Bedi, A., Ueno, K. & Hardwick, J.M. (1997). Conversion of Bcl-2 to a Bax-like death effector by caspases. Science. 278, 1966-1968. Cheng, E.H.-Y., Levine, B., Boise, L.H., Thompson, C.G.& Hardwick, J.M. (1996). Bax-independent inhibition of apoptosis by Bcl-xL. Nature. 379, 554-556. Chinnadurai, G. & Lutz, R.J. (1995). A conserved domain in Bak, distinct from BH1 and BH2, mediates cell death and protein binding functions. EMBO J. 14, 5589-5596. Chinnadurai, G., Lutz, R.J. & Cleary, M.L. (1986). Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation. Cell. 47, 19-28. Chinnaiyan, A.M., Chaudhary, D., O'Rourke, K., Koonin, E.V. & Dixit, V.M. (1997a). Role of CED-4 in the activation of CED-3. Nature 388, 728-729. Chinnaiyan, A.M., O'Rourke, K., Lane, B.R. & Dixit, V.M. (1997b). Interaction of CED-4 with CED-3 and CED-9: a molecular framework for cell death. Science 275, 1122-1126. Chinnaiyan, A.M., Orth, K., Orourke, K., Duan, H.J., Poirier, G.G. & Dixit, V.M. (1996). Molecular ordering of the cell death pathway--Bcl-2 and Bcl-xL function upstream of the ced-3-1ike apoptotic proteases. J. Biol. Chem. 271,4573-4576. Chittenden, T., Flemington, C., Houghton, A.B., Ebb, R.G., Gallo, G.J., Elangovan, B., Chinndurai, G & Lutz, RJ (1995) A conserved domain in Bak, distinct from BH1 and BH2, mediates cell death and protein binding functions. EMBO J 14:5589-5596. Clarke, P.G.H. (1990). Developmental cell death: morphological diversity and multiple mechanisms. Anat. Embryol. 81,195-213 Cleary, M.L., Smith, S.D. & Skar, J. (1986).Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bel-2/immunoglobulin transcript resulting from the t(14;18) translocation. Cell 47:19-28 Clem, R.J., Cheng, E.H., Karp, C.L., Kirsch, D.G., Ueno, K., Takahashi, A., Kastan, M.B., Griffin, D.E., Earnshaw, W.C., Veliuona, M.A. & Hardwick, J.M. (1998). Modulation of cell death by BcI-XL through

The Bcl-2 Protein Family and Cell Death

181

caspase interaction. Proc Natl Acad Sci U S A 95:554-559 Columbano, A. (1995). Cell death: current difficulties in discriminating apoptosis from necrosis in the context of pathological processes in vivo. J. Cell. Biochem. 58,181-190 Conradt, B. & Horvitz, H.R. (1998) The C. elegans protein EGL-1 is required for programmed cell death and interacts with the Bcl-2-1ike protein CED-9. Cell. 93,519-529. Cory, S. & Adams, J.M. (1998). Matters of life and death: programmed cell death at Cold Spring Harbor. Biochim. Biophys. Acta. 1377, R25-44. Cosulich, S.C., Worrall, V., Hedge, P.J., Green, S. &Clarke, P.R. (1997). Regulation of apoptosis by BH3 domains in a cell-free system. Curr. Biol. 7:913-920. Cotman, C.W. & Anderson, A.J. (1995). A potential role for apoptosis in neurodegeneration and Alzheimer's disease. Mol. Neurobiol. 10(1), 19-45. Cruz-Reyes, J, & Tata, J. (1995). Cloning, characterization and expression of two Xenopus bcl-2-1ike cell-survival genes. Gene 158:171-179. Davis, J.B. (1996). Oxidative mechanisms in beta-amyloid cytotoxicity. Neurodegeneration. 5(4),441-444. de Jong, D., Prins, F.A., Mason, D.Y., Reed, J.C., Van Ommen, G.B. & Kluin, P.M. (1994). Subcellular localization of the bcl-2 protein in malignant and normal lymphoid cells. Cancer Res. 54, 256-260. de Moissac, D., Mustapha, S., Greenberg, A.H. &Kirshenbaum, L.A. (1998). Bcl-2 activates the transcription factor NFkappaB through the degradation of the cytoplasmic inhibitor IkappaBalpha. J. Biol. Chem. 273, 23946-23951. Deckwerth, T.L., Elliot, J.L., Knudson, C.M., Johnson, E.M., Jr, Snider, W.D. & Korsmeyer, S.J. (1996). Bax is required for neuronal death after trophic factor deprivation and during development. Neuron 17,401-411. Degli,-Esposti, M. & McLennan, H. (1998). Mitochondria and cell produce reactive oxygen species in virtual anaerobiosis: relevance to ceramide-induced apoptosis. FEBS Lett. 430,338-342. del Peso, L, Gonzalez-Garcia, M, Page, C, Herrera, R. & Nunez, G (1997). lnterleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 278:687-689. Distelhorst, C.W., Lam, M. & McCormick, T.S. (1996). Bcl-2 inhibits peroxyde-induced ER Ca2÷ pool depletion. Oncogene. 12, 2051-2055. Dixon, S.C., Soriano, B.J., Lush, R.M., Borner, M.M. & Figg, W.D. (1997). Apoptosis: its role in the development of malignancies and its potential as a novel therapeutic target. Ann. Pharmacol. 31,76-82. Donovan, J.J., Simon, M.I. & Montal, M. (1982) Insertion of diphteria toxin into and across membranes: role of phosphoinositide asymmetry. Nature. 298,669-672. Dragunow, M., MacGibbon, G.A., Lawlor, P., Butterworth, N., Connor, B., Henderson, C., Walton, M., Woodgate, A., Hughes, P. & Faull, RL. (1997). Apoptosis, neurotrophic factors and neurodegeneration. Rev. Neurosci. 8(3-4), 223-265. Driscoll, M. (1992). Molecular genetics of cell death in the nematode Caenorhabditis elegans. J. Neurobiol. 23,1327-1351. Dubois-Dauphin, M., Frankowski, H., Tsujimoto, Y., Huarte, J., Martinou, J-C. (1994). Neonatal motoneurons overexpressing the bcl-2 protooncogene in transgenic mice are protected from axotomy-induced cell death. Proc. Natl. Acad. Sci. USA 91, 3309-3313 Dugan, L.L., Greedon, D.J., Johnson, E.M. & Holzman, D.M. (1997) Rapid suppression of free radical formation by nerve growth factor involves the mitogen-activated protein kinase pathway. Proc. Natl. Acad. Sci. U.S.A. 94, 4086-4091. Duke, R.C., Ojcius, D.M. & Young, J.D. (1996). Cell suicide in health and disease. Sci. Am. 275, 80-87. Eguchi, Y., Shimizu, S. & Tsujimoto, Y. (1997). Intracellular ATP levels determine cell fated by apoptosis

182

Q. Guo, S.L. Chan and I. Kruman

or necrosis. Cancer Res. 57, 1835-1840. Ellis, RE, Yuan, J. & Horvitz, H.R. (1991). Mechanisms and functions of cell death. Annu. Rev. Cell Biol. 7,663--698. Enari, M., Talanian, R.V., Wong, W.W. & Nagata, S. (1996). Sequential activation of ICE-like and CPP32-1ike proteases during Fas-mediated apoptosis. Nature 380,723-726. Escargueil-Blanc, I., Salvayre, R. & N6gre-Salvayre, A. (1994). Necrosis and apoptosis induced by oxidized low density lipoproteins occur through two calcium-dependent pathways in lymphoblastoid cells. FASEB J. 8,1075-1080. Farlie, P.G., Dringen, R., Rees, S.M., Kannourakis, G, & Bernard, O. (1995). bcl-2 transgene expression can protect neurons against developmental and induced cell death. Proc. Natl. Acad. Sci. USA 92,4397-4401. Farrow, S.N., White, J.H., Martinou, I., Raven, T., Pun, K.T., Grinham, C.J., Martinou, J.C. & Brown, R. (1995). Cloning of a bcl-2 homologue by interaction with adenovirus E1B 19K. Nature 374, 731-733. Ferrer, I., Tortosa, A., Condom, E., Blanco, R., Macaya, A. & Planas, A. (1994). Increased expression of bcl-2 immunoreactivity in the developing cerebral cortex of the rat. Neurosci. Lett. 179,13-16. Fisher, D.E. (1994). Apoptosis in cancer therapy: crossing the threshhold. Cell 78,539-542. Franke, T.F., Yang, S.I., Chan, T.O., Datta, K., Kazlauskas, A., Morrison, D.K., Kaplan, D.R. & Tsichlis, P.N. (1995). The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81:727-736. Frankowski, H., Missotten, M., Fernandez, P-A., Martinou, I., Michel, P., Sadoul, R. & Martinou, J.C. (1995). Function and expression of the Bcl-x gene in the developing and adult nervous system. NeuroReport 6,1917-1921. Fukuda, H. (1997). Programmed cell death during vascular system formation. Cell Death Differ. 5,684-688 Fukuda, K, Kojiro, M. & Chiu, J.F. (1993). Demonstration of extensive chromatin cleavage in transplanted Morris hepatoma 777 tissue: apoptosis or necrosis? Am. J. Pathol. 142, 935-946. Furukawa, K., Estus, S., Fu, W., Mark, R.J. & Mattson, M.P. (1997). Neuroprotective action of cycloheximide involves induction of Bcl-2 and antioxidant pathways. J. Cell Biol. 136, 1137-1149. Furukawa, K., Guo, Q., Schellenberg, G.D & Mattson M.P. (1998). Presenilin-1 mutation alter NGF-induced neurite outgrowth, calcium homeostasis, and transcription factor (AP-1) activation in PC12 cells. J. Neurosci. Res. 52, 618-624. Garcia, I., Martinou, I., Tsujimoto, Y. & Martinou, J-C. (1992). Prevention of programmed cell death of sympathetic neurons by the bcl-2 proto-oncogene. Science 258, 302-304. Gerlach, M., Riederer, P., Youdim, M.B. (1996). Molecular mechanisms for neurodegeneration. Synergism between reactive oxygen species, calcium, and excitotoxic amino acids. Adv. Neurol. 69, 177-194. Gillet, G., Guerin, M., Trembleau, A., Bran, G. (1995). A Bcl-2-related gene is activated in avian cells transformed by the Rous sarcoma virus. EMBO J. 14, 1372-1381. Givol, I., Tsarfaty, I., Resau, J., Rulong, S., da Silva, P.P., Nasioulas G, DuHadaway, J., Hughes, S.H. & Ewert, D.L. (1994). Bcl-2 expressed using a retroviral vector is localized primarily in the nuclear membrane and the endoplasmic reticulum of chicken embryo fibroblasts. Cell Growth Differ. 5, 419-429. Gonzalez-Garcia, M., Garcia, I., Ding, L., O'Shea, S., Boise, L.H., Thompson, C.B., Nunez, G. (1995). bcl-x is expressed in embryonic and postnatal neural tissues and functions to prevent neuronal cell death. Proc. Natl. Acad. Sci. USA 92, 4304-4308. Gonzalez-Garcia, M., Perez-Ballestero, F., Ding, L., Duan, L., Boise, L.H., Thompson, C.B. & Nunez, G. (1994). bcl-xL is the major bcl-x mRNA form expressed during murine development and its product

The Bcl-2 Protein Family and Cell Death

183

localizes to mitochondria. Development 120, 3033-3042. Gorman, A.M., McGoman, A., O'Neill, C. & Cotter, T. (1996). Oxidative stress and apoptosis in neurodegeneration. J. Neurol. Sci. 139 Suppl, 45-52. Gougeon, M-L. & Montagnier, L. (1993). Apoptosis in AIDS. Science 260,1269-1270. Grasl-Kraupp, B., Rullkay-Nedecky, B., Koudelka, H., Bukowska, K., Bursch, W., Schulte-Hermann, R. (1995). In situ dection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death, a cautionary note. Hepatology 21, 1465-1468. Green, D.R. & Reed, J.C. (1998). Mitochondria and apoptosis. Science. 281,1309-1312. Greenlund, L.J.S., Korsmeyer, S.J., Johnson, E.M. Jr. (1995a). Role of bcl-2 in the survival and function of developing and mature sympathetic neurons. Neuron 15,649-661. Greenlund, L.J.S., Deckwerth, T.L, & Johnson, E.M. (1995b). Superoxide dismutase delays neuronal apoptosis: A role for reactive oxygen species in programmed neuronal death. Neuron. 14, 303-315. Gross, A., Jockel, J., Wei, M.C. & Korsmeyer, S.J. (1998). Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J 17, 3878-3885. Gross, A., Yin, X.M., Wang, K., Wei, M.C., Jockel, J., Milliman, C., Erdjument-Bromage, H., Tempst, P. & Korsmeyer, S.J. (1999). Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death. J. Biol. Chem. 274, 1156-1163. Gross, A., Jockel, J., Wei, M.C. & Korsmeyer, S.J. (1998). Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J. 17, 3878-3885. Guo, Q., Sopher, B.L., Furukawa, K., Pham, D.G., Robinson, N., Martin, G.M. & Mattson, M.P. (1997). Alzheimer's presenilin mutation sensitizes neural cells to apoptosis induced by trophic factor withdrawal and amyloid B-peptide: involvement of calcium and oxyradicals. J. Neurosci. 17, 4212-4222. Guo, Q., Fu, W., Sopher, B.L., Miller, M.W., Ware, C.B., Martin, G.M. & Mattson, M.P. (1999a). Increased vulnerability of hippocampal neurons to excitotoxic necrosis in presenilin-I mutant knock-in mice. Nat. Med. 5, 101-107. Guo, Q., Fu, W., Xie, J., Luo, H., Sells, S.J., Geddes, J.W., Bondada, V., Rangnekar, V.M. & Mattson, M.P. (1998a). Par-4 is a new mediator of neuronal degeneration associated with the pathogenesis of Alzheimer's disease. Nat. Med. 4(8), 957-962. Guo, Q., Christakos, S., Robinson, N. & Mattson, M.P. (1998b). Calbindin D28K Blocks the Pro-Apoptotic Actions of Mutant Presenilin-h Reduced Oxidative Stress and Preserved Mitochondrial Function. Proc. Natl. Acad. Sci. USA. 95, 3227-3232. Guo, Q., Furukawa, K., Sopher, B.L., Pham, D.G., Xie, J., Robinson, N., Martin, G.M. & Mattson, M.P. (1996). Alzheimer's PS-1 mutation perturbs calcium homeostasis and sensitizes PC12 cells to death induced by amyloid B-peptide. NeuroReport 8, 379-383. Guo, Q., Sebastian, L., Sopher, B.L., Miller, M.W., Ware, C.B., Martin, G.M. & Mattson, M.P. (1999b). Increased vulnerability of hippocampal neurons from presenilin-I mutant knock-in mice to amyloid B-peptide toxicity: central roles of superoxide production and caspase activation. J. Neurochem. 72, 1019-1029. Guo, Q., Sebastian, L., Sopher, B.L., Miller, M.W., Glazner, G.W., Ware, C.B., Martin, G.M. & Mattson, M.P. (1999c). Neurotrophic factors (ADNF and bFGF) interrupt excitotoxic neurodegenerative cascades promoted by a PSI mutation. Proc. Natl. Acad. Sci. USA. In press. Guo, Q., Robinson, N. & Mattson, M.P. (1998c). Secreted B-amyloid precursor protein counteracts the proapoptotic action of mutant presenilin-1 by activation of NF-kB and stabilization of calcium homeostasis. J. Biol. Chem. 273 (20), 12341-12351.

184

Q. Guo, S.L. Chan and I. Kruman

Hakem, R., Hakem, A., Duncan, G.S., Henderson, J.T., Woo, M., Soengas, M.S., Elia, A., de la Pompa, J.L., Kagi, D., Khoo, W., Potter, J., Yoshida, R., Kaufman, S.A., Lowe, S.W., Penninger, J.M., & Mak, T.W. (1998). Differential requirement for caspase 9 in apoptosic pathways in vivo. Cell. 94, 339-352. Han, J., Sabbatini, P. & White, E. (1996). Induction of apoptosis by human Nbk/Bik, a BH3-containing protein that interacts with E1B 19K. Mol. Cell Biol 16, 5857-5864. Hedge, R., Srinivasula, S.M., Ahmad, M., Fernandes-Alnemri, T. & Alnemri, E.S. (1998). Blk, a BH3containing mouse protein that interacts with Bcl-2 and Bcl-xL, is a potent death agonist. J. Biol. Chem. 273, 7783-7786. Henderson, S., Huen, D., Rowe, M., Dawson, C., Johnson, G. & Rickinson, A. (1993). Epstein-Barr viruscoded BHRFI protein, a viral homologue of Bcl-2, protects human B cells from programmed cell death. Proc. Natl. Acad. Sci. U S A. 90, 8479-8483. Hengartner, M.O., Ellis, R.E. & Horvitz, H.R. (1992). Caenorhabditis elegans gene ced-9protects cells from programmed cell death. Nature 356, 494-499. Hengartner, M.O. (1997). CED-4 is a stranger no more. Nature 388, 714-715. Hengartner, M.O. & Horvitz, H.R. (1994). C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian protooncogene bcl-2. Cell. 76, 665-676. Henkart, P.A. (1996). ICE family proteases: mediators of all apoptotic cell death? Immunity 4, 195-201. Hennet, T., Richter, C. & Peterhans, E. (1993). Tumor necrosis factor-ct induces superoxide anion generation in mitochondria of L929 cells. Biochem. J. 289, 587-592. Hetts, S.W. (1998). To die or not to die: an overview of apoptosis and its role in disease. J.A.MA. 279(4), 300-307. Hirsch, E.C., Hunot, S., Damier, P. & Faucheux, B. (1998). Glial cells and inflammation in Parkinson's disease: a role in neurodegeneration? Ann. Neurol. 44(3 Suppl 1), S115-120. Hirsch, T., Marchetti, P., Susin, S.A., Dallaporta, B., Zamzami, N., Marzo, I., Geuskens, M. & Kroemer, G. (1997). The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene 15, 1573-1582. Hockenberry, D.M., Oltvai, Z.N., Yin, X.M., Milliman, C.L. & Korsmeyer, S.J. (1993). Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75,241-251. Hockenbery, D., Nunez, G., Millman, C., Schreiber, R.D. & Korsmeyer, S.J. (1990). Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348, 334-338. Hopcia, K.L., McCarey, Y.L., Sylvester, F.C. & Held, K.D. (1996). Radiation-induced apoptosis in HL60 cells: oxygen effect, relationship between apoptosis and loss of clonogenicity, and dependence of time to apoptosis on radiation dose. Radiat. Res. 145, 315-323. Hortnagl, H. & Hellweg, R. (1997). Pathophysiological aspects of human neurodegenerative diseases. Wien Klin Wochenschr. 109(16), 623-635. Hsu, S.Y. & Hsueh, A.J.W. (1998). A splicing variant of the Bcl-2 member Bok with a truncated BH3 domain induces apoptosis but does not dimerize with anti-apoptotic Bcl-2 proteins in vitro. J. Biol. Chem. 272, 30139-30146. Hsu, Y.T., Wolter, K.G. & Youle, R.J. (1997). Cytosol-to-membrane redistribution of Bax and BcI-X1 during apoptosis. Proc. Natl. Acad. Sci. U. S. A. 94, 3668-3672. Hu, Y., Benedict, M.A., WU, D., Inohara, N. & Nunez, G. (1998). Bcl-2-xL interacts with Apaf-1 and inhibits Apaf-l-dependent caspase-9 activation. Proc. Natl. Acad. Sci. U.S.A. 95, 4386-4391. Huang, D.C.G., Agams, J.M. & & Cory, S (1998). The conserved N-terminal BH4 domain of Bcl-2 homologues is essential for inhibition of apoptosis and interaction with CD-4. EMBO J. 17, 1029-1039. Hunter, J.J. & Parslow, T.G. (1996). A peptide sequence from Bax that converts Bcl-2 into an activator of

The Bcl-2 Protein Family and Cell Death

185

apoptosis. J. Biol. Chem. 271, 8521-8524. Ichas, F. & Mazat, J.-P. (1998). From calcium signaling to cell death: two conformations for the mitochondrial permeability transition pore. Switching from low- to high-conductance state. Biochim. Biophys. Acta. 1366, 33-50. Ichimiya, M., Chang, S.H., Liu, H., Berezesky, I.K., Trump, B.F. & Amstad, P.A. (1998). Effect of Bcl-2 on oxidant-induced cell death and intracellular Ca 2÷ mobilization. Am. J. Physiol. 275, C832-C839. Ink, B., Zornig, M., Baum, B., Hajibagheri, N., James, C., Chittenden, T. & Evan, G. (1997). Human Bak induces celldeath in Schizosaccharomyces pombe with morphological changes similar to those with apoptosis in mammalian cells. Mol. Cell Biol. 17, 2468-2474. Inohara, N., Ding, L., Chen, S. & Nunez, G. (1997). Harakiri, a novel regulator of cell death, encodes a protein that activates apoptosis and interacts selectively with survival-promoting proteins Bcl-2 and BcI-X(L). EMBO J. 16:1686-1694. Inohara, N., Ekhterae, D., Garcia, I., Carrio, R., Merino, J., Merry, A., Chen, S., & Nunez, G. (1998). Mtd, a novel Bcl-2 family member activates apoptosis in the absence of heterodimerization with Bcl-2 and BcI-XL. J. Biol. Chem. 273:8705-8710. Inohara, N., Gourley, T.S., Carrio, R., Muniz, M., Merino, J., Garcia, 1., Koseki, T., Hu, Y., Chen, S. & Nunez, G. (1998). Diva, a Bcl-2 homologue that binds directly to Apaf-1 and induces BH3-independent cell death. J. Biol. Chem. 272: 32479-32486. lrmler, M., Hoffman, K., Vaux, D.L. & Tschopp, J (1997). Direct physical interaction between the caenorhabditis elegans death proteins ced-3 and ced-4. FEBS Lett 406, 189-190. Ito, T., Deng, X., Carr, B. & May, W.S. (1997). Bcl-2 phosphorylation required for anti-apoptosis function. J. Biol. Chem. 272, 11671-11673. Itoh, G., Tamura, J., Suzuki, M., Suzuki, Y., Ikeda, H., Koike, M., Nomura, M., Jie, T. & Ito, K. (1995). DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis. Am. J. Pathol. 146, 1325-1331. Jacobson, M.D., Weil, M. & Raft, M.C. (1997). Programmed cell death in animal development. Cell 88, 347-354. James, C., Gschmeissner, S., Fraser, A. & Evan, G.I. (1997). CED-4 induces chromatin condensation in Schizosaccharomyces pombe and is inhibited by direct physical association with CED9. Curr. Biol. 7, 246-252. Johnson, E.M., J.R., Greenlund, L.J., Akins, P.T. & Hsu, C.Y. (1995). Neuronal apoptosis: current understanding of molecular mechanisms and potential role in ischemic brain injury. J. Neurotrauma 12, 843-852. Jurgensmeier, J.M., Krayewski, S., Armstrong, R.C., Wilson, G.M., Oltersdorf, T., Fritz, L.C., Reed, J.C. & Ottilie, S. (1997). Bax-and Bak-induced cell death in the fission yeast Schizosaccharomyces pombe. Mol. Biol. Cell 8, 325-339. Jurgensmeier, J.M., Xie, Z., Deveraux, Q., Ellerby, L., Bredesen, D. & Reed, J.C. (1998). Bax directly induces release of cytochrome c from isolated mitochondria. Proc. Natl. Acad. Sci. U.S.A. 95, 4997-5002. Kane, D.J., Ord, T., Anton, R. & Bredesen, D.E. (1995). Expression of bcl-2 inhibits necrotic neural cell death. J. Neurosci. Res. 40, 269-275. Kane, D.J., Sarafian, T.A., Anton, R., Hahn, H., Gralla, E.B., Valentine, J.S., Ord, T. & Bredesen, D.E. (1993). Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science 262,1274-1277. Kelekar, B.S., Chang, B.S., Harlan, J.E., Fesok, S.W. & Thompson, C.B. (1997). Bad is a BH3 domaincontaining protein that forms an activating dimer with Bcl-XL.Mol. Cell. Biol. 17, 1040-1046.

186

Q. Guo, S.L. Chan and I. Kruman

Keller, J.N., Guo, Q., Holtsberg, F.W., Bruce-Keller, A.J. & Mattson, M.P. (1998). Increased sensitivity to mitochondrial toxin-induced apoptosis in neural cells expressing mutant presenilin-1 in linked to perturbed calcium homeostasis and enhanced oxyradical production. J. Neurosci. 18(12), 4439-4450. Kerr, J.F.R., Wyllie, A.H. & Currie, A.R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239-257. Kerr, J.F.R. (1971). Shrinkage necrosis: a distinct mode of cellular death. J. Pathol. 105, 13-20. Kharabanda, S., Pandey, P., Schofield, L., Israels, S., Roncinske, R., Yoshida, K., Bharti, A., Yuan, Z.M., Saxena, S., Weichselbaum, R., Nalin, C. & Kufe, D. (1997). Role for Bcl-xL as an inhibitor of cytosolic cytochrome c accumulation in DNA damage-induced apoptosis. Proc. Natl. Acad. Sci. USA 94, 6939-6942. Kishkel, F.C., Hellbardt, Behrmann, I., Germer, M., Pawilita, M., Krammer, P.H., & Peter, M.E. (1995). Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 14, 5579-5588. Kitamoto, T. & Tateishi, J. (1996). Human prion disease and human prion protein disease. Curr. Top. Microbiol. Immunol. 207, 27-34. Kluck, R.M., Bossy-Wetzel, E., Green, D.R. & Newmeyer, D.D. (1997). The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275:1132-1136. Knudson, C.M., Tung, K.S., Tourtellotte, W.G., Brown, G.A. & Korsmeyer, S.J. (1995). Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science 270, 96-99. Krajewski, S., Krajewska, M., Shabaik, A., Miyashita, T., Wang, H-G. & Reed, J.C. (1994a). Immunohistochemical determination of in vivo distribution of bax, a dominant inhibitor of bcl-2. Am. J. Pathol. 145, 1323-13233. Krajewski, S., Krajewska, M., Shabaik, A., Wang, H.-G., Irie, S., Fong, L. & Reed, J.C. (1994b). Immunohistochemical analysis of in vivo patterns of Bcl-x expression. Cancer Res. 54, 5501-5507. Krajewski, S., Mai, J.K., Krajewska, M., Sikorska, M., Mossakowski, M.J. & Reed, J.C. (1995). Upregulation of Bax protein levels in neurons following cerebral ischemia. J. Neurosci. 15, 6364-6376. Krajewski, S., Tanaka, S., Takayama, S., Schibler, M.J., Fenton, W. & Reed, J.C. (1993). Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envlope, endoplasmic reticulum, and outer mitochondrial membranes. Cancer. Res. 53, 4701-4714. Kroemer. G. (1997). The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat. Med. 3,614-620. Kroemer, G., Petit, P., Zamzami, N., Vayssitre, J-L. & Mignotte, B. (1995). The biochemistry of programmed cell death. FASEB J. 9, 1277-1287 Kroemer, G., Zamzami, N. & Susin, S.A. (1997). Mitochondrial control of apoptosis. Immunol. Today 18, 44-51. Kroemer, G. (1995). The pharmacology of T cell apoptosis. Adv. Immunol. 58, 211-296. Kruman I, Guo, Q. & Mattson, M.P. (1998). Calcium and reactive oxygen species mediate staurosporineinduced mitochondrial dysfunction and apoptosis in PC12 cells. J. Neurosci. Res. 51,293-308. Kruman, I. & Mattson, M.P. (1999). Pivotal role of mitochondrial calcium uptake in neural cell apoptosis and necrosis. J. Neurochem. 72, 529-540 Kruman, I., Bruce-Keller, A.J., Bredesen, D.E., Waeg, G. & Mattson, M.P. (1997). Evidence that 4-hydroxynonenal mediates oxidative stress-induced neuroonal apoptosis. J. Neurosci. 17, 5089-5100. Kuida, K., Haydar, T., Kuan, C.-Y., Gu, Y., Taya, C., Harasuyama, H., Su, M.S.-S., Rakie, P. & Flavell, R. A.(1998). Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 94, 325-337. Kuida, K., Lippke, J.A., Ku, G., Harding, M.W., Livingston, D.J., Su, M.S. & Flavell, R.A. (1995). Altered

The Bcl-2 Protein Family and Cell Death

187

cytokine export and and apoptosis in mice deficient in interleukin-1 beta converting enzyme. Science. 267, 2000-2003. Kusiak, J.W., Izzo, J.A. & Zhao, B. (1996). Neurodegeneration in Alzheimer disease. Is apoptosis involved? Mol. Chem. Neuropathol. 28(1-3), 153-162. Lam, M., Bhat, M.B., Nunez, G., Ma, J. & Distelhorst, C.W. (1998). Regulation of Bcl-xL channel activity by calcium. J. Biol. Chem. 273, 17307-17310. Leist, M., Single, B., Castoldi, A.F., Kiihnle, S. & Nicotera, P. (1997). Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J. Exp. Med. 185,1481-1486. Leslie, C.C. (1997). Properties and regulation of cytosolic phospholipase A2. J. Biol. Chem. 272, 16709-16712. Li, H., Zhu, H., Xu, C.-J. & Yuan, J. (1998). Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94, 491-501. Li, P., Nijhavan, D., Brudihardjo, I., Srinivasula, S.M., Ahmad, M.,Alnemri, E.S. & Wang, X. (1997). Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91,479-489. Licinio, J. (1997). Central nervous system cytokines and their relevance for neurotoxicity and apoptosis. J. Neural. Transm. Suppl. 49, 169-175. Ligr, M., Madeo, F., Frohlich, E., Hilt, W., Frohlich, K.U. & Wolf, D.H. (1998) Mammalian Bax triggers apoptotic changes in yeast. FEBS Lett. 438, 61-65. Lin, E.Y., Orlofsky, A., Berger, M.S., Prystowsky, M.B. (1993). Characterization of A 1, a novel hemopoieticspecific early-response gene with sequence similarity to bcl-2. J. Immunol. 151, 1979-1988. Lin, E.Y., Orlofsky, A., Wang, H-G., Reed, J.C. & Prystowsky, M.B. (1996). A1, a bcl-2 family member, prolongs cell survival and permits myeloid differentiation. Blood 87, 983-992. Linden, R. (1994). The survival of developing neurons: a review of afferent control. Neuroscience 58(4), 671-682 Lithgow, T., van Driel, R., Bertram, J.F. & Strasser, A. (1994). The protein product of the oncogene bcl-2 is a component of the nuclear envelope, the endoplasmic reticulum, and the outer mitochondrial membrane. Cell Growth Differ. 5, 411-417. Liu, X., Kim, C.N., Yang, J., Jemmerson, R. & Wang, X. (1996). Induction of apoptotic program in cell free extracts: requirement for dATP and cytochrome c. Cell. 86, 147-157. Lo, A.C., Houenou, L.J. & Oppenheim, R.W. (1995). Apoptosis in the nervous system: morphological features, methods, pathology, and prevention. Arch. Histol. Cytol. 58(2), 139-149. Los, M., Van de Craen, M., Penning, LC., Schenk, H., Westendorp, M., Baeuerle, P.A., Droge, W., Krammer, P.H., Fiers, W. & Schulze-Osthoff, K (1993). Requirement of an ICE/CED-3 protease for Fas/APO-1 mediated apoptosis. Nature 375, 81-83. Lotem, J. & Sachs, L. (1996). Differential suppression by protease inhibitors and cytokines of apoptosis induced by wild-type p53 and cytotoxic agents. Proc. Natl. Acad. Sci. USA 93, 12507-12512. Luquin, M.R., Martinez-Vila, E. & Saldise, L. (1997). Mechanisms of cell death in Alzheimer's disease. Rev. Med. Univ. Navarra. 41(1), 34-45. Luschen, S., Ussat, S., Kronke, M. & Adam-Klages, S. (1998). Cleavage of human cytosolic phospholipase A2 by caspase-1 (ICE) and caspase-8 (FLICE). Biochem. Biophys. Res. Commmun. 253, 92-98. Macaya, A., Munell, F., Gubits, R.M. & Burke, R.E. (1994). Apoptosis in substantia nigra following developmental striatal excitotoxic injury. Proc. Natl. Acad. Sci. USA 91, 8117-8121. Mah, S.P., Zhong, L.T., Liu, Y., Roghani, A., Edwards, R.H. & Bredesen, D.E. (1993). The protooncogene

188

Q. Guo, S.L. Chan and 1. Kruman

bcl-2 inhibits apoptosis in PC12 cells. J. Neurochem. 60,1183-1186. Majno, G. & Joris, I. (1995). Apoptosis, oncosis, and necrosis. An overview of cell death.Am. J. Pathol. 146, 3-15. Mancini, M., Nicholson, D.W., Roy, S., Thornberry, N.A., Peterson, E.P., Casciola-Rosen, L.A. & Rosen, A. (1998). The caspase-3 precursor has a cytosolic and mitochondrial distribution: implications for apoptotic signaling. J. Cell Biol. 140, 1485-1495. Marchetti, P., Hirsch, T., Zamzami, N., Castedo, M., Decaudin, D., Susin, S.A., Masse, B. & Kroemer, G. (1996). Mitochondrial permeability transition triggers lymphocyte apoptosis. J. Immunol. 157, 4830-4836. Mark, R.J., Lovell, M.A., Markesbery, W.R. & Mattson, M.P. (1997). A role for 4-hydroxynonenal in disruption of ion homeostasis and neuronal death induced by amyloid l~-peptide. J. Neurochem. 68, 255-264. Marsden, C.D. & Olanow, C.W. (1998). The causes of Parkinson's disease are being unraveled and rational neuroprotective therapy is close to reality. Ann. Neurol. 44(3 Suppl 1), S189-196. Martin, L.J., AI-Abdulla, N.A., Brambrink, A.M., Kirsch, J.R., Sieber, F.E. & Portera-Cailliau, C. (1998). Neurodegeneration in excitotoxicity, global cerebral ischemia, and target deprivation: A perspective on the contributions of apoptosis and necrosis. Brain Res. Bull. 46(4), 281-309. Martinou, J.C., Dubois-Dauphin, M., Staple, J.K., Rodriguez, I., Frankowski, H., Missotten, M., Albertini, P., Talabot, D., Catsicas, S. & Pietra, C. (1994a). Overexpression of bcl-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia. Neuron 13, 1017-1030. Martinou, J-C., Frankowski, H., Missotten, M., Martinou, I., Potier, L. & Dubois-Dauphin, M. (1994b). Bcl-2 and neuronal selection during development of the nervous system. J. Physiol. 88,209-211 Marzo, I., Brenner, C. & Kroemer, G. (1998). The central role of mitochondrial megachannel in apoptosis: evidence obtained with intact cells, isolated mitochondria, and purified protein complexes. Biomed. Pharmacother. 52, 248-251. Masliah, E., Ge, N. & Mucke, L. (1996). Pathogenesis of HIV-1 associated neurodegeneration. Crit. Rev. Neurobiol. 10(1), 57-67. Mattson, M.P., Cheng, B., Davis, D., Bryant, K., Lieberburg, I. & Rydel, R.E. (1992). 13-amyloid peptides destabilize calcium homeostasis and render cortical neurons vulnerable to excitotoxicity. J. Neurosci. 12, 376-389. Mattson, MP. & Guo, Q. (1997a). Cellular and molecular neurobiology of presenilins: A role for the endoplasmic reticulum in the pathogenesis of Alzheimer's disease? J. Neurosci. Res. 50, 505-513. Mattson, M.P., Guo, Q., Furukawa, K. & Pederson, W. (1997b). Presenilins, the endoplasmic reticulum, and neuronal apoptosis in Alzheimer's disease. J. Neurochem. 70, 1-14. Mattson, MP., Robinson, N. & Guo, Q. (1997c). Estrogen stabilize mitochondrial function and protect neural cells against the pro-apoptotic action of mutant presenilin-1. NeuroReport 8, 3817-3821. McCarthy, N.J., Whyte, M.K.B., Gilbert, C.S. & Evan, G.I. (1997). Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak. J. Cell Biol. 136, 21-227. McConkey, D.J. (1996). Calcium-dependent, interleukin 1-converting enzyme inhibitor-insensitive degradation of lamin B1 and DNA fragmentation in isolated thymocyte nuclei. J. Biol. Chem. 271, 22398-22406. McConkey, D.J. & Orrenius, S. (1996). The role of calcium in the regulation of apoptosis. J. Leukoc. Biol. 59, 775-783. Merry, D.E., Veis, D.J., Hickey, W.F. & Korsmeyer, S.J. (1994). bcl-2 protein expression is widespread in the developing nervous system and retained in the adult PNS. Development 120,301-311.

The Bcl-2 Protein Family and Cell Death

189

Merry, D.E.& Korsmeyer, S.J. (1997). Bcl-2 gene family in the nervous system. Annu. Rev. Neurosci. 20, 245-267. Michaelidis, T.M., Sendtner, M., Cooper, J.D., Airaksinen, M.S., Holtmann, B., Meyer, M. & Thoenen, H. (1996). Inactivation of bcl-2 results in progressive degeneration of motoneurons,sympathetic and sensory neurons during early postnatal development. Neuron 17, 75-89. Miller, D.K., Myerson, J. & Becker, J.W. (1997). The interleukin-lB converting enzyme family of cysteine proteases. J. Cell. Biochem. 64, 2-10 Mills, E.M., Gunasekar, P.G., Pavlakovic, G., Isom, G.E. (1996). Cyanide-induced apoptosis and oxidative stress in differentiated PC12 cells. J. Neurochem. 67,1039-1046. Minn, A.J., Velez, P., Schendel, S.L., Liang, H., Muchmore, S.W., Fesik, S.W., Fill, M. & Thompson, C.B. (1997). Bcl-x(L) forms an ion channel in synthetic lipid membranes. Nature 385, 353-357. Miura, M., Zhu, H., Rotello, R., Hartwieg E.A. & Yuan, J. (1993). Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell. 75,653-660. Miyashita, T., Krajewski, S., Krajewska, M., Wang, H.G., Lin, H.K., Liebermann, D.A., Hoffman, B. & Reed, J.C. (1994). Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene 9,1799-1805. Monaghan, P., Robertson, D., Amos, T.A., Dyer, M.J., Mason, D.Y., Greaves, M.F. (1992). Ultrastructural localization of bel-2 protein. J. Histochem. Cytochem. 40, 1819-1825. Motoyama, N., Wang, F., Roth, K.A., Sawa, H., Nakayama, K., Negishi, I., Senju, S., Zhang, Q., Fujii, S. & Loh, D.Y. (1995). Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice. Science 267,1506-1510. Mountz, J.D., Wu, J., Cheng, J. & Zhou, T. (1994). Autoimmune disease: a problem of defective apoptosis. Arthritis Rheum. 37,1415-1420. Muchmore, S.W., Sattler, M., Liang, H., Meadows, R.P., Harlan, J.E., Yoon, H.S., Nettesheim, D., Chang, B.S., Thompson, C.B. & Wong, S.L. (1996). X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature 381,335-341. Murakami, M., Nakatani, Y., Atsumi, G-I., Inoue, K. & Kudo, I. (1997). Regulatory functions of phospholipase A2. Crit. Rev. Immunol. 17,225-283. Murphy, A. N., Bredesen, D.E., Cortopassi, G., Wang, E. & Fiskum, G. (1996). Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria. Proc. Natl. Acad. Sci. U.S.A. 93, 9893-9898. Nakayama, K., Nakayama,K-I., Negishi, 1., Kuida, K., Sawa, H. & Loh, D.Y. (1994). Targeted disruption of Bcl-2 alpha B in mice: occurrence of gray hair, polycystic kidney disease, and lymphocytopenia. Proc. Natl. Acad. Sci. USA 91,3700-3704. Nava, V.E., Cheng, E.H., Veliuona, M., Zou, S., Clem, R.J., Mayer, M.L. & Hardwick, J.M. (1997). Herpesvirus saimiri encodes a functional homolog of the human bcl-2 oncogene. J. Virol. 71, 4118-4122. Neilan, J.G., Lu, Z., Afonso, C.L., Kutish, G.F, Sussman, M.D. & Rock, D.L. (1993). An African swine fever virus gene with similarity to the proto-oncogene bcl-2 and the Epstein-Barr virus gene BHRF1. J. Virol. 67, 4391-4394. Ng, F.W., Nguyen, M., Kwan, T., Branton, P.E., Nicholson, D.W., Cromlish, J.A. & Shore, G.C. (1997). P28 Bap31, a Bcl-2/Bcl-xL-and procaspase-8 associated protein in the endoplasmic reticulum. J. Cell. Biol. 139, 327-338. Nguyen. M., Branton, P.E., Roy, S., Nicholson, D.W., Alnemri, E.S., Yeh, W.C., Mak, T.W. & Shore, G.C. (1998). E1A-induced processing of procaspase-8 can occur independently of FADD and is inhibited by

190

Q. Guo, S.L. Chan and L Kruman

Bcl-2. J. Biol. Chem. 273, 33099-33102. Nguyen, M., Branton, P.E., Walton, P.A., Oltvai, Z.N., Korsmeyer, S.J. & Shore, G.C. (1994). Role of membrane anchor domain of Bcl-2 in suppression of apoptosis caused by E1B-defective adenovirus. J. Biol. Chem. 269, 16521-16524. Nicholson, D.W., Ali, A., Thomberry, N.A., Vaillancourt, J.P., Ding, C.K., Gallant, M., Gareau, Y., Griffin, P.R., Labelle, M. & Lazebnik, Y.A. (1995). Identification and inhibition of the ICE/CED3 protease necessary for mammalian apoptosis. Nature 376, 3-43. Nicholson, D.W.& Thomberry, N.A. (1997). Caspases:killer proteases. Trends Biochem. Sci. 22, 299-306. Nunez, G., London, L., Hockenbery, D., Alexander, M., McKearn, J.P. & Korsmeyer, S.J. (1990). Deregulated Bcl-2 gene expression selectively prolongs survival of growth factor-deprived hemopoietic cell lines. J. Immunol.144, 3602-3610. O'Connor, L., Strasser, A., O'Reilly, L.A., Hausmann, G., Adams, J.M., Cory, S. & Huang, D.C. (1998). Bim: a novel member of the Bcl-2 family that promotes apoptosis. EMBO J. 17, 384-395. Oltvai, Z.N., Milliman, C.L. & Korsmeyer, S.J. (1993). Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74,609-619. Oppenheim, R.W. (1991). Cell death during development of the nervous system. Annu. Rev. Neurosci. 14, 453-501. Osborne, B.A. & Schwartz, L.M.. (1994). Essential genes that regulate apoptosis. Trends Cell Biol. 4, 394-398. Pan, G., O'Rourke, K. & Dixit, V.M. (1998). Caspase-9, BcI-XL, and Apaf-1 form a ternary complex. J. Biol. Chem. 273, 5841-5845. Peter, M.E., Heufelder, A.E. & Hengartner, M.O. (1977). Advances in apoptosis research. Proc. Natl. Acad. Sci. U.S.A. 94, 12736-12737. Pederson, W.A., Guo, Q., Hartman, B.K. & Mattson, M.P. (1997). Nerve growth factor-independent reduction in choline acetyltransferase activity in PC12 cells expressing mutant presenilin-1. J. Biol. Chem. 272, 22397-22400. Polyak, K., Xia, Y., Zweier, J.L., Kinzler, K.W. & Vogelstein, B. (1997). A model for p53-induced apoptosis. Nature. 389, 300-305. Qi, X.M., Simonson, M. & Distelhorst, C.W. (1997). Bcl-2 inhibits c-fos induction by calcium. Oncogene 15, 2849-2853. Quillet-Mary, A., Jaffrezou, J.P., Mansat, V., Bordier, C., Naval, J. & Laurent, G. (1997). Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis. J. Biol. Chem. 272, 21388-21395. Rao, D., Modha, D. & White, E. (1997). The E1B 19K protein associates with lamins in vivo and its proper localization is required for inhibition of apoptosis. Oncogene. 15, 1587-1597. Reed, J.C., Jurgensmeier, J.M & Matsuyama, S. (1998). Bcl-2 family proteins and mitochondria. Biochim. Biophys. Acta. 1366, 127-137. Robertson, A.M.G. & Thomson, J.N. (1982). Morphology of programmed cell death in the ventral nerve cord of Caenorhabditis elegans larvae. J. Embryol. Exp. Morph. 67, 89-100. Ruvolo, P.P., Deng, X., Carr, B.K., May, W.S. (1998). A functional role for mitochondrial protein kinase Calpha in Bcl2 phosphorylation and suppression of apoptosis. J. Biol. Chem. 273, 25436-25442. Ryan, J.J., Prochovnik, E., Gottlieb, C.E., Apel, l.J., Merino, R., Nunez, G. & Clarke, M.F. (1994). c-myc and bcl-2 modulate p53 function by altering p53 subcellular traffiking during the cell cycle. Proc. Natl. Acad. Sci. U.S.A. 91, 5878-5882. Sarid, R., Sato, T., Bohenzky, R.A., Russo, J.J. & Chang, Y. (1997). Kaposi's sarcoma-associated herpesvirus

The Bcl-2 Protein Family and Cell Death

191

encodes a functional bcl-2 homologue. Nat. Med. 3,293-298. Satin, A., Williams, M.S., Alexander-Miller, M.A., Berzofsky, J.A., Zacharchuk, C.M. & Henkart, P.A. (1997). Target cell lysis by CTL granule exocytosis is independent of ICE/Ced-3 family proteases. Immunity 6, 209-215. Sasaki, H., Matsuno, T., Tanaka, N. &Orita, K. (1996). Activation of apoptosis during the reperfusion phase after rat liver ischemia. Transpl. Proc. 28,1908-1909. Sattler, M., Liang, H., Nettesheim, D., Meadows, R.P., Harlan, J.E., Eberstadt, M., Yoon, H.S., Shuker, S.B., Chang, B.S., Minn, A.J., Thompson, C.B. & Fesil, S.W. (1997). Structure of Bcl-xu-Bak peptide complex: recognition between regulators of apoptosis. Science 275,983-986. Schendel, S.L., Xie, Z., Montal, M.O., Matsuyama, S., Montal, M. & Reed, J.C. (1997). Channel formation by antiapoptotic protein Bcl-2. Proc. Natl. Acad. Sci. U. S. A. 94, 5113-5118. Schlesinger, P.H., Gross, A., Yin, X.M., Yamamoto, K., Saito, M., Waksman, G. & Korsmeyer, S.J. (1997). Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2. Proc. Natl. Acad. Sci. U. S. A. 94, 11357-11362. Schwartzman, R.A. & Cidlowski, J.A. (1993). Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocrine Rev. 14, 133-151. Searle, J., Kerr, J.R.F.& Bishop, C.J. (1982). Necrosis and apoptosis: distinct modes of cell death with fundamentally different significance. Pathol. Annu. 17,229-259. Sedlak, T.W., Oltvai, Z.N., Yang, E., Wang, K., Boise, L.H., Thompson, C.B. & Korsmeyer, SJ. (1995). Multiple Bcl-2 family members demonstrate selective dimerizations with Bax. Proc. Natl. Acad. Sci. U.S.A. 15, 7834-7838. Server, A.C. & Mobley, W.C. (1991). Neuronal cell death and the role of apoptosis. In Apoptosis: The Molecular Basis of Cell Death, ed. LD Tomei, FO Cope, pp. 263-78. Plainview, NY: Cold Spring Harbor Lab. Seshagiri, S. & Miller, L.K. (1997). Caenorhabditis elegans CED-4 stimulates CED-3 processing and CED-3-induced apoptosis. Curr. Biol. 7,455-460. Shen, W., Kamendulis, L.M., Ray, S.D. & Corcoran, G.B. (1992), Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes: effects of Ca2+-endonuclease, DNA repair, and glutathione depletion inhibitors on DNA fragmentation and cell death. Toxicol. Appl. Pharmacol. 112, 32--40. Shibasaki, F., Kondo, E., Akagi, T. & McKeon, F. (1997). Suppression of signalling through transcription factor NF-AT by interactions between calcineurin and Bcl-2. Nature 386, 728-731. Shimizu, S., Eguchi, Y., Kamiike, W., ltoh, Y., Hasegawa, J., Yamabe, K., Otsuki, Y, Matsuda, H. & Tsujimoto, Y. (1996a). Induction of apoptosis as well as necrosis by hypoxia and predominant prevention of apoptosis by Bcl-2 and Bcl-Xt: Cancer Res. 56, 2161-2166. Shimizu, S., Eguchi, Y., Kamiike, W., Waguri, S., Uchiyama, Y., Matsuda, H. & Tsujimoto, Y. (1996b). Bcl-2 blocks loss of mitochondrial membrane potential while ICE inhibitors act at a different step during inhibition of death induced by respiratory chain inhibitors. Oncogene 13, 21-29. Shimizu, S., Eguchi, Y., Kamiike, W., Waguri, S., Uchiyama, Y., Matsuda, H. & Tsujimoto, Y. (1996c). Retardation of chemical hypoxia-induced necrotic cell death by Bcl-2 and ICE inhibitors: possible involvement of common mediators in apoptotic and necrotic signal transductions. Oncogene 12, 2045-2050. Shimizu, S., Eguchi, Y., Kosaka, H., Kamiike, W., Matsuda, H. & Tsujimoto, Y. (1995). Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-xL. Nature 374, 811-813. Shimuzu, S., Eguchi, W., Kamiike, Y., Funahashi, A., Mignon, V., Lacronique, H., Matsuda, H. & Tsujimoto, Y. (1998). Bcl-2 prevents apoptotic mitochondrial dysfunction by regulation proton flux. Proc. Natl. Acad.

192

Q. Guo, S.L. Chan and 1. Kruman

Sci. U.S.A. 95, 1455-1459. Shiraiwa, W., Inohara, N., Okada, S., Yuzaki, H., Shoji, S. & Ohm, S. (1996). An additional form of rat Bcl-x, Bcl-xbeta, generated by an unspliced RNA, promotes apoptosis in promyeloid cells. J. Biol. Chem. 271, 13258-13265. Shivers, B.D., Boxer, P.A., Keane, K.M., Kupina, N.C., Lynch, T., Schielke, G.P., Vartanian, M.G. & Vasilakos, J.P. (1998). Contribution of the ICE family to neurodegeneration. Ann. N. Y. Acad. Sci. 840, 59-64. Siman, R. & Scott, R.W. (1996). Strategies to alter the progression of Alzheimer's disease. Curr. Opin. Biotechnol. 7(6), 601-607. Simonian, P.L., Grillot, D.A., Andrews, D.W., Leber, B. & Nunez (1996). Bax homodimerization is not required for Bax to accelerate chemotherapy-induced cell death. J. Biol. Chem. 271, 32073-32077. Songyang, Z., Baltimore, D., Cantley, L.C., Kaplan, D.R. & Franke, T.F. (1997). lnterleukin 3-dependent survival by the Akt protein kinase. Proc. Natl. Acad. Sci. U. S. A. 94, 11345-11350. Spector, M.S., Desnoyers, S., Hoeppner, D.J. & Hengartner, M.O. (1997). Interaction between the, elegans cell-death regulators CED-9 and CED-4. Nature, 385, 653-656. Squier, M.K.T., Miller, A.C.K, Malkinson, A.M. & Cohen, J.J. (1994). Calpain activation in apoptosis. J. Cell. Physiol. 159, 229-237. Stellar, H. (1995). Mechanisms and genes of cellular suicide. Science 267, 1445-1449. Stewart, B.W. (1994). Mechanisms of apoptosis: integration of genetic, biochemical and cellular indicators. J. Natl. Cancer Inst. 86,1286-1296. Susin, S., Zamzami, N., Castedo, M., Daugas, E., Wang, H.-G., Geley, S., Fassy, F., Reed, J. & Kroemer, G. (1997). The central executioner of apoptosis: multiple connections between protease activation and mitochondria in FAS/APO-1/CD95-and ceramide-induced apoptosis. J. Exp. Med. 186, 25-37. Susin, S.A., Zamzami, N. & Kroemer, G. (1998). Mitochondria as regulators of apoptosis:doubt no more. Biochim. Biophys. Acta. 1366,151-165. Susin, S.A., Zamzami, N., Castedo, M., Hirsch, T., Marchetti, P., Macho, A., Daugas, E., Geuskens, M. & Kroemer, G. (1996). Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J. Exp. Med. 184,1331-1342. Takayama, S., Sato, T., Krajewski, S., Kochel, K. & Irie, S. (1995). Cloning and functional analysis of BAG-l: a novel Bcl-2-binding protein with anti-cell death activity. Cell 80, 279-284. Tanaka, M., Ito, H., Adachi, S., Akimoto, H., Nishikawa, T., Marumo, F. & Hiroe, M. (1994). Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes. Circ. Res, 75, 426-433. Tanaka, S., Saito, K. & Reed, LC. (1993). Structure-function analysis of the Bcl-2 oncoprotein. Addition of a heterologous transmembrane domain to portions of the Bcl-2 beta protein restores function as a regulator of cell survival..1. Biol. Chem. 268, 10920-10926. Tao, W., Kurschner, C.& Morgan, J.l. (1998). Bcl-xS and Bad potentiate the death suppressing activation of BcI-XI, Bcl-2 and A1 in yeast. J. Biol. Chem. 273, 23704-23708. Tomei, L.D.& Cope, F.O., eds. (1991). Apoptosis: The Molecular Basis of Cell Death. Curr. Commun. Cell Mol. Biol. Cold Spring Harbor, NY: Cold Spring Harbor Lab. Press pp. 246. Thornberry, N. & Lazebnik, Y. (1998). Caspases: enemies within. Science. 281, 1312-1316. Troost, D., Aten, J., Morsink, F. & de Jong, J.M. (1995). Apoptosis in ALS is not limited to motoneurons: Bcl-2 expression is increased in post-central cortex, adjacent to the affected motor cortex. J. Neurol. Sci. 129, 79-80. Tsujimoto, Y. & Croce, C.M. (1986). Analysis of the structure, transcripts, and protein products of bcl-2, the

The Bcl-2 Protein Family and Cell Death

193

gene involved in human follicular lymphoma. Proc. Natl. Acad. Sci. U.S.A. 83, 5214-5218. Vander Heiden, M.G., Chandel, N.S., Williamson, P.T., Schumacker, C.B. & Thompson, C.B. (1997). Bcl-x e regulates the membrane potential and volume homeostasis of mitochondria. Cell 91,627-637. Vanhaesebroeck, B., Reed, J.C., DeValck, D., Grooten, J. & Miyashita T. (1993). Effect of bcl-2 protooncogene expression on cellular sensitivity to tumor necrosis factor-mediated cytotoxicity. Oncogene 8, 1075-1081. Vasilakos, J. & Shivers, B. (1996). Watch for ICE in neurodegeneration. Mol Psychiatry. 1(1), 72-76. Vaux, D.L., Cory, S. & Adams, J.M. (1988). Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335,440-442. Vaux, D.L., Haecker, G. & Stmsser, A. (1994). An evolutionary perspective on apoptosis. Cell 76, 777-779. Vaux, D.L. & Strasser, A. (1996), The molecular biology of apoptosis. Proc. Natl. Acad. Sci. U.S.A. 93, 2239-2244. Vaux, D.L., Whitney, D. & Weissman, I.L (1996). Activation of physiological cell death mechanisms by a necrosis-causing agent. Microsc. Res. Tech. 34, 259-266. Vaux, D.L. (1997). CED-4--the third horseman of apoptosis. Cell 90, 389-390. Veis, D.J., Sorenson, C.M., Shutter, J.R. & Korsmeyer, S.J. (1993). Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 75, 229-240. Wang, H-G., Rapp, U.R. & Reed, J.C. (1996). Bcl-2 targets the protein kinase Raf-1 to mitochondria Cell 87; 629-638. Wang, K., Yin, X.M., Chao, D.T., Milliman, C.L & Korsmeyer, S.J. (1996). BID: a novel BH3 domain-only death agonist. Genes Dev. 10, 2859-2869. Wang, S., Miura, M., Jung, Y.-K., Zhu, H., Li, E. & Yuan, J.(1998). Murine caspase-11, an ICE-interacting protease, is essential for the activation of ICE. Cell 92, 501-509. White, E., Sabbatini, P., Debbas, M., Wold, W.S., Kusher, D.I. & Gooding, L. (1992). The 19-kilodalton adenovirus E1B transforming protein inhibits programmed cell death and prevents cytolysis by tumor necrosis factor alpha. Mol. Cell Biol. 12, 2570-2580. Williams, M.S. & Henkart, P.A. (1994). Apoptotic cell death induced by intracellular proteolysis. J. Immunol. 153, 4247-4255. Wolter, K.G., Hsu, Y.-T., Smith, C.L., Nechushtan, A., Xi, X.-G. & Youle, R.J. (1997). Movement of Bax from the cytosol to mitochondria during apoptosis. J. Cell Biol. 139, 1281-1292. Wu, D.Y., Wallen, H.D. & Nunez, G. (1997). Interaction and regulation of subcellular localization of ced-4 by ced-9. Science 275,1126-1129. Wyllie, A.H., Arends, M.J., Morris, R.G., Walker, S.W. & Evan, G. (1992). The apoptosis endonuclease and its regulation. Semin. Immunol. 4, 389-397. Wyllie, A.H., Duvall, E. & Blow, J.J. (1984). Intracellular mechanisms in cell death in normal and pathological tissues. In Cell Aging and Cell Death, ed. I Davies, DC Sigee,pp. 269-94. London: Cambridge Univ. Press. Wyllie, A.H., Kerr, J.F. & Currie, A.R.. (1980). Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251-306. Wyllie, A.H. (1981). Cell death: a new classification separating apoptosis from necrosis. In Cell Death in Biology and Pathology, ed. ID Bowen, RA Lockshin,pp. 9-34. London: Chapman & Hall. Wyllie, A.H. (1987a). Apoptosis: cell death in tissue regulation. J. Pathol. 153, 313-316. Wyllie, A.H. (1987b). Apoptosis: cell death under homeostatic control. Arch. Toxicol. 11, 3-10. Wyllie, A.H. (1995). The genetic regulation of apoptosis. Curr. Opin. Gen. Dev. 5, 97-104. Xiang, J., Chao, D.T. & Korsmeyer, S.J. (1996). Bax-induced cell death may not require interleukin

194

Q. Guo, S.L. Chan and I. Kruman

lb-converting enzyme-like proteases. Proc. Natl. Acad. Sci. U.S.A. 93, 14559-14563. Xu, Q. & Reed, J.C. (1998). Bax inhibitor-l, a mammalian apoptosis suppressor identified by functional screening in yest. Mol.Cell 1,337-346. Xue, D., Shaham, S. & Horvitz, H.R. (1996). The Caenorhabditis elegans cell-death protein CED-3 is a cysteine protease with substrate specificities similar to those of the human CPP32 protease. Genes Dev. 10, 1073-1083. Xue, D. & Horvitz, H.R. (1997). Caenorhabditis elegans CED-9 protein is a bifunctional cell death inhibitor. Nature 390, 305-308. Yamamoto, A.M., Eloy, L., Bach, J.F. & Garchon, H.J. (1998). N-terminus cleavage of bcl-2 by a novel cellular non-ICE cysteine proteinase. Leukemia 12, 1467-1472. Yang, E. & Korsmeyer, S.J. (1996). Molecular Thanatopsis: a discourse on the Bcl-2 family and cell death. Blood 88, 386-401. Yang, E., Zha, J., Jockel, J., Boise, L.H., Thomson, C.B. & Korsmeyer, S.J. (1995). Bad, a heterodimeric partner for Bcl-xL and Bcl-2, displaces Bax and promotes cell death. Cell 80, 285-291. Yang, J., Liu, X., Bhalla, K., Kim, C.N., Ibrado, A.M., Cai, J., Peng, T.I., Jones, D.P. & Wang, X. (1997). Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129-1132. Yin, X.M., Oltvai, Z.N. & Korsmeyer, S.J. (1994). BH 1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 369, 321-323. Yuan, J.Y. & Horvitz, H.R. (1990). The Caenorhabditis elegans genes ced-3 and ced-4 act cell autonomously to cause programmed cell death. Dev. Biol. 138, 33-41. Yuan, J.Y., Shaham, S., Ledoux, S., Ellis, H.M. & Horvitz, H.R. (1993). The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-l-beta-converting enzyme. Cell 75,641-652. Zamzami, N., Brenner, C., Marzo, I., Susin, S.A. & Kroemer, G. (1998). Subcellular and submitochondrial mode of action of Bcl-2-1ike oncoproteins. Oncogene 16, 2265-2282. Zamzami, N., Marchetti, P., Castedo, M., Zanin, P., Vassiere, J.-P., Vassiere, P.X., Petit, G. & Kroemer, G. (1995a). Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181, 1661-1672. Zamzami, N., Mrchetti, P., Castedo, M., Decaudin, D., Macho, A., Hirsch, T., Susin, S.A., Petit, P.X., Mignotte, B. & Kroemer, G. (1995b). Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J. Exp. Med. 182, 367-377. Zamzami, N., Susin, S.A., Marchetti, P., Hirsch, T., Gomez-Monterrey, I., Castedo, M. & Kroemer, G. (1996). Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183, 1533-1544. Zeitlin, S., Liu, J.P., Chapman, D.L., Papaioannou, V.E. &Efstratiadis, A. (1995) Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington's disease gene homologue. Nat. Genet. 11,155-163. Zha, H. & Reed, J.C. (1997). Heterodimerization-independent functions of cell death regulatory proteins Bax and Bcl-2 in yeast and mammalian cells. J. Biol. Chem, 272, 31482-31488. Zha, J., Harada, H., Yang, E., & Jockel, J. & Korsmeyer, S.J. (1996). Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-XL. Cell 87,619-626. Zhong, L-T., Sarafian, T., Kane, D.J., Charles, A.C., Mah, S.P., Edwards, R.H. & Bredesen, D.E. (1993). bcl-2 inhibits death of central neural cell induced by multiple agents. Proc. Natl. Acad. Sci. U.S.A 90, 4533-4537. Zhou. M., Demo, S.D., McClure, T.N., Crea, R. & Bitler. C.M. (1998) A novel splice variant of the cell death-promoting protein Bax. J. Biol. Chem. 272, 11930-11936.

The Bcl-2 Protein Family and Cell Death

195

Zhou, Q., Snipas, S., Orth, K., Muzio, M., Dixit, V.M. & Salvesen, G.S. (1997). Target protease specificity of the viral serpin CrmA. Analysis of five caspases. J. Biol. Chem. 272, 7797-7800. Zhu, W., Cowie, A., Wasfy, G.W., Penn, L.Z., Laber, B. & Andrews, D.W. (1996). Bcl-2 mutants with restricted subcellular location reveal spatially distinct pathways for apoptosis in different cell-types. EMBO J. 15, 4130-4141. Zou, H., Henzel, W.J., Liu, X., Lutschg, A. & Wang, X. (1997). Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90, 405-413.

THE R O L E OF JUN KINASES IN APOPTOSIS STEVEN P. T A M M A R I E L L O , G A R Y E. L A N D R E T H , and STEVEN ESTUS

Table of Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Role of c-Jun in Apoptosis and PCD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c-Jun in Survival, Proliferation, and Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c-Jun Activation is Phosphorylation Dependent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multiple Genes and Alternative Splicing Generate Complex JNK Family . . . . . . . . . . . . . . . JNK Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . JNKs Phosphorylate Multiple Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Substrate Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternate JNK Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . JNK and Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . JNK Activation in Sympathetic Neuron Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . JNK in Fas-mediated Apoptosis JNK in Cerebellar Granule Cell Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . JNK and TNF-induced Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative Stress Activates JNKs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . JNKs and Human Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction The c-Jun amino (N)-terminal __kinases (JNKs, also known as stress-activated protein kinases (SAPKs)) are one o f three subfamilies that belong to the mitogen-activated protein kinase ( M A P K ) family, along with the extracellular signal-regulated protein kinases 1 and 2 ( E R K I and ERK2) and p38. M A P K s are important regulatory proteins through which various extracellular signals are transduced into intracellular events (Davis, 1994; Robinson and Cobb, 1997). They are responsible for the phosphorylation of a variety of proteins, which include downstream kinases and transcription factors. In this chapter, we shall review the role of a primary JNK target, c-Jun in apoptosis, and discuss the activation and actions of JNKs, including downstream phosphorylation substrates in addition to c-Jun. We will conclude by discussing the role(s) o f the JNKs in models of apoptosis and human disease.

197 Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. 1 9 7 - - 2 1 4 © 2 0 0 1 Elsevier Science. Printed in the Netherlands.

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The Role of c-Jun in Apoptosis and PCD In many programmed cell death (PCD) models, one necessary hallmark is the phosphorylation and subsequent activation of the transcription factor c-Jun. PCD may be modeled as a sequential series of events wherein an initial cell death trigger is subsequently propagated (Figure 1). When this amplification reaches a critical threshold, apoptosis is initiated. Apoptosis is marked by blebbing of the plasma membrane, shrinkage of the cell body, condensation of nuclear chromatin, and fragmentation of nuclear DNA. These morphologic criteria are exhibited by both neuronal and nonneuronal cell-types, in response to both physiologic apoptotic stimuli, e.g. trophic factor deprivation or exposure to FAS ligand, and non-physiologic apoptotic stimuli, e.g. amyloid beta toxicity or staurosporine treatment (reviewed in Estus, 1998). Apoptosis is followed by a phase wherein cell debris is removed, often by cellular neighbors. Extensive cell death occurs during normal development of the tissues of vertebrates (Glucksman, 1951). In the nervous system, during normal mammalian development, approximately half of the neurons that are formed subsequently die by apoptosis (Oppenheim, 1991). Recent evidence suggests that apoptosis is involved not only during normal development, but in many neurodegenerative diseases and neurological conditions such as Alzheimer's Disease, Huntington's Disease, Batten's Disease, and stroke ( S u e t al., 1994; Lassmann et al., 1995; Portera-Calliau et al., 1995; Lane et al., 1996; Goto et al., 1990; Shigeno, 1990; Linnik et al., 1993; MacManus et al., 1993; 1994). The molecular events underlying PCD have been studied extensively; a model of the molecular events in sympathetic neurons undergoing PCD because of NGF deprivation has been proposed, and elements of this model may apply to many other apoptosis models (Figure 2). Following an initial stimulus, e.g. trophic factor withdrawal, the propagation phase includes a period of oxidative stress, which is followed by sequential JNK activation and c-Jun expression. When the apoptosis phase is initiated, cytochrome c is released from the mitochondria, leading to caspase activation and resultant chromatin condensation and fragmentation. Although c-Jun activation occurs in many examples of PCD (see below), c-Jun has also been extensively studied because of its role as an oncogene, c-Jun is a leucine zipper transcription factor that is part of the larger AP-1 transcription factor family (Angel and Karin, 1991). AP-1 transcription factors are composed of homodimeric complexes of members of the Jun family (c-Jun, JunB, JunD) or heterodimeric complexes of Jun family members alone or in combination with members of the Fos family (c-Fos, FosB, Fra-1, Fra-2). Activation of the AP-1 complex is caused by either increased expression of Fos and Jun proteins or by post-translational regulation, e.g. redox regulation and/or phosphorylation of Fos and/ or Jun family members (Abate et al., 1990; Derijard et al., 1994; Kyriakis et al., 1994; Xanthoudakis et al., 1992). The AP-1 family mediates an immediate-early gene response following cellular exposure to extemal stimuli. The activation of c-Jun occurs in many models of cell death (Miller and Johnson, 1996). In some neuronal and non-neuronal models, evidence has been presented implicating c-Jun as being necessary for apoptosis (Colotta et al., 1992; Estus et al., 1994; Schlingensiepen et al., 1994; Ham et al., 1995; Xia et al., 1995;

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P r o g r a m m e d Cell Death Figure 1, Apoptosis is a Stage in Programmed Cell Death (PCD). PCD can be summarized as a series of stages. Essentially, a trigger phase initiates a propagation phase. When the propagation phase reaches a critical threshold, apoptosis is initiated. Until apoptosis, the process is reversible. After apoptosis, cellular debris is removed in a "clean-up" process. Oxidative stress

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W a t s o n et al., 1998). Hence, c-Jun activation occurs during P C D in m a n y m o d e l s and, at least in s o m e instances, appears necessary for apoptosis. c-Jun in Survival, Proliferation, and Regeneration A l t h o u g h the primary focus o f this chapter will be the role o f c-Jun activation in cell death, several studies have attributed a functionally important role for c-Jun in areas such as cell cycle re-entry and oncogenesis (Angel & Karin, 1991; Cochran, 1993;

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Kovary and Bravo, 1991; Kovary and Bravo, 1992). These apparently disparate roles of c-Jun are not necessarily in conflict. Indeed, cell cycle re-entry has been implicated in apoptosis of post-mitotic cells, e.g. neurons or serum-deprived fibroblasts (Freeman et al., 1993); hence, if the actions of c-Jun are to encourage cell cycle re-entry, the ultimate result of this action, i.e. oncogenesis, normal cell cycle re-entry, or apoptosis, may depend upon other cellular constraints. The induction of c-jun per se has also been shown in response to many stimuli where a functional role for c-Jun has not been clearly defined. For example, c-jun is induced during axonal regeneration (Herdegen et al., 1993) and in PC12 cells induced to differentiate by NGF treatment (Wu et al., 1989). Although the axotomy-induced upregulation of c-jun appears to be conserved throughout vertebrates, suggesting that c-Jun plays a fundamental role in the evolutionary regeneration response, such a functional role has not been identified. c-Jun Activation is Phosphorylation Dependent Phosphorylation of c-Jun occurs on several residues. Although three sites of phosphorylation are found near the DNA-binding domain and inhibit this activity, and these sites may be phosphorylated by glycogen-synthase kinase-3 (Boyle et al., 1991), more recent intensive efforts examining c-Jun phosphorylation have focused on two phosphorylation sites in the amino-terminal transactivation domain (ser63 and ser73). This more recent work will be the subject of this review. The kinases which mediate this phosphorylation have been termed JNKs aka SAPKs (Derijard et al., 1994; Kyriakis et al., 1994). Since c-Jun phosphorylation at Ser73 occurs apparently only in a minority of neurons that express c-Jun, phosphorylation at Ser63 may be more important for c-Jun activation (Herdegen et al., 1997). Phosphorylation at these residues results in c-Jun activation, including translocation from the cytoplasm to the nucleus. While JNKs are the only proteins known to phosphorylate c-Jun at these activation sites in vivo, the MAP kinases ERK-1 and ERK-2 are capable of phosphorylating c-Jun in vitro (Pulverer et al., 1991). These results suggest that alternate kinase(s) may exist that are capable of phosphorylating c-Jun at Ser63 and/or Ser73. Consistent with this possibility, Watson et al. reported that Ser63 phosphorylation was increased in cerebellar granule cells undergoing apoptosis after survival signal withdrawal, but with no apparent increase in JNK activity (Watson et al., 1998). The alternative possibility, that c-Jun phosphatase activity may be modulated, has received relatively scant attention, although a possible Drosophila JNK phosphatase was described recently (Martin-Blanco et al., 1998).

Multiple Genes and Alternative Splicing Generate Complex JNK Family At least 10 isoforms of JNK are expressed in human brain, and each isoform differs in its interaction with downstream transcription factors, such as c-Jun, ATF-2, and ELK-1 (Gupta et al., 1996; Kyriakis et al., 1994) (See JNKs Phosphorylate Multiple Substrates). This indicates that the JNK group of protein kinases may interact selectively

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with transcription factors to mediate specific cellular roles (Kallunki et al., 1994). The 10 JNK isoforms identified in brain tissue arise from the alternative splicing of transcripts from three different genes, JNK1, JNK2, and JNK3 (Gupta et al., 1996). Although these genes are similar in amino acid sequence, all the isoforms have distinct in vitro biochemical properties, suggesting they are not functionally redundant (Gupta et al., 1996). Using an in-gel protein kinase assay, Hibi et al. originally demonstrated the electrophoretic separation of two classes of JNK activity having molecular masses of 46 and 54 kDa respectively (Hibi et al., 1993). At that time, Hibi proposed the 46 kDa isoform as JNK1 and the 54 kDa isoform as JNK2. However, Gupta et al. and Kyriakis et al. subsequently reported that these isoforms are actually encoded by both JNK1 and JNK2 by alternative splicing of the gene products (Gupta et al., 1996; Kyriakis et al., 1994). Indeed, since this alternative splicing was also detected in JNK3, the two isoforms originally isolated likely represent a mixture of JNK gene products.

JNK Activation JNKs are activated and participate in the induction of c-Jun by growth factors, proinflammatory cytokines, and multiple cellular stresses including uv-light, osmotic shock, alkylating agents, and protein synthesis inhibitors (Devary et al., 1991; Hibi et al., 1993; Kyriakis et al., 1994; Derijard et al., 1994). JNKs are activated by phosphorylation on threonine and tyrosine residues within the sequence Thr-Pro-Tyr by SEK1, also known as MAPK kinase 4 (Derijard et al., 1995). Unlike many kinases, SEK1 is not a dual specificity enzyme as it phosphorylates JNKs only on Tyr; the Thr on JNK is then autophosphorylated by JNK. Whether JNKs are activated prior to or following nuclear translocation is currently under debate, and is probably cell- or paradigm-specific. JNKs are translocated from the cytoplasm to the nucleus before their activation by SEK1 in models of ischemia, while following irradiation JNKs are activated without being translocated to the nucleus (Chen et al., 1996; Mizukami et al., 1997). SEK1 itself is activated by phosphorylation on Thr-223 by MEKK1 (Yan et al., 1994). Figure 3 shows a schematic diagram of the current model of the JNK signaling pathway. This pathway is likely relevant to neurons since microinjection of an expression vector that constituitively expresses MEKK1 results in increased activation of JNKs and c-Jun, and leads to apoptotic death in PC12 cells or sympathetic neurons, suggesting that MEKK1 can activate the JNK pathway in a SEKl-dependent manner (Xia, et al., 1995; Eilers et al., 1998). The activation of MEKK1 has been studied, but a definitive pathway has not been established at this point. MEKK1 may be activated through phosphorylation by the PAK protein kinase that is activated by the small GTP binding proteins Racl and Cdc42 (Bagrodia et al., 1995; Coso et al., 1995; Minden et al., 1995). Support of this model includes a recent report that constitutively activated Racl and Cdc42 activate the JNK pathway in a MEKKl-dependent manner. Moreover, this activation leads to increased transcriptional activity of c-Jun, ultimately causing apoptosis in NGF-maintained neurons (Bazenet et al., 1998). Further, the expression

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of dominant negative mutants of Cdc42 or Rac 1 blocks MEKK1 and JNK activation, as well as the apoptosis that normally occurs following NGF deprivation in sympathetic neurons (Bazenet et al., 1998). Once activated by phosphorylation, JNKs bind the amino-terminal transactivating domain of c-Jun, where residues Ser63 and Ser73 are phosphorylated. To increase the efficiency of the enzymatic reactions, c-Jun uses a docking site to attract JNK to itself (Karin and Hunter, 1995). Thus activated, c-Jun then modulates the transcription of many genes including the induction of c-jun itself (Smeal et al., 1991; Smeal et al., 1994). Although SEK1 is known to be an efficient JNK kinase, several reports suggest that JNK activation can occur via SEKl-independent pathways. Nagata et al. (1997) reported that the cytokines erythropoietin, thrombopoietin, and interleukin-3 activate JNK but not SEK1 during cell growth and differentiation in SKT6 cells (Nagata et al., 1997). Furthermore, JNKs can be activated even following the deletion of SEK1 activity by homologous recombination of mutant SEK1 (Nishina et al., 1997). Finally, the expression of SEKAL, a SEK1 dominant negative, is incapable of inhibiting JNK activation and subsequent c-Jun phosphorylation following NGF withdrawal in sympathetic neurons (Eilers et al., 1998). Recently, three alternate, SEKl-independent JNK kinases have been isolated, MKK7, JNKK2, and JNKK2-alpha (Lu et al., 1997;

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Tournier et al., 1997; Yang et al., 1998). All three have been found to phosphorylate JNK both in vitro and in vivo, but the role of each in the JNK-mediated apoptosis cascade remains to be elucidated.

JNKs Phosphorylate Multiple Substrates Although c-Jun is established as a JNK substrate, recent studies have led to the identification of additional JNK targets. Since many of these substrates are transcription factors, phosphorylation of these substrates often results in altered transcriptional activity (Hibi et al., 1993; Livingstone et al., 1995; van Dam et al., 1995; Gupta et al., 1996). Substrate Requirements Effective JNK substrates require two separate domains in order to be phosphorylated. These include a docking site which confers specificity and the phosphoacceptor site (Kallunki et al., 1996). The docking site increases the efficiency and specificity of the phosphorylation reaction. For example, JNKs phosphorylate c-Jun quite efficiently, JunD less efficiently, and JunB not at all. JunB contains a functional docking site, but lacks specificity residues, while JunD lacks a docking site but has a phosphoacceptor motif identical to c-Jun (Kallunki et al., 1996). Alternate JNK Substrates One additional JNK substrate is the transcription factor ATF-2, which, like c-Jun, is phosphorylated at the NH2-terminal activation domain (residues Thr69 and Thr71). This phosphorylation also occurs intracellularly as activation of the JNK pathway causes increased phosphorylation of ATF-2 on these sites in vivo, and increases ATF2 transcriptional activity (Livingstone et al., 1995). A second JNK target is the Ets-domain transcription factor Elk-1 (Zhang et al., 1998). Serum response factor and Elk-1 constitute part of a complex that binds the serum response element. Phosphorylation of Elk-1 by JNK causes increased transcription and expression of early immediate genes (Zhang et al., 1998). Interestingly, the substrate interaction is only apparent in neurons sensitive to hypoxia, where translocation of the substrate was accompanied by nuclear translocation of JNK. The tumor suppressor protein p53 is modulated by phosphorylation. Multiple kinases have been implicated in this action, including JNK1, JNK2, and JNK3. Hu et al. showed that each of the activated JNKs can phosphorylate mouse p53 (Ser34) in vitro (Hu et al., 1997). Furthermore, all three JNK isoforms associate with p53 in vivo, indicating that all three are likely p53 (Ser 34) kinases. This result is consistent with the data that p53 is activated in many apoptosis models. Lastly, JNKs are capable of phosphorylating the pro-life protein Bcl-2 in vitro (Maundrell et al., 1997). JNKs phosphorylate Bcl-2 within its flexible loop region, disabling Bcl-2 from its anti-apoptotic function, which, in turn, reinforces pro-

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apoptotic processes. Hence, JNKs may modulate apoptosis in transcription factor independent pathways.

JNK and Apoptosis JNK Activation and Neuronal Apoptosis JNK Upregulation Following NGF Withdrawal in Sympathetic Neurons

Neonatal sympathetic neurons undergo programmed cell death in the first post-natal week (Wright et al., 1983). This death can be accentuated by depriving the developing animal of the neurotrophin necessary for sympathetic neuron survival, i.e. NGF (Gorin and Johnson, 1979). This naturally occurring neuronal death can be recapitulated in vitro (Deckwerth and Johnson, 1993; Edwards et al., 1991). In this in vitro model, apoptosis is transcription and translation dependent, and apoptotic cells are visible 24-48 h after NGF deprivation (Martin et al., 1988). Two groups have shown that oxidative stress is an important early trigger in this model of death, and that death can be inhibited by overexpressing Cu/Zn-superoxide dismutase in the neurons (Greenlund et al., 1995; Jordan et al., 1995). Moreover, several years ago, we and others showed that c-Jun was induced during and necessary for death in this model (Estus et al., 1994; Ham et al., 1995). To investigate the role of c-Jun phosphorylation in this paradigm, we and others have performed immunofluorescent analyses of c-Jun phosphorylation as well as quantitative assays of JNK activity at different times after NGF deprivation (Eilers et al., 1998). By using an antibody raised against phospho-Jun (Ser 63) for immunofluorescent studies, we observed a robust increase in c-Jun phosphorylation in NGF deprived (Figure 4B, D), as compared to NGF maintained, neurons (Figure 4A, C). To correlate this increase in c-Jun phosphorylation with JNK activity, we then used an in vitro assay wherein JNK was immunoprecipitated and then exposed to a c-Jun fragment for a substrate. Here, we found that after a 2.5 hour delay, JNK activity was increased almost two-fold after NGF deprivation (Figure 5). Moreover, this increased activity was sustained until the neurons began to die, at approximately 25 h after NGF deprivation. Overall, these results essentially mirror the results of Ham and co-workers (Eilers et al., 1998). Hence, phosphorylation of c-Jun is increased in sympathetic neurons after NGF deprivation, and this correlates qualitatively with an increase in JNK activity. The Role of JNKs in PC12 Apoptosis

Activation of the JNK pathway also appears to be important in programmed cell death of PC12 cells (Xia et al., 1995). To characterize molecular mechanisms that regulate neuronal apoptosis, the contributions to cell death of MAP kinase family members, including ERK, JNK, and p38, were examined after withdrawal of nerve growth factor (NGF) from rat PC12 cells (Xia et al., 1995). NGF withdrawal-induced apoptosis is

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Figure 5. c-Jun Phosphorylation Increases in NGF-deprived Sympathetic Neurons: JNK Activity Assay. To assess changes in JNK activity, JNK was immunoprecipitated from sympathetic neurons at the indicated times of NGF deprivation. JNK activitiy was then assessed by exposing the immunoprecipitates to a c-Jun amino-terminal fragment and 32p-gamma--ATP; the radioactivity incorporated into the c-Jun fragment was determined by separating the c-Jun fragment from free radioactivity by polyacrylamide gel electrophoresis, followed by liquid scintillation counting of the c-Jun band. These data are from a representative experiment and depict the mean + SE of an analysis performed in triplicate.

p r e c e d e d by sustained activation o f the J N K and p38 e n z y m e s , and the inhibition o f E R K s . M o r e o v e r , these authors s h o w e d that overactivation o f the J N K led to apoptosis whereas activation o f the M A P K pathway protected f r o m apoptosis.

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JNK3-Deficient Hippocampal Cells are Protected from Excitotoxicity-Induced Delayed Apoptosis While JNK activity appears likely to contribute to neural cell apoptosis in vitro, the recent generation of murine "knockout lines" individually deficient for each of the JNK genes has allowed testing of the role of the JNKs in apoptosis more directly. In the first paper to be published from work with these mice, Yang et al. tested the hypothesis that since JNK3 is selectively expressed in neurons, lack of JNK3 would be neuroprotective to apoptosis-inducing stress (Yang et al., 1997a). Indeed, they reported that JNK3-deficient mice require higher concentrations of kainate to induce seizures, relative to wild-type mice. Moreover, these investigators correlated JNK3 deficiency with a decrease in kainate-induced AP-1 activity and apoptosis in hippocampal neurons. In contrast, JNK1 and JNK2 deficient mice lacked this protection. Somewhat surprisingly, the induction of c-Jun appeared equivalent between the JNK3 deficient and wild-type mice, suggesting that the induction of c-jun may be separable from JNK activity. However, these results do suggest that JNK3 may be critical for neuronal apoptosis.

Motor Neuron Apoptosis is Blocked by a Compound that Inhibits the JNK Signaling Cascade The semisynthetic compound CEP-1347 blocks apoptotic death in rat motorneurons and simultaneously inhibits JNK activation (Maroney et al., 1998). CEP-1347 was selected for testing because of its ability to induce choline acetyl transferase activity in spinal cord cultures, and because it promotes neuronal survival in chick dorsal root ganglion cultures (Borasio 1990; Kaneko et al., 1997). Following deprivation of trophic factor, JNK activity increases 4-fold in the first 24 h (Maroney et al., 1998). The addition of CEP-1347 inhibited this JNK activation and ultimately delayed death for up to 72 h after deprivation. The site of action of CEP-1347 was unclear, since CEP-1347 did not inhibit JNK directly. However, the ability of CEP-1347 to inhibit the JNK pathway was not specific to neurons, as CEP-1347 also inhibited osmotic or oxidative stress-induced JNK activation in Cos7 cells (Maroney et al., 1998). Hence, CEP-1347 appears to inhibit upstream of JNK in the JNK pathway.

Nitric Oxide Inhibits the JNK Pathway Nitric oxide (NO) is known to play an important anti-apoptotic role in hepatocytes, splenocytes, eosinophils, and B-lymphocytes (Kim et al., 1997; Melino et al., 1997). NO may modulate JNK2 activity by S-nitrosylation of the JNK2 protein in the apoptotic pathway (So et al., 1998). NO specifically blocks the phosphotransferase activity of JNK2 in vitro, and this effect can be reversed by the addition of thiol group reductants. This is thought to be one mechanism that might spare cells from apoptosis, and thus is currently being studied as a potential treatment modality.

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JNK in Fas-Mediated Apoptosis Fas is a widely expressed "cell death" receptor that is activated by Fas ligand (Nagata et al., 1997). Fas can activate the JNK pathway to induce apoptosis in a cell-specific manner; Fas-mediated apoptosis has been reported to involve JNK activation in SHEP but not Jurkat cells (Goillot et al., 1997; Lenczowski et al., 1997). The specific mechanism of JNK activation in Fas-dependent apoptosis has received little attention until recently. Yang et al. cloned Daxx, a novel protein that interacts with Fas both in vitro and in vivo, and this interaction potentiates Fas-mediated apoptosis in 293 cells and HeLa cells (Yang et al., 1997b). This represents the first example of a "missing link" between Fas and the JNK pathway. Daxx does not directly activate JNK, but appears to initiate the pathway by activating the JNK kinase kinase ASK1. The overexpression of a kinase-deficient ASK1 mutant inhibits Fas- and Daxx-induced apoptosis and JNK activation (Chang et al., 1998). JNK in Cerebellar Granule Cell Apoptosis Cerebellar granule cells undergo transcription-dependent apoptosis in vitro if deprived of serum, coupled with a decrease in K+ concentration in the medium from 25 to 5 mM (D'Mello et al., 1993). Following deprivation, c-Jun RNA and protein levels increase, as well as the phosphorylation of c-Jun at Ser63, similar to the NGF-deprived sympathetic neuron model (Eilers et al., 1998; Miller & Johnson, 1996; Watson et al., 1998). Surprisingly, JNK activity levels do not increase above the baseline level following deprivation, suggesting that c-Jun may be phosphorylated by a novel mechanism during cerebellar granule cell apoptosis (Gunn-Moore and Tavare, 1998; Watson et al., 1998). Moreover, treatment of cerebellar granule cells with glutamate promoted apoptosis via a JNK-independent pathway (Gunn-Moore & Tavare, 1998), again suggesting the existence of JNK-independent apoptosis pathways. JNK and TNF-Induced Apoptosis Tumor necrosis factor (TNF) is known to elicit multiple responses in cells, including proliferation, differentiation, and apoptosis (Tracey and Cerami, 1993; Vandenabeele et al., 1995). These responses are mediated by two cell surface receptors, TNFR1 and TNFR2 (Smith et al., 1994; Vandenabeele et al., 1995). Trimerization between TNFR1, TNF, and the TNFRl-associated death domain (TRADD) protein leads to apoptosis and JNK activation (Liu et al., 1996; Natoli et al., 1997). However, activation of JNK is not sufficient for apoptosis in this model, as a dominant negative Fas-associated death domain (FADD) protein blocks apoptosis but not JNK activation (Liu et al., 1996; Natoli et al., 1997). Oxidative Stress Activates JNKs As mentioned above, one hallmark of programmed cell death in many models is the production of superoxide radicals, which triggers the molecular cascade of events

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leading to apoptosis. Recent reports from different systems have correlated an oxidative stress event with JNK activation and apoptosis. Indeed, the activation of JNK by oxidative stress was one of the initial hallmarks of JNK (Derijard et al., 1994; Kyriakis et al., 1994). That oxidative stress may lead to JNK activation, and, in turn, apoptosis has been supported in published reports. For example, the neurotransmitter dopamine can exert a neurotoxic effect under certain pathologic conditions, including neurodegenerative diseases such as Parkinson's disease. Luo et al. reported that dopamine induces apoptosis in a time- and concentration-dependent manner in the 293 cell line and in primary neonatal rat postmitotic striatal neuron cultures (Luo et al., 1998). Prior to apoptosis, JNK activation, c-Jun phosphorylation, and a subsequent increase in c-Jun protein levels are observed, suggesting that dopamine is acting through a JNK activation signaling pathway. The addition of the antioxidants N-acetylcysteine and catalase effectively blocked DA-induced JNK activation and subsequent cellular apoptosis. JNKs and Human Pathogenesis Several reports since the mid 1990's suggest that JNK activation plays an active role in the apoptosis programs associated with multiple human pathological conditions. For example, JNK activation has been associated with stroke, i.e. 40 min of ischemia followed by as little as 5 min of reperfusion is sufficient to significantly increase JNK activity, with an activation as high as 7.5 fold after a 20 minute reperfusion (Pombo et al., 1994). Since ischemia alone is insufficient to induce this response, this induction is likely associated with the oxidative stress that accompanies reperfusion. JNK activation has also been implicated in a model of Huntington's disease. The expansion of a polyglutamine stretch at the amino terminus of huntingtin is associated with several neurodegenerative disorders, such as Huntington's Disease. Liu examined the expression of the full-length huntingtin with varying polyglutamine repeats and found that mutated huntingtin with 48 or 89 repeats induces JNK activity which precedes apoptotic death (Liu, 1998). The co-expression of a SEK1 dominantnegative mutant blocked the activation of JNKs and neuronal apoptosis almost completely, suggesting that huntingtin-related apoptosis is dependent on the SEK 1-pathway. That JNK may contribute to Parkinsons disease has also been suggested. For example, the SEK1/JNK pathway is utilized during manganese-induced apoptosis and manganese is known to induce neurological disorders similar to parkinsonisms. The application of manganese to PC12 ceils causes apoptotic characteristics such as D N A fragmentation which is preceded by the phosphorylation of c-Jun at Ser63 and Ser73, and SEK1 at Thr258 (Hirata et al., 1998). This suggests that the JNK pathway may contribute to manganese-associated apoptosis. Finally, recent papers demonstrate the ability of chemotherapeutic agents to induce apoptosis in a JNK-dependent manner. One example is the anti-cancer drug paclitaxel, which activates the JNK signaling pathway and ultimately leads to apoptosis in ovarian tumor cells (Lee et al., 1998). Another anti-cancer drug that activates the JNK apoptosis pathway is RRR-alpha-tocopheryl succinate (Yu et al., 1998). This drug induces apoptosis in human breast cancer ceils, following prolonged activation of JNK.

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Summary The c-Jun amino (N)-terminal kinases along with the ERKs and p38 represent the M A P K family. As we have discussed here, the JNKs modulate the transduction o f multiple extracellular signals into intracellular events (Davis, 1994; Robinson & Cobb, 1997). Indeed, since cell proliferation or cell death is typically modulated by extracellular signals, perhaps it is not surprising that the JNKs have been implicated in these processes. In considering the issues raised here, we have found several particularly interesting, including (i) the existence of JNK- independent and dependent apoptosis pathways, which suggests that the JNK pathway may be amenable to therapeutic modulation because apoptosis in every cell-type would not necessarily be altered. (ii) the recent discovery of non-transcription factor JNK substrates, which raises the possibility that this kinase family can act independently o f changes in gene expression and (iii) the relative dearth of attention accorded to possible JNK phosphatases, which may yet emerge as important in modulating the JNK pathway. In conclusion, in 1995, the year after the JNKs were identified, 43 scientific papers were published examining the JNKs. In 1998, at least 420 papers were published. Hence, in the four short years since the JNK family was initially identified, scientific investigators have very rapidly demonstrated the importance of this pathway in multiple cellular events. As for the next four years...

Acknowledgments The authors would like to acknowledge the help of H. Michael Tucker in preparing the figures and the financial support of NIH (NS-35607 to SE).

References Abate, C., Patel, L., Rauscher, F.J., III & Curran, T. (1990). Redox regulation of fos and jun DNA-binding activity in vitro. Science 249, 1157-1161. Angel, P. & Karin, M. (1991). The role of Jun, Fos and the Ap-1 complex in cell- proliferation and transformation. Biochim. Biophys. Acta 1072, 129-157. Bagrodia, S., Derijard, B., Davis, R.J. & Cerione, R.A. (1995). Cdc42 and PAK-mediated signaling leads to Jun kinase and p38 mitogen-activatedprotein kinase activation. J. Biol. Chem. 24, 27995-27998. Bazenet, C.E., Mota, M.A. & Rubin, L.L. (1998). The small GTP-binding protein Cdc42 is required for nerve growth factor withdrawal-inducedneuronal death. Proc. Natl. Acad. Sci. USA 95, 3984-3989. Borasio, G.D. (1990). Differential effects of the protein kinase inhibitor K-252a on the in vitro survival of chick embryonic neurons. Neurosci. Lett. 108, 207-212. Boyle, W.J., Smeal, T., Defize, L.H., Angel, P., Woodgett, J.R., Karin, M. & Hunter, T. (1991). Activation of protein kinase C decreases phosphorylationof c-Jun at sites that negatively regulate its DNA-binding activity. Cell 64, 573-584. Chang, H.Y., Nishitoh, H., Yang, X., Ichijo, H. & Baltimore, D. (1998). Activation of apoptosis signalregulating kinase 1 (ASK1) by the adapter protein Daxx. Science 281, 1860-1863.

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Chen, Y.R., Wang, X., Templeton, D., Davis, R.J. & Tan, T.H. (1996). The role of c-Jun N-terminal kinase (JNK) in apoptosis induced by ultraviolet C and gamma radiation. Duration of JNK activation may determine cell death and proliferation. J. Biol. Chem. 271, 31929-31936. Cochran, B.H. (1993). Regulation of immediate early gene expression. NIDA Res. Mono. Ser. 125, 3-24. Colotta, F., Polentarutti, N., Sironi, M. & Mantovani, A. (1992). Expression and involvement of c-fos and c-jun protooncogenes in programmed cell death induced by growth factor deprivation in lymphoid cell lines. J. Biol. Chem. 267, 18278-18283. Coso, O.A., Chiariello, M., Kalinec, G., Kyriakis, J.M., Woodgett, J. & Gutkind, J.S. (1995). Transforming G protein-coupled receptors potently activate JNK (SAPK). Evidence for divergence from the tyrosine kinase signaling pathway. J. Biol. Chem. 270, 5620-5624. D'Mello, S.R., Galli, C., Ciotti, T. & Calissano, P. (1993). Induction of apoptosis in cerebellar granule neurons by low potassium: Inhibition of death by insulin-like growth factor and cAMP. Proc. Natl. Acad. Sci. U.S.A. 90, 10989-10993. Davis, R.J. (1994). MAPKs: new JNK expands the group. Trends Biochem. Sci. 19, 470-473. Deckwerth, T.L. & Johnson, E.M., Jr. (1993). Temporal analysis of events associated with programmed cell death (apoptosis) of sympathetic neurons deprived of nerve growth factor (NGF). J. Cell Biol. 123, 1207-1222. Derijard, B., Hibi, M., Wu, I., Barrett, T., Su, B., Deng, T., Karin, M. & Davis, R. J. (1994). JNKI: A protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jan activation domain. Cell 76, 1025-1037. Derijard, B., Raingeaud, J., Barrett, T., Wu, I.H., Han, J., Ulevitch, R.J. & Davis, R.J. (1995). Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267, 682-685. Devary, Y., Gottlieb, R.A., Lau, L.F. & Karin, M. (1991). Rapid and preferential activation of the c-jun gene during the mammalian UV response. Mol. Cell. Biol. 11, 2804-2811. Edwards, S.N., Buckmaster, A.E. & Tolkovsky, A.M. (1991). The death programmed in cultured sympathetic neurons can be suppressed at the posttranslational level by nerve growth factor, cyclic AMP, and depolarization. J. Neurochem. 57, 2140-2143. Eilers, A., Whitfield, J., Babij, C., Rubin, L.L. & Ham, J. (1998). Role of the Jan kinase pathway in the regulation of c-Jun expression and apoptosis in sympathetic neurons. J. Neurosci. 18, 1713-1724. Estus, S. (1998). Gene induction during neuronal apoptosis, in Neuroprotective SignalTransduction. (M. Mattson, eds.), Humana Press, Totowa, N.J., pp. 83-94. Estus, S., Zaks, W., Freeman, R., Gruda, M., Bravo, R. & Johnson, E. (1994). Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis. J. Cell Biol. 127, 1717-1727. Freeman, R.S., Estus, S., Horigome, K. & Johnson, E.M., Jr. (1993). Cell death genes in invertebrates and (maybe) vertebrates. Curr. Opin. Neurobiol. 3, 25-31. Glucksman, A. (1951). Cell deaths in normal vertebrate ontogeny. Biol. Rev. 26, 59-86. Goillot, E., Raingeaud, J., Ranger, A., Tepper, R.I., Davis, R.J., Harlow, E. & Sanchez, I. (1997). Mitogenactivated protein kinase-mediated Fas apoptotic signaling pathway. Proc. Natl. Acad. Sci. USA 94, 3302-3307. Gorin, P.D. & Johnson, E.M. (1979). Experimental autoimmune model of nerve growth factor deprivation: effects on developing peripheral sympathetic and sensory neurons. Proc. Natl. Acad. Sci. USA 76, 5382-5386. Goto, K., Ishige, A., Sekiguchi, K., Iizuka, S., Sugimoto, A. & Yuzurihara, M. (1990). Effects of

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cycloheximide on delayed neuronal death in rat hippocampus. Brain Res. 534, 299-302. Greenlund, L.J.S., Deckwerth, T.L. & Johnson, E.M. (1995). Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death of protects sympathetic neurons from NGF deprivation induced apoptosis. Neuron 14, 303-315. Gunn-Moore, F.J. & Tavare, J.M. (1998). Apoptosis of cerebellar granule cells induced by serum withdrawal, glutamate or beta-amyloid, is independent of Jun kinase or p38 mitogen activated protein kinase activation. Neurosci. Lett. 250, 53-56. Gupta, S., Barrett, T., Whitmarsh, A.J., Cavanagh, J., Sluss, H.K., Derijard, B. & Davis, R.J. (1996). Selective interaction of JNK protein kinase isoforms with transcription factors. EMBO J. 15, 2760-2770. Ham, J., Babij, C., Whitfield, J., Pfarr, C.M., Lallemand, D., Yaniv, M. & Rubin, L.L. (1995). A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death. Neuron 14, 927-939. Herdegen, T., Brecht, S., Mayer, B., Leah, J., Kummer, W.R.B. & Zimmermann, M. (1993). Long-lasting expression of JUN and KROX transcription factors and nitric oxide synthase in intrinsic neurons of the rat brain following axotomy. J. Neurosci. 13, 4130-4145. Herdegen, T., Skene, P. & Bahr, M. (1997). The c-Jun transcription factor - bipotential mediator of neuronal death, survival, and regeneration. Trends Neurosci. 20, 227-231. Hibi, M., Lin, A., Smeal, T., Minden, A. & Karin, M. (1993). Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes. Dev. 7, 2135-2148. Hirata, Y., Adachi, K. & Kiuchi, K. (1998). Activation of JNK pathway and induction of apoptosis by manganese in PC12 cells. J. Neurochem. 71, 1607-1615. Hu, M.C., Qiu, W.R. & Wang, Y.P. (1997). JNK1, JNK2 and JNK3 are p53 N-terminal serine 34 kinases. Oncogene 15, 2277-2287. Jordan, J., Ghadge, G.D., Prehn, J.H., Toth, P.T., Roos, R.P. & Miller, R.J. (1995). Expression of human copper/zinc-superoxide dismutase inhibits the death of rat sympathetic neurons caused by withdrawal of nerve growth factor. Mol. Pharmacol. 47, 1095-1100. Kallunki, T., Deng, T., Hibi, M. & Karin, M. (1996). c-Jun can recruit JNK to phosphorylate dimerization partners via specific docking interactions. Cell 87,929-939. Kallunki, T., Su, B., Tsigelny, I., Sluss, H.K., Derijard, B., Moore, G., Davis, R. & Karin, M. (1994). JNK2 contains a specificity-determining region responsible for efficient c-Jun binding and phosphorylation. Genes Dev. 8, 2996-3007. Kaneko, M., Saito, Y., Saito, H., Matsumoto, T., Matsuda, Y., Vaught, J.L., Dionne, C.A., Angeles, T.S., Glicksman, M.A., Neff, N.T., Rotella, D.P., Kauer, J.C., Mallamo, J.P., Hudkins, R.L. & Murakata, C. (1997). Neurotrophic 3,9-bis[(alkylthio)methyl]-and-bis(alkoxymethyl)-K-252a derivatives. J. Med. Chem. 40, 1863-1869. Karin, M. & Hunter, T. (1995). Transcriptional control by protein phosphorylation: signal transmission from the cell surface to the nucleus. Curr. Biol. 5,747-757. Kim, Y.M., Talanian, R.V. & Billiar, T.R. (1997). Nitric oxide inhibits apoptosis by preventing increases in caspase-3-1ike activity via two distinct mechanisms. J. Biol. Chem. 272, 31138-31148. Kovary, K. & Bravo, R. (1991). The Jun and Fos protein families are both required for cell cycle progression in fibroblasts. Mol. Cell. Biol. 11, 4466-4472. Kovary, K. & Bravo, R. (1992). Existence of different Fos/Jun complexes during the G0-to-G1 transition and during exponential growth in mouse fibroblasts: differential role of Fos proteins. Mol. Cell. Biol. 12, 5015-5023.

212

S.P. Tarnmariello, G.E. Landreth, and S. Estus

Kyriakis, J.M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E.A., Ahmad, M.F., Avruch, J. & Woodgett, J. R. (1994). The stress-activated protein kinase subfamily of c-Jan kinases. Nature 369, 1546-160. Lane, S.C., Jolly, R.D., Schmechel, D.E., Alroy, J. & Boustany, R.M. (1996). Apoptosis as the mechanism of neurodegeneration in Batten's disease. J. Neurochem. 67,677-683. Lassmann, H., Bancher, C., Breitschopf, H., Wegiel, J., Bobinski, M., Jellinger, K. & Wisniewski, H.M. (1995). Cell death in Alzheimer's disease evaluated by DNA fragmentation in situ. Acta Neuropathol. (Bed.) 89, 3541. Lee, L.F., Li, G., Templeton, D.J. & Ting, J.P. (1998). Paclitaxel (Taxol)-induced gene expression and cell death are both mediated by the activation of c-Jun NH2-terminal kinase (JNK/SAPK). J. Bio. Chem. 273, 28253-28260. Lenczowski, J.M., Dominguez, L., Eder, A.M., King, L.B., Zacharchuk, C.M. & Ashwell, J.D. (1997), Lack of a role for Jan kinase and AP-1 in Fas-induced apoptosis. Mol. Cell. Biol. 17, 170-181. Linnik, M.D., Zobrist, R.H. & Hatfield, M.D. (1993). Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats. Stroke 24, 2002-2009. Liu, Y.F. (1998). Expression of polyglutamine-expanded Huntingtin activates the SEK1-JNK pathway and induces apoptosis in a hippocampal neuronal cell line. J. Biol. Chem. 273, 28873-28877. Liu, Z.G., Hsu, H., Goeddel, D.V. & Karin, M. (1996). Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kappaB activation prevents cell death. Cell 87, 565-576. Livingstone, C., Patel, G. & Jones, N. (1995). ATF-2 contains a phosphorylation-dependent transcriptional activation domain. EMBO J. 14, 1785-1797. Lu, X., Nemoto, S. & Lin, A. (1997). Identification of e-Jan NH2-terminal protein kinase (JNK). -activating kinase 2 as an activator of JNK but not p38. J. Biol. Chem. 272, 24751-24754. Luo, Y., Umegaki, H., Wang, X., Abe, R. & Roth, G.S. (1998). Dopamine induces apoptosis through an oxidation-involved SAPK/JNK activation pathway. J. Biol. Chem. 273, 3756-3764. MacManus, J.P., Buchan, A.M., Hill, I.E., Rasquinha, I. & Preston, E. (1993). Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain. Neurosci. Lett. 164, 89-92. MacManus, J.P., Hill, I.E., Huang, Z.G., Rasquinha, I., Xue, D. & Buchan, A.M. (1994). DNA damage consistent with apoptosis in transient ischemic neocortex. NeuroReport 5, 493-496. Maroney, A.C., Glicksman, M.A., Basma, A.N., Walton, K.M., Knight, E.J., Murphy, C.A., Bartlett, B.A., Finn, J.P., Angeles, T., Matsuda, Y., Neff, N.T. & Dionne, C.A. (1998). Motoneuron apoptosis is blocked by CEP-1347 (KT 7515). A novel inhibitor of the JNK signaling pathway. J. Neurosci. 18, 104-111. Martin, D.P., Schmidt, R.E., DiStefano, P.S., Lowry, O.H., Carter, J.G. & Johnson, E.M., Jr. (1988). Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation. J. Cell Biol. 106, 829-844. Martin-Blanco, E., Gampel, A., Ring, J., Virdee, K., Kirov, N., Tolkovsky, A.M. & Martinez-Arias, A. (1998). puckered encodes a phosphatase that mediates a feedback loop regulating JNK activity during dorsal closure in Drosophila. Genes Dev. 12, 557-570. Maundrell, K., Antonsson, B., Magnenat, E., Camps, M., Muda, M., Chabert, C., Gillieron, C., Boschert, U., Vial-Knecht, E., Martinou, J.C. & Arkinstall, S. (1997). Bcl-2 undergoes phosphorylation by c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTPbinding protein Rael. J. Biol. Chem. 272, 25238-25242. Melino, G., Bernassola, F., Knight, R.A., Corasaniti, M.T., Nistico, G. & Finazzi-Agro, A. (1997). S-nitrosylation regulates apoptosis. Nature 388, 432-433.

The Role of Jun Kinases in Apoptosis

213

Miller, T.M. & Johnson, E.M. (1996). Metabolic and genetic analysis of apoptosis in potassium/serumdeprived rat cerebellar granule cells. J. Neurosci. 16, 7487-7495. Minden, A., Lin, A., Claret, F.X., Abo, A. & Karin, M. (1995). Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell 81, 1147-1157. Mizukami, Y., Yoshioka, K., Morimoto, S. & Yoshida, K. (1997). A novel mechanism of JNK1 activation. Nuclear translocation and activation ofJNK1 during ischemia and reperfusion. J. Biol. Chem. 272, 16657-16662. Nagata, Y., Nishida, E. & Todokoro, K. (1997). Activation of JNK signaling pathway by erythropoietin, thrombopoietin, and interleukin-3. Blood 89, 2664-2669. Natoli, G., Costanzo, A., Ianni, A., Templeton, D.J., Woodgett, J.R., Balsano, C. & Levrero, M. (1997). Activation of SAPK/JNK by TNF receptor 1 through a noncytotoxic TRAF2-dependent pathway. Science 275,200-203. Nishina, H., Bachmann, M., Oliveira-dos-Santos, A.J., Kozieradzki, I., Fischer, K.D., Odermatt, B., Wakeham, A., Shahinian, A., Takimoto, H., Bernstein, A., Mak, T.W., Woodgett, J.R., Ohashi, P.S. & Penninger, J.M. (1997). Impaired CD28-mediated interleukin 2 production and proliferation in stress kinase SAPK/ERK 1 kinase (SEK 1)/mitogen-activated protein kinase kinase 4(MKK4)-deficient T lymphocytes. J. Exp. Med. 186, 941-953. Oppenheim, R.W. (1991). Cell death during development of the nervous system. Annu. Rev. Neurosci. 14, 453-501. Pombo, C.M., Bonventre, J.V., Avruch, J., Woodgett, J.R., Kyriakis, J.M. & Force, T. (1994). The stressactivated protein kinases are major c-Jun amino-terminal kinases activated by ischemia and reperfusion. J. Biol. Chem. 269, 26546-26551. Portera-Calliau, C., Hedreen, J.C., Price, D.L. & Koliatsos, V.E. (1995). Evidence for apoptotic cell death in Huntington disease and excitotoxic animal models. J. Neurosci. 15, 3775-3787. Pulverer, B.J., Kyriakis, J.M., Avruch, J., Nikolakaki, E. & Woodgett, J.R. (1991). Phosphorylation of c-jun mediated by MAP kinases. Nature 353,670-674. Robinson, M.J. & Cobb, M.H. (1997). Mitogen-activated protein kinase pathways. Curr. Opin. Cell. Biol. 9, 180-186. Schlingensiepen, K.H., Wollnik, F., Kunst, M., Schlingensiepen, R., Herdegen, T. & Brysch, W. (1994). The role of Jun transcription factor expression and phosphorylation in neuronal differentiation, neuronal cell death, and plastic adaptations in vivo. Cell. Mol. Neurobiol. 14, 487-505. Shigeno, T. (1990). Reduction of delayed neuronal death by inhibition of protein synthesis. Neurosci. Lett. 120, 117-119. Smeal, T., Binetruy, B., Mercola, D.A., Birrer, M. & Karin, M. (1991). Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73. Nature 354, 494-496. Smeal, T., Hibi, M. & Karin, M. (1994). Altering the specificity of signal transduction cascades: positive regulation of c-Jun transcriptional activity by protein kinase A. EMBO J. 13, 6006-6010. Smith, C.A., Farrah, T. & Goodwin, R.G. (1994). The TNF receptor superfamily of cellular and viral proteins: activation, co-stimulation, and death. Cell 76, 959-962. So, H.S., Park, R.K., Kim, M.S., Lee, S.R., Jung, B.H., Chung, S.Y., Jun, C.D. & Chung, H.T. (1998). Nitric oxide inhibits c-Jun N-terminal kinase 2 (JNK2) via S-nitrosylation. Biochem. Biophys. Res. Comm. 247, 809-813. Su, J.H., Anderson, A.J., Cummings, B.J. & Cotman, C.W. (1994). Immunohistochemical evidence for apoptosis in Alzheimer's disease. NeuroReport 5, 2529-2533. Tournier, C., Whitmarsh, A.J., Cavanagh, J., Barrett, T. & Davis, R.J. (1997). Mitogen-activated protein

214

S.P. Tammariello, G.E. Landreth, and S. Estus

kinase kinase 7 is an activator of the c-Jun NH2-terminal kinase. Proc. Natl. Acad. Sci. USA 94, 7337-7342. Tracey, K.J. & Cerami, A. (1993). Tumor necrosis factor, other cytokines and disease. Annu. Rev. Cell Biol. 9, 317-347. van Dam, H., Wilhelm, D., Herr, I., Steffen, A., Herrlich, P. & Angel, P. (1995). ATF-2 is preferentially activated by stress-activated protein kinases to mediate e-jan induction in response to genotoxic agents. EMBO J. 14, 1798-1811. Vandenabeele, P., Declercq, W., Vanhaesebroeck, B., Grooten, J. & Fiefs, W. (1995). Both TNF receptors are required for TNF-mediated induction of apoptosis in PCt0 cells. J. Immunol. 154, 2904-2913. Watson, A., Eilers, A., Lallemand, D., Kyriakis, J., Rubin, L.L. & Ham, J. (1998). Phosphorylation of c-Jun is necessary for apoptosis induced by survival signal withdrawal in cerebellar granule neurons. J. Neurosci. 18, 751-762. Wright, L.L., Cunningham, T.J. & Smolen, A.J. (1983). Developmental neuron death in the rat superior cervical sympathetic ganglion: cell counts and ultrastructure. J. Neurocytol. 12, 727-738. Wu, B.Y., Fodor, E.J., Edwards, R.H. & Rutter, W.J. (1989). Nerve growth factor induces the proto-oncogene c-jun in PCl2 cells. J. Biol. Chem. 264, 9000-9003. Xanthoudakis, S., Miao, G., Wang, F., Pan, Y.-C.E. & Curran, T. (1992). Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme. EMBO J. 11, 3323-3335. Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J. & Greenberg, M.E. (1995). Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270, 1326-1331. Yan, M., Dai, T., Deak, J.C., Kyriakis, J.M., Zon, L.I., Woodgett, J.R. & Templeton, D.J. (1994). Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEKI. Nature 372, 798-800. Yang, D.D., Kuan, C.Y., Whitmarsh, A.J., Rincon, M., Zheng, T.S., Davis, R.J., Rakic, P. & Flavell, R.A. (1997a). Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene. Nature 389, 865-870. Yang, J., New, L., Jiang, Y., Han, J. & Su, B. (1998). Molecular cloning and characterization of a human protein kinase that specifically activates c-Jun N-terminal kinase. Gene 212, 95-102. Yang, X., Khosravi-Far, R., Chang, H.Y. & Baltimore, D. (1997b). Daxx, a novel Fas-binding protein that activates JNK and apoptosis. Cell 89, 1067-1076. Yu, W., Simmons-Menchaca, M. You, H. Brown, P., Birrer, M.J., Sanders, B.G. & Kline, K. (1998). RRR-alpha-tocopheryl succinate induction of prolonged activation of c-jun amino-terminal kinase and c-jan during induction of apoptosis in human MDA-MB-435 breast cancer cells. Mol. Carcinog. 22, 247-257. Zhang, Z., Yang, X.Y. & Cohen, D.M. (1998). Hypotonicity activates transcription through ERK-dependent and -independent pathways in renal cells. Am. J. Physiol. 275, 1104-1112.

APOPTOSIS BY PAR-4 PROTEIN VIVEK M. R A N G N E K A R

Table of Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation and Identification of Par-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Expression of Par-4 in the Normal Prostate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Widespread Expression of Par-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Expression of Par-4 in In Vivo Models of Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Expression of Par-4 in Other In Vitro and In Vivo Paradigms of Apoptosis . . . . . . . . . . . . . . Structure-Function Analysis of Par-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of Par-4 Induced Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Expression in Aging-Related Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Is Par-4 a Tumor Suppressor? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Direction of Future Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

215 219 220 221 222 223 225 226 229 232 232 234

Introduction The processes of cell growth and programmed cell death (apoptosis) are carefully orchestrated to maintain homeostasis within the host tissue (Wyllie et al., 1980; Peter et al., 1997; Van Antwerp et al., 1998). The importance of a balance in these opposing processes is evident from the developmental anomalies that result when either process goes awry (Wylllie et al., 1980; Peter et al., 1997; Van Antwerp et al., 1998; Staunton and Gaffney, 1998; Chao and Korsmeyer, 1998). Excessive growth resulting from a defect in the apoptotic mechanisms is an harbinger of cancer, whereas excessive death is an overriding element in neuro-degenerative diseases like Alzheimer's, amyotrophic lateral sclerosis, Huntington's, Parkinsons, or in ischemia-reperfusion induced injury involved in cardiac arrest or stroke (Figure 1). Thus, apoptosis is a natural, physiologically relevant process that is essential to control the development of diseases like cancer but may prove detrimental or fatal when deregulated in neurons or reperfusion-injured tissue. The cascades of molecular events that lead to the morphological and biochemical features of apoptosis are currently under intense study (Williams, 1991; Barger et al., 1995; Beg and Baltimore, 1996; Liu et al., 1996; Van Antwerp et al., 1996; Wang et al., 1996; Finco et al., 1997; Hannun, 1997; Adam and Cory, 1998; Askenazi and Dixit, 1998; Green and Reed, 1998; Susin et al., 1998; Thornberry and Lazebnik, 1998). When the cell encounters exogenous insults, a cascade of secondmessenger signaling events is initiated at the plasma membrane that then triggers 215 Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. 215 -- 236 © 2001 Elsevier Science. Printed in the Netherlands.

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the expression o f immediate-early genes, many o f which are transcription regulators (Figure 2).

Survival of lntracellular Pathogen

Neurodegeneration

CANCER

IschemiaYreperfusion Injury

Development

The effects of apoptosis in naturally occurring processes. Apoptosis can block cancer progression and intracellular growth of pathogens within host ceils but is causal in the pathophysiology of neurodegenerative diseases and ischemia-reperfusioninduced injury. Figure 1.

growth factor withdrawal/ serum

c-fos c-jun c-mye egr-1 nur77

GROWTH STIMULATION

calcium elevation/UV

cytokines

p .4

c-jun egr-1

e-fos c-jun c.myc egr-1

l GROWTH ARREST

nur77

I ApO,TOSiS I

Immediate early genes in apoptosis. Immediate early genes represent the primary set of gene induction events that is important in the cascade that leads to phenotypic changes such as growth stimulation, growth arrest or apoptosis. The genes involved in these different processes are shown. Figure 2.

The immediate-early gene products then activate downstream genes that may ultimately lead to induction of a death pathway associated with the biochemical and molecular characteristics o f apoptosis. While a number o f exogenous signals utilize immediate-early gene induction events to trigger pathways that m a y lead to apoptosis, there are multiple examples of exogenous and endogenous signals that might operate independently o f gene induction events to cause apoptosis. Thus, while different insults

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may induce distinct private pathways, the downstream components of apoptosis are thought to commonly require activation of caspases (Figure 3), which cleave key survival proteins such as those involved in DNA repair or structural proteins in the cells (Porter et al., 1997; Thornberry and Lazebnik, 1998).

Private~l [ Judgement [

"~

Common 1easpases APOPTOSIS Figure 3. The privateandcommoncomponentsof apoptosis.The apoptotic pathway involvesa private component that includes early molecularchanges that are specific to the action of the inducers. The private pathway feeds into a commoncomponentthat is generallypresent in all normalcells.

My studies on the isolation and identification of pro-apoptotic genes used prostate cancer as an experimental model system. The normal or benign prostate, or prostate cancer, is an excellent model for studying apoptosis because the gland or tissue is composed of cells that are either dependent or independent of androgen for survival and/or growth, and withdrawal of androgen by castration (orchiectomy), for example, will lead to apoptotic death of the androgen-dependent cells, but not the androgen-independent cells (Stanisic et al., 1978; Buttyan et al., 1988; Connor et al., 1988; Kyprianou and Isaacs, 1988). The androgen-independent cells are particularly problematic in prostate cancer because the survival of these cells may lead to recurrent prostate cancer, which is the cause of mortality in the patients. The events that lead to apoptosis of the androgen dependent cells are not well characterized, but several lines of evidence point toward a role for intracellular calcium elevation after androgen-withdrawal (Figure 4). This presumably leads to induction of genes that trigger a cascade leading to apoptosis. On the other hand, the androgenindependent cells do not show an elevation in intracellular calcium levels, presumably because of a block in events downstream from the androgen receptor activation to calcium elevation. However, intracellular calcium can be artificially upregulated in the androgen independent cells in vitro by using calcium ionophores, which are expected to act by a primary pathway that is independent of the androgen-receptor in these cells. The pathway downstream of intracellular calcium elevation is therefore common to the androgen-dependent and -independent cells (Figure 4).

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ANDROGEN-DEPENDENT ANDROGEN-INDEPENDENT Androgen-withdrawal

Ca2+ GENE

I~XPRESSION

Androgen-withdrawal

Ca2+

GENEEXPRESSION

APOPTOSIS APOPTOSIS Figure 4.

The common component of the apoptotic machinery downstream of intracellular calcium

elevation in androgen-dependent and -independent cells. Androgen-withdrawal results in elevation of

intracellular calcium in androgen-dependent cells. This may result in gene expression-dependent apoptosis. By contrast, androgen-withdrawal does not result in intracellular calcium elevation in androgen-independent cells and therefore there is neither gene induction not apoptosis. However, in cell culture paradigms, one can upregulate intracellular calcium that leads to gene expression and apoptosis by using calcium ionophores or thapsigargin. I hypothesized that if one could identify gene components of the common pathway, a subset of the genes would represent pro-apoptotic genes that may be functionally relevant to apoptosis in both the androgen-dependent and -independent cells. In addition, because intracellular calcium elevation is central to apoptosis in a number of other model systems, such as: withdrawal of IL-3 from IL-3-dependent hematopoietic cells; activation of the B-cell receptor with anti-IgM; exposure of thymocytes to dexamethasone or T-cell receptor antibodies; or degenerating neurons in Alzheimer disease. I expected that some of the genes, identified as a part of the common pathway in prostatic cells, might serve as active components of private pathways induced by other diverse agents dependent on intracellular calcium elevation for apoptosis With this rationale, I decided to screen for genes that were upregulated in rat androgen-independent cells AT-3 when exposed to the apoptotic calcium ionophore ionomycin. However, before embarking on such an involved project, it was important to ascertain that gene expression was required for apoptosis induced by intracellular calcium elevation in the prostatic cells. AT-3 cells were left untreated or treated with ionomycin for various periods of time and then total DNA prepared from the cells was examined for oligonucleosomal fragmentation, a characteristic feature of apoptosis. These initial studies indicated that apoptosis was detectable at 4 to 5 h of exposure to ionomycin. The cells were then exposed to ionomycin for 5 h in the presence or absence of actinomycin D, a general inhibitor of gene transcription, or cycloheximide, a general inhibitor of new protein synthesis, and examined for morphological changes or for D N A laddering. These experiments indicated that exposure to ionomycin caused rounding of the normally fiat and attached cells. However, when exposed to ionomycin in the presence of actinomycin D or cycloheximide, the rounding of the ceils

Par-4 Mediated Apoptosis

219

was inhibited. Consistent with this observation, the D N A laddering seen with ionomycin alone was completely eliminated by the presence o f actinomycin D or cycloheximide. These observations offered a preliminary indication that gene expression was essential for apoptosis driven by intracellular calcium in prostatic ceils. Encouraged by this finding, differential hybridization (Figure 5) was performed on a c D N A library prepared from the AT-3 cells exposed to ionomycin plus cycloheximide for 5 h. The use of cycloheximide allowed the stabilization and selective enrichment of R N A s representing immediate-early genes and the 5 h time-point was based on the detection of early death in these cells upon exposure to ionomycin. DIFFERENTIAL

HYBRIDIZATION

Rat ANDROGEN-INDEPENDENT CELLS

l

Ionomycin + Cycloheximide

cDNA library

cDNA PROBE from:

Untreated Cells

D

cDNA for Induced Gene

Ionom ycin + Cycloheximide treated cells

Figure 5. Differential screening of AT-3 cell library. Differential hybridization was performed on a cDNA library prepared from AT-3 cells that had been treated with ionomycin plus cycloheximide for 5 h. The probes used for hybridization included radiolabeled cDNA from untreated or ionomycin plus cycloheximide treated AT-3 cells. Duplicate blots containing replicates of bacterial colonies representing clones from the cDNA library were probed with these radiolabeled cDNAs and the intensity of hybridization on each replicate filter was compared, cDNA clones showing stronger hybridization with the probe from ionomycin plus cycloheximide treated cells were used for further characterization.

Isolation and Identification of Par-4

Differential hybridization studies performed on about 20,000 clones from the c D N A library led to the identification of about 10 genes that were differentially expressed in response to ionomycin and cycloheximide. Further characterization by Northem blot analysis indicated that only five o f these genes were induced in response to ionomycin alone, and the others showed elevated R N A levels owing to the presence of cycloheximide. The genes (Figure 6) were referred to as prostate apoptosis response (par) genes (Sells et al., 1994). A more detailed analysis indicated that four o f the par genes (1, 3, 4, 5) were induced by apoptotic insults in both androgen-dependent and -independent cells. At this stage, faced with the challenge of deciding which genes to pursue, I decided

220

V.M. Rangnekar

Prostate Az)ootosls Resoonse (PAR~ GeneR par-1 : MAP-Kinase-Phosphatase-1 (MKP-1)

par-2

: EGF-Iike factor

par-3 :

serum-inducible

par~

: novel sequence

par-5

: novel sequence

cyr-61

Figure 6. Prostate apoptosis response (par) genes. Differential screening followed by Northern blot analysis led to the identification of five genes that were induced by ionomycinin rat AT-3 cells. These genes were called prostate apoptosisresponse (par) and further identified by nucleotide sequencing. Rat par-4 and -5 werejudged novel by GenBanksearches at the time. The humanpar-4 gene was later identified independently by the laboratories of Jorge Moscat in Spain and Yang Shi at Harvard Medical School.

to impose stringent criteria on the genes that would be further studied. These included the requirement that the gene(s) should be induced exclusively during apoptosis and not by growth-arresting, necrotic, or growth-stimulatory signals. Additionally, the deduced amino acid sequence should predict a protein structure of potential value in studying apoptosis. Only one of the five genes satisfied all of these criteria. The gene was judged as novel based on its nucleotide sequence (GenBank accession number U05989) and designated par-4. This gene is up-regulated by ionomycin or thapsigargin (which inhibits Ca-ATPase and increases influx of calcium by activation of the capacitative channel) in the androgen-independent prostate cancer cells AT-3. Most importantly, Par-4 protein levels were elevated after castration in the androgen-dependent cells of the rat ventral prostate (Figure 7). The par-4 gene is not up-regulated during growth stimulation, growth arrest, or necrosis in cell culture paradigms. Moreover, this gene is not induced after castration in organs such as the liver or the kidneys that contain androgen receptors, but that do not undergo involution after androgen ablation.

E x p r e s s i o n o f Par-4 in the N o r m a l Prostate

To determine whether Par-4 is upregulated in cells of the prostate that are expected to undergo apoptosis after androgen ablation, we performed immunohistochemistry on sections of the prostate from uncastrated and castrated rats (Boghaert et al., 1997). Interestingly, in intact rats Par-4 was present in the stromal compartment but not in the secretory cell compartment. By contrast, Par-4 protein is undetectable in the secretory epithelial cells of the prostate in uncastrated rats, but is induced within 24 h of castration in the secretory epithelial cells from castrated rats. The induced levels peak between 24 to 72 h of castration, coincident with the peak apoptotic phase in the involuting prostate after castration. Because the secretory epithelial ceils of the

Par-4 Mediated Apoptosis

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with testosterone

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Par-4 induction after castration in the rat ventral prostate is inhibited by testosterone.

Pellets that contained testosterone or no testosterone were placed in Sprague-Dawley rats and then the rats were either castrated or left uncastrated. At various time-points thereafter, the prostates were harvested and total protein extracts were prepared. Forty microgram amounts of each extract protein was subjected to Western blot analysis for Par-4. Castration caused induction of Par-4 and testosterone inhibited castrationinducible Par-4 expression.

prostate undergo apoptosis upon castration, these observations suggest a strong link between Par-4 induction and sensitivity to apoptosis. One surprising finding, however, is that although the rat prostate continues to undergo involution up to 12 days after castration, by which time it has shrunk to the minimal possible size, there is no detectable level of Par-4 protein in the secretory cells after 5 days o f castration. As the secretory epithelium is terminally differentiated, loss o f Par-4 positivity cannot be due to cell turnover. It is possible that the early induction of Par-4 is sufficient to trigger a long-term programmed cascade toward apoptosis. This would mean that, in the absence o f any interfering or antagonizing signal, the induction of Par-4 might commit cells to apoptosis. Alternately, it is possible that the number of apoptotic cells drops considerably to very low levels and the number o f cells with detectable Par-4 consequently drops.

Widespread Expression of Par-4

Par-4 protein expression is widespread in mammalian tissues. It is detected in cells originating from the endoderm, mesoderm, or ectoderm, suggesting that Par-4 is

222

V.M. Rangnekar

not likely involved in lineage determination during differentiation of the germinal embryonic layers (Boghaert et al., 1997). However, certain neurons and medulla of adrenal gland from ectodermal origin and dense connective tissue, thymocytes and lymphocytes, and some smooth muscle cells from mesodermal origin do not show Par-4 expression. Consistent with its pro-apoptotic role, Par-4 is expressed in apoptotic granulosa cells of atretic ovarian follicles and in terminally differentiated ceils, such as the cardiomyocytes, cerebellar Purkinje cells, and pyramidal cells in the hippocampus. On the other hand, tissues in which cells can be visually differentiated from their mature counterparts show the least intensity of Par-4 in the mature cells. For example, basal cells maintained higher levels of Par-4 than the hormone-sensitive terminally differentiated luminal cells of the mammary gland tissue. Also, cells of the stratum comeum of the skin or cells on top of the duodenal villi stained relatively less intensely for Par-4 as compared to the stem cells in the stratum basale and at the bottom of the crypts of Lieberkuhn, respectively. This may suggest that Par-4 has to be down regulated for successful differentiation and maintenance of the differentiated cells in these tissues; otherwise, sustained expression of Par-4 may lead to apoptosis of the differentiated cells. This hypothesis needs formal evaluation in experimental models of differentiation.

Expression of Par-4 in In Vivo Models of Apoptosis Besides the involuting prostate, we examined other in vivo models of apoptosis for the expression of Par-4 protein. The developing limb bud is a classical example of naturally occurring apoptosis in development. The cells of the web area between the digits in the developing limb bud undergo apoptosis and lead to the complete removal of the web. This web area stained positive for Par-4 expression. Of note here was the fact that only a few of the Par-4 expressors in the web area were apoptotic. This meant that Par-4 is likely to provide an early signal toward apoptosis in limb bud development. Another developmental model for apoptosis that was examined for the expression of Par-4 is the involuting tadpole tail. During metamorphosis, the tadpole tail undergoes involution via apoptosis and hence the adult frog lacks a tail. Apoptosis is thought to result from a surge in the levels of thyroid hormone. The different stages of the tadpole are: premetamorphosis (stage 52 ', 56) when the animal is simply growing and there are few, if any, metamorphic changes; prometamorphosis (stage 56 ', 59) when the thyroid hormone levels are rising and changes are beginning in some tissues, but not in the tail; and metamorphosis, which begins at stage 60 when the thyroid hormone levels peak and tail shrinkage begins. Low background levels of Par-4 protein are present in the pre- and prometamorphosis phase, but up-regulation of Par-4 is seen during the metamorphosis phase. Interestingly, unlike the developing limb bud or the involuting prostate, the apoptotic cells of the involuting muscle bundles in the tadpole tail continued to express Par-4. In any case, studies in all these models of apoptosis suggest that Par-4 expression or up-regulation is tightly linked to cells programmed for apoptosis. The association of Par-4 with these diverse evolutionarily

Par-4 MediatedApoptosis

223

distant models also suggested that the expression and function of Par-4 in apoptosis is likely to be conserved during evolution. Indeed, the expression o f Par-4 protein is conserved during evolution of vertebrates; it was detected by Western blot analysis of protein extracts prepared from various organs of birds, cow, dogs, sheep, horse, mouse, rat, and human. The ubiquitous expression of Par-4 protein suggests that it is likely to support an essential biological function(s) in vertebrates.

Expression of Par-4 in Other In Vitro and In Vivo Paradigms of Apoptosis To ascertain that par-4 up-regulation is apoptosis-response-specific, we tested the expression of Par-4 in the rat ventral prostate in response to castration and replenishment with testosterone. Sustained release pellets of testosterone were placed subcutaneously in rats either before castration or at 14 h postcastration, when the expression level of par-4 is low (Sells et al., 1994). For control, testosterone-free pellets were used and the rats were castrated to allow interpretation of the expression levels after androgen replacement. Prostates were harvested at different time-points and the levels of Par-4 were examined by Western blot analysis (as described in Boghaert et al., 1997; Sells et al., 1997). These experiments revealed that Par-4 expression is low in prostates from uncastrated rats and that Par-4 is induced after castration at the 24 h timepoint. Par-4 levels peak at 72 h postcastration; thereafter the levels of Par-4 drop to precastration levels by 120 h. By contrast, testosterone replacement completely prevented the induction of Par-4 in the castrated animals (see Figure 7). These data indicated that Par-4 is not induced by testosterone and that Par-4 induction after castration is inhibited by testosterone. Data for testosterone replacement at 14 h postcastration were similar to those shown in Figure 7 for testosterone pellets used before castration. To determine the type of cells in the prostate that show Par-4 induction, we performed immunohistochemical analysis for Par-4. These studies indicated that Par-4 was primarily expressed in the stromal cells and basal cells in intact rat prostates (Boghaert et al., 1997). After castration, Par-4 levels increased within 24 h in the ductal cells and the levels stayed high until 72 h but dropped at 120 h (Boghaert et al., 1997). Because the ductal cells of the prostate undergo apoptosis after androgen ablation, these findings are consistent with the induction of Par-4 in apoptotic situations. We did not examine whether Par-4 induction is downregulated by testosterone replacement in the castrated rats at 66 h postcastration, when the expression level of Par-4 is high (Boghaert et al., 1997). This experiment would have been relevant if the levels of Par-4 had continued to be high after 66 ', 72 h postcastration. However, because Par-4 levels were naturally decreased at postcastration time-points beyond 66 ', 72 h, i.e. at 96 ', 120 h, we did not see the relevance of performing this second experiment. The role of par-4 as an apoptosis-specific gene in diverse cell culture paradigms was also addressed. Par-4 induction was studied by Western blot analysis in nonprostatic apoptosis model systems that utilize intracellular Ca 2+ elevation-dependent or -independent pathways. A two-fold or higher induction that was reproducible in three separate experiments was considered; (+) less than two-fold induction, or that which was not reproducible was considered (-). Unless indicated otherwise, the cells were

224

V.M. Rangnekar

exposed to the indicated stimuli for various intervals of time (0.5, 1, 2, 3, 5, 8, or 24 h, or 2 I 16 days as appropriate). Apoptosis in the appropriate systems was verified by terminal transferase-mediated dUTP-nick end labeling (TUNEL) assays (method as described in Sells et al., 1997). These data (Table 1) suggest that although Ca 2÷ elevation is central to multiple apoptotic systems, par-4 is upregulated only in ionomycin-treated thymocytes and in IL-3-starved hematopoitic cells. In each of these non-prostatic model systems where Par-4 was not upregulated, we ascertained that the conditions (timings, concentration of stimulus, etc.) were optimal for the induction of other known positive control genes. Because Par-4 was not induced in non-apoptotic models (Table 1), these findings maintain our observation that Par-4 up-regulation is apoptosis-specific and further suggest that induction of the gene is restricted only to certain apoptotic situations. Table 1.

Induction of Par-4 Protein in Diverse Model Systems

Model system Activators that require intracellular Ca2~ elevation for apoptosis Mouse immature thymocytes exposed to the following stimuli: anti-CD3 antibody (100/~g/ml; total 1 ml per 60-mm plate) dexamethasone (1 X 10-7 M) ionomycin (200 nM) Mouse thymoma cell line, WEHI-17, exposed to dexamethasone (1 X 10-6 M) Mouse stem cell line 32D subjected to IL-3 withdrawal Activators not requiring intracellular Ca2÷elevation for apoptosis Mouse thymoma cell line, WEHI-17, exposed to: forskolin (up to 10 ,uM) 8-bromo-cAMP (up to 200/~M) Involuting mouse mammary tissue during post-lactational involution (i.e. days 1, 2, 3, 4, 6, 8, and 2 months after weaning): Other apoptotic or non-apoptotic situations tumor necrosis factor-ct (TNF-a) induced apoptosis in L929 fibroblast cells TNF-ct induced growth stimulation in glioblastoma cells or fibroblasts serum stimulation of serum-starved cultures of: human fibroblast cells WI-38 human melanoma cells A375-C6 growth arrest action of interleukin-1 in A375-C6 ceils heat shock induced stress response in human cervical carcinoma cells HeLa differentiation of human myoblastic leukemia cells HL-60: into granulocytes upon treatment with dimethyl sulfoxide (1.3%) into macrophages upon treatment with TPA (10 nM) TNF-a induced growth arrest in human melanoma ceils

Induction

225

Par-4 Mediated Apoptosis

Structure-Function Analysis of Par-4 The deduced amino acid sequence of Par-4 predicts a leucine zipper domain containing protein (Sells et al., 1997;Rangnekar, 1998) as depicted in Figure 8. About 70 amino acids at its carboxyl terminus constitutes a death domain homologous to that found in other apoptotic proteins such as Fas or TRADD (Diaz-Meco et al., 1996). Moreover, about 40 amino acids at the carboxy terminus conform to the canonical sequence for a leucine zipper domain, with a heptad repeat (abcdefg) n of hydrophobic residues at position "a" and leucine residues at position "d", with the exception of the hydrophobic residue methionine at position "d" in the third repeat. A number of other proteins that contain death domains also contain a number of leucine residues within the death domain, but none of them conforms to the requirements of the heptad repeats for a leucine zipper domain. Par-4 protein may be structurally novel with a leucine zipper domain located within a death domain. It is important to note that the death domain and the leucine zipper domain are conserved in rat and human Par-4. It may be necessary to formally determine whether a leucine zipper domain can coexist within a death domain structure and whether these two domains contribute independently or cooperatively to the pro-apoptotic action of Par-4. Alternately, it is possible that apoptotic signals may induce posttranslational changes to activate an active death domain conformation. In the yeast two-hybrid assay, Par-4 can form homodimers and this interaction requires the leucine zipper domain. The putative death domain was never tested for homodimerization in vitro or in vivo paradigms. The death domain does not seem to interact with the death domain of FADD, as a dominant negative mutant of FADD which blocks TNF-receptor 1 induced apoptosis does not block Par-4-dependent apoptosis (Berra et al., 1998).

1

NLS 20 - 25

PKC

PKC PKA

~ NLS 137-153

PKC

332

258

332

DD 290

Figure 8.

332

Structural domains of Par-4. Par-4 contains a leucine zipper domain and a death domain

sequence at its carboxy terminus. In addition, a number of putative phosphorylation sites for protein kinase C or protein kinase A and two nuclear localization sequences in Par-4 need functional evaluation. These domains are conserved in human and rat protein, which are functionally identical in causing apoptosis.

Par-4 protein is also predicted to contain nuclear localization sequences; one of these is a bipartite sequence similar to that found to confer nuclear localization in other proteins. However, whether these sequences are sufficient to confer nuclear translocation of Par-4 is uncertain. In our hands, a fusion protein containing the NLS KEKREK in frame with beta-galactosidase, showed localization around the nuclear envelope but failed to clearly localize inside the nucleus. Second, immunohistochemical analysis of

226

V.M. Rangnekar

a wide range of tissues performed by using the highly sensitive antigen-retrieval technique indicated cytoplasmic staining for Par-4. The nuclear localization sequences of Par-4 may confer nuclear membrane localization but not translocation into the nucleus. A more thorough analysis of this issue is required to elucidate the relevance of nuclear expression of Par-4 in certain tissue settings. Par-4 also contains a number of putative phosphorylation residues whose functional relevance is unclear, as neither hyperphosphorylation nor hypophosphorylation of the protein in response to apoptotic agents is obvious. However, Western blot analyses of Par-4 protein often show a doublet, with the slow migrating band that could potentially represent a phosphorylated form of the protein. The slow migrating band increases in intensity proportionally with the fast migrating and heavy 38 kDa band. The structural and functional relevance of the slow migrating band needs to be evaluated. Does expression or induction of Par-4 lead to apoptosis? A number of studies from my laboratory have suggested that expression or induction of a gene in any particular process does not necessarily guarantee the functional involvement of the protein in the process. Functional studies are, therefore, mandatory to ascertain the functional relevance of a gene product in the process. One approach to determine the function of genes is to cause their underexpression by using antisense oligomers or to inhibit their function by the use of dominant-negative proteins. The presence of the leucine zipper domain in Par-4 allowed us to design a dominant-negative inhibitor of Par-4 that would presumably act by direct binding to Par-4 protein and prevent its function. Our studies indicated that antisense inhibition of Par-4 with either a Par-4-antisense oligomer (Sells et al., 1997), or with the leucine zipper domain abrogated apoptosis induced by agents that elevate intracellular calcium or by serum-withdrawal (Figure 9). This finding implied that Par-4 protein levels are necessary to induce a cascade in response to apoptosis-producing agents. To address the question whether Par-4 is sufficient for apoptosis, we overexpressed this protein in various cell-types and then exposed the cells to apoptosis-producing treatments. Such experiments suggested that Par-4 expression sensitizes cells to apoptosis induced by agents that elevate intracellular calcium, by ultraviolet (UV) radiation, or by growth factor withdrawal (Diaz-Meco et al., 1996; Berra et al., 1997; Sells et al., 1997). However, tumor cell lines overproducing Par-4 failed to undergo apoptosis on their own implying that Par-4 is not sufficient to cause apoptosis and that a coactivating signal is necessary to fully utilize the potential of Par-4. The ability of Par-4 to sensitize cells to apoptosis is not restricted to prostate cancer cells; diverse other cell-types such as melanoma ceils, renal carcinoma cells, neuronal cells, and fibroblasts show sensitization to apoptosis with Par-4.

Mechanism of Par-4 Induced Apoptosis Par-4 induces apoptosis in a p53-independent manner (Berra et al., 1997; Qiu et al., 1999). Mouse embryo fibroblast cells derived from p53 null mice or those that are transiently transfected to express mutant forms of p53 protein show similar levels of sensitization to apoptosis as those that contain wild-type p53 (Qiu et al., 1999). The apoptotic pathway(s) activated by Par-4 can be blocked by overexpression of the anti-apoptotic

Par-4 Mediated Apoptosis

227

protein Bcl-2 and by synthetic inhibitors of caspases, suggesting that Par-4 function is positioned upstream of Bcl-2 and caspases in the apoptosis pathway (Figure 10). Because Par-4 contains a putative death domain, it was reasonable to anticipate that it may activate a death cascade analogous to that induced by other death-domaincontaining proteins such as Fas and TNF receptor. However, none of the components of this putative death pathway has been identified. Moreover, Par-4-induced apoptosis is not blocked by the dominant-negative FADD protein which can block both the Fas and TNF
292 332

PAR-4

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-

PAR-4.ACTH ~

PAR-4.Aleu.zip leu.zip

-

PAR-4

Figure 9.

+ leu.zip

The leucine zipper domain of Par-4 is essential to sensitize cells to apoptotic signals.

Deletion of the leucine zipper domain renders Par-4 ineffective in sensitizing cells to apoptosis. The leucine zipper domain when coexpressed with full length Par-4 functions as a dominant negative inhibitor of Par-4 action. GROWTH FACTORS

CELL DEATH

~

CYTOKIN~

t i B:l.;.~[f i pendent ,,"MVAPK' SURVIV.ZL/GROWTH

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Inhibition of PKC-~ by Par-4 leads to apoptosis. Binding of Par-4 to PKC-~ inactivates the cell survival functionsof PKC-~and results in apoptosis. Par-4 inhibition of PKC-~ results in inhibition of mitogen activated protein kinase activation. Par-4 also causes activation of p38. Figure 10.

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V.M. Rangnekar

Recent studies (Berra et al., 1998; Qiu et al., 1999) have identified Par-4 as an antisurvival protein because of its ability to inhibit the function of survival proteins such as the atypical protein kinase C isoforms, particularly PKC-~, and Bcl-2 (Figure 10). Studies utilizing PKC-~ as a bait in the yeast two-hybrid assay identified Par-4 as a potential partner. Further analysis confirmed the in vivo binding of Par-4 and PKC-~, and suggested that the leucine zipper domain of Par-4 is required for the interaction. Because activation of PKC-~ is required for activation of MAP kinases (ERK-1 or ERK-2) that are central to cell survival and growth, inactivation of PKC-~ by Par-4 causes a reduction in MAP kinase activity and leads to apoptosis. Additionally, Par-4 upregulates p38 kinase activity. Simultaneous downregulation of MAP kinase activity and upregulation of p38 activity are essential for the induction of an apoptotic program by Par-4 in response to UV radiation. These exciting observations need to be further explored at the mechanistic level, to identify the precise molecular components that differentially regulate the MAP kinase and p38 pathway. Bcl-2 is an anti-apoptotic protein that serves to protect cells from the action of multiple apoptotic cascades. This protein is also implicated in prolonging cell survival and providing cancer cells the opportunity to develop resistance to chemotherapeutic agents. Interestingly, regulated or constitutive expression of Par-4 causes diminished protein levels of Bcl-2 (Qiu et al., 1999). By contrast, Bax protein levels are unaltered in response to an increase in Par-4 levels. Consistent with this observation, Par-4 and Bcl-2 protein show inverse expression patterns in benign and cancerous prostate tissue. In benign prostate tissue, Par-4 is expressed in both the basal and secretory epithelium. By contrast, Bcl-2 is expressed only in the basal epithelium consistent with the fact that the secretory epithelium, but not the basal cells, show apoptosis. Tumor tissue, on the other hand, shows expression of Par-4, but not Bcl-2, and is apt to undergo apoptosis. A most striking feature of this inverse pattern is seen in recurrent androgenindependent xenografts, where pockets of Bcl-2 positive cells develop within a largely Bcl-2-negative tumor. Par-4 is absent within the Bcl-2-positive cells, but is strongly expressed within the Bcl-2-negative areas. Whether Par-4 and Bcl-2 show such opposite expression patterns in other normal or tumor tissues needs to be explored, and the precise relationship between these proteins in tissues needs to be deciphered. Of particular interest is the fact that in normal tissues where apoptotic cells are encountered only Par-4 is present, whereas in cells that survive apoptosis both Bcl-2 and Par-4 are expressed. Thus, if Par-4 downregulates Bcl-2 in tissues, the relative levels of these proteins is likely to determine the ultimate fate, i.e. survival or apoptosis of the cells within the tissue. Studies utilizing the Wilms' tumor suppressor protein WT1 as a bait in the yeast two hybrid assay identified Par-4 as a potential partner (Johnstone et al., 1996). Co-immunoprecipitation studies confirmed the interaction between WT1 and Par-4. The specificity of the WT1 and Par-4 binding was evident from the lack of interaction between another closely related protein, EGR-1, and Par-4. Like WT 1, EGR-1 containing zinc-finger domains that bind to the same consensus DNA binding site in target gene promoters and regulate gene transcription. Interestingly, the zinc finger-domain of WT1 but not of EGR1 binds to the leucine zipper domain of Par-4. The interaction with Par-4 alters the transcription activity of WTI: in promoter contexts in which WT1 acts as a

229

Par-4 Mediated Apoptosis

transcriptional activator, the interaction causes transcriptional repression, and in promoter contexts in which WT1 acts a transcriptional repressor Par-4 interaction causes stronger transcriptional repression. The ability of Par-4 to causes transcriptional repression was further confirmed by use of a GAL-4-Par-4 fusion protein, which when brought to the promoter GAL-4 binding sites by GAL-4, serves to cause transcriptional repression. Thus, when brought to the DNA by another DNA binding protein, Par-4 causes transcriptional repression. The biological relevance of the interaction with WT1 and the transcriptional repressor function of Par-4 was evident from the observation that coexpression of ectopic Par-4 relieves the anti-apoptotic effects of WT1 and enhances apoptosis in human melanoma cells (Figure 11). Most importantly, a recent study suggests that a KTS mutant of the WT1 protein fails to bind to Par-4 protein and that this WT1 mutant protein is expressed in small cell cancer cells in which it appears to be oncogenic (Kim et al., 1998). It appears that lack of binding to Par-4 frees up the WT1 mutant to alter the cell phenotype. A more detailed analysis of the structural and biological relevance of WT1 interaction with Par-4 may provide insights into the mechanisms that underscore WT1 mutant inducible oncogenesis. GROWTHARREST SIGNALS

CELLDEATH SIGNALS

pg3-independent ~

REVERSIBLE GROWTHARREST

bcl-2 caspase inhibitors

1

CELL DEATH

Figure 11. Effect of Par-4 and WT1 interaction in vivo. Binding of Par-4 via its leucine zipper domain to the zinc finger domain of WT1 leads to protection from the growth-arresting action of IL-1 and enhancement of the apoptotic action of thapsigargin in human melanoma cells.

Expression in Aging-Related Diseases Recent studies, in collaboration with the laboratory of Mark Mattson, have most elegantly linked Par-4 expression to degenerating hippocampal neurons. Immunohistochemical analysis of hippocampal tissue from Alzheimer patients indicated that Par-4 was expressed in about 30 ', 50% of the dying neurons (Guo et al., 1998). Most importantly, the Par-4 positive neurons also expressed the Tau protein, an indicator of in the hippocampus. Although the role of apoptosis in Alzheimer's disease is still being debated, Mattson's work clearly implicates Par-4 as a pro-apoptotic protein linked to degenerating neurons. This observation is substantiated by work in cell culture paradigms of apoptosis. The two key inducers of neuronal cell death, beta-amyloid protein and mutant presenilin-1, were found to induce the expression of Par-4 in

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primary neurons. A block in the expression of Par-4 by the use of antisense oligomers or dominant-negative Par-4 rescued the cells from beta-amyloid or mutant-presenilin-1induced apoptosis. Overexpression of Par-4 in the neuronal cells increased significantly the time frame and magnitude of the apoptotic response. The study also showed the involvement of free radicals, mitochondria, and caspase-1 in the sensitization to apoptosis by Par-4. Further studies that identify the direct targets of Par-4, in the aging neurons that determine the time frame of apoptosis, will be critical in deciding whether a block in Par-4 expression can prevent death of the neurons. If that is feasible and a role for apoptosis seems central in the death of neurons of Alzheimer's patients, Par-4 may provide an important physiologically relevant target for prevention of the disease. Since Par-4 overexpression is linked to the death of cancer cells, insights obtained from the Par-4-dependent downstream pathways in neuronal apoptosis, in these naturally occurring aging diseases, will be useful in the designing of strategies for the application of Par-4 in anticancer research. Recent studies suggest roles for Par-4 in the death of neurons that occurs in Parkinson's disease and amyotrophic lateral sclerosis (ALS). Levels of Par-4 protein are increased in substantia nigra dopaminergic neurons following administration of MPTP to monkeys and mice. MPTP is a toxin that selectively damages dopaminergic neurons affected in Parkinson's patients and induces a clinical profile essentially indistinguishable from Parkinson's patients. In the MPTP models, the increase in Par-4 levels occurs within 6 ', 24 h following MPTP administration, a timepoint many h to days prior to degeneration of dopaminergic neurons (Duan et al., 1999a). An increase in Par-4 levels also occurred in cultured human dopaminergic cells following exposure to the complex 1 inhibitor rotenone or iron, an oxidative insult. Treatment of the cultured dopaminergic cells with Par-4 antisense oligonucelotide prevents death induced by rotenone and iron (Duan et al., 1999a). In the case of ALS, studies of postmortem spinal cord tissue from ALS patients and transgenic mice expressing a copper zinc SOD mutation linked to an inherited from of ALS reveal increased levels of Par-4 in association with degenerating lower motor neurons in the ventral horn of spinal cord (Pedersen et al., 1999). Moreover, Par-4 levels increase rapidly following exposure of cultured motor neurons to oxidative insults relevant to the pathogenesis of ALS. Collectively these findings suggest a rather widespread role for Par-4 in the pathogenesis of many different human neurodegenerative disorders. The rapid increases in Par-4 protein levels following exposure of neurons to apoptotic insults (i.e. within 1 ', 4 h) suggested the possibility of upregulation at the translational level. Recent studies have indeed shown that Par-4 expression can be induced at the translational level. Duan et al. (1999b) have shown that exposure of synaptosomes, a preparation which consists of pre- and postsynaptic terminals and no nuclei, to apoptotic insults results in increased Par-4 protein levels. The latter studies showed that the protein synthesis inhibitor, cycloheximide, blocks the increase in Par-4 protein levels in synaptosomes, as well as in intact neurons. Interestingly, Par-4 induction plays a critical role in mitochondrial dysfunction and caspase activation in intact neurons and synaptosomes (Duan et al., 1999b). Such treatment of neurons or synaptosomes with Par-4 antisense oligonucleotides suppresses mitochondrial membrane depolarization, accumulation of intra-mitochondrial reactive oxygen species, and activation of caspase-3

231

Par-4 Mediated Apoptosis

following exposure to apoptotic insults such as staurosporine or iron. The ability of Par-4 to be induced locally in synaptic compartments at the translational level has intriguing implications for roles of Par-4 in synaptic degeneration and remodeling independent of cell death (Figure 12).

t Presy

"

"

N~TF ~a-" r GIut~ti~12~

Figure 12. Modelfor involvementof Par-4 in neuronal apoptosis.

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V.M. Rangnekar

Is Par-4 a T u m o r Suppressor?

The ability of Par-4 to suppress the growth of tumor cell lines has been extensively examined in cell culture models using high levels of inducible or constitutively expressed protein. These studies have suggested that Par-4 sensitizes cells to the action of apoptotic signals. Melanoma cells, prostate cancer cells, and renal cell carcinomaderived primary cell cultures are sensitized to apoptosis if they stably overexpress Par-4 protein. This sensitization requires the leucine zipper domain of Par-4 and can be overcome by coexpressing the dominant negative leucine zipper mutant of Par-4. Thus, unlike other tumor suppressors like p53 or retinoblastoma protein, Par-4 does not cause direct apoptosis or growth arrest of cells. However, renal cell carcinomas show downregulation of Par-4 expression (Cook et al., 1999). In a total of 27 renal cell carcinoma specimens examined, Par-4 was present in the proximal renal tubules in all specimens but was absent in the adjacent cancer areas, which arise from the proximal tubules, in 20 specimens. In addition to immunohistochemistry, the observation was confirmed quantitatively by Western blot analysis of normal and cancer tissue from within the same tumor. Reintroduction of Par-4 into the cell lines derived from the renal cell carcinomas rendered the cells highly sensitive to apoptosis by chemotherapeutic agents. Generally, renal cell carcinomas do not respond well to chemotherapy, and the patients succumb to the disease within a few years, greatly increasing the mortality rates from the disease. However, whether the loss of Par-4 is a deciding factor in the transformation process or progression of renal cell carcinoma needs to be carefully studied. Although Par-4 does not induce apoptosis on its own, the presence of Par-4 and environments cues in the proximal renal tubule vicinity may render the cells susceptible to apoptosis and offer protection from the events leading to tumorigenesis. Alternately, Par-4 may inhibit tumorigenesis by an apoptosis-independent mechanism (Figure 13). It would therefore not be too far fetched to hypothesize that Par-4 may serve as a tumor suppressor. SURVIVAL I I

PAR-4

PKC-lJla NF4~B

TUMORIGENESIS

APOPTOSIS

Figure 13. Model for Par-4 mediated apoptosis. Par-4 inhibits Bcl-2, PKC-zeta, and NF-KB functions and this results in inhibition of cell survival. Whether Par-4 also activates a p53-independent pathway downstream of p38 need further elucidation. Also, studies are ongoing to test whether Par-4 may function as a tumor suppressor independent of an apoptotic mechanism.

Direction of Future Studies

A model depicting antisurvival, antigrowth arrest, or pro-apoptotic actions of Par-4 is shown in Figure 10. Deciphering the mechanism(s) by which Par-4 activates p38 kinase and induces apoptotic death is critical for identifying the p53-independent pathway

Par-4 MediatedApoptosis

233

utilized by this widely expressed protein. In addition to functional inhibition of atypical PKC isoforms or WT1, Par-4 targets the central anti-apoptotic protein Bcl-2. However, it is becoming increasingly clear that the members of the Bcl-2 family of proteins confer redundant functions and operate in the cell-type or tissue-type specific manner. Whether Par-4 also downregulates the expression of the other members of the Bcl-2 family is currently being investigated. However, because the downregulation of the Bcl-2 family members may have different outcomes in different tissues, a study of all the members or their common targets is essential to obtain a true evaluation of the value of Par-4 to the processes. It is necessary, therefore, to identify other interactive proteins that influence Par-4 action in diverse cell-types. Such studies need to be appropriately designed to accommodate the likelihood that proteins may interact in a cell-type specific manner with Par-4 for a biologically relevant outcome. Characterization of the interactive partners of Par-4 should offer clues into why, despite the fact that Par-4 is widely expressed, it will not induce apoptosis unless a "coactivating" apoptotic signal is present. Perhaps, Par-4 is normally sequestered by another protein in order to maintain cell viability, and only when an appropriate insult is encountered, Par-4 dissociates from the complex and becomes activated. The leucine zipper domain and PKC- or PKA-mediated phosphorylation of Par-4 might facilitate such reversible interactions. It will be important to identify the cellular compartment(s) in which Par-4 is maximally active in accomplishing its function. In most tissues and cell lines, Par-4 shows cytoplasmic expression. On the other hand, both cytoplasmic and perinuclear or nuclear expression of Par-4 have been noticed in prostatic cells. It is possible that Par-4 may function as a transcriptional repressor either directly in the nucleus or indirectly by blocking the nuclear translocation of a transcription factor or activator in the cytoplasm. Thus, cytoplasmic Par-4 may be either translocated to the nucleus for direct transcriptional repression of target genes; or when confined to the cytoplasm, Par-4 may indirectly contribute to transcriptional repression of target genes by inhibiting a molecular pathway that is necessary for the activation (and translocation to the nucleus) of another transcription factor. The identification of downstream transcriptional targets of Par-4 that can transmit the apoptotic or antisurvival signals will provide valuable insights into the modus operandi of this pro-apoptotic protein. It would not be surprising if future studies discern both nuclear and cytoplasmic roles for this widely expressed protein. Because the par-4 gene is widely expressed and its upregulation is tightly linked with apoptosis, it is important to examine whether Par-4 protein levels are diminished in certain types of tumors. Once such tumors are identified, a more involved molecular study can address whether the diminished levels provide any growth or survival advantage to the tumor. By contrast, because prostate tumors show a higher incidence of apoptotic nuclei relative to unaffected normal or benign tissue, one can anticipate a role for Par-4 expression in apoptosis within such tumors. Besides cancer, studies of Par-4 expression and function in other diseases, such as neurodegenerative disorders that involve programmed cell death, may offer insights into the role of Par-4 in disease establishment and progression (Figure 14). Such studies may help design strategies based on Par-4 over- or underexpression for therapeutic intervention.

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Survival of lntraceUular

N e u r o d e g ~ Pathogen

. CASCe.R

Ischemia/reperfusion Injury

Development

Model for the roles of Par-4 in diverse apoptotie paradigms. Par-4 causes apoptosis in cancer cells thereby blocking cancer cell growth as well as neuronal degeneration contributing to Alzheimerdisease. Par-4 may also participatein ischemiareperfusioninjury,development,and inhibitionof host cell survivalleadingto abortiveinfectionwith intracellularpathogen. Figure 14.

Summary

Prostate apoptosis response-4 (Par-4) is a pro-apoptotic protein identified by differential hybridization in a screen for genes upregulated when prostate cancer cells are induced to undergo apoptosis. This protein is conserved in vertebrates and is expressed in almost all cell-types. Par-4 induction is associated with apoptosis in diverse cell culture and in vivo models of apoptosis. Par-4 is essential for apoptosis and enforced expression of Par-4 sensitizes cells to p53-independent apoptosis. Par-4 protein can abrogate cell survival function conferred by protein kinase C-~, Bcl-2, or NF-~:B. Par-4 is downmodulated in renal cell carcinomas and replenishment of Par-4 levels in primary renal cell carcinoma cultures sensitizes the cells to apoptosis by chemotherapeutic agents. Par-4 induces apoptosis in cancer, Alzheimer's disease, and developmental paradigms, and is likely to play a role in ischemia ', reperfusion injury. Future studies are being directed toward exploring the translational aspects of Par-4 in the design of therapeutics for cancer, neurodegenerative diseases, and ischemia ', reperfusion-induced cardiac injury.

Acknowledgments

The studies described here from my laboratory were supported by NIH/NCI grant R01 CA60872, CaPCURE grant, and the Council for Tobacco Research. I thank the current and past members of my laboratory especially Guofang (Shirley) Qiu, Sumathi Krishnan, Aysegul Nalca, Seong-Su Han, Stephen Sells, Swati Joshi-Barve, Scott Crist, Suzanne Humphreys, Prakash Nair, David Wood, Jr., Neeraj Maddiwar, and Jason Cook; and collaborators Yang Shi, Karl Heinz Scheidtmann, Michael Weinstein, Mansoor Ahmed, Erwin Boghaert, Neil Williams, Mark Mattson, and Robert Strange for contributing to the studies described here. Elegant studies from the

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235

laboratories of Jorge Moscat, Karl Heinz Scheidtmann, Mark Mattson, Jerry Pelletier, and Yang Shi have provided new insights into fundamental and biological aspects of Par-4.

References Adam, J.M. & Cory, S. (1998). The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322-1326. Askenazi, A. & Dixit, V.M. (1998). Death receptors: signaling and modulation. Science 281, 1305-1308. Barger, S.W., Horster, D., Furukawa, K., Goodman, Y. Krieglestein, J. & Mattson, M.P. (1995). Tumor necrosis factors ct and B protect neurons against amyloid B-peptide toxicity: evidence for involvement of a KB-binding factor and attenuation of peroxide and Ca 2+ accumulation. Proc. Natl. Acad. Sci. U.S.A. 92, 9328-9332. Beg, A.A. & Baltimore, D. (1996). An essential role for NF-KB in preventing TNF-ct induced cell death. Science 274, 782-784. Berra, E., Diaz-Meco, M.T. & Moscat, J. (1998). The activation of p38 and apoptosis by the inhibition of Erk is antagonized by the phosphoinositide 3-kinase/Akt pathway. J. Biol. Chem. 273, 10792-10797. Berra, E., Municio, M.M., Sanz, L., Frutos, S., Diaz-Meco, M.T. & Moscat, J. (1997). Positioning atypical protein kinase C isoforms in the uv-induced apoptotic signaling cascade. Mol. Cell. Biol. 17, 4346-4354. Boghaert, E.R., Sells, S.F., Walid, A-J., Malone, P., Williams, N.M., Weinstein, M.H., Strange, R. & Rangnekar, V.M. (1997). Immunohistochemical analysis of the proapoptotic protein Par-4 in normal rat tissues. Cell Growth Differ. 8,881-890. Buttyan, R., Zakeri, Z., Lochshin, R.& Wolgemuth, D. (1988). Cascade induction of c-fos, c-myc and heat shock 70K transcripts during regression of the rat ventral prostate gland. Mol. Endocrinol. 2, 650-657. Chao, D.T. & Korsmeyer, S.J. (1998). Bcl-2 family: Regulators of cell death. Annu. Rev. Immunol. 16, 395-419. Connor, J., Sawzchuk, I.S., Benson, M.C. Tomashefsky, P., O'Toole, K.M., Olsson, C.A. & Buttyan, R. (1988). Calcium channel antagonists delay regression of androgen-dependent tissues and suppress gene activity associated with cell death. Prostate 13,119-130. Cook, J., Krishnan, S., Ananth, S., Shi, Y., Walther, M.M., Linehan, M., Sukhatme, V.P., Weinstein, M.H. & Rangnekar, V.M. (1999). Decreased expression of the pro-apoptotic protein Par-4 in renal cell carcinoma. Oncogene. 18, 1205-1208. Diaz-Meco, M.T., Municio, M.M., Frutos, S., Sanchez, P., Lozano, J., Sanz, L. & Moscat, J. (1996). The product of par-4, a gene induced during apoptosis, interacts selectively with the atypical isoforms of protein kinase C. Cell 86, 777-786. Duan, W., Zang, Z., Gash, D.M. & Mattson, M.P. (1999a). Participation of Par-4 in degeneration of dopaminergic neurons in primate and rodent models of Parkinson's disease. Ann Neurol. 46, 587-597. Duan, W., Rangnekar, V. & Mattson, M.P. (1999b). Par-4 production in synaptic compartments following apoptotic and excitotoxic insults: evidence for a pivotal role in mitochondrial dysfunction and neuronal degeneration. J. Neurochem. 72:2312-2322. Finco, T.S., Westwick, J.K., Norris, J.L., Beg, A.A., Der, C.J. & Baldwin, A.S. Jr. (1997). Oncogenic Ha-Rasinduced signaling activates NF-KB transcriptional activity, which is required for cellular transformation. J. Biol. Chem. 272, 24113-24116. Green, D.G. & Reed, J.C. (1998). Mitochondria and apoptosis. Science 281, 1309-1312. Guo, Q., Fu, W., Xie, J., Luo, H., Sells S.F., Geddes, J.W., Bondada, V., Rangnekar, V.M. & Mattson, M.P. (1998). Par-4 is a novel mediator of neuronal degeneration associated with the pathogenesis of

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V.M. Rangnekar

Alzheimer's disease. Nat.Med. 4, 957-962. Hannun, Y.A. (1997). Apoptosis and the dilemma of cancer chemotherapy. Blood 89, 1845-1853. Johnstone, R.W., See, R.H., Sells, S.F., Wang, J., Muthukkumar, S., Englert, C., Haber, D.A., Licht, J.D., Sugrue, S.P., Roberts, T., Rangnekar, V.M. & Shi, Y. (1996). A novel repressor, par-4, modulates transcription and growth suppression functions of the Wilms' tumor suppressor WT1. Mol. Cell. Biol. 16, 6945-6956. Kim, J., Lee, K. & Pelletier, J. (1998). The DNA binding domains of the WT1 tumor suppressor gene product and chimeric EWS/WT1 oncoprotein are functionally distinct. Oncogene 16, 1021-1030. Kyprianou, N. & Isaacs, J.T. (1988). Activation of programmed cell death in the rat ventral prostate following castration. Endocrinol. 122, 552-562. Liu, Z.-G., Hsu, H., Goeddel, D.V. & Karin, M. (1996). Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-rB activation prevents cell death. Cell 87,565-576. Pedersen, W.A., Luo, H., Kruman, I., Kasarkis, E. & Mattson, M.P. (1999). The Par-4 protein participates in motor neuron degeneration in amyotrophic lateral sclerosis. FASEB J. 14,913-924. Peter, M.E., Heufelder, A.E. & Hengartner, M.O. (1997). Advances in apoptosis research. Proc. Natl. Acad. Sci. U.S.A. 94, 12736-12737. Porter, A.G., Ng P. & Janicke, R.U. (1997). Death substrates come alive. Bioessays 19, 501-507. Qiu, G., Ahmed, M., Sells, S.F., Mohiuddin, M., Weinstein, M.H. & Rangnekar, V.M. (1999). Mutually exclusive expression patterns of Bcl-2 and ParJ, in human prostate tumors consistent with downregulation of Bcl-2 by Par-4. Oncogene. 18, 7115-7123. Rangnekar, V.M. (1998). Apoptosis mediated by a novel leucine zipper protein Par-4. Apoptosis 3, 61-66. Sells, S.F., Wood, D.P. Jr., Barve, S.S.J-., Muthukumar, S., Jacob, R.J., Crist, S.A., Humphreys, S. & Rangnekar, V.M. (1994). Commonality of the gene programs induced by effectors of apoptosis in androgen-dependent and -independent prostate cells. Cell Growth Differ. 5, 457-466. Sells, S.F, Han, S.-S, Muthukkumar, S., Maddiwar, N., Johnstone, R., Boghaert, E., Gillis, D., Liu, G., Nair, P., Monnig, S., Collini, P., Mattson, M.P., Sukhatme, V.P., Zimmer, S.G., Wood Jr., D.P., McRoberts, W. J., Shi, Y. & Rangnekar, V.M. (1997). Expression and function of the leucine zipper protein par-4 in apoptosis. Mol. Cell. Biol. 17, 3823-3832. Stanisic, T., Sadlowsky, R., Lee, C. & Grayhack, J.T. (1978). Partial inhibition of castration-induced ventral prostate regression with actinomyein D and cycloheximide. Invest. Urol. 16, 19-22. Staunton, M.J. & Gaffney, E.F. (1998). Apoptosis: basic concepts and potential significance in human cancer. Pathol. Lab. Med. 122, 310-319. Susin, S.A., Zamzami, N. & Kroemer, G. (1998). Mitochondria as regulators of apoptosis: doubt no more. Biochimica et Biophya. Acta 1366, 151-165. Thornberry, N.A. & Lazebnik, Y. (1998). Caspases: Enemies within. Science 281, 1312-1316. Van Antwerp, D.J., Martin, S.J., Verma, I.M. & Green, D.R. (1998). Trends Cell Biol. 8, 107-111. Van Antwerp, D.J., Martin, S.J., Kafri, T., Green, D.R. & Verma, I.M. (1996). Suppression of TNF-ct-induced apoptosis by NF-KB. Science 274, 787-789. Wang C.-Y., Mayo, M.W. & Baldwin, A.S. Jr. (1996). TNF-a and cancer therapy-induced apoptosis: potentiation by inhibition of NF-KB. Science 274, 784-787. Williams, G.T. (1991). Programmed cell death: apoptosis and oncogenesis (minireview). Cell 65, 1097-1098. Wyllie, A.H., Kerr, J.F.R. & Currie, A.R. (1980). Cell death: The significance of apoptosis. Int. Rev. Cytol. 68, 251-306.

CYTOSKELETAL INVOLVEMENT IN APOPTOSIS RAKESH K. SRIVASTAVA, MARK P. MATI'SON and DAN L. LONGO

Table of Contents Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytoskeletal Structure and Dynamics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microtubule Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microtubule Nucleation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Relationship Between Microtubule Structure and Polarity . . . . . . . . . . . . . . . . . . . . . . . . . . Actin Filament Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytoskeleton and Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cytoskeletal Reorganization During Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modulation of Cell-Cell Contact During Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cleavage of 13-Cateninby Caspases During Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cleavage of Lamin B During Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cleavage of Integrins During Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Activation of Protein Kinases Due to Microtubule Damage . . . . . . . . . . . . . . . . . . . . . . . . . . Role of cAMP-Dependent Protein Kinase (PKA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Mitogen-Activated Protein Kinase (MAP Kinases) . . . . . . . . . . . . . . . . . . . . . . . . . Role of p34cdc2Kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Induction of Genes by Paclitaxel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

237 238 239 239 240 24 ! 241 243 243 246 247 248 250 251 253 254 255 256

Introduction The cytoskeleton of a cell is a network of protein filaments that influences the trafficking of molecules and organelles within the cell, the ability of the cell to move and migrate and change shape in response to signals from its environment, and the scrupulous control over chromosome segregation during mitosis. Three types of protein filaments comprise the cytoskeleton (Figure 1): intermediate filaments, microtubules, and actin filaments. Intermediate filaments are strong and durable structures that protect the cell against mechanical stress. Actin filaments are thin and flexible and are involved in cell movement (crawling) and muscle contraction. Microtubules are hollow tubes that organize the cell interior; they emanate from the centrosome in interphase cells and they form the mitotic spindle in dividing cells. Microtubules have been a useful target for cancer chemotherapy. Microtubules are the filaments that, when damaged, can initiate a signaling cascade leading to apoptosis.

237 Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. 237 -- 267 © 2001 Elsevier Science. Printed in the Netherlands.

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INTERMEDIATE FILAMENTS

MICROTUBULES

E ~,

.

ACTIN FILAMENTS

~

--

----- 5 _ - - - - - - ~

- •

t....._z 25 nm 1. Intermediate filaments. Intermediate filaments are ropelike fibers with a diameter of around 10 nm; they are made of intermediate filament proteins, which constitute a large and heterogeneous family. One type of intermediate filament forms a meshwork called the nuclear lamina just beneath the inner nuclear membrane. Other types extend across the cytoplasm, giving cells mechanical strength and carrying the mechanical stresses in an epithelial tissue by spanning the cytoplasm from one cell-cell junction to another. Mierotubules. Microtubules are long, hollow cylinders made of the protein tubulin. With an outer diameter of 25 nm, they are much more rigid than actin filaments. Microtubules are long and straight and typically have one end attached to a single microtubule organizing center (MTOC) called a centrosome. A e t i u f i l a m e n t s . Actin filaments are two-stranded helical polymers of the protein actin. They appear as flexible structures, with a diameter of 5-9 nm, that are organized into a variety of linear bundles, two-dimensional networks, and three-dimensional gels. Although actin filaments are dispersed through the cells, they are most highly concentrated in the cortex, just beneath the plasma membranes. Figure

Cytoskeletal

Structure

and

Dynamics

Cell adhesion to n e i g h b o r i n g cells and to the extracellular matrix ( E C M ) plays a central role in a variety of biological processes, i n c l u d i n g cell motility, growth, differentiation and apoptosis. These adhesion structures consist of protein complexes that are specific either to c e l l - E C M j u n c t i o n s (i.e. talin and paxillin) or to cell-cell j u n c t i o n s (et- and 13-catenin and plakoglobin), or shared b y both types of adhesion j u n c t i o n s (i.e. vinculin, ct-actinin, zyxin and tensin) (Table 1). A d h e s i v e interactions i n v o l v e a variety of t r a n s m e m b r a n e receptors that are linked via j u n c t i o n a l plaque proteins to the cytoskeleton. Recent studies have demonstrated that these t r a n s m e m b r a n e structural linkages are also i n v o l v e d in signal transduction (Clark and Brugge, 1995; Geiger et al., 1995; Schwartz et al., 1995; Y a m a d a and Miyamoto, 1995; G u m b i n e r , 1996; H u b e r et al., 1996a; Craig and Johnson, 1996). M a l i g n a n t transformation is characterized b y disruption o f cytoskeletal organization, decreased adhesion, and altered adhesiond e p e n d e n t responses. The growth o f m a n y cancer cells is ' a n c h o r a g e - i n d e p e n d e n t ' , and

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such cells have often lost the growth inhibitory effects conferred by cell-cell adhesion (i.e. have lost the 'contact inhibition of growth'). Table 1.

ExtracellularProteins Found in SpecificJunctions

Junctions

Proteins

CelI-ECMJunction Cell-Cell Junction CelI-ECMJunction and Cell-Cell Junction

talin, paxillin ct, f3-catenin,plakoglobin,cadherin vinculin, ct-actinin,zyxin, tensin, integrin, catenin

Microtubule Structure Microtubules are formed by the self-assembly of ctB tubulin heterodimers, along with a cohort of microtubule-associated proteins (Mitchison and Kirschner, 1984; Horio and Hotani, 1986). Thirteen parallel protofilaments, each of which is a linear arrangement of dimers, form the microtubule wall to which a variety of microtubule-associated proteins and motor proteins bind. Numerous ligands also bind to tubulin, affecting its assembly properties, among them several drugs that have proven to have anticancer properties. It has been suggested that intermediate filaments and microtubules are closely associated (Trevor et al., 1995; Svitkina et al., 1996); thus, disturbance of microtubules may also affect the organization of intermediate filaments. Microtubule Nucleation Microtubules are in a state of dynamic flux during the cell cycle. There are three distinct steps in the life cycle of a microtubule, namely, nucleation, assembly and disassembly. Nucleation is the formation of a tubulin ring composed of 13 ,/ tubulin molecules. A microtubule assembles or grows by addition of ctB tubulin heterodimers. Microtubules disassemble by loss of grouped subunits from the growing end. Even at steady state, microtubules undergo periods of prolonged growth and shrinkage and can switch rapidly between these states (Horio and Hotani, 1986; Mitchison and Kirschner, 1984). This process, known as dynamic instability, is thought to result from GTP hydrolysis by tubulin; a cap of GTP-containing tubulin subunits at the microtubule end is believed to stabilize the lattice, while exposure of tubulin-GDP at the ends destabilizes the polymer (Caplow and Shanks, 1996; Desai and Mitchison, 1997). This process gives rise to structures with a distinct polarity. The minus end is an ct tubulin component of an ctB tubulin heterodimer and the plus end, the direction of in vivo microtubule growth, has a B-tubulin molecule at its extreme end. There are clear differences in microtubule nucleation in vitro compared with nucleation in living ceils. In vitro, microtubules self-nucleate and grow from both ends, with the plus end growing more quickly. In vivo, they nucleate within the microtubule-organizing center (MTOC) to which the minus end remains anchored,

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whereas the so-called plus end grows into the cytoplasm. The tubulin assembles with GTP present on the 6-tubulin subunit exchangable site (E-site) and that GTP is hydrolyzed subsequently to produce GDP so that the bulk of the microtubule surface lattice is made up of tubulin-GDP. The y-tubulin molecule is a third form of tubulin localized to the centrosome from which microtubules originate. "/-tubulin forms a ring to which the czl3 heterodimers add. cd3 tubulin heterodimers bound to GTP are tightly bound to the growing microtubule; those bound to GDP are more loosely bound to the microtubule; thus, the GTP vs GDP status influences microtubule stability (Joshi, 1994). Relationship Between Microtubule Structure and Polarity Microtubules are ubiquitous cytoskeletal structures that are made of ctB tubulin heterodimers, ct and B tubulin are highly homologous (Mitchison and Kirschner, 1984; Horio and Hotani, 1986). Each monomer binds a guanine nucleotide, which is nonexchangeable in (x-tubulin (the nucleotide binding site in ct tubulin is known as the N-site) and exchangeable in B-tubulin (the binding site in 13-tubulin is known as the E-site). GTP at the E-site is required for microtubule assembly, and its hydrolysis follows addition of a dimer to the microtubule end, upon which it becomes non-exchangeable. The stability of the system is maintained by a cap of tubulin-GTP at the ends, and when this cap is lost the microtubule can come apart. This change in stability of heterodimer association with the phosphorylation state of the guanine nucleotide is the basis for the dynamic instability of microtubules. Both ct and I] subunits exist in several isotypic forms and undergo a variety of posttranslational modifications. The carboxy-terminal residues of both ct and 13 monomers are different among isotypes. Different isotypes are often found with different distributions in different cell-types, or in different sets of microtubules in a single cell, and yet they can often co-polymerize. These isotypes are partially interchangeable. Dynamic instability is crucial to microtubule function. New microtubules are continually growing outward from centrosomes looking for something, a specific molecule or organelle, to which to bind. If a tubule finds such a binding partner, it is stabilized. If a tubule does not find a binding partner, the tubule disassembles and retracts. During cell division, microtubules form the mitotic spindle. As prophase begins, two daughter centrosomes separate and move to opposite poles of the cell by walking along microtubules going in opposite directions. Each daughter centrosome sends out new microtubules to connect to sister chromatids via their kinetochores during prometaphase (after nuclear membrane breakdown). During metaphase, the chromosomes are aligned midway between the two centrosomes (now called spindle poles) and during anaphase the paired chromosomes separate and are brought close to the opposing centrosomes (spindle poles) by the shortening of the microtubules (that is, dynamic instability resulting in microtubule disassembly). During telophase, a new nuclear membrane reforms around each group of separated chromosomes and the centrosome and microtubules are excluded from the new nucleus. Cytokinesis separates the daughter cell cytoplasms by forming a contractile ring of actin and myosin halfway between the two centrosomes.

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The crucial role of microtubules in assuring the integrity of cell division results in the creation of a mitotic checkpoint. If microtubules are not functioning normally, disasterous cell division could ensue. Thus, the cell senses microtubule dysfunction and similar to the events that follow the detection of severe DNA damage, cells that cannot safely divide undergo apoptosis. Actin Filament Organization Actin filaments are highly dynamic structures that are intimately linked to the membrane through a variety of actin-binding proteins and integral membrane proteins. The linkage of actin filaments to integrins provides a structural link between the cytoskeleton and the extracellular matrix. Cell adhesion molecules linked through integrins to the actin filaments organize focal adhesion complexes that, in addition to providing structural stability and organization to cell form, also provide sites at which complex signaling processes are activated. For example, focal adhesion complexes are often sites where growth factor receptors are clustered and where signaling through such receptor tyrosine kinases occurs. Perturbation in the organization of focal adhesion complexes often occur in adherent cells undergoing apoptosis and changes in the actin cytoskeleton undoubtedly play a role in this process. In addition, data suggest that apoptosis-related changes in the actin cytoskeleton are critical for the formation of the cell surface blebs, a cell characteristic of apoptosis in most cell-types. Not only are the actin filaments important in controlling cell structure, particularly at the level of the plasma membrane and its relationships with extracellular matrix molecules and cell adhesion molecules, but the actin filaments also are increasingly recognized as playing important signaling roles. Through their interactions with various actin-binding proteins, actin filaments have been shown to modulate the function of ion channels and to regulate signaling through various kinase cascades. It is thought that actin filaments play a role in organizing spatial aspects of signal transduction from the plasma membrane to intracellular organelles including the endoplasmic reticulum and nucleus.

Cytoskeleton and Apoptosis Apoptosis is a fundamental cellular process in the development and homeostasis of all multicellular organisms (Raft, 1992; Wyllie, 1995). The aberrant regulation of apoptosis that has been observed in many disorders (such as neuronal diseases, AIDS, autoimmune diseases, and cancers) results from an imbalance between positive and negative regulators of cell survival (Thompson, 1995). Apoptotic morphological features are generally similar in all systems where they have been studied: the nucleus condenses and the cell shrinks and often fragments into apoptotic bodies that are rapidly phagocytosed by neighboring cells (Kerr et al., 1972; Wyllie, 1993). Apoptosis is always accompanied by alterations in the nuclear morphology, finally leading to nuclear fragmentation and formation of apoptotic bodies. Historically, pathologists called the nuclear fragmentation they observed under the microscope by the name "karyorrhexis",

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literally nucleus breakdown, a name that seems better suited to describe the process than the more recently coined "apoptosis". The activation of a cascade of cysteine proteases of the interleukin-ll3-converting enzyme (ICE)/ced3 (caspase) family exerts a pivotal role in the execution of apoptosis (Martin and Green, 1995; Nicholson et al., 1995). Mammalian cells express at least 10 such caspases, which cleave after aspartate residues (Alnemri, 1997). Overexpression of these caspases induces apoptosis, whereas their inhibition suppresses apoptosis (Lazebnik et al., 1994; Martin and Green, 1995; Nicholson et al., 1995). The regulatory mechanisms of the ICE family remain to be fully clarified (Chinnaiyan and Dixit, 1997). In general, a caspase exhibits full enzymatic activity only after proteolytic processing, which occurs either by self-cleavage or through cleavage by other proteases (FernandesAlnemri et al., 1994; Tewari and Dixit, 1995; Nicholson, 1996). The central role of the caspase proteases in mammalian apoptosis is supported by the existence of specific inhibitors encoded by viruses that need to keep their host cell alive. The viral proteins p35 and CrmA (Tewari et al., 1995; Xue and Horvitz, 1995), or chemical compounds such as aldehyde and fluoromethylketone derivatives of the target cleavage sequences suppress mammalian apoptosis triggered by a variety of stimuli (Miura et al., 1993; Beidler et al., 1995; Nicholson et al., 1995). If caspases play a crucial role in apoptosis, specific target proteins or death substrates become critically relevant for the execution-phase of the apoptotic program. Once the cell has determined that it has sustained irreversible DNA damage, it chooses not to waste energy attempting to repair the DNA damage. Thus, specific cleavage by caspases of the DNA repair enzymes poly(ADP-ribose) polymerase (PARP) and the catalytic subunit of the DNA-dependent protein kinase (DNA-PK), inactivates the DNA repair pathway (Lazebnik et al., 1994; Song et al., 1996). Cleavage of the nuclear lamins is dependent on Mch2 (caspase-6) and seems to be essential for disassembling the nuclear structure (Lazebnik et al., 1995; Takahashi et al., 1996). Other substrates, whose function is still unclear during apoptosis, are the 70-kD protein of the U1 small nuclear ribonucleoprotein (Casciola-Rosen et al., 1994), the sterol regulatory element-binding proteins SREBP-1 and SRBP-2 (Wang et al., 1996), D4 GDP dissociation inhibitor (Na et al., 1996), and pRb tumor suppressor (Janicke et al., 1996). Recently, several cytoskeletal proteins have been found to be the target of caspase(s). Two interesting substrates for caspases are protein kinase C and growth arrest-specific gene-2 (Gas2), the last being a component of the microfilament system (Brancolini et al., 1992). In both cases, proteolytic processing results in a gain of function which relates to increased kinase activity in the case of PKC (Emoto et al., 1995), or to an activity on the microfilament system and on cell morphology in the case of Gas2 (Brancolini et al., 1995). Recently, gain of function after caspase-3 processing has also been demonstrated for the p21-activated kinase 2 (Rudel and Bokoch, 1997), and the DNA fragmentation factor (Liu et al., 1997). Thus, major structural change in the nucleus and cleavage of DNA by cuts between nucleosomes follow as a consequence of caspase activation.

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Cytoskeletal Reorganization During Apoptosis Cytoskeletal reorganization is also an essential process for the execution of apoptosis. Anchorage of cells to the extracellular matrix through integrins (Frisch and Francis, 1994; Re et al., 1994) as well as cadherin-mediated homeotypic cell-cell interactions (Hermiston and Gordon, 1995), are thought to play crucial roles in cell survival. These adhesive interactions involve a variety of transmembrane receptors that are linked via junctional plaque~ proteins to the cytoskeleton. Cytoskeletal organization, rather than engagement or clustering of cell adhesion molecules, appears to be the critical determinant of life or death for endothelial cells (Chen et al., 1997). During apoptosis, a number of molecules involved in cytoskeletal regulation and signaling, such as ct-fodrin, gelsolin, growth arrest-specific gene 2 (Gas 2), extracellular-regulated kinase kinase-1 (MEKK-1), protein kinase C (PKC), and p21-activated kinase (PAK 2), have been shown to be substrates for proteolytic cleavage by caspases (Martin et al., 1995; Brancolini et al., 1995; Cryns et al., 1996; Alnemri, 1997; Kothakota et al., 1997; Nicholson and Thornberry, 1997; Rudel and Bokoch, 1997). Cells undergo apoptosis in response to removal of growth factors and exhibit classical biochemical and morphologic changes associated with apoptosis (Hase et al., 1994). The first morphologic changes observed after growth factor removal are cell retraction and membrane blebbing, with subsequent loss of cell-cell and cell-matrix contacts, resulting in cell detachment. Cytoskeletal disruption occurs with eventual loss of cell-cell contacts during apoptosis. An important element involved in endothelial cell-cell adhesion is the adherens junction, in which an endothelial-specific cadherin, vascular endothelial (VE)-cadherin, anchors the cytoskeleton of neighboring cells via the catenins, f~-Catenin and plakoglobin (T-catenin) are two related molecules that connect the intracellular domain of VE-cadherin to ct-catenin, a vinculin homolog that binds actin (Lampugnani et al., 1995). Similar changes in cell-cell and cell-matrix interaction structures are seen in other cell-types undergoing apoptosis. As a response to the absence of survival factors, cells apparently sever contacts with neighboring cells, retract from the adhesion substrate, and dismantle and lose stress fibers, extensively reorganizing their actin cytoskeleton (e.g. accumulation of actin in the perinuclear region). The actin cytoskeleton reorganizes into a ring-like structure near the nucleus. Nuclear fragments are external to the actin ring, suggesting that contraction of actin filaments present in the perinuclear region might be important in the sprouting of the nuclear bodies. At these stages the actin ring appeared to be constricted, with the nuclear fragments tending to drop away from the actin filaments. Such a phenotype could represent a final stage of the actin filament changes taking place during apoptosis. Modulation of Cell-Cell Contacts During Apoptosis Cell-cell contacts have been intensively studied in epithelial cells, where specialized domains of the plasma membrane and the adherens junctions play a critical role in cellular adhesiveness by providing a link between cell surface adhesion molecules and the cytoskeleton (Geiger et al., 1995). Such an adhesive role is dependent on members

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of the cadherin superfamily of adhesive receptors and the cytoplasmic adaptor proteins ct-, B-, and ?- catenin/plakoglobin, which connect the cadherins to the actin filaments (Clark and Brugge, 1995; Gumbiner, 1996; Huber et al., 1996b). Adherens junctions are found in many cell-types, including cardiac myocytes and fibroblasts, but they have been extensively studied in epithelial cells. Adherens junctions link cells together by organizing actin filaments to the plasma membrane. The transmembrane receptors are the cadherins, a family of homophilic Ca2+-dependent cell-cell adhesion molecules (Takeichi, 1995). Their crystal structures suggest that cadherin-mediated cell adhesion is not based solely on the stability of association among individual molecules, but rather by the generation of a cell adhesion zipper which provides a mechanism to form strong intercellular bonds (Shapiro et al., 1995). In vivo strong intercellular bonds are dependent upon the association of the cadherin carboxy-terminal cytoplasmic domain to the central region of B-catenin and ¥-catenin/ plakoglobin (Hulsken et al., 1994; Nagafuchi and Takeichi, 1988; Ozawa et al., 1989; Sacco et al., 1995). Deletion of the B-catenin-binding site from the cadherin cytoplasmic domain renders the cadherin nonfunctional in a cell aggregation assay (Nagafuchi and Takeichi, 1988; Ozawa et al., 1989). The central function of B-catenin as a regulator of adherens junctions has been further demonstrated in different organisms using different experimental approaches (Oyama et al., 1994; Peifer et al., 1993). B-catenin serves as an adapter to link cadherins to ct-catenin and thereby to the actin cytoskeleton, since et-catenin seems to interact with both actin and et-actinin (Knudsen et al., 1995; Rimm et al., 1995). In this context, B-catenin seems to be the critical component for cadherin-catenin complexes in the regulation of cellular adhesiveness (Hamaguchi et al., 1993). The fundamental roles of ct-catenin in cadherin cytoskeleton interactions have also been clearly established in various cell-types. Nonadhesive epithelial tumor cells lacking ct-catenin can be induced to form tightly adherent epithelia after reintroduction of ct-catenin (Watabe et al., 1994). Moreover, expression of E-cadherin-ct-catenin chimeric proteins has been reported to induce a strong and inflexible adhesive phenotype (Nagafuchi et al., 1994). A possible role for catenins in tumor suppression is suggested by studies showing decreased catenin levels in certain tumors without a significant change in E-cadherin expression (Navarro et al., 1993; Pierceall et al., 1995; Sommers et al., 1994; Vermeulen et al., 1995). In addition, restoration of a ct-catenin levels in prostate cancer cells resulted in the induction of E-cadherin function, cell-cell adhesion and suppression of tumorigenesis in nude mice (Ewing et al., 1995). In a gastric carcinoma cell line, a deletion in B-catenin that abolishes its binding to ct-catenin was shown to diminish E-cadherin function (Kawanishi et al., 1995). During apoptosis, there is disruption of the adherens junction complex, including metalloproteinase-mediated shedding of cadherins and caspase-mediated cleavage of B-catenin, plakoglobin, and focal adhesion kinase (FAK) (Levkau et al., 1998). Cleavage of FAK precedes cell detachment and coincides with loss of FAK and paxillin from focal adhesion sites and their redistribution into characteristic membrane blebs of dying cells. FAK cleavage prevents association of FAK with normal downstream targets, such as paxillin, and may actively interrupt survival signals and propagate the

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cell death program. A recent screen of cDNA pools for caspase substrates identified gelsolin as a substrate readily cleaved by CPP32 (Kothakota et al., 1997). Expression of the gelsolin cleavage product in multiple cell-types caused the cells to round up, detach from the plate, and undergo nuclear fragmentation. Further, neutrophils isolated from mice lacking gelsolin have delayed onset of apoptosis. Prolonged disruption of the adherens junction during apoptosis may be critical to the apoptotic process by providing a cell geometry permissive for subsequent irreversible apoptotic events. Thus, the rapid dissolution of components of the adherens junctions in apoptotic cells may be important for the activation of apoptotic signaling pathways. Recent studies of mechanisms of neuronal cell death have revealed novel roles for actin filaments and gelsolin in modulation of neuronal apoptosis and necrosis. Pretreatment of cultured embryonic rat hippocampal neurons with the actin-disrupting agent, cytochalasin D, results in resistance of the neurons to cell death induced by glutamate. Glutamate kills neurons by activating membrane receptors linked to membrane depolarization and calcium influx. Two types of ionotropic glutamate receptors are recognized, NMDA (N-methyl-D-aspartate) receptors have a high conductance for calcium and are activated upon glutamate-binding when the membrane is depolarized. Non-NMDA receptors flux sodium and are critical in membrane depolarization induced by glutamate. Whole cell patch-clamp analyses of currents through NMDA and non-NMDA receptors have shown that cytochalasin D enhances the rundown of NMDA currents. Calcium imaging studies demonstrate that cytochalasin D suppresses elevations in intracellular calcium levels induced by glutamate. This suppression of the calcium influx is linked to protection of neurons against excitotoxicity (Furukawa et al., 1995). Conversely, stabilization of actin filaments with the compound jasplakinolide enhances calcium influx and enhances cell death induced by glutamate. Cytochalasin D has also been shown to protect cultured hippocampal neurons against apoptosis induced by amyloid B-peptide (Furukawa and Mattson, 1995a). Gelsolin appears to play an important role in causing actin-depolymerization and reducing calcium influx. Thus, hippocampal neurons from gelsolin knock-out mice exhibit increased vulnerability to cell death induced by glutamate. Analyses of NMDA currents in neurons lacking gelsolin demonstrate a marked attenuation of current rundown and enhanced calcium influx following exposure to glutamate (Furukawa et al., 1997). Thus, the roles of gelsolin in modulating apoptosis appear to be complex, and may depend upon the cell-type and its particular array of cell death machinery components. For example, neurons, which express glutamate receptors, are vulnerable to excitotoxicity and, in these cells, activation of glutamate receptors plays a major role in both apoptotic and necrotic cell death, in many different settings. In contrast, most types of mitotic cells, including lymphocytes, do not express ionotropic glutamate receptors and therefore the signaling pathway may be less important in those cells. A particularly intriguing concept relevant to roles of the cytoskeleton in apoptosis has emerged from recent studies of apoptotic biochemical cascades activated in synapses, which are located a great distance from the cell body in neurons (Mattson et al., 1998a,b). The latter studies showed that, in synaptosome preparations that lack cell bodies, apoptotic stimuli such as staurosporine, or exposure to oxidative insults, could induce caspase activation, loss of plasma membrane phospholipid asymmetry,

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mitochondrial membrane depolarization, increased mitochondrial oxidative stress and calcium levels, and release of factors from mitochondria capable of inducing nuclear chromatin condensation and fragmentation. These studies have also shown that caspase activation in synaptic compartments can lead to mitochondrial dysfunction. Additional studies in cultured embryonic hippocampal neurons that are synaptically connected provide compelling evidence that apoptotic cascades can be engaged locally in synaptic compartments (Mattson et al., 1998a). While the role of the cytoskeleton in these processes in synapses has not been critically examined, it is of considerable interest that the cytoskeleton is highly organized in these synapses and therefore is likely to contribute to modulating apoptotic signaling. Interestingly, a recent study (Duan et al., 1999) has shown that Par-4 (prostate apoptosis response 4) can be induced at the translational level in synapses. Par-4 induction in intact cells is a very early event but appears to precede and be required for subsequent caspase activation and mitochondrial dysfunction (Guo et al., 1998). Thus, it appears that cells can compartmentalize apoptotic signaling cascades in a highly organized manner which undoubtedly is influenced greatly by cytoskeletal organization and cytoskeletal rearrangements associated with cell signaling pathways. Cleavage of B-Catenin by Caspases During Apoptosis B-Catenin is a ~92-kD protein component of cell-cell contacts and adherens junctions having both structural and signaling functions (Miller and Moon, 1996). Both B-catenin and plakoglobin are cleaved in human endothelial cells undergoing apoptosis after growth factor removal, and the cleavages appear to be partially mediated by caspases. In contrast to B-catenin and plakoglobin, ct-catenin remains intact, despite the presence of multiple potential caspase cleavage sites. B-Catenin cleavage during apoptosis was also observed in the presence of the proteosome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal-H, an inhibitor of the chymotryptic site on the proteosome (Pagano et al., 1995), thus suggesting that B-catenin cleavage is not mediated by the ubiquitin/proteosome system. Kinetics studies in vitro and functional studies in vivo indicate that B-catenin is cleaved at different sites, thus trimming both the amino- and carboxy-terminal regions of the protein (Brancolini et al., 1997). Primarily, two fragments of B-catenin are detected in apoptotic cells, whereas four fragments of plakoglobin are observed. The processing of B-catenin is suppressed by increased expression of Bcl-2 (Brancolini et al., 1997). The caspase family of proteases contains a QACXG pentapeptide in which the cysteine participates in catalysis and is characterized by the absolute requirement for an aspartic acid residue in the substrate P1 position (Thornberry and Lazebnik, 1998). In addition to the requirement for a P1 Asp, caspase-3 shows preference for an anionic Asp residue in the P4 residue (DXXD) (Nicholson, 1996). A DXXD consensus site is located in the carboxy-terminal part of B-catenin (aa 760-764: DLMDG), thus possibly explaining the rapid cleavage of the carboxy-terminal site of the protein as observed in vitro after incubation with caspase-3. This sequence is similar to the DQMDG site in p35, a baculovirus antiapoptotic protein that is efficiently cleaved by caspase-3 (Xue and Horvitz, 1995). It has been shown that within the amino-terminal domain

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of B-catenin, different aspartic acid residues are targets for caspase-3 cleavage. More specifically, aspartic acid residues at position 162 and/or 164 and 144 and/or 145 of B-catenin serve as caspase-3 targets within the amino-terminal region. Caspase-3 null mice (Kuida et al., 1996) demonstrate that caspase-3 plays a critical role during cell death in the mammalian brain but raises the possibility that other caspase proteases may have an important role during apoptosis in other tissues or in response to cell death-triggers. Caspase-3-dependent proteolytic cleavage of B-catenin could be prevented by addition of the specific caspase inhibitor. G-catenin could also be a target for other caspases; further studies are required to understand if other caspases are involved in B-catenin processing, and to identify the different aspartic acid residues cleaved during apoptosis. It has been demonstrated in vivo that proteolytic processing of the B-catenin at its amino- terminal domain impairs its ability to bind c~-catenin. Amino acid residues 120-151 of G-catenin, embedded in the amino-terminal domain and containing the first armadillo repeat, are required for interaction with c~-catenin (Aberle et al., 1994). This is confirmed in the human gastric cancer cell line HSC-39 where a truncated B-catenin originated from a homozygous in-frame deletion that removed aa 28-134, cannot interact with c~-catenin (Kawanishi et al., 1995; Oyama et al., 1994). Removal of the B-catenin domain responsible for interaction with c~-catenin should disassemble the connections among the adhesion receptors, cadherins, and the actin cytoskeleton, thereby impairing a sustained cell-cell adhesive interaction (Brancolini et al., 1997). In fact, B-catenin-related family member, ¥-catenin/plakoglobin, also plays a similar role in regulating cadherin adhesive activity in adherens junctions and in the specialized desmosomal junctions. The aspartic acid residues that are possible target sites for caspase activity in B-catenin are conserved among B-catenin, T-catenin, and armadillo (the Drosophila form of B-catenin). Indeed, T-catenin/plakoglobin is also proteolytically processed both in vitro after incubation with caspase-3, and in vivo during apoptosis in fibroblast and epithelial cells (Brancolini et al., 1997). It is interesting to note that E-cadherin complexes may promote cell survival and suppress cell death in epithelial cells (Hermiston and Gordon, 1995). In this context, cleavage of 13-catenin might also suppress transduction of survival signals, consequently contributing to the irreversibility of the apoptotic process. The evidence that some components of the actin filament system are substrates for the caspases (Brancolini et al., 1995; Martin et al., 1996; Na et al., 1996) suggests that these proteases coordinate actin architectural changes during apoptosis and that such changes are important for normal apoptotic progression (Cotter et al., 1992). Cleavage of Lamin B During Apoptosis The nuclear lamins are karyophilic proteins located at the nucleoplasmic surface of the inner nuclear membrane where they assemble in a polymeric structure referred to as the nuclear lamina (McKeon, 1991; Nigg, 1992). Lamins belong to the family of intermediate filaments. The lamina has been suggested to serve as a major chromatin anchoring site of nuclear scaffold-associated regions during interphase and to be

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involved in organizing higher order chromatin domains. The lamina is a dynamic structure regulated by phosphorylation. Phosphorylation by p34cdc2 kinase is key to the dissolution of the nuclear lamina during mitosis. Other lamin kinases include mitogen-associated protein kinases, c-AMP-dependent protein kinase (PKA) (Gerace and Burke, 1988) and protein kinase C (PKC) (Gerace and Burke, 1988; McKeon, 1991). Major PKC phosphorylation sites have been mapped to serine residues located in close proximity to the nuclear localization signal in the C-terminal region, and phosphorylation of these residues interferes with the nuclear transport of lamin B (Nigg, 1992). The p34 ~c2 phosphorylation sites are on both sides of the central-helical rod domain. Many mammalian cells contain three distinct lamins (lamins A, B, and C). Lamin proteolysis during apoptosis has been reported in various cell lines treated with different stimuli. Overexpression of mutant lamins A or B resistant to caspase cleavage delayed DNA fragmentation, suggesting that lamin cleavage participates in the activation of DNA fragmentation and nuclear apoptosis (Rao et al., 1996). Cleavage Of Integrins During Apoptosis Integrins are a family of cell adhesion receptors, each of which is a heterodimer complex of two transmembrane subunits, cx and B (Hynes, 1992), and are involved in both cell to cell and cell to matrix interactions. At least 15 different ct chains associate with 8 types of B chains to form a large number of heterodimeric integrins that can be classified into several major subfamilies based on their shared use of a particular B chain (Hynes, 1992). Members of three such subfamilies, the B1, B2, and B7 integrins, are commonly found on leukocytes. The expression of B1 integrins is widespread (for example, ~5B1, CD49e/CD29, is found on T cells, granulocytes, platelets, fibroblasts, endothelium, and epithelium), whereas the B2 and B7 integrins have a restricted pattern of expression. The major ligands of the integrins fall into two categories: cell surface molecules that are members of the immunoglobulin superfamily (such as vascular cell adhesion molecule [VCAM]-I, intracellular adhesion molecule [ICAM]-I, 2, 3, and mucosal addressin cell adhesion molecule [MadCAM]-I) and a variety of large extracellular proteins (such as fibronectin, vitronectin, fibrinogen, and complement component iC3b; (Hynes, 1992). A common feature of both groups is the presence of exposed acidic amino acids crucial for integrin binding (Bergelson and Hemler, 1995). Integrins bind to extracellular matrix (ECM) adhesion proteins such as vitronectin, fibronectin, collagen, and laminin, and regulate many cellular functions, including proliferation, differentiation, migration, and apoptosis (Brooks et al., 1994b; Clark and Brugge, 1995; Frisch and Francis, 1994; Meredith et al., 1993; Re et al., 1994). Ligand binding to integrins transduces signals across the plasma membrane, resulting in activation of protein kinases, influx of calcium, elevation of intracellular pH, and hydrolysis of membrane phospholipids and reorganization of the cytoskeleton (Schwartz et al., 1995). The interactions between integrin and various cytoplasmic components of the focal contact are shown in Figure 2. Integrin-mediated adhesion to extracellular matrix proteins is required for growth and survival of many cell-types (Meredith et al., 1993; Brooks et al., 1994b; Frisch and Francis, 1994; Re et al., 1994).

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Adhesion to ECM is required for progression of cells through the cell cycle by regulating cyclin DI, cyclin E-Cdk2, and Rb protein activities (Fang et al., 1996). Disruption of adhesion arrests cells in the G1 phase and causes apoptosis (Meredith et al., 1993; Howlett and Bissell, 1993; Frisch and Francis, 1994; Re et al., 1994; Ingber et al., 1995). The requirement of cell-ECM adhesive interactions for cell cycle progression and cell survival is likely to be important in tissue development and involution as a mechanism to regulate cell positioning and cell number (Lin and Bissell, 1993). In addition, anchorage dependence of survival may serve to limit tumor progression by preventing invasion or metastasis of tumor cells (Varner and Cheresh, 1996). Integrin-regulated survival properties have also been shown to be relevant in wound repair since integrin antagonists induced apoptosis of migrating endothelial cells, thereby blocking angiogenesis (Brooks et al., 1994a,b; Friedlander et al., 1996). Integrins interact with cytoskeletal proteins at focal adhesion sites and are critical for cytoskeletal organization and morphological characteristics of anchorage-dependent ceils (Ruoslahti and Reed, 1994).

Integrin

a-actinin

TalinTensl~n)a//Ai il in

a!in-~Z~l~: ~ I

I

Vinculin

I

I

[

i

i

I

I

Csk

c-src

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[

Proliferation, differentiation, migration and apoptosis ]

Figure 2. Interactions between integrin and intracelluar proteins. Model shows a number of different

signal transduction pathwaysproposedto result from integrin binding to ligand and subsequentactivation of kinases / cytoplasmic proteins. Arrows indicate direct interactions among various cytoplasmic components of integrin mediatedsignaling.

The mechanism by which integrin-mediated ECM adhesion is able to prevent apoptosis is not well understood. Focal adhesion kinase (FAK) is phosphorylated in response to cell adhesion, and constitutively activated membrane-targeted FAK was able to rescue cells from suspension-induced cell death (Frisch et al., 1996; Schlaepfer and Hunter, 1996). Engagement of integrins by the ECM causes organization of a

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complex structure, termed focal adhesion, on the cytoplasmic side of the plasma membrane (Schwartz et al., 1995). The tyrosine kinase FAK appears to play a central role in integrin signal transduction. FAK activation has been linked to cytoskeletal organization, regulation of small GTP-binding proteins like Rho and Ras, and interaction with intracellular proteins like c-Src and c-Fyn, Grb2/Sos, and PI-3 kinase (Scatena et al., 1998). FAK and its downstream targets are important relays in integrin-mediated signaling (Schaller and Parsons, 1994). Integrin engagement positively regulates expression of the antiapoptotic gene Bcl-2 in COS and endothelial cells (Zhang et al., 1995; Stromblad et al., 1996). In addition, ctvB3 engagement and clustering in endothelial cells, but not Bl or ctB 5 ligation, conferred an antiapoptotic phentoype to endothelial cells (Stromblad et al., 1996). Interestingly, the inhibition of angiogenesis by anti-ctvB3 antibody correlates with angiogenic endothelial cell apoptosis (Brooks et al., 1994b). Recently, it has been demonstrated that NF-KB mediates integrin-induced endothelial cell survival (Scatena et al., 1998). Various ECM molecules, including osteopontin, promote endothelial cell survival upon serum deprivation. Moreover, adhesion of endothelial cells to the ctB 3 ligand osteopontin increases nuclear NF-KB activity. Furthermore, 133integrin mediates osteopontin's protective effect and NF-KB activation. In addition, osteopontin- and vitronectin-mediated cell protective effects are abolished by inhibiting NF-•B nuclear translocation. Thus, NF-~B is an important signaling molecule in ctB 3 integrin-mediated endothelial cell survival. The cytoplasmic domain of the integrin 133 subunit is cleaved during endothelial cell apoptosis and calpain is likely to be the protease involved (Meredith et al., 1998). Calpain-mediated proteolysis of fodrin was also detected, indicating that calpain is activated during endothelial cell apoptosis. A phosphatase inhibitor, sodium orthovanadate, inhibited endothelial cell apoptosis and cleavage of the B3 subunit, suggesting that protein dephosphorylation preceded integrin cleavage in the apoptosis signaling pathway. B3 cleavage was observed in cells that were viable, suggesting that it is an early event and not the consequence of post-death proteolysis. The extent of B3 cleavage correlated with a loss in the capacity of cells to reattach to matrix proteins. Loss of reattachment capacity during apoptosis was significantly retarded by a calpain inhibitor. Since the integrin B-subunit cytoplasmic domain plays an important role in integrin-mediated cell adhesion and signaling (Reszka et al., 1992; Ylanne et al., 1993; LaFlamme et al., 1994; Akiyama et al., 1994; Ylanne et al., 1995; Chen et al., 1996; Hughes et al., 1996; Knezevic et al., 1996), its proteolysis may block integrin-mediated signals and disrupt cytoskeleton organization during apoptosis.

Activation of Protein Kinases Due to Microtubule Damage

At least two classes of drugs used to treat cancer attack the microtubules. The vinca alkaloids, vincristine, vinblastine, and vinorelban, bind tightly to free tubulin molecules and prevent polymerization of tubulin subunits into microtubules. Thus, mitotic spindle cannot form and cells cannot accurately divide the sister chromatids between the daughter cells. The taxanes, paclitaxel and docetaxel, bind tightly to microtubules and

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prevent their disassembly. Thus, mitotic spindle can form and bind to sister chromatids, but they cannot pull the chromosomes apart into the two daughter cells. Therefore, although these two classes of drugs have precisely the opposite mechanisms of action, their consequences for the cell are very similar. In each instance, mitosis is unable to proceed to completion because of microtubule dysfunction. Studies of human tumors that express Bcl-2, an anti-apoptotic member of the Bcl-2 gene family, have revealed that interfering with microtubule function elicits a damage response pathway within the cell just as DNA damage elicits a cellular response. And like DNA damage, microtubule damage is associated with the activation of signal transduction pathways that can lead to apoptosis. The first hint of such a microtubule damage response pathway was the detection of a phosphorylated form of Bcl-2 in tumor cells exposed to paclitaxel. The paclitaxel-induced phosphorylation of Bcl-2 implied that a kinase had been activated and that phosphorylated Bcl-2 could be functionally altered by phosphorylation. This section highlights the involvement of protein kinases in apoptosis in response to microtubule damage (Figure 3). Role of cAMP-dependent Protein Kinase (PKA) The actions of cAMP are well known in the regulation of various cellular functions including cell proliferation, differentiation and gene induction, through the activation of cAMP-dependent protein kinase (PKA) (Krebs and Beavo, 1979). We have demonstrated that downregulation of PKA type I and upregulation of PKA type II by cAMP analog 8-CI-cAMP, and Rlct antisense induce apoptosis in several cancer cell lines (Cho-Chung et al., 1997; Srivastava et al., 1998a,e). Recently, synergistic inhibition of growth and induction of apoptosis by 8-CI-cAMP and paclitaxel, cisplatin or retinoic acid in several human cancer cells has been demonstrated (Srivastava et al., 1998c; Tortora et al., 1998). Furthermore, intracytoplasmic microinjection of purified PKA catalytic subunit commits the cells to death (Vintermyr et al., 1993). In support of this hypothesis, PKA type II has been found to be associated with mammalian centromeres (Toumier et al., 1991) and upon microtubule disruption by paclitaxel, vincristine or vinblastine, activated PKA can phosphorylate Bcl-2 leading to apoptosis (Srivastava et al., 1998d). This suggests that activation of PKA due to microtubule damage is an important event in Bcl-2 phosphorylation and induction of apoptosis. We have demonstrated that paclitaxel or vincristine induced increased expression of Bax, while overexpression of Bcl-2 counteracted the effects of low doses of these drugs (Srivastava et al., 1998d). Phosphorylated Bcl-2 bound to Bax poorly and the majority of Bax was not associated with Bcl-2 in paclitaxel-treated cells (Srivastava et al., 1998d). In addition, paclitaxel and vincristine led to PKA-induced Bcl-2 phosphorylation and apoptosis which were blocked by the PKA inhibitor Rp-cAMP. This suggests that activation of PKA due to microtubule damage is an important event in Bcl-2 phosphorylation and induction of apoptosis. These microtubule-damaging drugs caused growth arrest in G2-M phase of the cell cycle, and had no effect on p53 induction, suggesting that phosphorylation mediated inactivation of Bcl-2 and apoptosis without the involvement of p53 (Srivastava et al., 1998d). By comparison, the DNA-damaging drugs methotrexate and doxorubicin had no effect on Bcl2 phosphorylation but induced

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Microtubule-damagingdrugs (Paclitaxel, docetaxel, vincdstine, vinblastine, colchicine)

p34 cdc2

JNKK/SEK1

.L

JNK/SAPK / ~ transcri)tionfactors (c-Jun, NFAT,ATF-4, Krox-24,NF~B,etc.). Bcl-2

Regulation of Apoptosis

StressResponses

Figure 3. Signal transduction pathways activated by microtubule-damaging drugs. Intracellular stress caused by microtubule-damagingdrugs activates Ras, ASK1, PKA and P34cdc2 signaling cascades. Activated kinase(s) may regulate apoptosis through phosphorylationof Bcl-2. Transcription factors may be induced/activated to mediate cellular responses to stress.

p53 expression. These data suggest that there may be a signaling cascade induced by agents that disrupt or damage the cytoskeleton that is distinct from (i.e., p53 independent), but perhaps related to (i.e., involves kinase activation and leads to apoptosis), the cellular response to DNA damage. Studies of mechanisms of cell death in neurons further suggest a role for microtubules in modulating the cell death process. Thus, treatment of cultured hippocampal neurons with the microtubule-stablizing agent paclitaxel protects the neurons against excitotoxicity and apoptosis (Furukawa and Mattson, 1995b). Conversely, the microtubuledisrupting agent, colchicine, promotes neuronal cell death. Colchicine destabilizes calcium homeostasis in neurons whereas paclitaxel stabilizes homeostasis. In addition,

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oxidative modification of microtubules and microtubule-associated proteins is suggested to play a role in neuronal cell death associated with several neurodegenerative disorders, including Alzheimer's disease. For example, 4-hydroxynonenal, an aldehyde product of lipid peroxidation can modify the microtubule-associated protein tau in a manner similar to that which occurs in degenerating neurons in Alzheimer's disease (Mattson et al., 1997a). 4-hydroxynonenal covalently modifies proteins on cysteine, lysine, and histidine residues, and can promote aggregation of many different proteins including cytoskeletal proteins. Thus, cell death cascades lead to modification of the cytoskeleton, on the one hand, and changes in the cytoskeleton can influence the outcome (i.e. life or death), on the other hand. Role of Mitogen-Activated Protein Kinases (MAP Kinases) Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine protein kinases that include: (i) the extracellular signal-regulated kinases or ERK subfamily represented by p42MAPK/ERK2 and p44MAPK/ERK1 (Cowley et al., 1994), (ii) the c-Jun NH2-terminal kinases/stress-activated protein kinases or JNK/SAPK subfamily, represented by the p46 JNK/SAPK and p54 JNK/SAPK isoforms and their variants (Davis, 1994; Gupta et al., 1995; Kyriakis et al., 1994), and (iii) the p38MAPK subfamily (Raingeaud et al., 1995). MAPKs transduce signals from the cell membrane to the nucleus in response to a variety of different stimuli and participate in various intracellular signaling pathways that control a wide spectrum of cellular processes including cell growth, differentiation, and stress responses (Cowley et al., 1994; Davis, t994). In contrast to ERK1 and ERK2, which are activated by mitogenic stimuli, JNK/SAPK and p38 are activated by inflammatory cytokines and cellular stresses such as osmotic and heat shock, UV- and y-irradiation, protein synthesis inhibitors, metabolic poisons, lipopolysaccharide (LPS), or proinflammatory cytokines, growth factor deprivation and surface immunoglobulin crosslinking in human B lymphocytes (Derijard et al., 1994; Kyriakis et al., 1994; Liu et al., 1995; Raingeaud et al., 1995; Rosette and Karin, 1996; Sluss et al., 1994; Verheij et al., 1996). In general, ERKs act as survival signals whereas JNKs participate in cell death signaling (Xia et al., 1995). In fact, the stimulation of JNK was a prerequisite for cell death under various conditions, and a blockade of JNK activation resulted in the prevention of cell death (Verheij et al., 1996; Xia et al., 1995). MAPKs can be dephosphorylated and inactivated by dual-specificity phosphatases (Verheij et al., 1996; Xia et al., 1995). These findings imply that JNK may function in an intracellular signaling pathway leading to cell death. Once activated, JNK/SAPK phosphorylates several transcription factors including c-Jun (Kallunki et al., 1994; Kyriakis et al., 1994), ATF-2 (Gupta et al., 1995), and ELK-1 (Cavigelli et al., 1995), thereby regulating gene expression. We and others have shown that microtubule-damaging agents cause growth arrest at the G2/M phase of the cell cycle and induce apoptosis (Tishler et al., 1995; Vogt et al., 1996; Bonfoco et al., 1996; Huschtscha et al., 1996; Kawamura et al., 1996; Wahl et al., 1996; Srivastava et al., 1998d). It has been proposed, however, that G2/M arrest may not be sufficient to induce apoptosis and that additional phosphoregulatory pathways may be required (Tishler et at., 1995; Haldar et al., 1996; Blagosklonny et al.,

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1997; Srivastava et al., 1998d). On the other hand, evidence is also accumulating to indicate that JNK/SAPK activation may regulate the cell cycle (Robinson and Cobb, 1997) and apoptosis (Xia et al., 1995; Cuvillier et al., 1996; Karin et al., 1997; Seimiya et at., 1997; Wang et al., 1998). We have demonstrated that JNK is activated in response to microtubule damage by paclitaxel, and JNK activation leads to inactivation of anti-apoptotic functions of Bcl-2 through phosphorylation (Srivastava et al., 1998b). Deletion of the loop region of Bcl-2 completely abrogated paclitaxel-induced Bcl-2 phosphorylation, cytochrome c release from the mitochondria, caspase-3 activation, PARP cleavage and apoptosis (Srivastava et al., 1998b). However, the serine7°alanine mutation was 1/3rd as active as wild type Bcl-2 (Srivastava et al., 1998b). These data suggest that activation of JNK/SAPK pathway plays a central role in transducing signals arising from microtubule damage. Apoptosis signal-regulating kinase (ASK1) is a recently characterized MAPK kinase kinase (Ichijo et al., 1997). Overexpression of ASK1 induces apoptosis in mink lung epithelial cells, and ASK1 is activated in cells treated with tumor necrosis factor-a, suggesting a role of ASK1 in stress- and cytokine-induced apoptosis (Ichijo et al., 1997). Microtubule damaging drugs activate JNK/SAPK through signal transduction by both Ras and ASK1, indicating that multiple signal transduction pathways may be required for this type of cellular stress response (Wang et al., 1998). Hence the involvement of Ras, ASK1, and JNK/SAPK in signal transduction pathways may be critical for microtubule damage and subsequent apoptosis. The mechanism(s) by which microtubular disarray activates both Ras and ASK1 remains to be elucidated. Role of p34 c°c2 Kinase The p34 cat2 kinase is universally required as the master control enzyme during mitosis in eukaryotes (Nurse, 1990). Its activity controls the G2-M-phase transition by promoting breakdown of the nuclear membrane, chromatin condensation, and microtubule spindle formation. This kinase is activated by Thr-161 phosphorylation and its association with other proteins, primarly cyclin B. During G2-phase of the cell cycle, Cdc25 phosphatase binds to and activates cyclin B/Cdc2 complex by dephosphorylating Cdc2 at phosphotyrosine 15 and phosphothreonine 14 residues, thus allowing catalysis of ATP within an ATP binding pocket (Nigg, 1993; Solomon, 1993). Exit from mitosis requires the inactivation of p34 ode2 by degradation of the cyclin B-regulatory subunit (Gallant and Nigg, 1992) and dephosphorylation of p34 ~d~2at Thr-161 (Lorca et al., 1992). Recently, paclitaxel induced p34 ¢dc2 kinase activity in human breast carcinoma MCF-7 cells was shown (Shen et al., 1998). Olomoucine, a potent p34 ~ac2 inhibitor, effectively prevented paclitaxel-induced p34 ¢dc2 kinase activation and subsequent apoptosis (Shen et al., 1998). Thus p34 ~de2 kinase plays a crucial role in paclitaxel induced apoptosis. However, the precise mechanism of its activation remains to be defined and the substrates it modifies to initiate apoptosis are not yet known.

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Induction of Genes by Paclitaxel Pharmacological features of paclitaxel may originate in part from its effects on gene expression and not simply from its effects on microtubule assembly. In addition to induction of cytokine genes (Bogdan and Ding, 1992; Manthey et al., 1992; Burkhart et al., 1994; Kirikae et al., 1996; Lee et al., 1997), paclitaxel also induces early-response genes encoding transcription factors with tumor suppressor effects (krox-24) and enzymes that govern proliferation, apoptosis, and inflammation [2'5'-oligoadenylate synthase, cyclooxygenase-2 (COX-2), and an IKB kinase termed CHUK (Moos and Fitzpatrick, 1998a,b)]. Changes in gene expression appear relevant to the pharmacological and toxicological properties of the individual taxanes. Docetaxel, a potent analog of paclitaxel, did not induce any of these genes, implying that paclitaxel modulates gene expression by a mechanism that is distinct from microtubule stabilization and cell cycle arrest. COX-2 catalyzes the formation of prostaglandins, a family of biologically active lipid mediators (Fletcher et al., 1992). Induction of cytokines and COX-2 appears relevant to the pharmacology of paclitaxel because these gene products can modulate inflammation and apoptosis. By stimulating formation of cytoprotective prostaglandins, COX-2 could either blunt apoptosis (Tsujii and DuBois, 1995) or aggravate immediate hypersensitivity reactions associated with paclitaxel administration (Rowinsky et al., 1993; Rowinsky, 1997). ATF-4 and Krox-24 are transcription factors induced by paclitaxel (Gashler and Sukhatme, 1995). 2'-5'-Oligoadenylate synthetase catalyzes formation of purine oligomers that activate the nucleases necessary for the antiviral and cytopathic actions of interferons (Slattery et al., 1979). CHUK is a conserved helix-loop-helix ubiquitous kinase (Connelly and Marcu, 1995), recently identified as an inhibitor of KB (I~:B) kinase. CHUK phosphorylates IKB, releasing NF~:B from its cytosolic, inactive I~:B:NFKB complex (DiDonato et al., 1997; Regnier et al., 1997). Krox-24, a zinc-finger transcription factor (also known as EGR-1, zif268, or NGF1A), is especially notable because its expression is deficient in several tumor cells and this defect correlates with tumor formation (Huang et al., 1995; 1997). Enhanced expression of krox-24 can prevent oncogenic transformation (Huang et al., 1994). Thus, induction of krox-24 might augment the antineoplastic effects of paclitaxel in some cases (Von Hoff, 1997). It is interesting to note that prostate cancer deviates from this pattern. Increased expression of EGR-1 in prostate cancer promotes the growth and invasiveness of this cancer (Thigpen et al., 1996). Compared with other types of cancer, prostate cancer is not responsive to paclitaxel (Rowinsky, 1997; Von Hoff, 1997). The relative cytotoxic potency of the microtubule-stabilizing agents against various cell lines or tumors is complex and difficult to predict because it depends on at least three effects: (i) their potency as microtubule-stabilizing agents, (ii) their potency as cell-cycle inhibitors, and (iii) the net effects of gene induction on cell survival and apoptosis. A comprehensive understanding of the scope of gene induction may facilitate optimal clinical application of the taxanes. Paclitaxel can influence transcriptional pathways in several ways: (a) it can act indirectly, via induction of proteins like CHUK that govern a transcriptional pathway

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without changing the levels of the transcription factor (e.g. NFKB); (b) it can act directly, by increasing the expression of transcription factors (e.g. ATF-4 and Krox-24); and (c) it can induce genes that activate latent nucleases in cells (e.g. 2'-5'-oligoadenylate synthetase). ATF-4 and 2'-5'-oligoadenylate synthetase often are part of a general "stress-response" mechanism of cells. The induction of 2'-5'-oligoadenylate synthetase is particularly striking because of its established role in mediating the pleiotropic effects of interferon, including its antineoplastic effects. Activation of nucleases is accompanied by induction of apoptosis; however, the precise nucleases and how they are regulated have not yet been defined.

Conclusions

Progress in our understanding of cell biology has identified the critical role the cytoskeleton plays in a host of normal functions including growth, differentiation, and more recently noted, cell death. The process of neoplastic transformation often targets cytoskeletal control mechanisms (for example, loss of cadherins, loss of contact inhibition) to overcome natural growth controls. Apoptosis also involves breakdown of adherens junctions, integrins, cadherins, catenins, and redistribution of actin filaments to facilitate the extrusion of apoptotic bodies from the nucleus. Anticancer drugs that target microtubules appear to promote apoptosis. The inability of the cell to progress through mitosis due to drug-induced microtubule dysfunction activates a newly described stress response pathway involving, at least in some instances, protein kinase A, c-jun kinase, and p34 cdc2kinase and the initiation of the apoptosis cascade (Figure 3). An understanding of the dynamic changes in the cytoskeleton under distinct physiologic and pathologic conditions may give rise to new insights about disease treatment and prevention. The complex reorganization of cell morphology during apoptosis seems to be achieved by specific proteolytic processing of different cytoskeletal proteins. Our developing knowledge of the importance of actin and actin-associated proteins in cancer could well lead to a similar expansion of interest in the therapeutic potential of compounds that target the actin cytoskeleton. Finally, an important emerging issue in studies of apoptosis concerns the extent to which mechanisms are similar and different in mitotic and postmitotic cells and whether therapeutic approaches that benefit, for example, cancer, might be a detriment to postmitotic cells such as neurons. As described previously, for example, cytoskeletonaffecting agents such as paclitaxel and cytochalasin D that may suppress growth or promote death of cancer cells may actually provide benefits to neurons. Another example comes from studies of the transcription factor NF-KB and its role in apoptosis. It was shown by Barger and coworkers (1995) that activation of NF-rB can prevent neuronal apoptosis. It was subsequently shown that activation of NF-KB also prevents apoptosis of cancer cells (Beg and Baltimore, 1996). In neurons and tumor cells, activation of NF-nB leads to induction of expression of manganese SOD, an antioxidant enzyme (Mattson et al., 1997b). Manganese SOD suppresses oxyradical production in mitochondria and stabilizes mitochondrial function (Mattson et al., 1997b;

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K e l l e r et al., 1998). T h u s , c o n s i d e r i n g t h e r a p e u t i c a p p r o a c h e s , o n e w o u l d w a n t to b l o c k a c t i v a t i o n o f N F - K B to p r o m o t e t u m o r cell death, b u t o n e w o u l d w a n t to a c t i v a t e NF-IcB to p r e v e n t n e u r o n a l cell d e a t h in n e u r o d e g e n e r a t i v e disorders. T h e r e f o r e , an a g e n t t h a t i n h i b i t s NF-IcB m i g h t b e b e n e f i c i a l t o w a r d s a b r a i n t u m o r b u t m i g h t , at t h e s a m e time, p r o m o t e n e u r o n a l d e g e n e r a t i o n . It will t h e r e f o r e b e i m p o r t a n t to d e v e l o p cell-type-specific therapeutic approaches based on apoptotic signaling mechanisms.

References Aberle, H., Butz, S., Stappert, J., Weissig, H., Kemler, R. & Hoschuetzky, H. (1994). Assembly of the cadherin-catenin complex in vitro with recombinant proteins. J. Cell Sci. 107, 3655-3663. Akiyama, S.K., Yamada, S.S., Yamada, K.M. & LaFlamme, S.E. (1994). Transmembrane signal transduction by integrin cytoplasmic domains expressed in single-subunit chimeras. J. Biol. Chem. 269, 15961-15864. Alnemri, E.S. (1997). Mammalian cell death proteases: a family of highly conserved aspartate specific cysteine proteases. J. Cell. Biochem. 64, 33-42. Barger, S.W., Horster, D., Furukawa, K., Goodman, Y., Krieglstein, J. & Mattson, M.P. (1995). Tumor necrosis factors ct and 13 protect neurons against amyloid 13-peptide toxicity: Evidence for involvement of a KB-binding factor and attenuation of peroxide and Ca 2+ accumulation. Proc. Natl. Acad. Sci. USA 92, 9328-9332. Beg, A.A. & Baltimore, D. (1996). An essential role for NF-IcB in preventing TNF-alpha-induced cell death. Science 274, 782-784. Beidler, D.R., Tewari, M., Friesen, P.D., Poirier, G. & Dixit, V.M. (1995). The baculovirus p35 protein inhibits Fas- and tumor necrosis factor- induced apoptosis. J. Biol. Chem. 270, 16526-16528. Bergelson, J.M. & Hemler, M.E. (1995). Integrin-ligand binding. Do integrins use a 'MIDAS touch' to grasp an Asp? Curr. Biol. 5, 615-617. Blagosklonny, M.V., Giannakakou, P., EI-Deiry, W.S., Kingston, D.G., Higgs, P.I., Neckers, L. & Fojo, T. (1997). Raf-1/bcl-2 phosphorylation: a step from microtubule damage to cell death. Cancer Res. 57, 130-135. Bogdan, C. & Ding, A. (1992). Taxol, a microtubule-stabilizing antineoplastic agent, induces expression of tumor necrosis factor a and interleukin-1 in macrophages. J. Leukoc. Biol. 52, 119-121. Bonfoco, E., Leist, M., Zhivotovsky, B., Orrenius, S., Lipton, S.A. & Nicotera, P. (1996). Cytoskeletal breakdown and apoptosis elicited by NO donors in cerebellar granule cells require NMDA receptor activation. J. Neurochem. 67, 2484-2493. Brancolini, C., Benedetti, M. & Schneider, C. (1995). Microfilament reorganization during apoptosis: the role of Gas2, a possible substrate for ICE-like proteases. Embo J. 14, 5179-5190. Brancolini, C., Bottega, S. & Schneider, C. (1992). Gas2, a growth arrest-specific protein, is a component of the microfilament network system. J. Cell Biol. 117, 1251-1261. Brancolini, C., Lazarevic, D., Rodriguez, J. & Schneider, C. (1997). Dismantling cell-cell contacts during apoptosis is coupled to a caspase- dependent proteolytic cleavage of beta-catenin. J. Cell. Biol. 139, 759-771. Brooks, P.C., Clark, R.A. & Cheresh, D.A. (1994a). Requirement of vascular integrin av133for angiogenesis. Science 264, 569-571. Brooks, P.C., Montgomery, A.M., Rosenfeld, M., Reisfeld, R.A., Hu, T., Klier, G. & Cheresh, D.A. (1994b). Integrin Cry133antagonists promote tumor regression by inducing apoptosis of angiogenic

258

R.K. Srivastava M.P. Mattson and D.L. Longo

blood vessels. Cell 79, 1157-1164. Burkhart, C.A., Berman, J.W., Swindell, C.S. & Horwitz, S.B. (1994). Relationship between the structure of taxol and other taxanes on induction of tumor necrosis factor-ct gene expression and cytotoxicity. Cancer Res. 54, 5779-5782. Caplow, M. & Shanks, J. (1996). Evidence that a single monolayer tubulin-GTP cap is both necessary and sufficient to stabilize microtubules. Mol. Biol. Cell. 7, 663-675. Casciola-Rosen, L.A., Miller, D.K., Anhalt, G.J. & Rosen, A. (1994). Specific cleavage of the 70-kDa protein component of the U1 small nuclear ribonucleoprotein is a characteristic biochemical feature of apoptotic cell death. J. Biol. Chem. 269, 30757-30760. Cavigelli, M., Dolfi, F., Claret, F.X. & Karin, M. (1995). Induction of c-fos expression through JNK-mediated TCF/EIk-1 phosphorylation. Embo J. 14, 5957-5964. Chen, C.S., Mrksich, M., Huang, S., Whitesides, G.M. & lngber, D.E. (1997). Geometric control of cell life and death. Science 276, 1425-1428. Chen, G., Branton, P.E., Yang, E., Korsmeyer, S.J. & Shore, G.C. (1996). Adenovirus E1B 19-kDa death suppressor protein interacts with Bax but not with Bad. J. Biol. Chem. 271, 24221-24225. Chinnaiyan, A.M. & Dixit, V.M. (1997). Portrait of an executioner: the molecular mechanism of FAS/APO-1induced apoptosis. Semin. Immunol. 9, 69-76. Cho-Chung, Y.S., Nesterova, M., Kondrashin, A., Noguchi, K., Srivastava, R. & Pepe, S. (1997). Antisense-protein kinase A: a single-gene-based therapeutic approach. Antisense Nucleic Acid Drug Dev. 7, 217-223. Clark, E.A. & Brugge, J.S. (1995). lntegrins and signal transduction pathways: the road taken. Science 268,233-239. Connelly, M.A. & Marcu, K.B. (1995). CHUK, a new member of the helix-loop-helix and leucine zipper families of interacting proteins, contains a serine-threonine kinase catalytic domain. Cell Mol. Biol. Res. 41,537-549. Cotter, T.G., Lennon, S.V., Glynn, J.M. & Green, D.R. (1992). Microfilament-disrupting agents prevent the formation of apoptotic bodies in tumor cells undergoing apoptosis [published erratum appears in Cancer Res 1992 Jun 15;52(12):3512]. Cancer Res. 52, 997-1005. Cowley, S., Paterson, H., Kemp, P, & Marshall, C.J. (1994). Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell 77, 841-852. Craig, S.W. & Johnson, R.P. (1996). Assembly of focal adhesions: progress, paradigms, and portents. Curr. Opin. Cell Biol. 8, 74-85. Cryns, V.L., Bergeron, L., Zhu, H., Li, H. & Yuan, J. (1996). Specific cleavage of alpha-fodrin during Fasand tumor necrosis factor- induced apoptosis is mediated by an interleukin-lB-converting enzyme/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease. J. Biol. Chem. 271, 31277-31282. Cuvillier, O., Pirianov, G., Kleuser, B., Vanek, P.G., Coso, O.A., Gutkind, S. & Spiegel, S. (1996). Suppression of ceramide-mediated programmed cell death by sphingosine-1- phosphate. Nature 381, 800-803. Davis, R.J. (1994). MAPKs: new JNK expands the group. Trends Biochem. Sci. 19, 470-3. Derijard, B., Hibi, M., Wu, I.H., Barrett, T., Su, B., Deng, T., Karin, M. & Davis, R.J. (1994). JNKI: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76, 1025-1037. Desai, A. & Mitchison, T.J. (1997). Microtubule polymerization dynamics. Annu. Rev. Cell. Dev. Biol. 13, 83-117. DiDonato, J.A., Hayakawa, M., Rothwarf, D.M., Zandi, E. & Karin, M. (1997). A cytokine-responsive I•B kinase that activates the transcription factor NF-KB. Nature 388,548-554.

Apoptosis Regulation by Cytoskeleton

259

Duan, W., Rangnekar, V. & Mattson, M.P. (1999). Par-4 production in synaptic compartments following apoptotic and excitotoxic insults: Evidence for a pivotal role in mitochondrial dysfunction and neuronal degeneration. J. Neurochem. In press. Emoto, Y., Manome, Y., Meinhardt, G., Kisaki, H., Kharbanda, S., Robertson, M., Ghayur, T., Wong, W.W., Kamen, R., Weichselbaum, R. & et al. (1995). Proteolytic activation of protein kinase C6 by an ICE-like protease in apoptotic cells. Embo J. 14, 6148-6156. Ewing, C.M., Ru, N., Morton, R.A., Robinson, J.C., Wheelock, M.J., Johnson, K.R., Barrett, J.C. & Isaacs, W.B. (1995). Chromosome 5 suppresses tumorigenicity of PC3 prostate cancer cells: correlation with re-expression of ct-catenin and restoration of E-cadherin function. Cancer Res. 55, 4813-4817. Fang, F., Orend, G., Watanabe, N., Hunter, T. & Ruoslahti, E. (1996). Dependence of cyclin E-CDK2 kinase activity on cell anchorage. Science 271,499-502. Fernandes-Alnemri, T., Litwack, G. & Alnemri, E.S. (1994). CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-lB-converting enzyme. J. Biol. Chem. 269, 30761-30764. Fletcher, B.S., Kujubu, D.A., Perrin, D.M. & Herschman, H.R. (1992). Structure of the mitogen-inducible TIS10 gene and demonstration that the TIS10-encoded protein is a functional prostaglandin G/H synthase. J. Biol. Chem. 267, 4338-4344. Friedlander, M., Theesfeld, C.L., Sugita, M., Fruttiger, M., Thomas, M.A., Chang, S. & Cheresh, D.A. (1996). Involvement of integrins O~vl~3 and ctvl35 in ocular neovascular diseases. Proc. Natl. Acad. Sci. USA 93, 9764-9769. Frisch, S.M. & Francis, H. (1994). Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell. Biol. 124, 619-626. Frisch, S.M., Vuori, K., Ruoslahti, E. & Chan-Hui, P. Y. (1996). Control of adhesion-dependent cell survival by focal adhesion kinase. J. Cell. Biol. 134, 793-799. Furukawa, K. & Mattson, M.P. (1995a). Cytochalasins protect hippocampal neurons against amyloid 13-peptide toxicity: evidence that actin depolymerization suppresses Ca 2÷influx. J. Neurochem. 65, 1061-1068. Furukawa, K. & Mattson, M.P. (1995b). Taxol stabilizes [Ca2+]i. and protects hippocampal neurons against excitotoxicity. Brain Res. 689, 141-146. Furukawa, K., Fu, W., Witke, W., Kwiatkowski, D.J. & Mattson, M.P. (1997). The actin-severing protein gelsolin modulates calcium channel activity and vulnerability to excitotoxicity in hippocampal neurons. J. Neurosci. 17, 8178-8186. Furukawa, K., Smith-Swintosky, V.L. & Mattson, M.P. (1995). Evidence that actin depolymerization protects hippocampal neurons against excitotoxicity by stabilizing [Ca2+]~Exp. Neurol. 133, 153-163. Gallant, P. & Nigg, E.A. (1992). Cyclin B2 undergoes cell cycle-dependent nuclear tmnslocation and, when expressed as a non-destructible mutant, causes mitotic arrest in HeLa cells. J. Cell Biol. 117,213-224. Gashler, A. & Sukhatme, V.P. (1995). Early growth response protein 1 (Egr-1): prototype of a zinc-finger family of transcription factors. Prog. Nucleic Acid Res. Mol. Biol. 50, 191-224. Geiger, B., Yehuda-Levenberg, S. & Bershadsky, A.D. (1995). Molecular interactions in the submembrane plaque of cell-cell and cell-matrix adhesions. Acta. Anat. 154, 46-62. Gerace, L. & Burke, B. (1988). Functional organization of the nuclear envelope. Annu. Rev. Cell Biol. 4, 335-374. Gumbiner, B.M. (1996). Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 84, 345-357. Guo, Q., Fu, W., Xie, J., Luo, H., Sells, S.F., Geddes, J.W., Bondada, V. Rangnekar, V. & Mattson, M.P. (1998). Par-4 is a novel mediator of neuronal degeneration associated with the pathogenesis of Alzheimer's

260

R.K. Srivastava M.P. Mattson and D.L. Longo

Gupta, S., Campbell, D., Derijard, B. & Davis, R. J. (1995). Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science 267, 389-393. Haldar, S.Chintapalli, J. & Croce, C. M. (1996). Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res. 56,1253-1255. Hamaguchi, M., Matsuyoshi, N., Ohnishi, Y., Gotoh, B., Takeichi, M. & Nagai, Y. (1993). p60v-src causes tyrosine phosphorylation and inactivation of the N-cadherin-catenin cell adhesion system. Embo J. 12, 307-314. Hase, M., Araki, S., Kaji, K. & Hayashi, H. (1994). Classification of signals for blocking apoptosis in vascular endothelial cells. J. Biochem. (Tokyo) 116, 905-909. Hermiston, M.L. & Gordon, J.I. (1995). In vivo analysis of cadherin function in the mouse intestinal epithelium: essential roles in adhesion, maintenance of differentiation, and regulation of programmed cell death. J. Cell Biol. 129, 489-506. Horio, T. & Hotani, H. (1986). Visualization of the dynamic instability of individual microtubules by dark-field microscopy. Nature 321,605-607. Howlett, A.R. & Bissell, M.J. (1993). The influence of tissue microenvironment (stroma and extraceflular matrix) on the development and function of mammary epithelium. Epithelial Cell Biol. 2, 79-89. Huang, R.P., Darland, T., Okamura, D., Mercola, D. & Adamson, E.D. (1994). Suppression of v-sis-dependent transformation by the transcription factor, Egr-1. Oncogene 9, 1367-1377. Huang, R.P., Fan, Y., de Belle, I., Niemeyer, C., Gottardis, M.M., Mercola, D. & Adamson, E.D. (1997). Decreased Egr-1 expression in human, mouse and rat mammary cells and tissues correlates with tumor formation. Int. J. Cancer 72, 102-109. Huang, R.P., Liu, C., Fan, Y., Mercola, D. & Adamson, E.D. (1995). Egr-1 negatively regulates human tumor cell growth via the DNA-binding domain. Cancer Res 55, 5054-5062. Huber, O., Bierkamp, C. & Kemler, R. (1996a). Cadherins and catenins in development. Curt. Opin. Cell Biol. 8,685-691. Huber, O., Korn, R., McLaughlin, J., Ohsugi, M., Herrmann, B.G. & Kemler, R. (1996b). Nuclear localization of beta-catenin by interaction with transcription factor LEF-1. Mech. Dev. 59, 3-10. Hughes, P.E., Diaz-Gonzalez, F., Leong, L., Wu, C., McDonald, J.A., Shattil, S.J. & Ginsberg, M.H. (1996). Breaking the integrin hinge. A defined structural constraint regulates integrin signaling. J. Biol. Chem. 271, 6571-6574. Hulsken, J., Behrens, J. & Birchmeier, W. (1994). Tumor-suppressor gene products in cell contacts: the cadherin-APC-armadillo connection. Curr. Opin. Cell Biol. 6, 711-716. Huschtscha, L.I., Bartier, W.A., Ross, C.E. & Tattersall, M.H. (1996). Characteristics of cancer cell death after exposure to cytotoxic drugs in vitro. Br. J. Cancer. 73, 54-60. Hynes, R.O. (1992). Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25. Ichijo, H., Nishida, E., Irie, K., ten Dijke, P., Saitoh, M., Moriguchi, T., Takagi, M., Matsumoto, K., Miyazono, K. & Gotoh, Y. (1997). Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275, 90-94. Ingber, D.E., Prusty, D., Sun, Z., Betensky, H. & Wang, N. (1995). Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis. J. Biomech. 28, 1471-1484. Janicke, R.U., Walker, P.A., Lin, X.Y. & Porter, A.G. (1996). Specific cleavage of the retinoblastoma protein by an ICE-like protease in apoptosis. Embo J. 15, 6969-6978. Joshi, H.C. (1994). Microtubule organizing centers and gamma-tubulin. Curr. Opin. Cell Biol. 6, 54-62. Kallunki, T., Su, B., Tsigelny, I., Sluss, H.K., Derijard, B., Moore, G., Davis, R. & Karin, M. (1994). JNK2 contains a specificity-determining region responsible for efficient c-Jun binding and phosphorylation.

Apoptosis Regulation by Cytoskeleton

261

Genes Dev. 8, 2996-3007. Karin, M., Liu, Z. & Zandi, E. (1997). AP-1 function and regulation. Curr. Opin. Cell Biol. 9, 240-246. Kawamum, K.I., Grabowski, D., Weizer, K., Bukowski, R. & Ganapathi, R. (1996). Modulation of vinblastine cytotoxicity by dilantin (phenytoin) or the protein phosphatase inhibitor okadaic acid involves the potentiation of anti-mitotic effects and induction of apoptosis in human tumour cells. Br. J. Cancer 73, 183-188. Kawanishi, J., Kato, J., Sasaki, K., Fujii, S., Watanabe, N. & Niitsu, Y. (1995). Loss of E-cadherin-dependent cell-cell adhesion due to mutation of the beta-catenin gene in a human cancer cell line, HSC-39. Mol. Cell. Biol. 15, 1175-1181. Keller, J,N, Kindy, M.S. Holtsberg, F.W. St. Clair, D.K.,Yen, H.C., Germeyer, A., Steiner, S.M., BruceKeller, A.J., Hutchins, J.B.,& Mattson, M.P. (1998). Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction. J. Neurosci. 18(2):687-697. Kerr, J.F., Wyllie, A.H. & Currie, A.R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239-257. Kirikae, T., Ojima, I., Kirikae, F., Ma, Z., Kuduk, S.D., Slater, J.C., Takeuchi, C.S., Bounaud, P.Y. & Nakano, M. (1996). Structural requirements of taxoids for nitric oxide and tumor necrosis factor production by murine macrophages. Biochem. Biophys. Res. Commun. 227,227-235. Knezevic, I., Leisner, T.M. & Lam, S.C.T. (1996). Direct binding of the platelet integrin t~133 (GPIII3-IIIa) to talin. Evidence that interaction is mediated through the cytoplasmic domains of both cd3 and 133. J. Biol. Chem. 271, 16416-16421. Knudsen, K.A., Soler, A.P., Johnson, K.R. & Wheelock, M.J. (1995). Interaction of ct-actinin with the cadherin/catenin cell-cell adhesion complex via ct-catenin. J. Cell Biol. 130, 67-77. Kothakota, S., Azuma, T., Reinhard, C., Klippel, A., Tang, J., Chu, K., McGarry, T. J., Kirschner, M.W., Koths, K., Kwiatkowski, D.J. & Williams, L.T. (1997). Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science 278, 294-298. Krebs, E.G. & Beavo, J.A. (1979). Phosphorylation-dephosphorylation of enzymes. Annu. Rev. Biochem. 48, 923-959. Kuida, K., Zheng, T.S., Na, S., Kuan, C., Yang, D., Karasuyama, H., Rakic, P. & Flavell, R.A. (1996). Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 384, 368-372. Kyriakis, J.M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E.A., Ahmad, M.F., Avruch, J. & Woodgett, J.R. (1994). The stress-activated protein kinase subfamily of c-Jan kinases. Nature 369, 156-160. LaFlamme, S.E., Thomas, L.A., Yamada, S.S. & Yamada, K.M. (1994). Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly. J. Cell Biol. 126, 1287-1298. Lampugnani, M.G., Corada, M., Caveda, L., Breviario, F., Ayalon, O., Geiger, B. & Dejana, E. (1995). The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin, 13-catenin, and ct-catenin with vascular endothelial cadherin (VE-cadherin). J. Cell Biol. 129, 203-217. Lazebnik, Y.A., Kaufmann, S. H., Desnoyers, S., Poirier, G.G. & Earnshaw, W.C. (1994). Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 371,346-347. Lazebnik, Y.A., Takahashi, A., Poirier, G.G., Kaufmann, S.H. & Earnshaw, W.C. (1995). Characterization of the execution-phase of apoptosis in vitro using extracts from condemned-phase cells. J. Cell Sci. Suppl. 19, 41-49. Lee, L.F., Haskill, J.S., Mukaida, N., Matsushima, K. & Ting, J.P. (1997). Identification of tumor-specific paclitaxel (Taxol)-responsive regulatory elements in the interleukin-8 promoter. Mol. Cell. Biol. 17, 5097-5105.

262

R.K. Srivastava M,P. Mattson and D.L. Longo

Levkau, B., Herren, B., Koyama, H., Ross, R. & Raines, E.W. (1998). Caspase-mediated cleavage of focal adhesion kinase pp125FAK and disassembly of focal adhesions in human endothelial cell apoptosis. J. Exp. Med. 187, 579-586. Lin, C.Q. & Bissell, M.J. (1993). Multi-faceted regulation of cell differentiation by extracellular matrix. FASEB J. 7,737-743. Liu, X., Zou, H., Slaughter, C. & Wang, X. (1997). DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89, 175-184. Liu, Y., Gorospe, M., Yang, C. & Holbrook, N.J. (1995). Role of mitogen-activated protein kinase phosphatase during the cellular response to genotoxic stress. Inhibition of c-Jun N-terminal kinase activity and AP-l-dependent gene activation. J. Biol. Chem. 270, 8377-8380. Lorca, T., Labbe, J.C., Devault, A., Fesquet, D., Capony, J.P., Cavadore, J.C., Le Bouffant, F. & Doree, M. (1992). Dephosphorylation of cdc2 on threonine 161 is required for cdc2 kinase inactivation and normal anaphase. Embo J. 11, 2381-2390. Manthey, C.L., Bmndes, M.E., Perera, P.Y. & Vogel, S.N. (1992). Taxol increases steady-state levels of lipopolysaccharide-inducible genes and protein-tyrosine phosphorylation in murine macrophages. J. Immunol. 149, 2459-2465. Martin, S.J., Amarante-Mendes, G.P., Shi, L., Chuang, T.H., Casiano, C.A., O'Brien, G.A., Fitzgerald, P., Tan, E.M., Bokoch, G.M., Greenberg, A.H. & Green, D.R. (1996). The cytotoxic cell protease granzyme B initiates apoptosis in a cell- free system by proteolytic processing and activation of the ICE/CED-3 family protease, CPP32, via a novel two-step mechanism. Embo J. 15, 2407-2416. Martin, S.J. & Green, D.R. (1995). Protease activation during apoptosis: death by a thousand cuts? Cell 82, 349-352. Martin, S.J., O'Brien, G.A., Nishioka, W.K., McGahon, A. J., Mahboubi, A., Saido, T,C. & Green, D.R. (1995). Proteolysis of fodrin (non-erythroid spectrin) during apoptosis. J. Biol. Chem. 270, 6425-6428. Mattson, M.P., Fu, W., Waeg, G. & Uchida, K. (1997a). 4-hydroxynonenal, a product of lipid peroxidation, inhibits dephosphorylation of the microtubule-associate protein tau. NeuroReport 8, 2275-2281. Mattson, M.P., Goodman, Y., Luo, H., Fu, W. & Furukawa, K. (1997b). Activation of NF-KB protects hippocampal neurons against oxidative stress-induced apoptosis: evidence for induction of Mn-SOD and suppression of peroxynitrite production and protein tyrosine nitration. J. Neurosci. Res. 49, 681-697. Mattson, M.P., Keller, J.N. & Begley, J.G. (1998a). Evidence of synaptic apoptosis. Exp. Neurol. 153, 35-48. Mattson, M.P., Partin, J. & Begley, J.G. (1998b). Amyloid B-peptide induces apoptosis-related events in synapses and dendrites. Brain Res. 807, 167-176. McKeon, F. (1991). Nuclear lamin proteins: domains required for nuclear targeting, assembly, and cell-cycle-regulated dynamics. Curr. Opin. Cell Biol. 3, 82-86. Meredith, J., Jr., Mu, Z., Saido, T. & Du, X. (1998). Cleavage of the cytoplasmic domain of the integrin beta3 subunit during endothelial cell apoptosis. J. Biol. Chem. 273, 19525-19531. Meredith, J.E., Jr., Fazeli, B. & Schwartz, M.A. (1993). The extracellular matrix as a cell survival factor. Mol. Biol. Cell. 4, 953-961. Miller, J.R. & Moon, R.T. (1996). Signal transduction through beta-catenin and specification of cell fate during embryogenesis. Genes Dev. 10, 2527-2539. Mitchison, T. & Kirschner, M. (1984). Dynamic instability of microtubule growth. Nature 312, 237-242. Miura, M., Zhu, H., Rotello, R., Hartwieg, E.A. & Yuan, J. (1993). Induction of apoptosis in fibroblasts

Apoptosis Regulation by Cytoskeleton

263

by IL-IB-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell 75,653-660. Moos, P. J. & Fitzpatrick, F.A. (1998a). Taxanes propagate apoptosis via two cell populations with distinctive cytological and molecular traits. Cell Growth Differ. 9, 687-697. Moos, P.J. & Fitzpatrick, F.A. (1998b). Taxane-mediated gene induction is independent of microtubule stabilization: induction of transcription regulators and enzymes that modulate inflammation and apoptosis. Proc. Natl. Acad. Sci. U S A 95, 3896-3901. Na, S., Chuang, T.H., Cunningham, A., Turi, T.G., Hanke, J.H., Bokoch, G.M. & Danley, D.E. (1996). D4-GDI, a substrate of CPP32, is proteolyzed during Fas-induced apoptosis. J. Biol. Chem. 271, 11209-11213. Nagafuchi, A., Ishihara, S. & Tsukita, S. (1994). The roles of catenins in the cadherin-mediated cell adhesion: functional analysis of E-cadherin-ct catenin fusion molecules. J. Cell. Biol. 127,235-245. Nagafuchi, A. & Takeichi, M. (1988). Cell binding function of E-cadherin is regulated by the cytoplasmic domain. Embo J. 7, 3679-3684. Navarro, P., Lozano, E. & Cano, A. (1993). Expression of E- or P-cadherin is not sufficient to modify the morphology and the tumorigenic behavior of murine spindle carcinoma cells. Possible involvement of plakoglobin. J. Cell Sci. 105, 923-934. Nicholson, D.W. (1996). ICE/CED3-1ike proteases as therapeutic targets for the control of inappropriate apoptosis. Nat. Biotechnol. 14, 297-301. Nicholson, D.W., Ali, A., Thornberry, N.A., Vaillancourt, J.P., Ding, C.K., Gallant, M., Gareau, Y., Griffin, P.R., Labelle, M., Lazebnik, Y.A. & et al. (1995). Identification and inhibition of the 1CE/CED-3 protease necessary for mammalian apoptosis. Nature 376, 37-43. Nicholson, D.W. & Thornberry, N.A. (1997). Caspases: killer proteases. Trends Biochem. Sci. 22, 299-306. Nigg, E.A. (1992). Assembly-disassembly of the nuclear lamina. Curr. Opin. Cell Biol. 4, 105-109. Nigg, E.A. (1993). Targets of cyclin-dependent protein kinases. Curr. Opin. Cell Biol. 5, 187-193. Nurse, P. (1990). Universal control mechanism regulating onset of M-phase. Nature 344, 503-508. Oyama, T., Kanai, Y., Ochiai, A., Akimoto, S., Oda, T., Yanagihara, K., Nagafuchi, A., Tsukita, S., Shibamoto, S., lto, F. & et al. (1994). A truncated beta-catenin disrupts the interaction between E-cadherin and alpha-catenin: a cause of loss of intercellular adhesiveness in human cancer cell lines. Cancer Res. 54, 6282-6287. Ozawa, M., Baribault, H. & Kemler, R. (1989). The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. Embo J. 8, 1711-1717. Pagano, M., Tam, S.W., Theodoras, A.M., Beer-Romero, P., Del Sal, G., Chau, V., Yew, P.R., Draetta, G.F. & Rolfe, M. (1995). Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 269, 682-685. Peifer, M., Orsulic, S., Sweeton, D. & Wieschaus, E. (1993). A role for the Drosophila segment polarity gene armadillo in cell adhesion and cytoskeletal integrity during oogenesis. Development 118, 1191-1207. Pierceall, W.E., Woodard, A.S., Morrow, J.S., Rimm, D. & Fearon, E.R. (1995). Frequent alterations in E-cadherin and ct- and B-catenin expression in human breast cancer cell lines. Oncogene l l, 1319-1326. Raft, M.C. (1992). Social controls on cell survival and cell death. Nature 356, 397-400. Raingeaud, J., Gupta, S., Rogers, J.S., Dickens, M., Han, J., Ulevitch, R.J. & Davis, R.J. (1995).

264

R.K. Srivastava M.P. Mattson and D.L. Longo

Pro-inflammatory cytokines and environmental stress cause p38 mitogen- activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J. Biol. Chem. 270, 7420-7426. Rao, L., Perez, D. & White, E. (1996). Lamin proteolysis facilitates nuclear events during apoptosis. J. Cell Biol. 135, 1441-1455. Re, F., Zanetti, A., Sironi, M., Polentarutti, N., Lanfrancone, L., Dejana, E. & Colotta, F. (1994). Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells. J. Cell Biol. 127, 537-546. Regnier, C.H., Song, H. Y., Gao, X., Goeddel, D.V., Cao, Z. & Rothe, M. (1997). Identification and characterization of an IkappaB kinase. Cell 90, 373-383. Reszka, A.A., Hayashi, Y. & Horwitz, A.F. (1992). Identification of amino acid sequences in the integrin beta 1 cytoplasmic domain implicated in cytoskeletal association. J. Cell Biol. 117, 1321-1330. Rimm, D.L., Koslov, E.R., Kebriaei, P., Cianci, C.D. & Morrow, J.S. (1995). Alpha l(E)-catenin is an actin-binding and -bundling protein mediating the attachment of F-actin to the membrane adhesion complex. Proc. Natl. Acad. Sci. U S A 92, 8813-8817. Robinson, M.J. & Cobb, M.H. (1997). Mitogen-activated protein kinase pathways. Curr. Opin. Cell Biol. 9, 180-186. Rosette, C. & Karin, M. (1996). Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors. Science 274, 1194-1197. Rowinsky, E.K. (1997). The development and clinical utility of the taxane class of antimicrotubule chemotherapy agents. Annu. Rev. Med. 48, 353-374. Rowinsky, E.K., Eisenhauer, E.A., Chaudhry, V., Arbuck, S.G. & Donehower, R.C. (1993). Clinical toxicities encountered with paclitaxel (Taxol). Semin. Oncol. 20, 1-15. Rudel, T. & Bokoch, G.M. (1997). Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. Science 276, 1571 - 1574. Ruoslahti, E. & Reed, J.C. (1994). Anchorage dependence, integrins, and apoptosis. Cell 77,477-478. Sacco, P.A., McGranahan, T.M., Wheelock, M.J. & Johnson, K.R. (1995). Identification of plakoglobin domains required for association with N- cadherin and alpha-catenin. J. Biol. Chem. 270, 20201-20206. Scatena, M., Almeida, M., Chaisson, M.L., Fausto, N., Nicosia, R.F. & Giachelli, C.M. (1998). NF-KB mediates ctvB3 integrin-induced endothelial cell survival. J. Cell. Biol. 141, 1083-1093. Schaller, M.D. & Parsons, J.T. (1994). Focal adhesion kinase and associated proteins. Curr. Opin. Cell Biol. 6, 705-710. Schlaepfer, D.D. & Hunter, T. (1996). Signal transduction from the extracellular matrix--a role for the focal adhesion protein-tyrosine kinase FAK. Cell Struct. Funct. 21,445-450. Schwartz, M.A., Schaller, M.D. & Ginsberg, M.H. (1995). Integrins: emerging paradigms of signal transduction. Annu. Rev. Cell Dev. Biol. 11,549-599. Seimiya, H., Mashima, T., Toho, M. & Tsuruo, T. (1997). c-Jan NH2-terminal kinase-mediated activation of interleukin-lbeta converting enzyme/CED-3-1ike protease during anticancer drug-induced apoptosis. J. Biol. Chem. 272, 4631-4636. Shapiro, L., Fannon, A.M., Kwong, P.D., Thompson, A., Lehmann, M.S., Grubel, G., Legrand, J.F., Als-Nielsen, J., Colman, D.R. & Hendrickson, W.A. (1995). Structural basis of cell-cell adhesion by cadherins. Nature 374, 327-337. Shen, S.C., Huang, T.S., Jee, S.H. & Kuo, M.L. (1998). Taxol-induced p34cdc2 kinase activation and apoptosis inhibited by 12-O- tetradecanoylphorbol-13-acetate in human breast MCF-7 carcinoma cells. Cell Growth Differ. 9, 23-29.

Apoptosis Regulation by Cytoskeleton

265

Slattery, E., Ghosh, N., Samanta, H. & Lengyel, P. (1979). Interferon, double-stranded RNA, and RNA degradation: activation of an endonuclease by (2'-5')An. Proc. Natl. Acad. Sci. U S A 76, 4778-4782. Sluss, H.K., Barrett, T., Derijard, B. & Davis, R.J. (1994). Signal transduction by tumor necrosis factor mediated by JNK protein kinases. Mol. Cell. Biol. 14, 8376-8384. Solomon, M.J. (1993). Activation of the various cyclin/cdc2 protein kinases. Curr. Opin. Cell Biol. 5, 180-186. Sommers, C.L., Gelmann, E.P., Kemler, R., Cowin, P. & Byers, S.W. (1994). Alterations in B-catenin phosphorylation and plakoglobin expression in human breast cancer cells. Cancer Res. 54, 3544-3552. Song, Q., Lees-Miller, S.P., Kumar, S., Zhang, Z., Chan, D.W., Smith, G.C., Jackson, S.P., Alnemri, E.S., Litwack, G., Khanna, K.K. & Lavin, M.F. (1996). DNA-dependent protein kinase catalytic subunit: a target for an ICE- like protease in apoptosis. Embo J. 15, 3238-3246. Srivastava, R.K., Lee, Y.N., Noguchi, K., Park, Y.G., Ellis, M.J., Jeong, J.S., Kim, S.N. & Cho-Chung, Y.S. (1998a). The RIIB regulatory subunit of protein kinase A binds to cAMP response element: an alternative cAMP signaling pathway. Proc. Natl. Acad. Sci.U S A 95, 6687-6692. Srivastava, R.K., Qing-Sheng Mi, Q.-S., Hardwick, J.M. & Longo, D.L. (1999). Deletion of loop region of Bcl-2 completely blocks paclitaxel-induced apoptosis. Proc. Natl. Acad. Sci. USA 96, 3775-3780. Srivastava, R.K., Srivastava, A.R. & Cho-Chung, Y.S. (1998c). Synergistic effects of 8-chlorocyclic-AMP and retinoic acid on induction of apoptosis in Ewing's sarcoma CHP-100 cells. Clin. Cancer Res. 4, 755-761. Srivastava, R.K., Srivastava, A.R., Korsmeyer, S.J., Nesterova, M., Cho-Chung, Y.S. & Longo, D.L. (1998d). Involvement of microtubules in the regulation of Bcl-2 phosphorylation and apoptosis through cyclic AMP-dependent protein kinase. Mol. Cell Biol. 18, 3509-3517. Srivastava, R.K., Srivastava, A.R., Park, Y.G., Agrawal, S. & Cho-Chung, Y.S. (1998e). Antisense depletion of Rlct subunit of protein kinase A induces apoptosis and growth arrest in human breast cancer cells. Breast Cancer Res. Treat. 49, 97-107. Stromblad, S., Becker, J.C., Yebra, M., Brooks, P.C. & Cheresh, D.A. (1996). Suppression of p53 activity and p21WAF1/CIP1 expression by vascular cell integrin alphaVbeta3 during angiogenesis. J. Clin. Invest. 98,426-433. Svitkina, T.M., Verkhovsky, A.B. & Borisy, G.G. (1996). Plectin sidearms mediate interaction of intermediate filaments with microtubules and other components of the cytoskeleton. J. Cell Biol. 135, 991-1007. Takahashi, A., Alnemri, E.S., Lazebnik, Y.A., Femandes-Alnemri, T., Litwack, G., Moir, R.D., Goldman, R.D., Poirier, G.G., Kaufmann, S.H. & Earnshaw, W.C. (1996). Cleavage of lamin A by Mch2a but not CPP32: multiple interleukin 1g-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis. Proc. Natl. Acad. Sci. U S A 93, 8395-8400. Takeichi, M. (1995). Morphogenetic roles of classic cadherins. Curr. Opin. Cell Biol. 7, 619-27. Tewari, M. & Dixit, V. M. (1995). Fas- and tumor necrosis factor-induced apoptosis is inhibited by the poxvirus crmA gene product. J. Biol. Chem. 270, 3255-3260. Tewari, M., Telford, W.G., Miller, R.A. & Dixit, V.M. (1995). CrmA, a poxvirus-encoded serpin, inhibits cytotoxic T-lymphocyte- mediated apoptosis. J. Biol. Chem. 270, 22705-22708. Thigpen, A.E., Cala, K.M., Guileyardo, J.M., Molberg, K.H., McConnell, J.D. & Russell, D.W. (1996). Increased expression of early growth response-1 messenger ribonucleic acid in prostatic adenocarcinoma. J. Urol. 155,975-981. Thompson, C.B. (1995). Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456-1462.

266

R.K. Srivastava M.P. Mattson and D.L. Longo

Thornberry, N.A. & Lazebnik, Y. (1998). Caspases: enemies within. Science 281, 1312-1316. Tishler, R. B., Lamppu, D.M., Park, S. & Price, B.D. (1995). Microtubule-active drugs taxol, vinblastine, and nocodazole increase the levels of transcriptionally active p53. Cancer Res. 55, 6021-6025. Tortora, G., Caputo, R., Damiano, V., Bianco, R., Pepe, S., Pomatico, G., Bianco, A.R., Jiang, Z., Agrawal, S. & Ciardiello, F. (1998). Cooperative antitumor effect of mixed backbone oligonucleotides targeting protein kinase A in combination with cytotoxic drugs or biologic agents. Antisense Nucleic Acid Drug Dev. 8, 141-145. Tournier, S., Raynaud, F., Gerbaud, P., Lohmann, S. M., Doree, M. & Evain-Brion, D. (1991). Association of type II cAMP-dependent protein kinase with p34cdc2 protein kinase in human fibroblasts. J. Biol. Chem. 266, 19018-19022. Trevor, K.T., McGuire, J.G. & Leonova, E.V. (1995). Association of vimentin intermediate filaments with the centrosome. J. Cell Sci. 108, 343-356. Tsujii, M. & DuBois, R.N. (1995). Alterations in cellular adhesion and apoptosis in epithelial ceils overexpressing prostaglandin endoperoxide synthase 2. Cell 83,493-501. Varner, J.A. & Cheresh, D.A. (1996). Integrins and cancer. Curr. Opin. Cell Biol. 8,724-730. Verheij, M., Bose, R., Lin, X.H., Yao, B., Jarvis, W.D., Grant, S., Birrer, M.J., Szabo, E., Zon, L.I., Kyriakis, J.M., Haimovitz-Friedman, A., Fuks, Z. & Kolesnick, R.N. (1996). Requirement for ceramide-initiated SAPK/JNK signaling in stress- induced apoptosis. Nature 380, 75-79. Vermeulen, S.J., Bruyneel, E.A., Bracke, M.E., De Bruyne, G.K., Vennekens, K.M., Vleminckx, K L., Berx, G.J., van Roy, F.M. & Mareel, M.M. (1995). Transition from the noninvasive to the invasive phenotype and loss of alpha-catenin in human colon cancer cells. Cancer Res. 55, 4722-4728. Vintermyr, O.K., Gjertsen, B.T., Lanotte, M. & Doskeland, S. O. (1993). Microinjected catalytic subunit of cAMP-dependent protein kinase induces apoptosis in myeloid leukemia (IPC-81) ceils. Exp. Cell Res. 206, 157-161. Vogt, A., Qian, Y., McGuire, T.F., Hamilton, A.D. & Sebti, S.M. (1996). Protein geranylgeranylation, not farnesylation, is required for the G1 to S phase transition in mouse fibroblasts. Oncogene 13, 1991-1999. Von Hoff, D.D. (1997). The taxoids: same roots, different drugs. Semin. Oncol. 24, S13-3-S13-10. Wahl, A.F., Donaldson, K.L., Fairchild, C., Lee, F.Y., Foster, S.A., Demers, G.W. & Galloway, D.A. (1996). Loss of normal p53 function confers sensitization to Taxol by increasing G2/M arrest and apoptosis. Nat. Med. 2, 72-79. Wang, T.H., Wang, H.S., Ichijo, H., Giannakakou, P., Foster, J.S., Fojo, T. & Wimalasena, J. (1998). Microtubule-interfering agents activate c-Jun N-terminal kinase/stress- activated protein kinase through both Ras and apoptosis signal-regulating kinase pathways. J. Biol. Chem. 273, 4928-4936. Wang, X., Zelenski, N.G., Yang, J., Sakai, J., Brown, M.S. & Goldstein, J.L. (1996). Cleavage of sterol regulatory element binding proteins (SREBPs) by CPP32 during apoptosis. Embo J. 15, 1012-1020. Watabe, M., Nagafuchi, A., Tsukita, S. & Takeichi, M. (1994). Induction of polarized cell-cell association and retardation of growth by activation of the E-cadherin-catenin adhesion system in a dispersed carcinoma line. J. Cell Biol. 127,247-256. Wyllie, A.H. (1993). Apoptosis (the 1992 Frank Rose Memorial Lecture). Br J Cancer 67,205-208. Wyllie, A.H. (1995). The genetic regulation of apoptosis. Curr. Opin. Genet. Dev. 5, 97-104. Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J. & Greenberg, M.E. (1995). Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270, 1326-1331. Xue, D. & Horvitz, H.R. (1995). Inhibition of the Caenorhabditis elegans cell-death protease CED-3 by a CED-3 cleavage site in baculovirus p35 protein. Nature 377,248-251.

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Yamada, K.M. & Miyamoto, S. (1995). lntegrin transmembrane signaling and cytoskeletal control. Curr. Opin. Cell Biol. 7,681-689. Ylanne, J., Chert, Y., O'Toole, T.E., Loftus, J.C., Takada, Y. & Ginsberg, M.H. (1993). Distinct functions of integrin alpha and beta subunit cytoplasmic domains in cell spreading and formation of focal adhesions. J. Cell. Biol. 122, 223-233. Ylanne, J., Huuskonen, J., O'Toole, T.E., Ginsberg, M.H., Virtanen, I. & Gahmberg, C.G. (1995). Mutation of the cytoplasmic domain of the integrin 133 subunit. Differential effects on cell spreading, recruitment to adhesion plaques, endocytosis, and phagocytosis. J. Biol. Chem. 270, 9550-9557. Zhang, Z., Vuori, K., Reed, J.C. & Ruoslahti, E. (1995). The %13~ integrin supports survival of cells on fibronectin and upregulates Bcl-2 expression. Proc. Natl. Acad. Sci. U S A 92, 6161-6165.

ANTI-APOPTOTIC ROLE OF THE TRANSCRIPTION FACTOR NF-I~B

MARK P. MATTSON

Table of Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NF-•B Structure and Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . From Bad Guy to Hero . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms Underlying the Anti-Apoptotic Action of NF-•B . . . . . . . . . . . . . . . . . . . . . . . . Novel Insight Into the Roles of NF-nB in Apoptosis: Lessons from Neurons . . . . . . . . . . . . Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

269 270 272 276 284 286 289

Introduction

As can be appreciated from the other chapters in this volume, many different signaling pathways are activated in cells undergoing apoptosis. Considerable effort has been placed on linking such signaling pathways to the cell death process, and important findings have arisen from this approach. For example, activation of members of the caspase family of cysteine proteases is now recognized as an important necessary event in many different cell-types undergoing apoptosis (Cohen, 1997; Miller, 1997; Yuan, 1997). The signaling mechanisms of cytotoxic cytokines such as tumor necrosis factor (TNF) and Fas ligand are being rapidly revealed by elegant molecular approaches (see Schulze-Osthoff et al., 1998 for review). In general, the latter cell death effector pathways involve the activation and/or aggregation of various proteins with characteristic "death domains" that (directly or indirectly) activate downstream caspases which, in turn, appear to execute key mitochondrial and nuclear events that culminate in cell death. While such work has certainly been informative, it has also inhibited progress in identifying and understanding signaling mechanisms that prevent apoptosis. The reason is that leaders in the cell death field jumped to the (incorrect) conclusion that if a particular enzyme or transcription factor is activated in cells undergoing apoptosis, then its activation must contribute to the cell death process. This "guilt by association" reasoning has been shown to be flawed in many cases for the simple reason that many (perhaps most) signaling pathways activated in injured and dying ceils are designed to prevent cell death (Mattson and Furukawa, 1996). As we shall see in this chapter, the transcription factor NF-KB provides a classic example of the transformation of a "mediator of apoptosis" (implied, based on guilt-by-association) to an anti-apoptotic factor (clearly established, in cause -- effect studies).

269 Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. 2 6 9 - - 2 9 5 © 2 0 0 1 Elsevier Science. Printed in the Netherlands.

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M.P. Mattson

NF-KB Structure and Regulation NF-~:B (nuclear factor ~cB) was originally identified in B lymphocytes where it stimulates transcription of the immunoglobulin ~c light chain (Sen and Baltimore, 1986; Baeuerle and Henkel, 1994; Baeuerle and Baltimore, 1996). Since then, a variety of genes have been shown to be responsive to NF-~:B, including those for cytokines, cell surface receptors and antioxidant enzymes (Table 1).

Table 1.

Establishedand candidate KB-responsivegenes

Gene

Cell-type

Response

TNFct IL-6 TGFB Mn-SOD calbindin l-JcB(x

lymphocytes, microglia, neurons, astrocytes lymphocytes, glial ceils macrophages, glial cells tumor cells, neurons, astrocytes, many others neurons, astrocytes many

induction induction induction induction induction induction

NF-KB exists in the cytosol as an inducible 3 subunit complex consisting of two (prototypical) subunits of 50 kDa (p50) and 65 kDa (p65; RelA), and an inhibitory subunit called I-KB. However, depending upon cell-type, developmental stage and environmental factors, cells may express other NF-~:B DNA-binding subunits (e.g. p52, c-Rel and RelB) and I-~cBs (e.g. Bcl-3 and I-~Be) (Table 2). Table 2.

Examplesof NF-KBproteins, I-KBproteins and their cellular expression

Subunit

Function/Partners

Cellular Expression

1965(RelA) p50 p52 c-Rel ReI-B I-KBct I-KBI3 l-•Be Bcl-3

transcription activation/p50, transcription activation/p65,p50,p52 transcription activation/p50 transcription activation/p65 transcription activation/p50 inhibitory subunit/p50,p65 modulatory/p65,p50 inhibitory subunit/p65,c-Rel inhibitory subunit/p50,p52

widespread widespread lymphocytes,epithelial cells immune system immune system widespread immune system, elsewhere widespread lymph nodes, spleen

See Verma et al., 1996 for further details and references. NF-IcB activation occurs when I-~:B is induced to dissociate from the complex (Figure 1). The p50-p65 dimer then translocates to the nucleus and binds to 5' regulatory elements of genes responsive to NF-~:B; consisting of a decameric sequence ( 5 ' - G G G A C T T F C C - 3 ' ) .

NF- KB and Apoptosis 271

=lutamate Ca z+ ~t~ [~11~ sM

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Figure 1. Mechanisms of regulation of NF-nB activity. The inactive form of NF-KB exists in the cytosol as a three subunit complex, with the prototypical components being p65 and p50 (transcription factor dimer) and I-rBct (inhibitory subunit). Signals that activate NF-KB do so by inducing phosphorylation of I-KBct (which may target I-KBc~for degradation in the proteosome) which, in turn, causes its dissociation from the p65-p50 dimer. The p65-p50 dimer then translocates to the nucleus and binds to consensus KB sequences in the enhancer region of KB-responsive genes. Anti-apoptotic genes induced by NF-KB include those encoding Mn-SOD, calbindin & inhibitors of apoptosis (IAP). Many different stimuli result in the activation of NF-KB including activation of the TNF receptor (p55) occurs when TNF binds and induces receptor trimerization. A dimer of the protein TRADD (TNF receptor associated death domain) then associates with a "death domain" in the cytoplasmic portion of the receptor which, in turn, causes association of TRAF2 (TNF receptor-associated protein 2) and IAP (inhibitor of apoptosis) with TRADD. A kinase cascade is thus activated which results in phosphorylation of I-KB. Calcium and reactive oxygen species (ROS) are important inducers of NF-KB in cells exposed to a variety of apoptotic insults. T h e m e c h a n i s m o f activation o f NF-KB m a y i n v o l v e phosphorylation and proteolysis o f I-KB ( W o r o n i c z et al., 1997). Signals that activate NF-KB include: T N F , IL-1, lipopolysaccharide, and phorbol esters (Table 3). Interestingly, antioxidants prevent NF-~cB activation by each o f the a f o r e m e n t i o n e d signals, suggesting that reactive o x y g e n species ( R O S ) are i n v o l v e d in the activation process. This is particularly interesting because increasing e v i d e n c e suggests that T N F , and other stimuli that activate NF-KB, induce expression o f antioxidant e n z y m e s , most notably M n - s u p e r o x i d e dismutase ( M n S O D ) ( L e w i s - M o l o c k et al., 1994; Bruce et al., 1996; Mattson et al., 1997).

272

Table 3.

M.P. Mattson

Examples of stimuli that activate NF-KB

Stimulus

Cell-type

Response

Genes Modulated

TNF

Lymphocytes Hepatocytes Vascular endothelial Neurons Astrocytes Microglia

apoptosis, cytokine prod. apoptosis, cytokine prod. apoptosis, cytokine prod. prevention of apoptosis prevention of apoptosis cytokine production

interleukins, lAP cytokines, adhesion cytokines, adhesion MnSOD, calbindin MnSOD, calbindin TNF, IL-6, TGFI3

Glutamate

Neurons

Synaptic plasticity prevention of apoptosis

glutamate receptors? MnSOD, calbindin

NGF

Neurons

prevention of apoptosis

???

Oxyradicals

Many diff cells

prevention of apoptosis

MnSOD, calbindin

Knockout of the p65 subunit of NF-KB by targeted gene disruption in embryonic stem cells results in embryonic lethality, apparently as the result of liver failure (Beg et al., 1995). In contrast, mice develop normally in the absence of the p50 subunit of NF-KB, although such p50-/- mice do exhibit altered lymphocyte responses when challenged with lipopolysaccharide and infectious agents (Sha et al., 1995; Snapper et al., 1996). The latter findings demonstrate that p65 is essential for normal development, whereas p50 is not. The data also indicate, at least with regards to normal development, that •B-binding proteins other than p50 may heterodimerize with p65 and thereby substitute for p50 (Baeuerle and Baltimore, 1996).

From Bad Guy to Hero Until recently a prevailing view was that NF-~B is a pro-apoptotic transcription factor. This view was based upon the strong correlation of NF-KB activation and cell death in many different cell death paradigms (e.g. Grilli et al., 1996a). There was, however, no evidence for a cause-effect relationship between NF-KB activation and apoptosis, and it was never established that NF-~zB did play a direct role in the cell death process. We had been studying cytoprotective signaling pathways, and had developed the concept of "programmed cell life" to explain the involvement of growth factor signaling pathways in preventing cell death (Mattson and Furukawa, 1996). During the course of this work we discovered that TNF can prevent the death of cultured embryonic rat hippocampal neurons following exposure to several apoptotic insults including glucose deprivation and amyloid B-peptide (Cheng et al., 1994; Barger et al., 1995). Moreover, the vulnerability of neurons in the brains of mice lacking the p55 TNF

NF-tcB and Apoptosis 273 receptor to excitotoxic and ischemic brain injury was greatly increased (Bruce et al., 1996; Gary et al., 1998), suggesting an anti-death role for endogenous injury-induced T N F and NF-~cB. W e found that T N F induced activation of NF-v:B in the neurons, and that the anti-apoptotic action of T N F was mimicked by treatment with I-KB antisense oligonucleotides (Barger et al., 1995) and was abolished by treatment o f the cells with v:B decoy D N A (Mattson et al., 1997). An example of the anti-apoptotic action of TNF, and abolition o f the protective effect by ~:B decoy D N A treatment, is shown in Figure 2. Further evidence that NF-KB activation is sufficient to confer cellular resistance to apoptosis comes from studies showing that treatment of hippocampal cultures with C2-ceramide (at concentrations that activate NF-KB) protects neurons against apoptosis induced by oxidative and excitotoxic insults (Goodman and Mattson, 1996).

.~+A1~25-35

l c B D e c o y + T N F c c + A 1325-35

Figure 2. TNF prevents neuronal apoptosis induced by amyloid B-peptide by a mechanism involving NF-•B activation. Images of cultured hippocampal neurons stained with the fluorescent DNA-binding dye Hoechst 33342. The cultures had been exposed to the following conditions: Control, 34 h exposure to saline; Af525-35, 24 h pretreatment with saline followed by a 10 h exposure to 5 pM AI325-35;TNFct+AI325-35,24 h pretreatment with 100 ng/ml TNFa followed by a 10 h exposure to A1325-35; KBdecoy+TNFct+A1325-35, 24 h pretreatment with 25 pM KB decoy DNA plus 100 ng/ml TNFa followed by a 10 h exposure to Af525-35. A1325-35 induced nuclear DNA condensation and fragmentation (e.g. arrowheads) which was largely prevented in cultures pretreated with TNFct, but not in cultures pretreated with KB decoy DNA plus TNFct. Modified from Mattson et al. (1997).

NF-KB is activated when cells are subjected to oxidative stress (Schreck et al., 1991). Initially it was proposed that the mechanism for activation of NF-~B in response to oxidative stress involved oxidative damage to I-KB (Schmidt et al., 1995). Other data

274

M.P. Mattson

pointed to a role for protein kinase C (Folguiera et al., 1996). More recently several I-KB kinases have been identified, and their activity appears to be required for activation of NF-KB in response to a variety of stimuli (DiDonato et al., 1997). Activation of NF-KB in cells subjected to oxidative stress appears to be part of a cytoprotective response. As evidence, we found that treatment of cultured hippocampal neurons with KB decoy DNA increases their vulnerability to cell death induced by glutamate and Fe 2÷, two insults that kill neurons by oxidative stress pathways (Mattson et al., 1997). These data demonstrate a role for activation of N F - r B in enhancing resistance of neurons to excitotoxic and oxidative insults. Our discovery of an anti-apoptotic role for NF-nB in neurons has recently been confirmed in several studies of non-neuronal cells (Table 4). Treatment of a cultured lymphoma cell line with agents that prevent activation of NF-nB induced apoptosis and microinjection of IKBct or a p65 antibody induced apoptosis whereas overexpression of p65 prevented apoptosis (Wu et al., 1996). Whereas p65-deficient mouse fibroblasts and macrophages were killed by TNFct, cells containing p65 were not killed (Beg and Baltimore, 1996). Reintroduction of p65 into the p65-deficient cells restored their resistance TNFct-induced apoptosis. Overexpression of the p50 and p65 subunits of NF-KB in fibrosarcoma cells conferred resistance to killing by TNFct and chemotherapeutic agents, and inhibition of NF-nB nuclear translocation by overexpression of the super-repressor I-KBct enhanced cell killing (Wang et al., 1996). Expression of a dominant-negative I-KBct in cultured human fibroblasts, lymphoma cells and bladder carcinoma cells enhanced apoptosis induced by TNFct (Van Antwerp et al., 1996). In another study manipulation of downstream effectors of p55 TNF receptor activation (FADD, TRAF2 and RIP) modulated apoptosis in a manner consistent with an anti-apoptotic role for NF-KB (Liu et al., 1996). Collectively, these data suggest a widespread anti-apoptotic role for NF-KB. Table 4.

Chronologyof first evidence that activation of NF-KBcan prevent apoptosis.

Evidence

Apoptotic Insult

Reference

TNF prevents neuron death I-~B antisense prevents neuron death Neuron death enhanced in TNF receptor knockout mice I-KBctand p65 antibody induce apoptosis p65 expression prevents apoptosis

glutamate; glucoprivation amyloid peptide ischemia, kainate

Cheng et al., 1994 Barger et al., 1995 Bruce et al., 1996

none TNF TNF Chemotherapeutic agents TNF oxidative agents, amyloid

Wu et al., 1996 Beg and Baltimore, 1996 Van Antwerp et al., 1996 Wang et al., 1996 Liu et al., 1996 Mattson et al., 1997

I-rBt~ superrepressor promotes apoptosis TNFR-1 cascade componentsalter apoptosis rB decoy DNA prevents neuron death

Recent studies of injury-induced neuronal death in mice lacking p50 suggest that, while this NF-KB subunit does not play a critical role in modulating apoptosis during

NF- tcB and Apoptosis

275 normal development, it does serve an anti-apoptotic role following tissue injury. The extent of neuronal death in the brain following experimentally-induced epileptic seizures or focal cerebral ischemia was significantly increased in p50 knockout mice compared to wild-type mice (Z.F.Yu and M.P.Mattson, unpublished data). Studies of hippocampal cell cultures showed that spontaneous apoptotic cell death in culture was significantly increased in neurons from the p50-deficient mice (Figure 3). Additional data suggest that at least one cytoprotective response to injury, upregulation of Mn-SOD, is impaired in mice lacking p50 (Z.F. Yu and M.P. Mattson, unpublished data). 120 100--

._~ P

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60

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40

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20

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In addition to its role in modulating apoptosis of neurons and tumor cells, NF-•B is believed to play important roles in injury responses of vascular endothelial cells that are involved in the process of atherosclerosis. NF-•B is activated in smooth muscle cells, endothelial cells and macrophages of atherosclerotic lesions, but not in normal blood vessels. A variety of cytokines including TNF are increased in the microenvironment of atherosclerotic plaques, as are levels of proteins known to be responsive to NF-~B (e.g. cell adhesion molecules and cytokines) (Gerritsen and Bloor, 1993). Experimental studies have shown that TNF can induce dysfunction and apoptosis in cultured vascular endothelial cells, particularly when protein synthesis is inhibited, and that certain fatty acids and oxidized lipoproteins may exacerbate TNFs actions (Toborek et al., 1996, 1997). Whether NF-KB's role in atherosclerosis is contributory or beneficial remains unclear and, indeed, recent findings suggest that whereas TNF and linoleic acid cross-amplify each other's effects on endothelial cell damage, they do not synergize in activation of NF-~cB (Hennig et al., 1996; Toborek et al., 1996). Because oxidative stress and calcium overload play major roles in apoptosis of vascular endothelial cells in various paradigms (Blanc et al., 1997; Toborek et al., 1997), it seems likely that

276

M.P. Mattson

NF-IcB activation serves a cytoprotective function in endothelial cells as is the case in other cell-types (see next section). Indeed, it was recently reported that induction of lAP expression in response to NF-~B protects endothelial cells against TNF-induced apoptosis (Stehlik et al., 1998). A well-known means of preventing apoptosis in a variety of cell-types is to treat them with the protein synthesis cycloheximide. The ability of cycloheximide to prevent cell death has been interpreted to indicate that the cell death process requires synthesis of "killer" proteins. Interestingly, levels of cycloheximide that cause only a small impairment of protein synthesis can also prevent apoptosis by a mechanism involving induction of neuroprotective gene products including the anti-apoptotic gene bcl-2 and antioxidant enzymes (Furukawa et al., 1997). Anti-apoptotic concentrations of cycloheximide suppressed accumulation of ROS suggesting activation of antioxidant pathways. Treatment of cultures with Bcl-2 antisense reduced the neuroprotective action of cyloheximide suggesting that increased Bcl-2 expression was mechanistically involved in the anti-apoptotic actions= of cycloheximide. In addition, activity levels of Cu/Zn-SOD, Mn-SOD and catalase were significantly increased in cells exposed to anti-apoptotic concentrations of cycloheximide. More recently, we have found that NF-nB activity is increased in cultured neurons treated with anti-apoptotic concentrations of cycloheximide, and that KB decoy DNA largely abolishes the neuroprotective effects of cycloheximide (unpublished data). These findings suggest potential cooperative anti-apoptotic mechanisms involving NF-~:B and Bcl-2 because data suggest that NF-lcB may play a role in the anti-apoptotic actions of Bcl-2 (Ivanov et al., 1995).

Mechanisms Underlying the Anti-Apoptotic Action of NF-~:B Increased oxidative stress and perturbed cellular calcium homeostasis appear to play important roles in the apoptotic process in many different cell death paradigms (McConkey and Orrenius, 1996; Kruman et al., 1997, 1998). Studies in different laboratories have identified several genes induced by NF-~cB that likely play important roles in increasing cellular resistance to apoptosis. Wong and coworkers (Wong and Goeddel, 1988; Wong et al., 1989) observed that tumor cells resistant to TNF-induced apoptosis exhibit increased expression of the antioxidant enzyme Mn-SOD following TNF treatment, whereas cells vulnerable to TNF-induced apoptosis dO not increase their levels of Mn-SOD. Recent studies of the effects of TNF on different types of brain cells further support a widespread and important role for Mn-SOD production in the cytoprotective action of TNF (Bruce-Keller et al., 1998). The latter study showed that TNF induces Mn-SOD expression in neurons and astrocytes, but not in oligodendrocytes; TNF protected the neurons and astrocytes, but not the oligodendrocytes, against apoptosis induced by a mitochondrial toxin. Moreover, when cultured neurons were treated with TNF for 24-48 h and then mitochondria were isolated from the cells and their vulnerability to mitochondrial toxins examined, the mitochondria from TNF-treated cells were more resistant to the insults compared to mitochondria from cells not treated with TNF (Figure 4).

NF-KB and Apoptosis

277 150'

100 tO

O

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0

Figure 4.

Transmembrane potential

Calcium release

Treatment of intact tumor cells with TNF increases resistance of isolated mitochondria to a

metabolic toxin. Cultured PC12 cells were treated with saline (Control) or 100 ng/ml TNF for 24 h, and were then exposed to 10 mM 3-nitropropionic acid for 90 min. Mitochondrial transmembrane potential (assessed using the fluorescent probe JC-I) and calcium release (assessed using the fluorescent probe calcein green-4N) were quantified. Values are the mean and SE of 3-4 experiments. Modified from Bruce-Keller et al. (1998).

Mn-SOD is induced in cultured hippocampal neurons and other cell-types by TNF in an NF-l
278

M.P. Mattson

4O

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Figure 5. Pheochromocytoma cells overexpressing mitochondrial Mn-SOD are resistant to mitochondrial dysfunction and apoptosis. (Upper graph) A PC6 cell line overexpressing human Mn-SOD, and a control PC6 cell line transfected with empty vector (PC6-V) were exposed to FeSO4 or amyloid 13-peptide (A1325-35) for 24 h and the percentage of cells with apoptotic nuclei was quantified. Values are the mean and SE of determinations made in 6 cultures. *p < 0.001 compared to value for vehicle-treated cultures. **p < 0.001 compared to corresponding value for PC6-V cultures. (Lower graph) A PC6 cell line overexpressing human Mn-SOD, and a control PC6 cell line transfected with empty vector (PC6-V) were exposed to 4-hydroxynonenal (an aldehydic product of membrane lipid peroxidation) for the indicated time periods, and relative mitochondrial transmembrane potential was quantified using the fluorescent probe JC-1. Values are the mean and SE of determinations made in 6 cultures.

M n - S O D in h i p p o c a m p u s is correlated with resistance to i s c h e m i a - i n d u c e d d a m a g e (Akai et al., 1990; Liu et al., 1993; Ohtsuki et al., 1993). T h e s e findings suggest that m i t o c h o n d r i a l superoxide production is a critical e v e n t in the apoptotic cascade. S u p e r o x i d e interacts with nitric o x i d e resulting in f o r m a t i o n o f peroxynitrite, a d a m a g i n g reactive o x y g e n m o l e c u l e that p r o m o t e s m e m b r a n e lipid peroxidation (Keller et al., 1998), and can induce apoptosis ( E s t e v e z et al., 1995). T h e ability o f S O D m i m i c k s

NF- 1¢Band Apoptosis

279

A

FSa-II

+

NEO_

SOD-L

SOD-H

i pH 7.3

pH 8.3

11

Figure 6. Mn-SOD suppresses alkaline pH-induced reactive oxygen species production in fibrosarcoma cells. Confocal images of 2,7-dichlorofluorescein fluorescence, an indicator of cellular reactive oxygen species, in four different fibrosarcoma cell lines (untransfected, empty vector-transfected, overexpressing Mn-SOD at a low level, and overexpressing Mn-SOD at a high level) exposed to medium at two different pHs. Note that alkaline pH induces a large increase in oxidative stress in cells lacking Mn-SOD, but not in cells expressing Mn-SOD. Modifed from Majima et al. (1998).

and gene therapy to prevent or reverse disease processes in animal models or several human disorders attest to the importance of superoxide anion radical in human disease (Black et al., 1994; Baker et al., 1998; Epperly et al., 1998). Several studies have shown that Mn-SOD, in particular, is highly effective in preventing neuronal apoptosis in experimental models of Alzheimer's disease and stroke (Mattson et al., 1997; Keller et al., 1998). Recent findings suggest that, in addition to enhancement of antioxidant defenses, NF-~B may protect cells against apoptosis by modulating the expression of proteins involved in the regulation of cellular calcium homeostasis. TNF induced the expression of calbindin D28k in embryonic hippocampal neurons (Cheng et al., 1994) and in astrocytes (Mattson et al., 1995), and protected those cells against death induced by agents that elevate intraeellular calcium levels (e.g. excitatory amino acids and a calcium ionophore). Treatment of astrocytes with KB decoy DNA blocked induction of calbindin and the anti-apoptotic effect of TNF (Figure 7). PC12 cell lines stably overexpressing calbindin D28k exhibit increased resistance to apoptosis induced by a calcium ionophore, trophic factor withdrawal, and an apoptosis-enhancing mutation in the presenilin-1 gene linked to Alzheimer's disease (Guo et al., 1998a; Wemyj et al., 1999). Calbindin D28k appears to interupt the apoptotic cascade in the very early stages prior to increases in superoxide production and mitochondrial membrane depolarization. There are several different neurodegenerative disorders in which an association between selective neuronal vulnerability and lack of calbindin expression has been noted (Iacopino et al., 1992). For example, dentate granule neurons

280

M.P. Mattson

in the hippocampus contain high levels of calbindin and are spared in Alzheimer's disease and stroke, whereas pyramidal neurons in region CA1 of the hippocampus contain little or no calbindin and are vulnerable. In amyotrophic lateral sclerosis the spinal cord lower motor neurons that degenerate lack calbindin, whereas resistant cranial nerve motor neurons contain calbindin (Alexianu et al., 1994). Overactivation of glutamate receptors in neurons in the brain and spinal cord can induce neuronal apoptosis by a mechanism involving calcium overload and oxyradical production (Ankarcrona et al., 1995; Mattson and Mark, 1996). We recently discovered that NF-~cB can modulate neuronal excitability by altering whole-cell currents through voltagedependent calcium channels and ionotropic glutamate receptor channels (Furukawa et al., 1998a). Treatment of cultured rat hippocampal neurons with TNF for 24-48 h resulted in an increase in Ca 2+ current density, and a decrease in currents induced by glutamate. The effect of TNF on calcium current and glutamate-induced currents were mimicked by C2-ceramide and blocked by treatment with KB decoy DNA (Furukawa et al., 1998a). Measurements of intracellular calcium levels showed that neurons pretreated with TNF exhibited i n c r e a s e d [Ca2+]i following membrane depolarization, but reduced [Ca2+]i responses to glutamate, compared to neurons in untreated control cultures or cultures co-treated with KB decoy DNA (Figure 8). Levels of the GluR4 subunit of the AMPA subtype of glutamate receptor was decreased in neurons treated with TNF, suggesting a possible negative regulation by NF-KB of transcription of this glutamate receptor subunit. These findings suggest that modulation of expression of proteins involved in regulation of cellular calcium homeostasis may be an important mechanism whereby NF-KB protects cells against apoptosis. 100

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apoptosis. Primary rat cortical astrocytes were pretreated for 24 h in the presence of 100 ng/ml TNF, 20 p M ~cB decoy DNA, or a combination of r B decoy DNA and TNF. The percentage of cells with apoptotic nuclei was quantified 16 h following exposure to vehicle (0.5% dimethylsulfoxide) or 1 ,uM calcium ionophore A23187. In additional, cultures relative levels of calbindin were quantified following immunostaining with calbindin antibody. Values are the mean of determinations made in 2 cultures/condition.

NF-KB and Apoptosis

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In addition to being stimulated by activation of cell surface receptor signaling pathways, NF-KB is responsive to potentially cytotoxic agents within the cell. ROS and calcium are two prominent (potentially neurotoxic) agents that can activate NF-KB. For example, exposure of cultured neurons to amyloid B-peptide (a 40-42 amino acid peptide that forms insoluble plaques in the brain tissue of Alzheimer's patients) results in oxyradical (superoxide, hydrogen peroxide, and peroxynitrite) production (Goodman and Mattson, 1994; Mattson et al., 1997; Keller et al., 1998; Guo et al., 1999). The oxidative stress results in NF-KB activation which protects the neurons against apoptosis (Mattson et al., 1997; Guo et al., 1998b). We recently discovered that regulation of NF-KB activity is perturbed in neural cells expressing a mutated form of presenilin-1 linked to early-onset inherited Alzheimer's disease (Guo et al., 1998b). Presenilin-1 mutations had been shown to increase neuronal vulnerability to apoptosis by a mechanism involving perturbed calcium regulation in the endoplasmic reticulum, and increased levels of oxidative stress (Guo et al., 1997). Cells expressing presenilin-1 mutations exhibited an aberrant pattern of NF-~:B activation following exposure to apoptotic insults characterized by enhanced early activation with a subsequent prolonged depression of NF-KB activity that was associated with sensitivity to apoptosis (Guo et al., 1998b). Related studies have elucidated a feed-forward neuroprotective pathway involving NF-KB is that activated by the secreted form of amyloid precursor protein (sAPP) (Figure 9). sAPP is known to exhibit potent anti-excitotoxic and

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anti-apoptotic effects in neurons (Mattson et al., 1993; Furukawa et al., 1996). sAPP activates NF-KB in cultured hippocampal neurons by a cGMP-mediated mechanism (Barger and Mattson, 1996). The latter study also used cultured neuroblastoma cells expressing a KB reporter gene, to show that sAPP can induce KB-mediated transcription. Pretreatment of cells expressing mutant presenilin-1 with sAPP restored the normal pattern of activation of NF-KB following exposure to apoptotic insults and prevented cell death (Figure 10; Guo et al., 1998b). Interestingly, Grilli et al. (1996b) reported that the enhancer region 5' to the gene encoding BAPP contains KB-binding sites, and that NF-KB induces transcription of APP. This suggests a scenario in which NF-KB activation in cells under stress leads to APP (and sAPP) production, resulting in further activation of NF-KB.

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Additional mechanisms whereby NF-KB may suppress apoptosis are being elucidated. For example, activation of NF-KB in tumor cells was shown to block activation of caspase-8 by a mechanism involving induction of expression of TNF receptor-associated factors I and 2, and the inhibitor of apoptosis proteins c-IAP1 and c-IAP2 (Wang et al.,

NF- tcB and Apoptosis

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Figure 10. NF-KBactivity following exposure of PCI2 cells to an apoptotic insult is altered by mutant presenilin-1 and restored by secreted form of amyloid precursor protein. Gel-shift analysis of NF-KB DNA binding activity in cell extracts from PC12 cells expressing mutant presenilin-1 that had been exposed to vehicle for 4 h (Control), or to the indicated treatments for the indicated time periods. Amyloid 13-peptide(AB), 50 uM; sAPPa, 10 nM; KB decoy DNA, 25 ,uM. Modified from Guo et al. (1998b). 1998). IEX-1L is an NF-KB target that appears to play an important role in prevention of apoptosis in tumor cells (Wu et al., 1998). Interestingly, well-known modulators of apoptosis are being linked to NF-KB signaling. For example, it was recently shown that levels of NF-KB activity are increased in cultured myocytes that overexpress the antiapoptotic gene Bcl-2 by a mechanism involving enhanced degradation of I-KB (de Moissac et al., 1998). In addition, caspase-3 was shown to cleave I-KBct, and evidence was provided that the cleavage transforms I-KBc~ into a constitutive inhibitor of NF-KB thereby promoting apoptosis (Barkett et al., 1997). Whereas activation of NF-KB appears to be a powerful anti-apoptotic mechanism, inhibition of NF-KB may represent an important mechanism for inducing or enhancing apoptosis. Glucocorticoids are a potent trigger for apoptosis of T lymphocytes. Glucocorticoids have been shown to suppress NF-KB activity by a mechanism involving induction of I-KBB expression (Ramdas and Harmon, 1998). Apoptosis of macrophages induced by Y. enterocolitica was associated with impaired activation of NF-KB, and this suppression of NF-KB appears to play a key role in apoptosis induced by this infectious agent (Ruckdeschel et al., 1998). Another example comes from the studies of the pro-apoptotic adenoviral E1A protein, which indicate that inhibition of NF-KB plays a central in sensitization of cells to radiation-induced apoptosis (Shao et al., 1997).

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Finally, recent studies suggest a possible role for suppression of NF-~B activation in the pro-apoptotic action of Par-4, a novel leucine zipper- and death domain-containing protein recently linked to apoptotsis of tumor cells and of neurons in several different neurodegenerative disorders (Sells et al., 1997; Guo et al., 1998c). Par-4 has been shown to inhibit atypical isoforms of protein kinase C, which appear to be positioned upstream of NF-KB activation in anti-apoptotic cascades (Berra et al., 1997). Complex cytokine cascades that are activated in response to tissue injury are orchestrated, in part, by NF-rB. The TNF gene is highly responsive to NF-KB, and this provides a means for immune cells in injured tissues to rapidly produce large quantities of TNF. That NF-KB is an important mediator of TNF production in vivo is demonstrated by experiments showing that, following severe epileptic seizures, the normal increase in TNF levels in affected brain cells is attenuated in mice lacking p50 (Figure 11). In addition to inducing TNFet expression, NF-KB stimulates expression of interleukin-6 which, in turn, induces interleukin-ll3 expression (Collins et al., 1995). The extensive literature on the involvement of NF-KB in regulating cytokine cascades suggests that, although NF-KB activation can prevent apoptosis of the cell in which it is activated, it can indirectly lead to apoptosis of other cells by promoting production of cytotoxic agents such as nitric oxide. Indeed, the gene encoding inducible nitric oxide synthase contains KB binding sites in its enhancer region (Taylor et al., 1998). Cytokine-mediated activation of microglia may explain the ability of inhibitors of NF-KB to protect against cell damage in certain experimental paradigms (Qin et al., 1998). Another example of cell death-enhancing actions of NF-KB comes from a recent study in which it was shown that NF-~B is an essential mediator of Fas ligand expression in response to DNA-damaging agents, and that NF-KB thereby contributes to stress-induced apoptosis (Kasibhatla et al., 1998).

Novel Insight Into the Roles of NF-~B in Apoptosis: Lessons from Neurons Because of its cytoplasmic localization, and its ability to be activated in response to increases of intracellular calcium and ROS levels, NF-KB is in a unique position to mediate long-term, spatially-defined, changes in nerve cell plasticity and survival. Studies have shown that NF-KB is present in synaptic terminals located great distances from the neuronal cell body, and that NF-KB activation can occur locally in such synapses (Kaltschmidt et al., 1993; Meberg et al., 1996). Synaptosomal preparations from rat cerebral cortex contained considerable inducible NF-rB, and double-labelling immunohistochemical analyses showed co-localization of NF-KB subunits and the synaptic vesicle-associated protein synaptophysin. NF-KB is therefore located at sites such as growth cones and postsynaptic densities where processes mediating developmental and synaptic plasticity are initiated. NF-KB is activated in response to excitatory neurotransmitters, membrane depolarization and synaptic activity (Guerrini et al., 1995). Exposure of cultured cerebellar granule neurons to excitatory amino acid transmitters or to KC1 (depolarization) results in activation of NF-KB, as determined at the single cell level using an antibody that selectively reacts with the activated nuclear form of p65 (Kaltschmidt et al., 1995). The latter study showed that activation of

NF-tcB and Apoptosis

285

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Figure I1. Seizure-inducedproduction of TNF is suppressed in brain cells of mice lacking the p50 subunit of NF-~:B. Wild-type mice (p50+/+) and mice lacking p50 (p50-/-) were administered the seizure-inducing excitotoxin kainic acid into the dorsal hippocampus. Six hours later the mice were killed, perfused with fixative, and coronal brain sections were cut at 30 ,um. Sections were immunostained with a TNFct antibody. TNF immunoreactivity was intense in hippocampal neurons in the wild-type mouse, but not in the p50-/mouse (arrowheads).

NF-KB could be blocked by an antioxidant indicating that free radicals were involved in glutamate receptor- and depolarization-induced activation of NF-KB. Whether NF-nB is activated at glutamatergic synapses during normal activity throughout the brain remains to be determined although, in contrast to other cell-types, NF-KB is normally present in the cytoplasm of neurons in both constitutively-active and inducible forms (Korner et al., 1989; Kaltschmidt et al., 1993; Kaltschmidt et al., 1994; Kaltschmidt et al., 1995). There is considerable heterogeneity in constitutive NF-KB activity among neuronal populations suggesting a relationship to cell function (Kaltschmidt et al., 1994). Increasing data suggest that pathways that normally regulate synaptic transmission and structural plasticity in the nervous system are also involved in neuronal apoptosis, both during normal development and in neurodegenerative disorders. NF-nB provides an excellent example of a transcription factor regulating plasticity and death. NF-KB is activated in association with long-term potentiation of synaptic transmission (LTP), a process believed to be central to learning and memory (Meberg et al., 1996). Interestingly, NF-KB is also activated in response to low frequency stimulation, in contrast to other transcription factors (e.g. c-fos, and NGFI-A) that are induced by high-frequency (LTP-inducing) stimulation parameters but not by low frequency stimulation (Cole et al., 1989; Dragunow et al., 1989; Worley et al., 1993). Excessive neuronal activity, as occurs during epileptic seizures, results in marked

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NF-KB activation in cortex and hippocampus of adult rats (Unlap and Jope, 1995). It is believed that synaptic activity during seizures and during LTP may share many features (Baudry, 1986). NF-KB may therefore represent a signaling pathway designed to protect neurons against the potentially damaging effects of excessive neuronal activity. Apoptotic death signals in neurons may be activated at remote synaptic sites, and overactivation of glutamate receptors and underactivation of trophic factor receptors are implicated in such synaptically controlled apoptosis. We have found that biochemical changes consistent with apoptosis are activated locally in synapses (Mattson et al., 1998a,b). Thus, exposure of cortical synaptosomes (a preparation consisting of connected pre- and post-synaptic terminals, but no cell bodies) to insults known to induce apoptosis in intact neurons resulted in loss of membrane phospholipid assymetry, caspase activation, and mitochondrial alterations characteristic of apoptosis. A caspase inhibitor prevented mitochondrial membrane depolarization in synaptosomes. Cytosolic extracts from synaptosomes exposed to apoptotic insults cause chromatin condensation and fragmentation in isolated nuclei, demonstrating that signals capable of inducing nuclear apoptosis are generated locally in synapses (Figure 12A; Mattson et al., 1998a,b). Studies of intact neuronal circuits in hippocampal cultures have confirmed that apoptotic events can occur locally in synapses, and have further indicated that such signals can propagate to the cell body (Figure 12B; Mattson et al., 1998a,b). These findings that "synaptic apoptosis" can occur independently of the cell body, a possibility that has far-reaching implications for mechanisms of synaptic "remodeling" and neuronal death in both physiological and pathophysiological settings (Figure 13). During development of the nervous system, many neurons undergo apoptosis; such natural cell neuronal death appears to be regulated by competition for a limited supply of target-derived trophic factor (Vogel, 1993; Houenou et al., 1994). Nerve growth factor, which is known to play a pivotal role in preventing programmed cell death in several types of neurons, has been shown to activate NF-KB in neurons (Carter et al., 1996; Tong and Perez-Polo, 1996; Furukawa et al., 1998b). Synapse loss that occurs in both acute and chronic neurodegenerative disorders might also involve apoptotic cascades in the synapses that occur well prior to cell death (Martin et al., 1994; Anglade et al., 1996; DeKosky et al., 1996; Homer et al., 1996). There is considerable evidence that intracellular triggers of apoptosis, including calcium and ROS, can progress from postsynaptic dendritic regions to the cell body (Mattson, 1996). The ability of NF-KB to modulate gene expression in ways that suppress oxyradical accumulation and stabilize calcium homeostasis (Barger et al., 1995; Mattson et al., 1997, Furukawa et al., 1998a), suggests that this transcription factor is likely to play a major role in relaying antiapoptotic signals to the cell body, which then transforms the signal via modulation of gene expression and then sends protective proteins back to the synapse.

Future Directions

NF-•B is now firmly established as a transcription factor intimately involved in modulating cell death pathways. The preponderance of evidence indicates that NF-rB induces the expression of anti-apoptotic gene products. Future studies should be aimed

NF- tcB and Apoptosis 287

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at identifying upstream regulators of N F - r B activation, on the one hand, and gene targets of NF-KB that confer resistance to apoptosis, on the other hand. Elucidation of functional interrelationships between pro-apoptotic factors (e.g. Par-4, Bax, and caspases), anti-apoptotic factors (e.g. Bcl-2, cyclic GMP, and PKC~), and NF-•B will greatly improve our understanding of the "yin-yang" orchestration of programmed cell death and programmed cell life. The development of pharmacological and genetic manupulations that activate or suppress NF-KB activity will likely have broad applicability to treatment of various human diseases. For example, agents that inhibit NF-~zB activity can promote apoptosis of tumor cells, and are therefore potentially useful in various cancers. Conversely, agents that activate NF-KB may prove effective in preventing neuron death in the many neurodegenerative disorders that involve apoptosis.

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apoptosis and cell death. Glutamate receptor activation or disengagement of neurotrophic factor (NTF) receptors results in oxidative stress and elevation of intracellular calcium levels which, in turn, induce caspase activation and mitochondrial membrane depolarization (MD) and permeability transition (PT). Mitochondria release apoptotic factors (AF) capable of inducing nuclear chromatin condensation and DNA fragmentation. In addition, caspase activation results in exposure of phosphatidylserine (PS) on the synaptic membrane surface, which may be a trigger for local microglial responses. Apoptotic biochemical cascades initiated locally in synaptic compartments may propogate to the cell body and elicit mitochondrial and/or nuclear apoptotic events. Modified from Mattson et al. (1998a).

NF-tcB and Apoptosis

289 Finally, a better understanding of the physiological roles of NF-~B will likely also further our understanding of links between adaptive responses of cells to environmental demands and cell death.

Summary The transcription factor NF-rB has moved to the forefront in the field of apoptosis, because of recent findings showing that activation of NF-KB prevents apoptosis of many different cell-types in a variety of cell culture and in vivo paradigms. Activation of NF-KB was first shown to mediate anti-apoptotic actions of tumor necrosis factor (TNF) in cultured neurons, and was subsequently shown to prevent death of cancer cells induced by chemotherapeutic agents. NF-nB is activated in response to engagement of several different cytokine and growth factor receptors, and in response to a variety of cell stressors. Oxidative stress and elevation of intracellular calcium levels are particularly important inducers of NF-KB activation. Activation of NF-nB can interupt apoptotic biochemical cascades at relatively early steps, prior to mitochondrial dysfunction and oxyradical production. Gene targets for NF-KB that may mediate its anti-apoptotic actions include the antioxidant enzyme Mn-superoxide dismutase and the calcium-binding protein calbindin D28k. The available data identify NF-KB as an important effector of "programmed cell life" a set of evolutionarily conserved biochemical and molecular cascades designed to prevent cell death in a variety of physiological and pathological settings. Because NF-KB may play roles in a range of diseases that involve abnormalities in regulation of cell death, pharmacological and genetic manipulations of NF-KB signaling are being developed in order to either enhance (e.g. cancer) or prevent (e.g. Alzheimer's disease) apoptosis.

Acknowledgments I thank S.W. Barger, A.J. Bruce-Keller, K. Furukawa, D. Gary, Q. Guo, B. Hennig, and M. Toborek for their valuable contributions to original research in this laboratory. This work was supported by grants from the NIH (NIA and NINDS), the Alzheimer's Association, and the Kentucky Spinal Cord and Head Injury Research Trust.

References Akai, F., Maeda, M., Suzuki, K., Inagaki, S., Takagi, H. & Taniguchi, N. (1990). Immunocytochemical localization of manganese superoxide dismutase (Mn-SOD) in the hippocampus of the rat. Neurosci. Lett. 115, 19-23. Alexianu, M.E., Ho, B.K., Mohamed, A.H., La Bella, V., Smith, R.G. & Appel, S.H. (1994). The role of calcium-binding proteins in selective motoneuronvulnerability in amyotrophiclateral sclerosis. Ann. Neurol. 36, 846-858. Anglade, P., Mouatt-Prigent, A., Agid, Y. & Hirsch, E. (1996). Synapticplasticity in the caudate nucleus of

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patients with Parkinson's disease. Neurodegeneration 5, 121-128. Ankarcrona, M., Dypbukt, J.M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S.A. & Nicotem, P. (1995). Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15, 961-973. Baeuerle, P.A. & Henkel, T. (1994). Function and activation of NF-J(B in the immune system. Annu. Rev. Immunol. 12, 141-179. Baeuerle, P.A. & Baltimore, D. (1996). NF-~(B: ten years after. Cell 87, 13-20. Baker, K., Marcus, C.B., Huffman, K., Kruk, H., Malfroy, B. & Doctrow, S.P. (1998). Synthetic combined superoxide dismutase/catalase mimetics are protective as a delayed treatment in a rat stroke model: a key role for reactive oxygen species in ischemic brain injury. J. Pharm. Exp. Therap. 284, 215-221. Barger, S.W., Horster, D., Furukawa, K., Goodman, Y., Krieglestein, J. & Mattson, M.P. (1995). Tumor necrosis factors ct and B protect neurons against amyloid B-peptide toxicity: evidence for involvement of a •B-binding factor and attenuation of peroxide and Ca2+ accumulation. Proc. Natl. Acad. Sci. U.S.A. 92, 9328-9332. Barger, S.W. & Mattson, M.P. (1996). Induction of neuroprotective KB-dependent transcription by secreted forms of the Alzheimer's B-amyloid precursor. Mol. Brain Res. 40, 116-126. Barkett, M., Xue, D., Horvitz, H.R. & Gilmore, T.D. (1997). Phosphorylation of I•B-alpha inhibits its cleavage by caspase CPP32 in vitro. J. Biol. Chem. 272, 29419-29422. Baudry, M. (1986). Long-term potentiation and kindling: similar biochemical mechanisms? Adv. Neurol. 44, 401-410. Beg, A.A., Sha, W.C., Bronson, R.T., Ghosh, S. & Baltimore, D. (1995) Embryonic lethality and liver degeneration in mice lacking the relA component of NF-~cB. Nature 376, 167-170. Beg, A.A. & Baltimore, D. (1996). An essential role for NF-KB in preventing TNFct-induced cell death. Science 274, 782-784. Berra, E., Municio, M.M., Sanz, L., Frutos, S., Diaz-Meco, M.T. & Moscat, J. (1997). Positioning atypical protein kinase C isoforms in the UV-induced apoptotic signaling cascade. Mol. Cell. Biol. 17, 4346-4354. Black, S.C., Schasteen, C.S., Weis, R.H., Riley, D.P., Driscoll, E.M. & Luchesi, B.R. (1994). Inhibition of in vivo myocardial ischemic and reperfusion injury by a synthetic manganese-based superoxide dismutase

mimetic. J. Pharm. Exp. Therap. 270, 1208-1215. Blanc, E.M., Toborek, M., Mark, R.J., Hennig, B. & Mattson, M. P. (1997). Amyloid B-peptide disrupts barrier and transport functions and induces apoptosis in vascular endothelial cells. J. Neurochem. 68, 1870-1881. Bruce, A.J., Boling, W., Kindy, M.S., Peschon, J., Kraemer, P.J., Carpenter, M.K., Holtsberg, F.W. & Mattson, M.P. (1996). Altered neuronal and microglial responses to brain injury in mice lacking TNF receptors. Nature Medicine 2, 788-794. Bruce-Keller, A.J., Geddes, J.W., Knapp, P.E., Keller, J.N., Holtsberg, F.W., Steiner, S.M. & Mattson, M.P. (1998). Anti-apoptotic effects of TNF against metabolic poisoning: mitochondrial stabilization by MnSOD. J. Neuroimmunol. In press. Carter, B.D., Kaltschmidt, C., Kaltschmidt, B., Offenhauser, N., Bohm-Matthaei, R., Baeuerle, P.A. & Barde, Y.A. (1996). Selective activation of NF-KB by nerve growth factor through the neurotrophin receptor p. 75. Science 272, 542-545. Cheng, B., Christakos, S. & Mattson, M.P. (1994). Tumor necrosis factors protect neurons against excitotoxic/metabolic insults and promote maintenance of calcium homeostasis. Neuron 12, 139-153. Cohen, G.M. (1997). Caspases: the executioners of apoptosis. Biochem. J. 326, 1-16.

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291 Cole, A.J., Saffen, D.W., Baraban, J.M. & Worley, P.F. (1989). Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation. Nature 340, 474-476. Collins, T., Read, M.A., Neish, A.S., Whitley, M.Z., Thanos, D. & Maniatis, T. (1995). Transcriptional regulation of endothelial cell adhesion molecules: NF-~B and cytokine-inducible enhancers. FASEB J. 9, 899-909. DeKosky, S.T., Scheff, S.W. & Styren, S.D. (1996). Structural correlates of cognition in dementia: quantification and assessment of synapse change. Neurodegeneration 5,417-421. de Moissac, D., Mustapha, S., Greenberg, A.H. & Kirshenbaum, L.A. (1998). Bcl-2 activates the transcription factor NF-KB through the degradation of the cytoplasmic inhibitor I-KBct. J. Biol. Chem. 273, 23946-23951. DiDonato, J.A., Hayakawa, M., Rothwarf, D.M., Zandi, E. & Karin, M. (1997). A cytokine-responsive hoB kinase that activates the transcription factor NF-KB. Nature 388,548-554. Dragunow, M., Abraham, W.C., Goulding, M., Mason, S.E., Robertson, H.A. & Faull, R.L. (1989).Long-term potentiation and the induction of c-fos mRNA and proteins in the dentate gyrus of unanesthetized rats. Neurosci. Lett. 101,274-280. Epperly, M., Bray, J., Kraeger, S., Zwacka, R., Engelhardt, J., Travis, E. & Greenberger, J. (1998). Prevention of late effects of irradiation lung damage by manganese superoxide dismutase gene therapy. Gene Ther. 5, 196-208. Estevez, A.G., Radi, R., Barbeito, L., Shin, J.T., Thompson, J.A. & Beckman, J.S. (1995). Peroxynitriteinduced cytotoxicity in PC12 cells: evidence for an apoptotic mechanism differentially modulated by neurotrophic factors. J. Neurochem. 65, 1543-1550. Folgueira, L., McElhinny, J.A., Bren, G.D., MacMorran, W.S., Diaz-Meco, M.T., Moscat, J. & Paya, C.V. (1996). Protein kinase C-zeta mediates NF-KB activation in human immunodeficiency virus-infected monocytes. J. Virol. 70, 223-231. Furukawa, K., Barger, S.W., Blalock, E. & Mattson, M.P. (1996). Activation of K + channels and suppression of neuronal activity by secreted g-amyloid precursor protein. Nature 379, 74-78. Furukawa, K., Estus, S., Fu, W. & Mattson, M. P. (1997). Neuroprotective action of cycloheximide involves induction of Bcl-2 and antioxidant pathways. J. Cell Biol. 136, 1137-1150. Furukawa, K. & Mattson, M.P. (1998a). The transcription factor NF-~cB mediates increases in calcium currents and decreases in NMDA and AMPA/kainate-induced currents in response to TNFct in hippocampal neurons. J. Neurochem. 70, 1876-1886. Furukawa, K., Guo, Q., Schellenberg, G. D. & Mattson, M.P. (1998b). Presenilin-1 mutation alters NGFinduced neurite outgrowth, calcium homeostasis, and transcription factor (AP-1) activation in PCI2 cells. J. Neurosci. Res. 52, 618-624. Gary, D.S., Bruce-Keller, A.J., Kindy, M.S. & Mattson, M.P. (1998). Ischemic and excitotoxic brain injury is enhanced in mice lacking the p55 tumor necrosis factor receptor. J. Cereb. Blood Flow Metab. In press. Gerritsen, M.E. & Bloor, C.M. (1993). Endothelial cell gene expression in response to injury. FASEB J. 7,523-532. Goodman, Y. & Mattson, M.P. (1994). Secreted forms of B-amyloid precursor protein protect hippocampal neurons against amyloid B-peptide-induced oxidative injury. Exp. Neurol. 128, 1-12. Goodman, Y. & Mattson, M.P. (1996). Ceramide protects hippocampal neurons against excitotoxic and oxidative insults, and amyloid B-peptide toxicity. J. Neurochem. 66, 869-872. Grilli, M., Pizzi, M., Memo, M. & Spano, P. (1996). Neuroprotection by aspirin and sodium salicylate through

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blockade of NF-kappaB activation. Science 274, 1383-1385. Grilli, M., Goffi, F., Memo, M. & Spano, P. (1996b). Interleukin-ll3 and glutamate activate the NF-•B/Rel binding site from the regulatory region of the amyloid precursor protein gene in primary neuronal cultures. J. Biol. Chem. 271, 15002-15007. Guerrini, L., Blasi, F. & Denis-Donini, S. (1995). Synaptic activation of NF-kappa B by glutamate in cerebellar granule neurons in vitro. Proc. Natl. Acad. Sci. U. S. A. 92, 9077-9081. Guo, Q., Sopher, B., Furukawa, K., Pham, D., Robinson, N., Martin, G. & Mattson, M.P. (1997). Alzheimer's presenilin mutation sensitizes neural cells to apoptosis induced by trophic factor withdrawl and amyloid 8-peptide: involvement of calcium and oxyradicals. J. Neurosci. 17, 4212-4222. Guo, Q., Christakos, S., Robinson, N. & Mattson, M.P. (1998a). Calbindin blocks the pro-apoptotic actions of mutant presenilin-l: reduced oxidative stress and preserved mitochondrial function. Proc. Natl. Acad. Sci. U.S.A. 95, 3227-3232. Guo, Q., Robinson, N. & Mattson, M.P. (1998b). Secreted APP¢~ counteracts the pro-apoptotic action of mutant presenilin-1 by activation of NF-KB and stabilization of calcium homeostasis J. Biol. Chem. 273, 12341-12351. Guo, Q., Fu, W., Xie, J., Luo, H., Sells, S.F., Geddes, J.W., Bondada, V., Rangnekar, V.M. & Mattson, M.P. (1998c). Par-4 is a mediator of neuronal degeneration associated with the pathogenesis of Alzheimer's disease. Nature Med. 4, 957-962. Guo, Q., Sebastian, L., Sopher, B.L., Miller, M.W., Ware, C.B., Martin, G.M. & Mattson, M.P. (1999). Increased vulnerability of hippocampal neurons from presenilin-1 mutant knock-in mice to amyloid B-peptide toxicity: central roles of superoxide production and caspase activation. J. Neurochem. in press. Hennig, B., Toborek, M., Joshi-Barve, S., Barger, S.W., Barve, S., Mattson, M.P. & McClain, C.J. (1996). Linoleic acid activates nuclear transcription factor-KB (NFKB) and induces NFKB-dependent transcription in cultured endothelial cells. Am. J. Clin. Nutr. 63,322-328. Homer, C.H., Davies, H.A., Brown, J. & Stewart, M.G. (1996). Reduction in numerical synapse density in chick (Gallus domesticus) dorsal hippocampus following transient cerebral ischaemia. Brain Res. 735, 354-359. Houenou, L.J., L. Li, Lo, A.C., Yan, Q. & Oppenheim, R.W. (1994). Naturally occurring and axotomyinduced motoneuron death and its prevention by neurotrophic agents: a comparison between chick and mouse. Prog. Brain Res. 102, 217-226. Iacopino, A., Christakos, S., German, D., Sonsalla, P.K. & Altar, C.A. (1992). Calbindin-D28K-containing neurons in animal models of neurodegeneration: possible protection from excitotoxicity. Mol. Brain Res. 13,251-261. Ivanov, V.N., Deng, G., Podack, E.R. & Malek, T.R. (1995). Pleiotropic effects of Bcl-2 on transcription factors in T cells: potential role of NF-~B p50-p50 for the anti-apoptotic function of Bcl-2. Int. Immunol. 7, 1709-1720. Jones, P.L., Ping, D. & Boss, J.M. (1998). Tumor necrosis factor a and interleukin-lbeta regulate the murine manganese superoxide dismutase gene through a complex intronic enhancer involving C/EBP-beta and NF-kappaB. Mol. Cell. Biol. 17, 6970-6981. Kaltschmidt, C., Kaltschmidt, B. & Baeuerle, P.A. (1993). Brain synapses contain inducible forms of the transcription factor NF-KB. Mech. Dev. 43, 135-147. Kaltschmidt, C., Kaltschmidt, B., Neumann, H., Wekerle, H. & Baeuerle, P.A. (1994). Constitutive NF-KB activity in neurons. Mol. Cell. Biol. 14, 3981-3992. Kaltschmidt, C., Kaltschmidt, B. & Baeuerle, P.A. (1995). Stimulation of ionotropic glutamate receptors

NF-KB and Apoptosis

293 activates transcription factor NF-•B in primary neurons. Proc. Natl. Acad. Sci. U.S.A. 92, 9618-9622. Kasibhatla, S., Brunner, T., Genestier, L., Echeverri, F., Mahboubi, A. & Green, D.R. (1998). DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-~B and AP-1. Mol. Cell 1,543-551. Keller, J.N., Kindy, M.S., Holtsberg, F.W., St. Clair, D.K., Yen, H.-C., Germeyer, A., Steiner, S.M., Bruce-Keller, A.J., Hutchins, J.B. & Mattson, M.P. (1998). Mitochondrial MnSOD prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation and mitochondrial dysfunction. J. Neurosci. 18, 687-697. Korner, M., Rattner, A., Mauxion, F., Sen, R. & Citri, Y. (1989). A brain-specific transcription activator. Neuron 3,563-572. Kruman, I., Bruce-Keller, A.J., Bredesen, D.E., Waeg, G. & Mattson, M.P. (1997). Evidence that 4-hydroxynonenal mediates oxidative stress-induced neuronal apoptosis. J. Neurosci. 17, 5089-5100. Kruman, I., Guo, Q. & Mattson, M.P. (1998). Calcium and reactive oxygen species mediate staurosporineinduced mitochondrial dysfunction and apoptosis in PC12 cells. J. Neurosci. Res. 51,293-308. Lewis-Molock, Y., Suzuki, K., Taniguchi, N., Nguyen, D.H., Mason, R.J. & White, C.W. (1994). Lung manganese superoxide dismutase increases during cytokine-mediated protection against pulmonary oxygen toxicity in rats. Am. J. Respir. Cell Mol. Biol. 10, 133-141. Liu, Z.G., Hsu, H., Goeddel, D.V. & Karin, M. (1996). Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-•B activation prevents cell death. Cell 87, 565-576. Liu, X.H., Kato, H., Nakata, N., Kogure, K. & Kato, K. (1993). An immunohistochemical study of copper/zinc superoxide dismutase and manganese superoxide dismutase in rat hippocampus after transient cerebral ischemia. Brain Res. 625, 29-37. Majima, H.J., Oberley, T.D., Furukawa, K., Mattson, M.P., Yen, H.-C., Szweda, L.1. & St Clair, D.K. (1998). Prevention of mitochondrial injury by manganese superoxide dismutase reveals a primary mechanism for alkaline-induced cell death. J. Biol. Chem. 273, 8217-8224. Martin, L.J., Pardo, C.A., Cork, L.C. & Price, D.L. (1994). Synaptic pathology and glial responses to neuronal injury precede the formation of senile plaques and amyloid deposits in the aging cerebral cortex. Am. J. Pathol. 145, 1358-1381. Mattson, M.P., Cheng, B., Culwell, A., Esch, F., Lieberburg, I. & Rydel, R.E. (1993). Evidence for excitoprotective and intraneuronal calcium-regulating roles for secreted forms of B-amyloid precursor protein. Neuron 10, 243-254. Mattson, M.P., Cheng, B., Baldwin, S., Smith-Swintosky, V.L., Keller, J., Geddes, J.W., Scheff, S.W. & Christakos, S. (1995). Brain injury and tumor necrosis factors induce expression of calbindin D-28k in astrocytes: a cytoprotective response. J. Neurosci. Res. 42, 357-370. Mattson, M.P. (1996). Calcium and free radicals: mediators of neurotrophic factor- and excitatory transmitterregulated developmental plasticity and cell death. Perspect. Dev. Neurobiol. 3, 79-91. Mattson, M.P. & Furukawa, K. (1996). Programmed cell life: anti-apoptotic signaling and therapeutic strategies for neurodegenerative disorders. Restorative Neurol. Neurosci. 9, 191-205. Mattson, M.P. & Mark, R.J. (1996). Excitotoxicity and excitoprotection in vitro. Adv. Neurol. 71, 1-37. Mattson, M.P., Goodman, Y., Luo, H., Fu, W. & Furukawa, K. (1997). Activation of NF-•B protects hippocampal neurons against oxidative stress-induced apoptosis: evidence for induction of Mn-SOD and suppression of peroxynitrite production and protein tyrosine nitration. J. Neurosci. Res. 49, 681-697. Mattson, M.P., Keller, J.N. & Begley, J.G. (1998a). Evidence for synaptic apoptosis. Exp. Neurol. 153, 35-48. Mattson, M.P., Partin, J. & Begley, J.G. (1998b). Amyloid B-peptide induces apoptosis-related events in synapses and dendrites. Brain Res. 807, 167-176.

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M.P. Mattson

McConkey, D.J. & Orrenius, S. (1996). The role of calcium in the regulation of apoptosis. J. Leukoc. Biol. 59, 775-783. Meberg, P.J., Kinney, W.R., Valcourt, E.G. & Routtenberg, A. (1996). Gene expression of the transcription factor NF-KB in hippocampus: regulation by synaptic activity. Mol. Brain Res. 38, 179-190. Miller, D.K. (1997). The role of the caspase family of cysteine proteases in apoptosis. Semin. Immunol. 9, 35-49. Ohtsuki, T., Matsumoto, M., Suzuki, K., Taniguchi, N. & Kamada, T. (1993). Effect of transient forebrain ischemia on superoxide dismutases in gerbil hippocampus. Brain Res. 620, 305-309. Qin, Z.H., Wang, Y., Nakai, M. & Chase, T.N. (1998). Nuclear factor-kappa B contributes to excitotoxininduced apoptosis in rat striatum. Mol. Pharmacol. 53, 33-42. Ramdas, J. & Harmon, J.M. (1998). Glucocorticoid-induced apoptosis and regulation of NF-rB activity in human leukemic T cells. Endocrinology 139, 3813-3821. Ruckdeschel, K., Harb, S., Roggenkamp, A., Hornef, M., Zumbihl, R., Kohler, S., Heesemann, J. & Rouot, B. (1998). Yersinia enterocolitica impairs activation of transcription factor NF-KB: involvement in the induction of programmed cell death and in the suppression of the macrophage tumor necrosis factor alpha production. J. Exp. Med. 187, 1069-1079. Schmidt, K.N., Traenckner, E.B., Meier, B. & Baeuerle, P.A. (1995). Induction of oxidative stress by okadaic acid is required for activation of the transcription factor NF-rB. J. Biol. Chem. 270, 27136-27142. Schreck, R., Albermann, K. & Baeuerle, P. A. (1991). Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells (a review). Free Rad. Res. Commun. 17, 221-237. Schulze-Osthoff, K., Ferrari, D., Los, M., Wesselborg, S. & Peter, M.E. (1998). Apoptosis signaling by death receptors. Eur. J. Biochem. 254, 439-459. Sells, S.F., Han, S.-S., Muthukkumar, S., Maddiwar, N., Johnstone, R., Boghaert, E., Gillis, D., Liu, G., Nair, P., Monnig, S., Collins, P., Mattson, M.P., Sukhatme, V.P., Zimmer, S.G., Wood, D.P., McRoberts, J.W., Shi, Y. & Rangnekar, V.M. (1997). Expression and function of the leucine zipper protein Par-4 in apoptosis. Mol. Cell. Biol. 17, 3823-3832. Sen, R. & Baltimore, D. (1986). Inducibility of K immunoglobulin enhancer-binding protein NFIcB by a postranslational mechanism. Cell 47,921-928. Sha, W.C., Liou, H.C., Tuomanen, E.I. & Baltimore, D. (1995). Targeted disruption of the p50 subunit of NF-KB leads to multifocal defects in immune responses. Cell 80, 321-330. Shao, R., Karunagaran, D., Zhou, B.P., Li, K., Lo, S.S., Deng, J., Chiao, P. & Hung, M.C. (1997). Inhibition of nuclear factor-KB activity is involved in E1A-mediated sensitization of radiation-induced apoptosis. J. Biol. Chem. 272, 32739-32742. Snapper, C.M., Zelazowski, P., Rosas, F.R., Kehry, M.R., Tian, M., Baltimore, D. & Sha, W.C. (1996). B cells from p50/NF-rB knockout mice have selective defects in proliferation, differentiation, germ-line CH transcription, and Ig class switching. J. Immunol. 156, 183-191. Sovak, M.A., Bellas, R.E., Kim, D.W., Zanieski, G.J., Rogers, A.E., Traish, A.M. & Sonenshein, G.E. (1997), Aberrant nuclear factor-kappaB/Rel expression and the pathogenesis of breast cancer. J. Clin. Invest. 100, 2952-2960. Stehlik, C., de Martin, R., Kumabashiri, I., Schmid, J.A., Binder, B.R. & Lipp, J. (1998). Nuclear factor (NF)-~cB-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor a-induced apoptosis. J. Exp. Med. 188, 211-216. Taylor, B.S, de Vera, M.E., Ganster, R.W,, Wang, Q., Shapiro, R.A., Morris, S.M. Jr, Billiar, T.R., Geller, D.A. (1998). Multiple NF-KB enhancer elements regulate cytokine induction of the human inducible nitric oxide synthase gene. J. Biol. Chem. 273, 15148-15156.

NF-~cB and Apoptosis 295 Toborek, M., Barger, S.W., Mattson, M.P., Barve, S., McClain, C.J. & Hennig, B. (1996). Linoleic acid and TNF-ct cross-amplify oxidative injury and dysfunction of endothelial cells. J. Lipid Res. 37, 123-135. Toborek, M., Blanc, E.M., Kaiser, S., Mattson, M.P. & Hennig, B. (1997). Linoleic acid potentiates TNF~-mediated oxidative stress, disruption of calcium homeostasis and apoptosis of cultured vascular endothelial cells. J. Lipid Res. 38, 2155-2167. Tong, L. & Perez-Polo, J.R. (1996). Effect of nerve growth factor on AP-1, NF-•B and Oct DNA binding activity in PC12 cells: extrinsic and intrinsic elements. J. Neurosci. Res. 45, 1-12. Unlap, T. & Jope, R.S. (1995). Inhibition of NF-•B DNA binding activity by glucocorticoids in rat brain. Neurosci Lett. 198, 41-44. Van Antwerp, D.J., Martin, S.J., Kafri, T., Green, D.R. & Verma, I.M. (1996). Suppression of TNF-et-induced apoptosis by NFKB. Science 274, 787-789. Verma, I.M., Stevenson, J.K., Schwarz, E.M., Van Antwerp, D. & Miyamoto, S. (1996). Rel/NF-KB/IKB family: intimate tales of association and dissociation. Genes Dev. 9, 2723-2735. Vogel, K.S. (1993). Development of trophic interactions in the vertebrate peripheral nervous system. Mol. Neurobiol. 7,363-382. Wang, C.-Y., Mayo, M.W. & Baldwin, A.S. Jr. (1996). TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-KB. Science 274, 784-787. Wang, C.Y., Mayo, M.W., Korneluk, R.G., Goeddel, D.V., Baldwin, A.S. Jr. (1998). NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 281, 1680-1683. Wernyj, R.P., Mattson, M.P. & Christakos, S. (1999). Expression of calbindin D28k in C6 glial cells stabilizes intracellular calcium levels and protects against apoptosis induced by calcium inophore and amyloid B-peptide. Mol. Brain. Res. In press. Wong, G.H. & Goeddel, D.V. (1988). Induction of manganous superoxide dismutase by tumor necrosis factor: possible protective mechanism. Science 242, 941-944. Wong, G.H., Elwell, J.H., Oberley, L.W. & Goeddell, D.V.(1989). Manganous superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor. Cell 58,923-931. Worley, P.F., Bhat, R.V., Baraban, J.M., Erickson, C.A., McNaughton, B.L. & Barnes, C.A. (1993). Thresholds for synaptic activation of transcription factors in hippocampus: correlation with long-term enhancement. J. Neurosci. 13, 4776-4786. Woronicz, J.D., Gao, X., Cao, Z., Rothe, M. & Goeddel, D.V. (1997). IkappaB kinase-beta: NF-KB activation and complex formation with I~(B kinase-ot and NIK. Science 278, 866-869. Wu, M., Lee, H., Bellas, R.E., Schauer, S.L., Arsura, M., Katz, D., Fitzgerald, M.F., Rothstein, T.L., Sherr, D.H. & Sonenshein, G.E. (1996). Inhibition of NF-KB/Rel induces apoptosis of murine B cells. EMBO J. 15, 4682-4690. Wu, M.X., Ao, Z., Prasad, K.V., Wu, R. & Schlossman, S.F. (1998). IEX-1L, an apoptosis inhibitor involved in NF-~(B-mediatedcell survival. Science 281,998-1001. Yen, H.C., Oberley, T.D., Vichitbandha, S., Ho, Y.S. & St Clair, D.K. (1996). The protective role of manganese superoxide dismutase against adriamycin-induced acute cardiac toxicity in transgenic mice. J. Clin. Invest. 98, 1253-1260. Yuan, J. (1997). Transducing signals of life and death. Curr. Opin. Cell Biol. 9, 247-251. Zhong, W., Oberley, L.W., Oberley, T.D. & St Clair, D.K. (1997). Suppression of the malignant phenotype of human glioma cells by overexpression of manganese superoxide dismutase. Oncogene 14, 481-490.

INHIBITOR OF APOPTOSIS PROTEINS (lAPS) QUINN L. DEVERAUX AND JOHN C. REED

Table of Contents Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IAP Family Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure and Chromosomal Location of the Human lAP Genes . . . . . . . . . . . . . . . . . . . . . . Expression of lAP Family Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IAPs and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . lAPs and the Caspase-Family Cell Death Proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . lAP and Signal Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Insights into lAPs from Insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

297 297 300 301 302 304 308 310 314

Introduction The Inhibitor of Apoptosis (IAP) family of proteins was first discovered in baculoviruses, where they were shown to be involved in suppressing the host cell death response to viral infection. Interestingly, ectopic expression of some baculoviral lAPs blocks apoptosis in mammalian cells, suggesting conservation of the cell death program among diverse species and commonalities in the mechanism used by the IAPs to inhibit apoptosis. Since the initial discovery of the baculoviral IAPs, homologs have been found in numerous species, including humans. Though the mechanisms by which the IAPs suppress cell death remain debated, several studies have provided insights into the biochemical functions of these intriguing proteins. Moreover, a variety of reports have suggested an important role for the IAPs in some human diseases.

IAP Family Proteins IAP-family proteins are characterized by a novel domain of ~70 amino acids termed the Baculoviral IAP Repeat (BIR), the name of which derives from the original discovery of these apoptosis suppressers in the genomes of baculoviruses by Lois Miller and her colleagues (Bimbaum et al., 1994; Crook et al., 1993). Up to 3 tandem copies of the BIR domain can occur within the known IAP-family proteins of animal species (Figure 1). The conserved presence and spacing of cysteine and histidine residues present within BIR domains (Cx2Cx6Wx3DxsHx6C) may suggest that this structure represents a novel zinc-binding fold, but formal proof of this has yet to be obtained. 297 Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. 2 9 7 - - 321 © 2 0 0 1 Elsevier Science. Printed in the Netherlands.

Q.L. Deveraux and J.C. Reed

298

Mammalian 497 II

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Structures of BIR-domain containing proteins. The topologies of the known BIR-containing

proteins are represented from various species, highlighting the locations of the BIR (black), CARD (light-grey), RING (dark-grey), and UBC (dotted) domains. Some of these proteins have not been demonstrated to block apoptosis and thus do not yet qualify to be considered lAPs, including Ac-IAP, BRUCE, the porcine plAP, the nematode and yeast lAPs. Amino acid length is indicated to the right of each protein. The mammalian proteins are based on human cDNA cloning results.

lAP-Family Proteins 299 Proteins containing BIR domains have been identified in a wide range of eukaryotic species, including the fission yeast Schizosaccharomyces pombe, the budding yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, the fly Drosophila melanogaster, and several mammalian species including mice, rats, chickens, pigs, and humans. However, many of these BIR-containing proteins are untested where suppression of apoptosis is concerned and some BIR-containing proteins such as Ac-IAP do not appear to block cell death. Thus, it seems likely that BIR domains are not devoted exclusively to apoptosis suppression but rather function as protein-interaction domains that may have evolved to suit a variety of purposes. Structure-function studies of IAP family proteins performed to date have uniformly demonstrated a requirement for at least one BIR domain for suppression of apoptosis. Some reports, however, have indicated that the baculoviral IAPs require both N-terminal BIR domains and a C-terminal RING (Really Interesting New Gene) domain for their anti-apoptotic function in insect cells (Clem and Miller, 1994; Harvey et al., 1998). Several of the mammalian, fly, and viral IAPs have a RING domain located near their carboxyl-termini (Figure 1). NMR analysis has determined that RING domains represent a metal binding motif capable of coordinating two zinc atoms. However, RING motifs appear significantly different from other zinc-binding domains, in terms of both their zinc-ligation scheme and their three-dimensional structures (Barlow et al., 1994). Similar zinc finger motifs are present in numerous proteins and can facilitate DNA and protein interactions which, in some cases, can be altered to provide functional specificity (Borden and Freemont, 1996). The relevance of the RING domain for IAPmediated suppression of apoptosis, however, appears to depend upon cellular context. Some reports have indicated that a region encompassing a single BIR, such as BIR2 of baculovirus Op-IAP and Drosophila D-IAP1, can be sufficient for inhibition of apoptosis induced by the fly apoptosis protein HID in insect cells (Vucic et al., 1998b). In a separate study, cell death suppressing activity mapped to the N-terminal BIR domains in the Drosophila IAPs, D-IAP1 or D-IAP2, and removal of the C-terminal RING domain, actually enhanced their ability to suppress developmental programmed cell death and cell death induced by ectopic expression of the fly apoptosis gene RPR (see below) in the developing fly eye (Hay et al., 1995). Similarly, the human IAP-family proteins, c-IAPI, c-IAP2 and XIAP have been reported to retain anti-apoptotic function in the absence of their C-terminal RING domains (Deveraux et al., 1997; Roy et al., 1997; Takahashi et al., 1998). Thus, the relationship between BIR domains and the RING motif may not be exclusively linked to anti-apoptotic function. The human c-IAP1 and c-IAP2 proteins contain a Caspase Recruitment Domain (CARD) located between the BIR and RING domains (Figure 1). The functional significance of this domain for the anti-apoptotic function of IAPs is largely untested, but N-terminal fragments of human c-IAP1 and c-IAP2 which retain only the BIR domains are sufficient to block apoptosis, implying that the CARD domain is not absolutely required (Roy et al., 1997). c-IAP1, a CARD-containing IAP-family member, has been reported to bind CARDIAK/RIP2/RICK, a protein that contains a CARD domain and a serine/threonine kinase domain similar to that found in the NF-KB-inducing protein RIP (McCarthy et al., 1998; Thome et al., 1998). CARDIAK/RIP2/RICK has been reported to bind and induce activation of pro-caspase-1, a member of the caspase

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family of cysteine proteases (Thome et al., 1998). Pro-caspase-1 contains an N-terminal CARD domain and is required for proteolytic processing of certain proinflammatory cytokines (pro-IL-113; pro-IL-18). Caspase-1 can also participate in apoptosis in some circumstances. For example, though caspase-1 knock-out mice are phenotypically normal and possess no overt signs of failed programmed cell death, they do exhibit resistance to pathological neuronal cell death induced by ischemia (Schielke et al., 1998). To date, however, it remains untested whether IAPs interfere with CARDIAKinduced activation of pro-caspase-1. Additional domains of potential interest are also found in some of the other mammalian lAP-family proteins. The NAIP protein, for example, contains a P-loop consensus sequence similar to some ATP/GTP-binding proteins, but whether this IAP member binds purine nucleotides or requires this domain for apoptosis suppression remains undetermined (Roy et al., 1995). BRUCE, a large ~528 kDa BIR-containing protein contains a functionally intact Ubiquitin Conjugating (UBC) domain (Hauser et al., 1998b). However, it unknown whether BRUCE suppresses apoptosis. Taken together, the conserved domain structure of lAPs, the BIR domain, can be linked with other BIR domains and/or with a variety of distinct motifs including RING, CARD, UBC and ATP/GTP-binding domains. These non-BIR motifs presumably either diversify the functions of IAPs or provide ways of regulating individual members or subgroups of the family of lAP proteins.

Structure and C h r o m o s o m a l Location of the H u m a n l A P Genes

To date, six lAP relatives have been identified in humans, including NAIP, c-IAP1/HIAP-2, c-IAP2/HIAP-1, XIAP/hlLP, Survivin and BRUCE (Ambrosini et al., 1997; Duckett et al., 1996; Hauser et al., 1998a; Liston et al., 1996; Rothe et al., 1995). Mouse orthologues of most human lAPs have also been identified, implying conservation of the lAP gene family in mammals. The human XIAP, c-IAP1, c-IAP2, NAIP and Survivin genes have been assigned to chromosomal locations Xq25, 1 lq22-q23, 1 lq22-23, 5q13.1 and 17q25, respectively (Ambrosini et al., 1998; Liston et al., 1996; Rajcan-Separovic et al., 1996; Roy et al., 1995). Interestingly, the human c-IAP1 and c-IAP2 genes are located within ~7 kbp of each other on 1 lq22-23 (Young et al., 1998). The NAIP gene locus is complex, containing several tandem copies of NAIP sequences, most representing pseuodgenes that vary in number among individuals (Roy et al., 1995). Another intriguing peculiarity of gene structure is found in Survivin (Ambrosini et al., 1998). The coding strand of the Survivin gene is entirely complementary (antisense) to a previously characterized gene encoding the Effector Cell Protease Receptor-1 (EPR-1). Separate promoters, oriented in opposing directions, control expression of ERP-1 and Survivin in an exclusionary fashion, wherein transcripts produced for one of these appear to inhibit the expression of the other through an antisense RNA-based mechanism (Ambrosini et al., 1998). It remains to be determined whether any of these gene loci are directly involved in genetic alterations found in tumors.

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Expression of lAP Family Genes Over-expression of XIAP, c-IAP1, c-IAP2, NAIP or Survivin has been shown to suppress apoptosis induced by a variety of stimuli, including Tumor Necrosis Factor (TNF), Fas, menadione, staurosporin, etoposide (VP16), Taxol and growth factor withdrawal (Ambrosini et al., 1997; Duckett et al., 1996; Li et al., 1998a; Liston et al., 1996). Expression of the mammalian lAP family genes appears to be differentially regulated in vivo, thus presumably providing mechanisms for controlling the presence or absence of lAPs in response to particular cellular or environmental signals. XIAP mRNA was observed in all adult and fetal tissues examined except peripheral blood leukocytes, indicating that it is a ubiquitously expressed member of the family (Liston et al., 1996). However, analysis of XIAP expression at the single cell level by immunohistochemistry or in situ hybridization is needed to determine whether differentiation or cell lineage-specific expression occurs. Expression of c-IAP1 and c-IAP2 is highest in the kidney, small intestine, liver, and lung and lowest in the central nervous system (Young et al., 1998). Although the tissue distributions of c-IAP1 and c-IAP2 are reportedly similar, the relative expression of c-IAP1 is generally higher (Young et at., 1998). NAIP mRNA appears to be expressed at levels sufficient for detection by Northern blot analysis only in adult liver and in placenta, but can be detected in brain by RT-PCR (Roy et al., 1995). Though most tissues have not been examined in detail, lAP expression has been investigated in the ovary, where apoptosis is thought to play an important role in ovulation. In granulosa cells from preantral and early antral follicles, extensive apoptosis was associated with reduced protein levels of c-IAP2 and XIAP (Li et al., 1998b). Gonadotropin treatment increased c-IAP2 and XIAP protein content and suppressed apoptosis in granulosa cells, resulting in the development of follicles to the antral and preovulatory stages. Thus, these lAPs may play an important role in determining the fate of the granulosa cells, and thus, the eventual follicular destiny (Li et al., 1998b). Survivin exhibits the most restricted expression of an lAP-family member in adult tissues. Survivin mRNA is found by Northern blotting only in occasional normal adult human or mouse tissues (Ambrosini et al., 1997). The Survivin promoter contains four copies of G] repressor elements that have been implicated in controlling cell cycle periodicity in some G2/M-regulated genes (Li et al., 1998a). Moreover, when studied in reporter gene assays, the Survivin promoter exhibits typical M-phase-inducible transactivation, suggesting that Survivin is a bona fide cell cycle regulated gene and raising the possibility that Survivin expression may be induced in dividing cells (Li et al., 1998a). In HeLa cells, Survivin mRNA is upregulated ~40 fold at G2/M (Li et al., 1998a). Immunohistochemical analysis and in situ hybridization studies have demonstrated expression of Survivin in several fetal tissues (Adida et al., 1998). In the mouse embryo, prominent and nearly ubiquitous distribution of Survivin was found at embryonic day (E) 11.5, whereas at El5 to E21, Survivin expression was restricted to only a few locations. Expression of Survivin in embryonic and fetal development may contribute to tissue homeostasis and differentiation, with the gene then becoming quiescent in most normal adult tissues (Adida et al., 1998).

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lAPs and Disease

Because gene knockout experiments have yet to be reported, the in vivo physiological roles of individual lAP family genes are presently unclear. The NAIP gene, however, was first identified because of its apparent deletion in patients with Spinal Muscular Atrophy (SMA), a hereditary motoneuron degenerative disease (Liston et al., 1996; Roy et al., 1995). Though the primary genetic defect in SMA has been ascribed to an adjacent gene (Liu et al., 1997), SMN, rather than NAIP, patients with the most severe forms of this disease appear to harbor deletions at 5q13.1 that encompass both the SMN and NAIP genes. Intriguingly, the SMN protein has been reported to bind Bcl-2 and enhance Bcl-2-mediated protection from apoptosis (Iwahashi et al., 1997), raising the possibility that two survival genes may be lost in more severely affected individuals, and suggesting that NAIP may be required for the survival in vivo of some specific types of compromised neurons. NAIP may also be involved in adaptive responses to ischemia. Transient forebrain ischemia selectively elevates levels of NAIP in rat neurons that are resistant to ischemia-reperfusion (Xu et al., 1997). Upregulation of endogenous NAIP expression or intracerebral injection of NAIP-encoding adenoviruses reportedly reduces ischemic damage in vivo in the rat hippocampus, suggesting that NAIP may play a role in conferring resistance to ischemia-induced cell death (Xu et al., 1997). However, in cell culture experiments, transfection of primary cerebellar granule cell neurons with adenoviruses encoding NAIP, XIAP, c-IAP1, or c-IAP2 delayed but did not prevent apoptosis induced by K+ depolarization and serum deprivation. Non-apoptotic cell death induced by L-glutamate was unimpaired by these lAP-family proteins (Simons et al., 1998). Thus, lAPs are apparently insufficient to protect some types of neurons from insults often associated with ischemia. In addition to lAP involvement in neurological disease, several studies have implicated lAPs in cancer biology. For example, the oncoprotein v-Rel, a member of the Rel/NF-kappaB family of transcription factors, induces malignant transformation and inhibits apoptosis. The chicken homolog of c-IAP1 (ch-IAP1) was found to be upregulated following expression of v-Rel in fibroblasts, a B-cell line, and in spleen cells (You et al., 1997). ch-IAP1 reportedly suppresses mammalian cell apoptosis induced by the overexpression of caspase-1. Expression of exogenous ch-IAP1 in temperature-sensitive v-Rel-transformed spleen cells also inhibited apoptosis of these cells at the nonpermissive temperature. Based upon these results, it appears that ch-IAP1 is induced and functions as a suppressor of apoptosis in the v-Rel-mediated transformation process (You et al., 1997). The lAP member Survivin is expressed in a high proportion of the most common human cancers but not in normal, terminally differentiated adult tissues, thus making Survivin an exciting new tumor marker (Ambrosini et al., 1997). The assessment of Survivin expression in human tumor specimens included both in situ RNA hybridization and immunohistochemical analysis, confirming expression in tumor cells but not admixed stromal cells or adjacent normal tissues (Ambrosini et al., 1998). Thus, altered expression of Survivin appears to define a common event associated with the pathogenesis of most human cancers. Moreover, reductions in Survivin expression

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303 achieved using antisense strategies cause apoptosis and sensitization to anticancer drugs, at least in some tumor cell lines, implying that Survivin expression can be important for cell survival or chemoresistance in carcinomas (Ambrosini et al., 1998). Not all tumors, however, express Survivin. Moreover, even within a given type of cancer, heterogeneity in Survivin expression may be observed. Immunohistochemical assessments of Survivin expression in tumors where immunointensity, percentage immunopositivity, or both have been measured for purposes of segregating Survivin negative from positive (Survivin low from high) tumors suggest that expression of Survivin (or higher levels of Survivin expression) is associated with worse clinical outcome or other unfavorable prognostic features in neuroblastomas, colon and gastric cancers (Adida et al., 1998; Ambrosini et al., 1997; 1998; Lu et al., 1998). Though preliminary, assessments of Survivin expression therefore may be of prognostic significance for patients with some types of cancer. Survivin may have relevance to cancer for other reasons as well. Not only is the expression of the Survivin gene enhanced at the G2/M phase of the cell cycle but the Survivin protein reportedly binds tightly (K D ~5-7 uM) with polymerized microtubules (Li et al., 1998a). Deletion of a predicted carboxyt-terminal coiled-coil region (A 100-142) from Survivin impaired microtubule binding and abrogated Survivin's ability to protect against taxol-induced apoptosis, implying that association with microtubules is critical for Survivin function. In this regard, gene transfer-mediated over-expression of wild-type Survivin failed to protect tumor cell lines against apoptosis induced by microtubuledisrupting agents such as nocodazole or vincristine, further implicating binding of Survivin to polymerized microtubules as a necessary requirement for activating the anti-apoptotic function of Survivin or for targeting it to critical locations that require its protection (Li et al., 1998b). Interestingly, mutation of a conserved cysteine in the Survivin BIR domain (Cys84-Ala) also abolished Survivin's cytoprotective abilities. However, the BIR (Cys84-Ala) mutant retained the ability to associate with microtubules similar to wild-type Survivin and, indeed, appeared to interfere with the function of endogenous Survivin by competing for microtubute binding (Li et al., 1998a). Over-expression of the BIR mutant also resulted in a progressive increase in caspase activity, occurring predominantly at the G2/M border. Based upon these collective results, the authors suggest that Survivin may counteract an apoptotic pathway that becomes active during a G2/M checkpoint (Li et al., 1998a). Thus, these findings further underscore the interdependence of cell cycle regulation and programmed cell death. Murine TIAP (named based upon its high expression in thymus and testis), exhibits 84% sequence identity with human survivin (Kobayashi et al., 1999). Similar to survivin, TIAP is expressed in growing tissues such as thymus, testis, and intestine of adult mice and many tissues of embryos. Expression of TIAP was upregulated in synchronized NIH 3T3 cells at S to G2/M phase of the cell cycle and high expression of TIAP was detected specifically in proliferating cells in vitro and in vivo. The expression pattern of TIAP in proliferating cells suggests that high expression of TIAP may be a factor in the generation of neoplasms, however, high levels of TIAP may also be the result of rapid growth (Kobayashi et al., 1999). Nevertheless, the authors hypothesize that during cell proliferation, inducible lAPs such as TIAP, may be important factors for cellular protection (Kobayashi et al., 1999).

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lAPs and the Caspase-Family Cell Death Proteases At present, it remains controversial how the lAPs suppress apoptosis. Recent studies, however, have demonstrated that several of the human lAPs (XIAP, c-IAP1 and c-IAP2) directly inhibit caspases (Deveraux et al., 1997; Roy et al., 1997). Caspases are a family of cysteine proteases with substrate specificity for aspartic acid that represent highly conserved components of apoptotic programs throughout the animal kingdom (Reviewed in Alnemri et al., 1996; Salvesen and Dixit 1997). XIAP, c-IAP1 and c-IAP2 bind and potently inhibit caspases 3, 7 and 9 but not caspases 1, 6, 8 or 10 or CED3 (Deveraux et al., 1998,1997; Roy et al., 1997). Survivin also can be co-immunoprecipitated with caspases-3, -7, and -9 and it suppresses apoptosis induced by over-expression of these caspases, implying that Survivin also is a caspase inhibitor (Tamm et al., 1998). Consistent with this idea, TIAP (mouse survivin) can bind to active caspase 3 in vitro and inhibits caspase-induced cell death of transfected Rat-1 cells (Kobayashi et al., 1999). Thus, IAPs appear to represent the first identified family of endogenous cellular inhibitors of caspases in mammals. Caspases are initially synthesized as single polypeptide chains representing latent precursors (zymogens) that undergo proteolytic processing at specific aspartic acid residues to produce subunits which form the active heterotetrameric protease. In mammalian cells, activation of the caspase zymogens has been reported to occur through at least three independent mechanisms: (a) cleavage by upstream active caspases; (b) cleavage by Granzyme B, an aspartate-specific serine protease found in the granules of cytolytic T-cells; and (c) auto-processing of zymogens with assistance from other caspase-interacting proteins which can occur in either a cis- or trans-manner (Reviewed in Salvesen and Dixit, 1997; Stennicke and Salvesen, 1998). One mechanism for triggering auto-processing and activation of pro-caspase-8 entails its recruitment to plasma membrane receptor complexes, such as the Fas, a member of tumor necrosis factor (TNF) family of cell death receptors. Pro-caspase-8 zymogens possess ~1% the activity of the processed fully-active protease. When brought into close apposition by oligimerization around Fas receptor complexes, these zymogens trans-process each other, yielding autonomous, active proteases (Juo et al., 1998; Martin et al., 1998; Stennicke et al., 1998). Once activated, caspase-8 can then directly or indirectly activate pro-caspase-3 and other downstream caspases, which function as the ultimate effectors of apoptosis by cleaving a variety of substrate proteins in cells. Another identified mechanism for initiating caspase activation requires the participation of mitochondria and involves a protein known as the Apoptosis Protease Activating Factor-1 (Apaf-1) (Li et al., 1997; Liu et al., 1996; Reed 1997; Zou et al., 1997). Apaf-1 is a cytosolic protein that rests in a latent state until bound by cytochrome c. Cytochrome c is commonly released from the mitochondria during apoptosis induced by many, but probably not all, cell death stimuli (Reviewed in Green and Reed, 1998; Reed, 1997). The resulting Apaf-1/cyto-c complex associates with the zymogen form of caspase-9 in the presence of dATP or ATP, promoting the auto-catalytic activation of caspase-9. Once activated, caspase-9 can then directly cleave and activate procaspase-3, resulting in additional caspase activation and apoptosis.

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305 Caspases-8 and -9 thus represent the pinnacle caspases in the Fas/TNF-family death receptor and cytochrome c/Apaf-1 pathways, respectively (Figure 2). Studies with cells derived from caspase-8 and caspase-9 knock-out mice indicate that caspase-8 is absolutely required for Fas-induced apoptosis; whereas caspase-9 is necessary for apoptosis induced by multiple stimuli known to trigger cytochrome c release from mitochondria (Hakem et al., 1998; Juo et al., 1998; Kuida et al., 1998; Varfolomeev et al., 1998). Though lAPs do not bind or inhibit caspase-8, they do bind to and inhibit its substrate, caspase-3, thus arresting the cascade of proteolysis and providing protection from Fas/caspase-8-induced apoptosis (Deveraux et al., 1998, 1997; Roy et al., 1997). In contrast, in the mitochondrial pathway for caspase activation, XIAP, c-IAP1, and c-IAP2 directly bind to the pinnacle caspase, pro-caspase-9, and prevent its processing and activation induced by cytochrome c, both in intact cells and in cell extracts where caspase activation is induced by addition of exogenous cytochrome c (Deveraux et al., 1998). Because they can also directly bind to and potently inhibit the next caspase in the cytochrome c/Apaf-1 induced cascade, caspase-3 (Li et al., 1997), these lAP-family proteins also presumably interfere with a reported amplification loop in which active caspase-3 cleaves and activates additional pro-caspase-9 molecules (Srinivasula et al., 1998). These observations are consistent with reports that overexpression of lAP-family proteins inhibits apoptosis induced by Bax and other pro-apoptotic Bcl-2 family proteins, which are known for their ability to target mitochondria and induce cytochrome c release (Bossy-Wetzel et al., 1998; Deveraux et al., 1997; Jtirgensmeier et al., 1998; Mahajan et al., 1998; Wolter et al., 1997). The lAPs, however, do not interfere with Bax-mediated release of cytochrome c in vitro using isolated mitochondria as well as intact cells (Finucane et al., 1998; Jtirgensmeier et al., 1998), an observation that is consistent with other data indicating that the human IAPs (at least XIAP, c-IAP1, c-IAP2, and Survivin) block caspase activation and apoptosis downstream of Bax, Bik, Bak and cytochrome-c (Deveraux et al., 1998, 1997; Duckett et al., 1998; Orth and Dixit, 1997; Roy et al., 1997; Tamm et al., 1998). The failure of lAPs to prevent cell death stimuli from triggering cytochrome c release has important implications for determining whether cell death will be prevented in the long-term versus merely delayed (see below). Apoptosis induced by over-expression of pro-caspases-3, -7, or -9 is also suppressible by co-expression of XIAP, c-IAP1, c-IAP2, or Survivin (Deveraux et al., 1997; Roy et al., 1997; Tamm et al., 1998). The over-expression of these zymogens presumably results in a few molecules becoming active proteases, resulting in feedforward amplification in which the active caspases cleave and activate more of their zymogens or other caspase zymogens. Consequently, in co-transfection experiments, lAPs generally prevent the appearance of cleaved caspases, most likely by squelching this amplification loop. In this regard, the ratio of caspase to lAP is likely to be critical in determining whether apoptosis is successfully prevented in such experiments, possibly explaining the reported failure of lAPs to inhibit cell death induced by over-expression of caspase-7 in transient transfection studies in which the relative levels of protein production were not determined (Hawkins et al., 1996). Interestingly, binding of XIAP, c-IAP1, c-IAP2, and Survivin to caspases 3 and 7 requires the proteolytic processing and activation of these caspases. In contrast, the lAPs

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Figure 2. Several members of the TNF family of death receptors (exemplified here by Fas) recruit caspase-8 to their cytosolic domains upon binding their respective ligands, resulting in proteolytic activation of this proximal caspase (Wallaeh et al., 1997). Once activated, caspase-8 can induce, either directly or indirectly, the activation of a number of distal caspases such as caspase-3 (Muzio et al., 1997; Stennicke et al., 1998). IAPs can suppress this pathway by directly inhibiting caspase-3 activity (Deveraux et al., 1998, 1997; Roy et al., 1997). Another, although not mutually exclusive, pathway for caspase activation involves cytochrome-c (tyro-c) which, at least in mammalian cells, is often released from the mitochondria into the eytosol as an early event in apoptosis (Bossy-Wetzel et al., 1998; Kharbanda et al., 1997; Kluck et al., 1997; Liu et al., 1996; Yang et al., 1997). Upon entering the cytosol, cyto-c induces the ATP or dATP-dependent formation of a complex of proteins that results in the proteolytic activation of the executioner easpases (Liu et al., 1996). Among the members of this complex are the CED-4 homolog Apaf-1, and caspase-9 (Li et al., 1997; Liu et al., 1996; Zou et al., 1997). IAPs suppress these mitochondrial dependent pathways by inhibiting the activation of caspase-9--the apical caspase in the cytochrome c pathway for cell death (Deveraux et al., 1998,1997; Roy et al., 1997; Tamm et al., 1998).

bind both the u n p r o c e s s e d and processed forms o f caspase-9 ( D e v e r a u x et al., 1998, 1997; R o y et al., 1997). R e c e n t data indicate that significant differences exist b e t w e e n the activation m e c h a n i s m o f caspase-9 c o m p a r e d to other caspases. A c t i v a t i o n o f caspase-9 does not absolutely require its cleavage, but does require association with active Apaf-1 (Stennicke et al., 1999). T h e apparent ability o f u n p r o c e s s e d caspase-9 to a s s u m e an active c o n f o r m a t i o n m a y , therefore, explain w h y I A P s can b i n d to it.

lAP-Family Proteins 307 The BIR regions (BIR 1, 2 and 3) of human XIAP, c-IAP1 and c-IAP2 were found to be necessary and sufficient for their caspase inhibitory and anti-apoptotic activities (Deveraux et al., 1997; Roy et al., 1997). Subsequently, the caspase inhibitory activity and anti-apoptotic activity of XIAP was localized specifically to the second (BIR2) of its three tandem BIR domains (Takahashi et al., 1998), indicating that a single BIR domain can be sufficient to potently inhibit caspases (Ki < 2 nM for XIAP-BIR2 measured against caspases-3 and -7). Similarly, the smallest of the known lAP family proteins, Survivin, which contains a single BIR domain, can bind caspases and prevent caspase-induced apoptosis (Tamm et al., 1998). Surprisingly, the BIR1 and BIR3 domains of XIAP apparently lack caspase-binding capability, despite their striking amino-acid similarity to BIR2 (42% for BIR1; 32% for BIR3). Assuming these results cannot be ascribed to trivial explanations such as misfolding of protein fragments taken out of their normal context of the intact protein, these observations suggest that not all BIR domains are created equal. Thus, it is plausible that BIR domains within the same protein may have distinct functions. An analogous situation can be found in equistatin a sea anemone protein composed of three thyroglobulin-typel domains known to inhibit papain-like cysteine proteinases, papain, and cathepsins. Only the first (N-terminal domain) of these three thyroglobulin-typel motifs inhibits papain whereas cathepsin D inhibitory activity maps to the C-terminal domains. The lAPs appear to bind and inhibit their caspase targets at either a 1:1 or 2:1 molar ratio, possibly reflecting the presence of two active sites per enzyme (Deveraux et al., 1997; Roy et al., 1997). The inhibitory constants (Ki's) for XIAP, c-IAP1 and c-IAP2 measured against caspases-3 and -7 range from -0.2 nM to 10 nM, indicating that the lAPs are quite potent protease inhibitors. Although the mechanism of caspase inhibition by the human lAPs is currently unknown, one might suspect they function like other caspase inhibitors, such as the viral proteins CrmA or p35 (discussed below). However, the inhibitory mechanism of the lAPs does not appear to involve peptide bond hydrolysis (Deveraux et al., 1997; Roy et al., 1997). In contrast, p35 is hydrolyzed by its caspase targets and remains tightly bound to the active site (Zhou et al., 1998). lAPs, however, may still function as competitive inhibitors of caspases -- similar to the Kunitz, Kazal and Eglin families of serine protease inhibitors which possess loops that conform to the catalytic pocket of their target protease(s)(Bode and Huber, 1991). Likewise, the cystatins contain loops that adapt to active sites of the papain family of cysteine proteases (Turk and Bode, 1991). In these mechanisms, the loop region binds tightly to the catalytic groove of the protease, yet no peptide bond hydrolysis is observed. An alternative possibility is suggested by the observation that caspases use cysteine and histidine residues as part of their catalytic mechanism and these proteases can be inhibited by metals such as zinc (Perry et al., 1997; Stennicke and Salvesen, 1997). Thus, it is possible that metal binding by the BIR domain may play a role in the caspase inhibitory mechanism employed by the lAP-family proteins.

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IAP and Signal Transduction lAP family of proteins have been linked to signal transduction pathways utilized by members of the TNF Receptor (TNFR) family as well as some other types of receptors. Though the extracellular domains of all members of the TNFR family share amino acid sequence homology and a characteristic spacing of cysteines involved in disulfide bond formation, their intracellular domains can vary. TNFR1 and TNFR2 are the prototypical representatives of the two major branches of this receptor family. The cytosolic domain of TNFR1 contains a Death Domain (DD) which has been linked to pro-caspase-8 activation via the adapter protein Tradd. The Tradd protein also contains a DD and interacts not only with TNFR1 and itself, but also with Fadd, another DD-containing protein which in turn binds pro-caspase-8 via a homophilic interaction motif called the Death Effector Domain (DED) (Reviewed in Singh et al., 1998). In this way, TNFR1 can trigger caspase-8 activation and apoptosis, analogous to Fas. Studies have shown that activation of NF-KB prevents apoptosis by inducing expression of Mn-SOD and other protective gene products (Cheng et al., 1994; Barger et al., 1995; Mattson et al., 1997). In addition to binding Fadd, Tradd also binds another type of signaling protein that induces NF-KB activation and promotes cell survival. These signaling proteins are called TRAFs, for TNF Receptor Associated Factors (reviewed in (Arch et al., 1998; Baker and Reddy, 1996). Several TRAF-family proteins have been identified and at least three of them, TRAF-2, TRAF-5, and TRAF-6, have been shown to activate NF-•B apparently through their ability to bind an NF-~B inducing kinase, NIK. Through the concerted actions of additional kinases, the ultimate result is phosphorylation of I-KB on serine 32 and 36, thus targeting this NF-icB inhibitor for polyubiquitination and degradation by the 26S proteasome (Mercurio et al., 1997; Regnier et al., 1997; Zandi et al., 1998). Unlike TNFR1, which requires an adapter protein, the cytosolic domains of TNFR2 and a variety of other TNF-family cytokine receptors, including CD27, CD30, and CD40, directly bind TRAFs involved in NF-lcB activation. However, these TNFR2-1ike-family members do not bind Tradd-like adapter proteins and do not recruit cell death proteases. Thus, while TNFRI directly triggers caspase-8 recruitment and activation, the TNFR2-1ike subgroup of receptors does not. Moreover, the TNFR2-branch of the TNF-family receptors typically stimulates cell proliferation and survival rather than death. How are the IAPs relevant to these TNF-family receptor signaling complexes? The human c-IAP1 and c-IAP2 proteins were first discovered by virtue of their association with TNFR2 receptor complexes (Rothe et al., 1995). c-IAP1 and c-IAP2 do not directly contact TNFR2, but are recruited to the receptor by binding to TRAF-1/TRAF-2 heterocomplexes (Rothe et al., 1995). The N-terminal BIR-containing region of these lAPs is required for interactions with TRAFs. The interaction of c-IAPI and c-IAP2 with TRAF-1 and TRAF-2 appears to be specific, in that (a) these lAPs do not bind to TRAF-3, 4, 5, or 6 and (b) other lAPs (XIAP, NAIP) reportedly fail to bind TRAFs altogether (Roy, et al., 1997). Thus, TRAF binding is not a universal feature of lAP-family proteins.

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309 c-IAP2 has been functionally implicated in TNF-induction of NF-KB and protection from apoptosis (Chu et al., 1997). First, TNF has been shown to induce expression of c-IAP2 though stimulation of NF-•B. Second, over-expression of c-IAP2 reportedly can also lead to NF-KB activation. Third, c-IAP2 expression suppresses cell death induced by TNF through the receptor TNFR1. These c-IAP2 activities are reportedly blocked in cells by co-expressing a dominant form of I-KB that is resistant to TNF-induced degradation, implying that c-IAP2 participates in a positive feedback mechanism regulating NF-KB activation by targeting I-~cB degradation. Moreover, a mutant of c-IAP2 lacking the C-terminal RING domain inhibited NF-KB induction by TNF and enhanced TNF killing. Based upon these findings, the authors suggested that c-lAP2 is critically involved in TNF signaling events that induce NF-KB and that are required for suppression of TNF-induced apoptosis (Chu et al., 1997). In this regard, despite the ability of TNFR1 to induce activation of caspase-8, TNFc~ is not cytotoxic in most types of cells unless inhibitors of macromolecular synthesis are also applied, thus preventing gene expression. This paradox has been attributed to parallel activation of the aforementioned NF-KB pathway that induces the expression of several anti-apoptotic genes, including certain IAPs. The question that remains is whether induction of lAP-family genes is critical for the anti-apoptotic effect of NF-~B. Studies of the effects of TNF on lAP-family gene expression in endothelial cells suggests the answer to the first of these questions may be difficult to obtain due to redundancy in IAP family genes. Transcription of c-IAP1, c-IAP2, and XIAP genes was found to be strongly upregulated upon treatment of endothelial cells with the TNFct, interleukin 113, and LPS-reagents that lead to NF-KB activation (Stehlik et al., 1998). In these studies, overexpression of I-KB suppressed NF-KB activation and prevented the induction of all these lAP-family genes. I-KB over-expression also sensitized endothelial cells to TNF-induced apoptosis. Ectopic expression of at least one of the IAPs, XIAP, suppressed the I-KB effect, thereby protecting endothelial cells from TNF-induced apoptosis, suggesting that XIAP represents one of the NF-KB-regulated genes that can counteract the apoptotic signals caused by TNF-induced activation of caspase-8 (Stehlik et al., 1998). Thus, while we do not know whether lAP expression is necessary for NF-~B-mediated protection against TNF, it is sufficient. These findings also indicate that it may be worth considering whether dysfunctional regulation of the IAPs occurs in sepsis and some inflammatory conditions, where cytokine-induced endothelial cell death occurs. At what point or points in the apoptotic cascade do the lAPs interfere with TNF-induced apoptosis? Recently, NF-KB was reported to block TNF-induced activation of pro-caspase-8 (Wang et al., 1998). Under conditions in which NF-KB activation was prevented with dominant-negative I-KB, gene transfection studies suggested that the combination of TRAF-1, TRAF-2, c-IAP1 and c-IAP2 was needed to substitute for NF-KB and fully suppress TNF-induced apoptosis. In the same cells, however, either c-IAP1 or c-IAP2 alone was sufficient to suppress apoptosis induced by etoposide -- a stimulus that appears to indirectly enter the apoptosis pathway primarily at the level of mitochondria (Wang et al., 1998). The implication is that c-IAP1 and c-IAP2 require TRAF-1 and TRAF-2 to interfere with the upstream cell death protease, caspase-8, but not for inhibiting caspases that operate downstream of mitochondria.

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Moreover, these and other data (Deveraux et al., 1998; McCarthy and Dixit, 1998), suggest that the lAPs have the ability to regulate different apoptotic pathways or distinct steps within the same apoptotic pathway, possibly depending on what additional proteins the lAPs bind, such as TRAFs. In addition to NF-KB, one study has provided evidence that some IAP-family proteins can regulate the Jun N-terminal kinase (JNK) pathway (Sanna et al., 1998). Specifically, JNK1 activation induced by over-expression of pro-caspase-1 is reportedly augmented by co-expression of XIAP. Moreover, XIAP blocked cell death induced by over-expression of pro-caspase-1. Intriguingly, XIAP's ability to suppress cell death in this context was abrogated by co-expression of a dominant-negative form of JNK1 (Sanna et al., 1998). However, the mechanism by which activated JNK1 might suppress apoptosis in this or other systems has not been elucidated. Moreover, since JNK1 has been shown to be a pro-apoptotic component in several instances (Schulze-Osthoff et al., 1998), it seems unlikely that JNK activation is the predominant mechanism used by lAP-family proteins to suppress cell death. Though unexplored to date, it is tempting to speculate that the functional connection of XIAP to pro-caspase-1 may somehow be related to the observation that at least some lAPs can interact with a pro-caspase-1 activating protein, CARDIAK/RIP-2 (McCarthy et al., 1998). A further connection between lAPs and receptor signaling comes from recent studies in Xenopus embryos (Yamaguchi et al., 1999). A member of the MAP kinase kinase family TAK1 and its activator, TAB1, participate in the bone morphogenetic protein (BMP) signaling pathway involved in mesoderm induction and patterning in early Xenopus embryos. XIAP reportedly associates not only with TAB1 but also with the BMP receptors (Yamaguchi et al., 1999). Interestingly, while the BIR domains of XIAP are required for TAB 1 binding, the C-terminal RING domain mediates interaction of XIAP with the BMP receptor. Thus, XIAP appears to function as an adapter, linking TABI to the BMP receptor. Receptor bound TAB1 then activates TAK1 -- thereby potentiating the signal(s) leading to mesoderm induction. In support of this idea, the authors found that injection of XIAP mRNA into dorsal blastomeres enhanced the ventralization of Xenopus embryos in a TAB1-TAKl-dependent manner, and a truncated form of XIAP lacking the TAB 1-binding domain partially blocked the expression of ventral mesoderrnal marker genes induced by a constitutively active BMP receptor. Interestingly, overexpression of TAK1 induces apoptosis that can be suppressed by co-expression of XIAP (Yamaguchi et al., 1999). Therefore, XIAP may control the decision between cell death and differentiation -- thus potentiating mesoderm induction signaling while blocking apoptosis.

Insights into IAPs from Insects lAPs were first discovered in insect viruses (Birubaum et al., 1994; Crook et al., 1993). Possibly one of the most ancient evolutionary pressures for a cell suicide program can be attributed to viruses. Death of infected host cells stymies viral propagation -- thereby protecting uninfected neighboring cells. However, many viruses have coevolved strategies for promoting cell survival by targeting conserved steps in the hosts

lAP-Family Proteins 311 cell death program (Bump et al., 1995; Komiyama et al., 1994; Zhou et al., 1997). An example of a viral regulator of apoptosis is the coxpox virus protein CrmA, a serpin-family protease inhibitor that exhibits specificity for caspases-1 and 8, with inhibitory constants (Ki's) of 0.01 and 0.95 nM, respectively. Another viral inhibitor of caspases is the baculovirus protein p35, which is structurally distinct from CrmA and has no apparent cellular homolog. The p35 protein has broad inhibitory activity against most of the caspase-family enzymes, with Ki's typically of ~ 1 nM (Komiyama et al., 1994; Zhou and Salvesen, 1997). In addition to p35, baculoviruses also encode lAPs (Clem and Miller, 1994). The BIR, RING and other sequences in the lAPs exhibit no similarity to p35. Despite their apparent lack of structural similarity to p35, the lAP proteins encoded by the Orgyia pseudotsugata (Op) and Cydia pomonella (Cp) baculoviruses, have been shown by genetic complementation analysis to functionally overlap with p35 with respect to suppression of the insect cell death response to viral infection. Similar to p35, ectopic expression of baculoviral OplAP or CplAP protects both insect and mammalian cells from apoptosis induced by a variety of stimuli, including caspase overexpression -- observations that are consistent with the idea that the IAPs block apoptosis at an evolutionary conserved point common to many apoptotic programs (Clem and Miller, 1994; Hawkins et al., 1996). Though lAPs can complement p35-deficient baculoviruses (Clem and Miller, 1994), some studies suggest that IAPs and p35 suppress apoptosis by distinct mechanisms. For example, OplAP and p35 were reported to exhibit differences in their abilities to protect PC12 cells from apoptosis induced by serum withdrawal, leading the authors to conclude that they function by distinct mechanisms (Hawkins et al., 1998). Moreover, in insect cells from the fall armyworm Spodoptera frugiperda (Sf 21 cells), p35 reportedly blocks apoptosis induced by transfection of plasmids encoding active SF-caspase-1, an insect caspase that becomes activated during the course of baculoviral infection (Sheshagiri and Miller, 1997). Although overexpression of Op-IAP can prevent processing of the pro-form of SF-caspase-1 and suppress apoptosis induced by viral infection, it does not block cell death induced by direct gene transfer-mediated expression of active Sf-caspase-1 (Sheshagiri and Miller, 1997). This observation implies that Op-IAP can interfere with upstream mechanisms responsible for activation of Sf pro-caspase-1 but, unlike p35, it is not a direct inhibitor of this particular protease. These results can be interpreted at least two ways: either Op-IAP is not a caspase inhibitor or Op-IAP is a caspase inhibitor but Sf-caspase-1 is downstream of the caspases it targets. This latter argument has its basis in the observation that the human lAP-family proteins XIAP, c-IAPI,c-IAP2, and Survivin reportedly bind and inhibit selected caspases but not all caspases, unlike p35, which is a broad-specificity inhibitor of these enzymes (Figure 3). Additional insights into the mechanisms of lAP-family proteins have come from genetic and biochemical analysis of the cell death pathway in Drosophila. The baculoviral Op-IAP and Cp-IAP proteins and the Drosophila lAP-family proteins DIAP-1 and DIAP-2 have been shown to bind the Drosophila cell death proteins reaper (RPR), HID and GRIM (Vucic et al., 1997, 1998a). Expression of RPR, HID or GRIM in the fly or in cultured insect cells promotes apoptosis that can be suppressed by co-expression of viral Op-IAP or Cp-IAP or of cellular lAPs, DIAP-1 and DIAP-2. The RPR, HID, and

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lAP-Family Proteins 313 GRIM proteins contain a conserved N-terminal 14 amino acid sequence that has been shown to be necessary and sufficient for the inducing apoptosis and for binding to either the baculoviral or endogenous cellular lAPs (Vucic et al., 1998a). Thus, binding to lAPs appears to be the common feature that unifies these Drosophila cell death proteins (FIGURE 3). The RPR, HID, and GRIM proteins, however, do not bind the baculovirus p35 protein, indicating that the lAPs and p35 either suppress death through distinct mechanisms or are subject to differential regulation, at least with respect to HID, GRIM, and RPR. In this regard, it was recently shown that DIAP-1 can block apoptosis induced by the Drosophila drlCE, insect Sf-caspase-1, and mammalian caspase-3, in insect SF-21 cells (Kaiser et al., 1998). DIAP-1 inhibited apoptosis induced by the active forms of all three caspases and physically interacted with the active, but not the proform of drlCE. The mouse homolog of XIAP also interacted with and blocked apoptosis induced by active drlCE but was relatively ineffective in blocking Sf-caspase-1 (Kaiser et al., 1998). In these assays, Op-IAP and DIAP-2 were unable to inhibit effectively any of the active caspases tested and failed to interact with drlCE. These observations, therefore, provide further evidence that caspase inhibition is an evolutionarily conserved mechanism by which at least some of the lAPs block cell death and reinforce the idea that lAPs inhibit some members of the caspase family but not others (Deveraux et al., 1997). Another more recently described cell death gene identified in the fly, DOOM also encodes an lAP-binding protein (Harvey et al., 1997), Thus, the lAPs appear to define a major point of convergence for regulation of the cell death pathway in the fly (Figure 3). It is unclear, however, whether the various Drosophila proteins described above are cell death inducers that are inhibited by the lAPs, or conversely whether they are trans-dominant inhibitors of the lAPs which free an IAP-inhibitable protein(s) so that it can induce apoptosis (e.g. a caspase). Evolutionary conservation of the mechanisms used by RPR and GRIM to induce apoptosis has been suggested by recent studies indicating that these fly cell death proteins can induce apoptosis in mammalian cells (McCarthy and Dixit, 1998). RPR or GRIM-induced apoptosis of mammalian cells was reportedly inhibited by a broad range of protein and peptidyl caspase inhibitors, as well as by the human c-IAP1 and c-IAP2 proteins. Additionally, in vivo binding studies demonstrated that both RPR and GRIM physically interacted with human lAPs, and required the homologous 14-amino acid N-terminal segment for lAP binding. When expressed in mammalian cells, RPR and GRIM co-localized with c-IAP1 to a perinuclear location. Deletion of the N-terminal 15 amino acids of RPR or GRIM abolished co-localization with c-IAP1. However, these N-terminal deletion mutants still promoted apoptosis, which was also still suppressed by co-expression of c-IAP1 or c-IAP2. The authors suggested that even though these RPR and HID mutants fail to bind lAPs, the c-lAPs are nevertheless able to attenuate death, presumably by inhibiting downstream active caspases (McCarthy and Dixit, 1998). The lAPs, therefore, may possess multiple mechanisms for inhibiting cell death pathways at several distinct steps. Interestingly, an effector (DREDD) of RPR, HID and GRIM-mediated apoptosis was recently discovered and appears to be a caspase (Chen et al., 1998a). Heterozygosity at the DREDD locus suppressed RPR- and GRIM-induced apoptosis in transgenic flies

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implying that the relative abundance of the DREDD protein can significantly influence the extent to which RPR and GRIM retain pro-apoptotic function. In co-transfection experiments, RPR, GRIM, and HID also trigger caspase-like processing of DREDD. Based upon the observation that RPR is sufficient to initiate caspase activity in a cell free system (Evans et al., 1997), the authors suggested that RPR, HID and GRIM might engage the apoptotic pathway via this novel caspase (Chen et al., 1998a). It would be interesting to determine if the viral and fly lAPs bind to and inhibit the caspase DREDD. Since it has not been shown that lAPs can directly bind HID, GRIM and RPR using purified proteins, it is conceivable that the lAPs actually bind DREDD and that their reported association with HID, GRIM and RPR is an indirect consequence of the binding of these death proteins to DREDD rather than the lAPs within a multiprotein complex. Other mechanisms, however, may be involved in apoptosis induced by HID, GRIM and RPR, including modulation of voltage-gated potassium (K+) channels (Avdonin et al., 1998) and interactions with other apoptosis regulating proteins such as Scythe (Thress et al., 1998). If RPR, HID and GRIM do trigger apoptosis through effects on K+ channels or interactions with SCYTHE, these events result in caspase activation and caspase inhibitors, such as p35, potently suppresses apoptosis induced by these Drosophila death proteins both in cell culture models and in transgenic flies. Thus, an lAP connection to caspase inhibition might still be involved.

Summary IAP family proteins are conserved throughout animal evolution and can block apoptosis when expressed in cells derived from multiple species often suppressing apoptosis across species barriers (Reviewed in Clem and Duckett, 1998). Thus, while the details of their regulation may vary, these proteins evidently target a common mechanism involved in programmed cell death. Until proven otherwise, the most likely explanation for how lAPs prevent apoptosis is by binding to and inhibiting caspases, as indicated by recent studies (Deveraux et al., 1998, 1997; Roy et al., 1997; Takahashi et al., 1998; Tamm et al., 1998). (Kaiser et al., 1998; Kobayashi et al., 1999). Though all lAP-family proteins require at least one BIR domain for their anti-apoptotic function, it should be emphasized that not all BIR-containing proteins are necessarily involved in apoptosis regulation, as indicated by the failure of the Ac-IAP protein to suppress apoptosis despite harboring a BIR-domain. Although these, apparently non-functional, BIR domains may be evolutionary remnants, the high degree of primary sequence conservation among BIR domains indicates constraints on variance, even for BIR domains that are not anti-apoptotic. Thus, it seems likely that BIR domains play roles in aspects of cellular biology distinct from cell death or in facets of apoptosis not revealed by contemporary assays. In this regard, it is interesting to note the occurrence of sequences with high similarity to BIR domains in yeast, since it is debatable as to whether yeast possess an apoptosis program, and if they do, it does not appear to involve caspases (Fraser and James, 1998; J0rgensmeier et al., 1997; Madeo et al., 1997; Shaham et al., 1998; Zha et al., 1996).

lAP-Family Proteins 315 If lAPs function primarily as inhibitors of caspases, then we can anticipate from other experiments where artificial means were used to suppress these proteases that lAPs will be capable of rescuing cells from some cell death signals, but not others, depending on the type of cell under investigation and the specific cell death stimulus involved (Green and Reed, 1998). Mitochondrial involvement appears to be one of the key variables that determines whether caspase inhibitors are sufficient to provide long-term protection and preservation of clonigenic potential, versus merely delaying death by converting an apoptotic stimulus into a necrotic one (Green and Reed, 1998; Reed, 1997). In many types of cells, loss of cytochrome c from mitochondria, for example, has two ways of killing cells: (a) activation of caspases via Apaf-1; or (b) cessation of mitochondrial electron-chain transport with subsequent ATP depletion, generation of reactive oxygen species, and related sequellae. If the role of IAPs is relegated to caspase suppression, this may prevent cytochrome c-induced apoptosis but not necessarily stop cell death induced by caspase-independent mechanisms. Recent studies in which release of cytochrome c was found to be a potentially reversible event suggest that whether cytochrome c loss from mitochondria defines a cell death commitment point will likely vary among cell-types and depending on a variety environmental factors (Chen et al., 1998b). These factors may include the extent to which cells are able to produce sufficient ATP from anaerobic glycolysis in the cytosol and the method by which mitochondrial membrane barrier function was altered to allow for exodus of cytochrome c (i.e. reversible versus irreversible/rupture). Defining the in vivo requirements for IAPs in the maintenance of cell survival may be difficult because of potential redundancy. Humans have at least five and possibly more lAP family genes and lower organisms, such as Drosophila, appear to contain at least two lAP genes, implying evolutionary pressure to ensure adequate back up if one of these genes were to become inactivated. If lAPs do indeed function predominantly as caspase inhibitors, then one could imagine a very important role for these endogenous protease inhibitors in ensuring that the small amounts of adventitial caspase activation, that must surely occur on a routine basis, do not amplify out of control, resulting in inappropriate cell death. In this regard, virtually every other protease system studied to date contains molecules whose sole function is directed toward dampening proteolysis through the cascade (Reviewed in Colman et al., 1994), thus, ensuring that biologically appropriate triggering of the pathway only occurs when certain thresholds are surpassed. By analogy, it is attractive to consider lAP-family proteins in the same way, as proteins that set thresholds for how much caspase activation is necessary to successfully trigger apoptosis. Through alterations in the levels of lAP-family gene expression, stability, localization or interactions of lAPs with other proteins, this lAP-dependent threshold for caspase-induced apoptosis could be varied to suit various physiological needs. Dysregulation of these normal control mechanisms then could be a contributor to various diseases characterized by excessive (ischemia, AIDS, SMA) or inadequate (cancer, autoimmunity) cell death.

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Acknowledgments We thank Guy Salvesen and numerous members of our laboratory for their stimulating comments, T. Brown and E. Smith for manuscript and figure preparation, and the Leukemia Society of American (QLD; Fellow) as well as the NCI, NIA, and California Breast Cancer Research Program (JCR) for their generous financial support.

References Adida, C., Crotty, P.L., McGrath, J., Berrebi, D., Diebold, J. & Altieri, D.C. (1998). Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation. Am. J. Pathol. 152, 43-49. Alnemri, E.S., Livingston, D. J., Nicholson, D. W., Salvesen, G., Thornberry, N.A., Wong, W.W. & Yuan, J. (1996). Human ICE/CED-3 Protease Nomenclature. Cell 87, 171. Ambrosini, G., Adida, C. & Altieri, D. (1997). A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat. Med. 3, 917-921. Ambrosini, G., Adida, C., Sirugo, G. & Altieri, D.C. (1998. Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting. J. Biol. Chem. 273, 11177-11182. Arch, R.H., Gedrich, R.W. & Thompson, G.B. (1998). Tumor necrosis factor receptor-associated factors (TRAFs)--a family of adapter protein that regulates life and death. Genes and Dev. 12, 2821-30. Avdonin, V., Kasuya, J., Ciorba, M.A., Kaplan, B., Hoshi, T. & Iverson, L. (1998). Apoptotic proteins reaper and grim induce stable inactivation in voltage-gated K+ channels. Proc. Natl. Acad. Sci. U.S.A. 95, 11703-11708. Baker, S.J. & Reddy, E.P. (1996). Transducers of life and death: TNF receptor superfamily and associated proteins. Oncogene 12, 1-9. Barger, S.W., Horster, D., Furukawa, Y., Goodman, Y., Krieglstein, J. & Mattson, M.P. (1995). Tumor necrosis factors ct and 13 protect neurons against amyloid 13-peptide toxicity: evidence for involvement of ~:B-binding factor and attenuation of peroxide and Ca 2+ accumulation. Proc. Natl. Acad. Sci. U.S.A. 92, 9328-9332. Barlow, P.N., Luisi, B., Milner, A., Elliott, M., Everett, R. (1994). Structure of the C3HC4 domain by 1H-nuclear magnetic resonance spectroscopy. A new structural class of zinc-finger. J. Mol. Biol. 237, 201-211. Birnbaum, M.J., Clem, R.J. & Miller, L.K. (1994). An apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs. J. Virol. 68, 2521-2528. Bode, W. & Huber, R. (1991). Proteinase-protein inhibitor interaction. Biomed. Biochim. Acta 50, 437-446. Borden, K.L. & Freemont, P.S. (1996). The RING finger domain: a recent example of a sequence-structure family. Curr. Opin. Struct. Biol. 6, 395-401. Bossy-Wetzel, E., Newmeyer, D.D. & Green, D.R. (1998). Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. Embo J. 17, 37-49. Bump, N.J., Hackett, M., Hugunin, M., Seshagiri, S., Brady, K. Chen, P., Ferenz, C., Franklin, S., Ghayur, T. & Li, P. (1995). Inhibition of ICE family proteases by baculovirus antiapoptotic protein p35. Science 269, 1885-1888. Chen, P., Rodriguez, A., Erskine, R., Thach, T. & Abrams, J.M. (1998a). Dredd, a novel effector of the

lAP-Family Proteins 317 apoptosis activators reaper, grim, and hid in drosophila. Dev. Biol. 210, 202-16. Chen, Q., Takeyama, N., Brady, G., Watson, A.J.M. & Dive, C. (1998b). Blood cells with reduced mitochondrial membrane potential and cytosolic cytochrome c can survive and maintain clonogenicity given appropriate signals to suppress apoptosis. Blood 92, 4545-4553. Cheng, B., Christakos, S. & Mattson, M.P. (1994). Tumor necrosis factors protect neurons against excitotoxic/metabolic insults and promote maintenance of calcium homeostasis. Neuron 12, 139-153. Chu, Z.L., McKinsey, T.A., Liu, L., Gentry, J.J., Malim, M.H. & Ballard, D.W. (1997). Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-lAP2 is under NF-~B control. Proc. Natl. Acad. Sci. U.S.A. 94, 10057-10062. Clem, R.J. & Duckett, C.S. (1998). The lAP genes: unique arbitrators of cell death. TIBS 23, 159-162. Clem, R.J. & Miller, LK. (1994). Control of programmed cell death by the baculovirus genes p35 and iap. Mol.Cell.Biol. 14, 5212-5222. Colman, R.W., Marder, V.J., Salzman, E.W. & Hirsh, J. (1994). Overview of Hemostasis. Hemostasis and Thrombosis 3rd Edition, 3-18. Crook, N.E., Clem, R.J. & Miller, L.K. (1993). An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif. J. Virol. 67, 2168-74. Deveraux, Q.L., Roy, N., Stennicke, H.R., Van Arsdale, T., Zhou, Q. Srinivascula, S.M., Alnemri, E.S., Salvensen, G.C. & Reed, J.C. (1998). lAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J. 17, 2215-2223. Deveraux, Q.L., Takahashi, R., Salvesen, G.S. & Reed, J.C. (1997). X-linked lAP is a direct inhibitor of cell death proteases. Nature 388,300-303. Duckett, C.S., Li, F., Tomaselli, K.J., Thompson, C.B. & Armstrong, R.C. (1998). Human lAP-like protein regulates programmed cell death downstream of BcI-XL and cytochrome c. Mol. Cell. Biol. 18, 608-15. Duckett, C.S., Nava, V.E., Gedrich, R.W., Clem, R.J., Van Dongen, J.L. Gilfallan, M.C., Shiels, H., Harwick, J.M. & Thompson, C.B. (1996). A conserved family of cellular genes related to the baculovirus iap gene and encoding apoptosis inhibitors. EMBO J. 15, 2685-2689. Evans, E.K., Kuwana, T., Strum, S.L., Smith, J.J., Newmeyer, D.D. & Kornbluth, S. (1997). Reaper-induced apoptosis in a vertebrate system. EMBO J. 16, 7372-81. Finucane, D.M., Bossy-Wetzel, E., Cotter, T.G. & Green, D.R. (1998). Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by BcI-XL. J. Biol. Chem. 1999 274, 2225-33. Fraser, A. & James, C. (1998). Fermenting debate: do yeast undergo apoptosis? Trends Cell Biol. 8, 219-21. Green, D. & Reed, J. (1998). Mitochondria and apoptosis. Science 281, 1309-1312. Hakem, R., Hakem, A., Duncan, G.S., Henderson, J.T., Woo, M. Soongas, M.S. Elia, A., de la Pompa, J.L, Kagi, D. Khoo, W. Potter, J., Yoshida, R., Kaufman, S.A., Lowe, S.W., Penninger, J.M. & Mak, T.W. (1998). Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94, 339-352. Harvey, A.J., Bidwai, A.P. & Miller, LK. (1997). Doom, a product of the Drosophila mod(mdg4) gene, induces apoptosis and binds to baculovirus inhibitor-of-apoptosis proteins. Mol. Cell. Biol. 17, 2835-43. Harvey, A.J., Soliman, H., Kaiser, W. & Miller, L.K. (1997). Anti- and Pro-apoptotic activities of baculovirus and Drosophila lAPs in an insect cell line. Cell Death & Diff. 4, 733-744. Hauser, H.P., Bardroff, M., Pyrowolakis, G. & Jentsch, S. (1998a). A giant ubiquitin-conjugating enzyme related to lAP apoptosis inhibitors. J. Cell. Biol. 141, 1415-22. Hauser, H.P., Bardroff, M., Pyrowolakis, G. & Jentsch, S. (1998b). A giant ubiquitin-conjugating enzyme related to lAP apoptosis inhibitors. J. Cell. Biol. 141, 1415-1422. Hawkins, C.J., Ekert, P.G., Uren, A.G., Holmgreen, S.P. & Vaux, D.L. (1998). Anti-apoptotic potential of

318

Q.L. Deveraux and J.C. Reed

insect cellular and viral IAPs in mammalain cells. Cell Death Differ. 5,569-576. Hawkins, C.J., Uren, A.G., Hacker, G., Medcalf, R.L. & Vaux, D.L. (1996). Inhibition of interleukin lb-converting enzyme-mediated apoptosis of mammalian cells by baculovirus lAP. Proc. Natl. Acad. Sci. U.S.A. 93, 13786-13790. Hay, B.A., Wassarman, D.A. & Rubin, G.M. (1995). Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell 83, 1253-1262. Higgins, D.G. & Sharp, P.M. (1989). Fast and sensitive multiple sequence alignments on a microcomputer. CABIOS 5, 151-153. Iwahashi, H., Eguchi, Y., Yasuhara, N., Hanafusa, T., Matsuzawa, Y. & Tsujimoto, Y. (1997). Synergistic anti-apoptotic activity between bcl-2 and smn implicated in spinal muscular atrophy. Nature 390, 413-417. Juo, P., Kuo, C.J., Yuan, J. & Blenis, J. (1998). Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade. Curr. Biol. 8, 1001-1008. Jiirgensmeier, J.M., Krajewski, S., Armstrong, R., Wilson, G.M., Oltersdorf, T., Fritz, L.C., Reed, J.C. & Ottilie, S. (1997). Bax- and Bak-induced cell death in the fission yeast Schizosaccharomyces pombe. Mol. Biol. Cell 8,325-229. Jiirgensmeier, J.M., Xie, Z., Deveraux, Q., Ellerby, L., Bredesen, D. & Reed, J.C. (1998). Bax directly induces release of cytochrome c from isolated mitochondria. Proc. Natl. Acad. Sci. U S A 95, 4997-5002. Kaiser, W.J., Vucic, D. & Miller, L.K. (1998). The Drosophila inhibitor of apoptosis D-IAP1 suppresses cell death induced by the caspase drlCE. FEBS Letters 440, 243-248. Kharbanda, S., Pandey, P., Schofield, L., Israel, S., Roncinske, R. Yoshida, K., Bharti, A., Yuan, Z.M., Saxena, S., Weichselbaum, R., Nalin, C. & Kufe D. (1997). Role for Bcl-XL as an inhibitor of cytosolic cytochrome C accumulation in DNA damage-induced apoptosis. Proc. Natl. Acad. Sci. U.S.A. 94, 6939-42. Kluck, R.M., Bossy-Wetzel, E., Green, D.R. & Newmeyer, D.D. (1997). The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 1132-1136. Kobayashi, K., Hatano, M., Otaki, M., Ogasawara, T. & Tokuhisa, T. (1999). Expression of a murine homologue of the inhibitor of apoptosis protein is related to cell proliferation. Proc. Natl. Acad. Sci. U.S.A. 96, 1457-1462. Komiyama, T., Ray, C.A., Pickup, D.J., Howard, A.D., Thornberry, N.A., Peterson, E.P. & Salvesen, G. (1994). Inhibition of interleukin-1 beta converting enzyme by the cowpox virus serpin CrmA. An example of cross-class inhibition. J. Biol. Chem. 269, 19331-19337. Kuida, K., Haydar, T.F., Kuan, C.Y., Gu, Y., Taya, C. Karasuyama, H., Su, M.S. Rakic, P. & Flavell, R.A. (1998). Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 94, 325-337. Li, F., Ambrosini, G., Chu, E.Y., Plescia, J., Tognin, S., Marchisio, P.C. & Alteri, D.C. (1998a). Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396, 580-584. Li, J., Kim, J.M., Liston, P., Li, M., Miyazaki, T., Mackenzie, A.E., Komeluk, R.G. & Tsang, B.K. (1998b). Expression of inhibitor of apoptosis proteins (IAPs) in rat granulosa cells during ovarian follicular development and atresia. Endocrinology 139, 1321-1328. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S., Ahmad, M., Alnemri, E.S. & Wang, X. (1997). Cytochrome c and dATP-dependent formation of Apaf-1/Caspase-9 complex initiates an apoptotic protease cascade. Cell 91,479-489. Liston, P., Roy, N., Tamai, K., Lefebvre, C., Baird, S., Cherton-Horvat, G., Farahani, R., McLean, M., lkeda, J.E., MacKenzie, A. & Korneluk, R.G. (1996). Suppression of apoptosis in mammalian ceils by NAIP

lAP-Family Proteins 319 and a related family of IAP genes. Nature 379, 349-353. Liu, Q., Fischer, U., Wang, F. & Dreyfuss, G. (1997). The spinal muscular atrophy disease gene product, SMN, and its associated protein SIP1 are in a complex with spliceosomal snRNP proteins. Cell 90, 1013-1021. Liu, X., Kim, C.N., Yang, J., Jemmerson, R. & Wang, X. (1996). Induction of apoptotic program in cell-free extracts: requirement for dATP and Cytochrome C. Cell 86, 147-157. Lu, C.D., Altieri, D.C. & Tanigawa, N. (1998). Expression of a novel anti-apoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res 58, 1808-1812. Madeo, F., Frohlich, E. & Frohlich, K. U. (1997). A yeast mutant showing diagnostic markers of early and late apoptosis. J. Cell Biol 139, 729-734. Mahajan, N., Linder, K., Berry, G., Gordon, G., Heinm, R. & Herman, B. (1998). Bcl-2 and Bax Interactions in Individual Mitochondria Probed with Mutant Green Fluorscent Proteins and Fluorescence Resonance Energy Transfer. Nature - Biotechnology 16, 547-552. Martin, D., Siegel, R., Zheng, L. & Lenardo, M. (1998). Membrane oligomerization and cleavage activates the caspase-8 (FLICE/MACHal) death signal. J. Biol. Chem. 273, 4345-4349. Mattson, M.P., Goodman, Y., Huo, H., Fu, W. & Furukawa, K. (1997). Activation of NF-•B protect hippocampal neurons against oxidative stress-induced apoptosis: evidence for induction of Mn-SOD and suppression of peroxynitrite production and protein tyrosine nitration. J. Neurosci. Res. 49: 681-697. McCarthy, J.V. & Dixit, V.M. (1998). Apoptosis induced by Drosophila reaper and grim in a human system. Attenuation by inhibitor of apoptosis proteins (clAPs). J. Biol. Chem. 273, 24009-15. McCarthy, J.V., Ni, J. & Dixit, V.M. (1998). RIP2 Is a Novel NF-B-activating and Cell Death-inducing Kinase. J. Biol. Chem. 273, 16968-16975. Mercurio, F., Zhu, H., Murray, B.W., Shevchenko, A., Bennett, B.L., Li, J., Young, D.B., Barbosa, M., Mann, M., Manning, A. & Rao, A. (1997). IKK-1 and IKK-2: cytokine-activated IKB kinases essential for NF-KB activation. Science 278, 860-866. Muzio, M., Salvesen, G.S. & Dixit, V.M. (1997). FLICE induced apoptosis in a cell-free system. J. Biol. Chem. 272, 2952-2956. Orth, K. & Dixit, V.M. (1997). Bik and Bak induce apoptosis downstream of CrmA but upstream of inhibitor of apoptosis. J. Biol. Chem. 272, 8841-8844. Perry, D., Smyth, M., Stennicke, H., Salvesen, G., Duriez, P., Poirer, G.G., & Hannum, Y.W. (1997). Zinc is a potent inhibitor of the apoptotic protease, caspase-3. J. Biol. Chem. 272, 18530-18533. Rajcan-Separovic, E., Liston, P., Lefebvre, C. & Korneluk, R.G. (1996). Assignment of human inhibitor of apoptosis protein (lAP) genes xiap, hiap-1, and hiap-2 to chromosomes Xq25 and llq22-q23 by fluorescence in situ hybridization. Genomics 37,404-406. Reed, J.C. (1997). Cytochrome C: Can't live with it; Can't live without it. Cell 91,559-562. Regnier, C.H., Song, H.Y., Gao, X., Goeddel, D.V., Cao, Z. & Rothe, M. (1997). Identification and characterization of an IkappaB kinase. Cell 90, 373-83. Rothe, M., Pan, M.-G., Henzel, W.J., Ayres, T.M. & Goeddel, D.V. (1995). The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell 83, 1243 - 1252. Roy, N., Deveraux, Q.L., Takashashi, R., Salvesen, G.S. & Reed, J.C. (1997). The c-IAP-1 and c-IaP-2 proteins are direct inhibitors of specific caspases. EMBO J. 16, 6914-6925. Roy, N., Mahadevan, M.S., McLean, M., Shutler, G., Yaraghi, Z., Farahani, R., Baird, S. Besner-Johnston, A., Lefebvre, C. & Kang, X. (1995). The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy. Cell 80, 167-178.

320

Q.L. Deveraux and J.C. Reed

Salvesen, G.S. & Dixit, V.M. (1997). Caspases: intmcellular signaling by proteolysis. Cell 91,443-446. Sanna, M.G., Duckett, C.S., Richter, B.W., Thompson, C.B. & Ulevitch, R.J. (1998). Selective activation of JNK1 is necessary for the anti-apoptotic activity of hILP. Proc. Natl. Acad. Sci. U.S.A. 95, 6015-6020. Schielke, G.P., Yang, G.Y., Shivers, B.D. & Betz, A.L. (1998). Reduced ischemic brain injury in interleukin-1 beta converting enzyme-deficient mice. J. Cereb. Blood Flow Metab 18, 180-185. Schulze-Osthoff, K., Ferrafi, D., Los, M., Wesselborg, S. & Peter, M.E. (1998). Apoptosis signaling by death receptors. Eur. J. Biochem. 254, 439-459. Shaham, S., Shuman, M.A. & Herskowitz, I. (1998). Death-defying yeast identify novel apoptosis genes. Cell 92, 425-427. Sheshagiri, S. & Miller, L.K. (1997). Baculovirus inhibitors of apoptosis (IAPs) block activation of Sf-caspase-1. Proc. Natl. Acad. Sci. U.S.A. 94, 13606-13611. Simons, M., Beinroth, S., Gleichmann, M., Liston, P., Komeluk, R. G., MacKenzie, A.E., Bahr, M., Klockgether, T., Robertson, G.C., Weller, M. & Schulz, J.B. (1998). Adevovirus-Mediated Gene Transfer of lnhibitors of Apoptosis Proteins Delays Apoptosis in Cerebellar Granule Neurons. J. Neurochem. 1999 72, 292-301. Singh, A., Ni, J. & Aggarwal, B.B. (1998). Death domain receptors and their role in cell demise. Interferon Cytokine Res. 18,439-450. Srinivasula, S., Ahmad, M., Femandes-Alnemri, T. & Alnemri, E. (1998). Autoactivation of procaspase-9 by apaf-l-mediated oligomerization. Mol. Cell 1,949-957. Stehlik, C., Martin, R.D., Kumabashiri, I., Binder, B. R. & Lipp, J. (1998). NF-~cB regulated x-chromosomelinked IAP gene expression protects endothelial cells from TNF-a induced apoptosis. J. Exp. Med. 188, 211-216. Stennicke, H.R., Deveraux, Q.L., Humke, E. W., Reed, J.C., Dixit, V.M. & Salvesen, G.S. (1999). Caspase-9 can be activated without proteolytic processing. J. Biol. Chem. 1999 274, 8359-62. Stennicke, H.R., Jtirgensmeier, J.M., Shin, H., Deveraux, Q., Wolf, B.B., Yang, X., Shou, Q., Ellerby, H.M., EIlerby, L.M., Bredesen, D., Green, D.R., Reed, J.C., Froelich, C.J. & Salvesen, G.C. (1998). Pro-caspase-3 is a major physiologic target of caspase-8. J. Biol. Chem. 273, 27084-27090. Stennicke, H.R. & Salvesen, G.S. (1997). Biochemical characteristics of caspase-3, -6, -7, and -8. J. Biol. Chem. 272, 25719-25723. Stennicke, H.R. & Salvesen, G.S. (1998). Properties of the caspases. Biochim. Biophys. Acta 1387, 17-31. Takahashi, R., Deveraux, Q., Tamm, I., Welsh, K., Assa-Munt, N., Salvesen, G.S. & Reed, J.C. (1998). A single BIR domain of XIAP sufficient for inhibiting caspases. J.B.C. 273, 7787-7790. Tamm, I., Wang, Y., Sausville, E., Scudiero, D.A., Vigna, N., Oltersdorf, T. & Reed, J.C. (1998). IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas(CD95), Bax, and anticancer drugs. Cancer Res. 58, 5315-5320. Thome, M., Hofmann, K., Burns, K., Martinon, F., Bodmer, J.L., Mattmann, G. & Tschopp, J. (1998). Identification of CARDIAK, a RIP-like kinase that associates with caspase-1. Curr. Biol. 8,885-888. Thress, K., Henzel, W., Shillinglaw, W. & Kornbluth, S. (1998). Scythe, a novel reaper-binding apoptotic regulator. EMBO J. 17, 6135-6143. Turk, V. & Bode, W. (1991). The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett. 285, 213-219. Varfolomeev, E.E., Schuchmann, M., Luria, V., Chinnilkulchai, N., Beckmann, J.S., Mett, I.L., Rebfikov, D., Brodianski, V.M., Kemper, O.C., Kollet, O., Lapidot, T., Softer, D., Sobe, T., Avraham, K.B., Goncharov, T. Holtman, H., Lonai, P. & Wallach, D. (1998). Targeted disruption of the mouse caspase

lAP-Family Proteins 321 8 gene ablates cell death induction by the TNF receptors, Fas/Apol, and DR3 and is lethal prenatally. Immunity 9, 267-276. Vucic, D., Kaiser, W.J., Harvey, A.J. & Miller, L.K. (1997). Inhibition of reaper-induced apoptosis by interaction with inhibitor of apoptosis proteins (lAPs). Proc. Natl. Acad. Sci. U.S.A. 94, 10183-10188. Vucic, D., Kaiser, W.J. & Miller, L.K. (1998a). Inhibitor of apoptosis proteins physically interact with and block apoptosis induced by Drosophila proteins HID and GRIM. Mol. Cell Biol. 18, 3300-3309. Vucic, D., Kaiser, W.J. & Miller, L.K. (1998b). A Mutational Analysis of the Baculovirus Inhibitor of Apoptosis Op-IAP. J. Biol. 1998 273, 33915-31. Wallach, D., Boldin, M., Varfolomeev, E., Beyaert, R., Vandenabeele, P. & Fiers, W. (1997). Cell death induction by receptors of the TNF family: towards a molecular understanding. FEBS Lett 410, 96-106. Wang, C.Y., Mayo, M.W., Korneluk, R.G., Goeddel, D.V. & Baldwin, A.S. (1998). NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-lAP2 to suppress caspase-8 activation. Science 281, 1680-1683. Wolter, K.G., Hsu, Y.T., Smith, C.L., Nechushtan, A., Xi, X.G. & Youle, R.J. (1997). Movement of bax from the cytosol to mitochondria during apoptosis. J. Cell Biol. 139, 1281-1292. Xu, D.G., Crocker, S.J., Doucet, J.P., St-Jean, M., Tamai, K., Hakim, A.M., Ikeda, J.E., Liston, P., Thompson, C.S., Korneluk, R.G., MacKenzie, A. & Robertson, G.S. (1997). Elevation of neuronal expression of NAIP reduces ischemic damage in the rat hippocampus. Nat. Med. 3,997-1004. Yamaguchi, K., Nagai, S., Ninomiya-Tsuji, J., Nishita, M., Tamai, K., Irie, K., Ueno, N. Nishida, E., Shibuya, H. & Matsumoto, K. (1999). XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TAB 1-TAK1 in the BMP signaling pathway. EMBO J. 18, 179-187. Yang, J., Liu, X., Bhalla, K., Kim, C.N., Ibrado, A.M., Cai, J., Peng, T.I., Jones, D.P. & Wang, X. (1997). Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129-1132. You, M., Ku, P.T., Hrdlickova, R. & Bose, J.H.R. (1997). ch-IAP1, a member of the inhibitor-of-apoptosis protein family, is a mediator of the antiapoptotic activity of the v-rel oncoprotein. Mol. Cell. Biol. 17, 7328-7341. Young, S.S., Liston, P., Xuan, J.Y., McRoberts, C., Lefebvre, C.A. & Kerneluk, R.G. (1998). Genomic Organization of the Physical map of the Human Inhibitors of Apoptosis: HIAP1, HIAP2. Genomics. M. Gerome 1999 10, 44-48. Zandi, E., Chen, Y. & Karin, M. (1998). Direct phosphorylation of IkB by IKKa and IKKa: Discrimination between free and NF-KB-bound substrate. Science 281, 1360-1363. Zha, H., Fisk, H.A., Yaffe, M.P., Mahajan, N., Herman, B. & Reed, J.C. (1996). Structure-function comparisons of the proapoptotic protein Bax in yeast and mammalian cells. Mol. Cell Biol. 16, 6494-6508. Zhou, Q., Krebs, J.F., Snipas, S.J., Price, A., Alnemri, E.S., Tomaselli, K.J. & Salvensen, G.S. (1998). Interaction of the baculovirus anti-apoptotic protein p35 with caspases. Specificity, kinetics, and characterization of the caspase/p35 complex. Biochemistry 37, 10757-10765. Zhou, Q. & Salvesen, G.S. (1997). Activation of pro-caspase-7 by serine proteases includes a non-canonical specificity. Biochem. J. 324, 361-364. Zhou, Q., Snipas, S., Orth, K., Muzio, M., Dixit, V.M. & Salvesen, G.S. (1997). Target protease specificity of the viral serpin CrmA: analysis of five caspases. J. Biol. Chem. 272, 7797-7800. Zou, H., Henzel, W.J., Liu, X., Lutschg, A. & Wang, X. (1997). Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90, 405-413.

EXCITOTOXINS, NITRIC OXIDE AND PROGRAMMED NEURONAL DEATH M A R C E L LEIST and PIERLUIGI N I C O T E R A

Table of Contents Self-Amplifying Loops in Programmed Neuronal Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ischemia and Excitotoxicity: Apoptosis or Necrosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Tuning of Cell Death: Recruitment of Different Subroutines . . . . . . . . . . . . . . . . . . . . . Therapeutic Implications of a Branched Death Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Tuning of Cell Death by Energy Metabolism: ATP and the Shape of Neuronal Demise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Indirect Excitotoxicity -- Apoptosis by Energy Depletion . . . . . . . . . . . . . . . . . . . . . . . . . . . Nitric Oxide as a Mediator of Neuronal Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nitric Oxide: Dr. Jekyll or Mr. Hyde? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

323 325 326 327 328 330 334 336 337 339

Self-Amplifying Loops in Programmed Neuronal Death Cell death is frequently classified according to morphological or conceptual criteria (Kerr et al., 1972). Programmed neuronal death, as opposed to accidental cell lysis is a scheduled event during development and tissue turnover (Raft et al., 1993; Oppenheim, 1991). Nonetheless, compelling evidence shows that execution of a program leading to cell death is also the ultimate consequence of neuropathologic conditions. Frequently, programmed cell death results in morphological changes, typical of apoptosis (Kerr et al., 1972). However, the concept of a death program is not necessarily linked to a morphological appearance. For example, developmental cell death does not always exhibit an apoptotic-like morphology (Schwartz et al., 1993). Also, certain stimuli can elicit death via a series of well-defined and controllable steps that lead to a morphology (i.e., shape) of cell death, best described by necrosis (Nicotera et al., 1986; Bonetti and Raine, 1997 Sohn et al., 1998; Vercammen et al., 1998;), while a large number of "accidental" traumatic or toxic insults can lead to rapid cell demise with an apoptotic shape (Leist and Nicotera, 1997; Kroemer et al., 1997). This implies that the shape of cell death is not necessarily linked to a single program. Although classical execution of apoptosis via caspases is likely the "default" death program, retained from the developmental settings, other pathways may contribute to increase the effectiveness of programmed cell death in adult mammalian cells. According to this view, death signals could potentially activate distinct and sometimes diverging pathways (i.e. execution subroutines). Each subroutine may become predominant 323 Cellular and Molecular Mechanisms Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. 3 2 3 - - 3 4 7 © 2 0 0 1 Elsevier Science. Printed in the Netherlands.

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depending on the cell-type, the local metabolic conditions and co-stimulatory or inhibitory factors. Some subroutines of the death program may not be active in all cases. In particular, under pathological conditions, stimuli like reactive oxygen species, nitric oxide or toxins may trigger the cell death program in some cases, and inhibit some of its subroutines in others. While some execution pathways are closely associated with the decision on life or death, others may mainly determine the ultimate appearance of cell death. This is exemplified by the fact that inhibition of some subroutines of the cell death program does not always result in cell survival. Frequently, inhibition of individual pathways of the death program that are mainly responsible for the apoptotic morphology switches death into necrosis (Leist et al., 1997b; Melino et al., 1997; Hirsch et al., 1997; Green and Kroemer, 1998). Known core elements of the death program include a class of cysteine proteases (caspases) related to the C. elegans death gene, ced-3 (Cohen, 1997), mitochondrial alterations (Kroemer et al., 1997), and alterations of the cellular ion homeostasis or redox balance (Leist and Nicotera, 1997). Although each of these conditions may lead to cell death independently, often they interact in a complex system of positive self-amplifying loops. An example encountered in neuropathology is the injury following a primary mitochondrial failure. Mitochondrial dysfunction has been implicated in the development of neurodegenerative diseases such as Huntington's and Parkinson's disease, ischemic brain damage, and Alzheimer's disease. Genetic mitochondrial defects are also a possible pathogenic cause of other neurodegenerative disorders. Mutations in mitochondrial protein-coding genes, or in nuclear genes, which encode for mitochondrial proteins can result in neurodegenerative conditions such as the Leber hereditary optic neuropathy (LHON) and the mitochondrial encephalophathy, lactic acidosis, and stroke-like episodes (MELAS syndrome). It is generally assumed that mutations in genes encoding mitochondrial proteins results in energy impairment. Energy failure, would in turn sensitize neurons to excitotoxicity (see below). The ensuing Ca2÷overload could then promote further mitochondrial damage, and oxidative stress in a selfpropagating process eventually leading to cell death. In addition to genetic defects, stressful stimuli (for example Ca 2÷ overload) affect mitochondria in different ways. A frequently described phenomenon encountered in cell pathology is the loss of permeability of the mitochondrial inner membrane (Zoratti and Szabo, 1995). This event is defined as permeability transition (PT) and probably involves the opening of a proteinaceous pore (Kroemer et al., 1997). Pore opening is associated with (i) the release into the cytosol of mitochondrial intermembrane proteins, including cytochrome c and the apoptosis inducing protein AIF (ii) a breakdown of mitochondrial ATP-generation, and (iii) a loss of mitochondrial Ca 2÷. A number of secondary reactions would then be activated including (i) the activation of caspases by cytochrome c in conjunction with the cytosolic factor Apaf-l, (ii) uncoupling of the respiratory chain, associated with production of reactive oxygen species, due to the loss of cytochrome c, and (iii) increased stress on residual functioning mitochondria by Ca 2÷ overload (White and Reynolds, 1996). Different proteases can also trigger mitochondrial alterations, by cleaving mitochondrial structural or regulatory proteins (Green and Kroemer, 1998). Ca 2÷ activated proteases, calpains can induce

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PT (Aguilar et al., 1996), while execution caspases cleave the Bcl-2 related protein Bid (Luo et al., 1998), which results in release of cytochrome c. Permeabilization of either the outer or inner mitochondrial membrane, inhibition of energy metabolism and oxidative stress would again ensue. Counter-regulatory mechanisms are also important. PT, the release of mitochondrial proteins and the decision on life or death are often controlled by the interplay of proapoptotic (e.g. Bcl-2) and antiapoptotic (e.g. Bax) proteins of the Bcl-2 family (Kroemer, 1997). Accordingly, deletion of Bax or overexpression of Bcl-2 can protect neurons from otherwise lethal stimuli (Martinou et al., 1994; Sagot et al., 1995; Xiang et al., 1998; Miller et al., 1997). Although they have been mainly characterised in models of apoptotic demise, Bcl-2-1ike proteins have been shown to prevent neuronal necrosis (Zhong et al., 1993; Shimizu et al., 1996). Along this line, triggering of cell death via a Bax-dependent mechanism can result in cerebellar granule cell (CGC) necrosis, when caspase activity is inhibited (Miller et al., 1997).

Ischemia and Excitotoxicity: Apoptosis or Necrosis? Many neurodegenerative conditions are linked to extensive neuronal loss, which may occur by either apoptosis or necrosis. A condition probably common to a variety of neurological disorders is excitotoxicity (Meldrum and Garthwaite, 1990; Choi, 1988). This term describes the supra-physiological stimulation of glutamate receptor subtypes such as the NMDA-receptor. These ionotropic receptors act as ligand-gated Ca z+ channels, and their prolonged activation results in intracellular Ca 2÷ overload and cell death. Until recently, neuronal demise due to excitotoxicity or to neurotoxicants has been regarded as a type of death different from that encountered in development. Injury by neurotoxins or glutamate was supposed to lack the regulated series of events involved in a death program, and thus it would invariably ensue in necrosis. Recent evidence suggests that key regulators of programmed cell death, i.e. p53 (Xiang et al., 1998), Bax (Miller et al., 1997), Bcl-2 (Martinou et al., 1994) and caspases are also involved in excitotoxic-ischemic neuronal injury. For instance, prevention of neuronal death in cerebral ischemia by caspase inhibitors (Hara et al., 1997; Loddick et al., 1996) suggests that at least some mechanisms of this demise are similar to those of other forms of programmed cell death. This concept is directly corroborated by in vitro experiments showing that excitotoxicity may result in apoptosis or necrosis (Ankarcrona et al., 1995) and that caspase inhibitors significantly reduce the extent of excitotoxic damage (Leist et a1.,1997c, D u e t al., 1997; Jordan et al., 1997; Tenneti et al., 1998). Perhaps more evidently in excitotoxicity than in other stressful conditions, several alternative execution subroutines can be activated depending on the conditions of stimulation, and the neuronal subpopulation. For example, the intensity of insult (Ankarcrona et al., 1995; Bonfoco et al., 1995) or the participation of individual receptors (Portera-Cailliau et al., 1997) may determine the mode of cell death. In some cases, excessive Ca 2÷ entry may convert the mode of cell death from apoptosis to necrosis (Gwag et al., 1995). Even the source of the excitotoxic stimulus may be important.

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Enhanced synaptic release of excitotoxins, such as that elicited in culture systems by NO, can trigger autocrine excitotoxicity, which predominantly leads to apoptosis, at least in cerebellar granule neurons (Leist et al., 1997c, 1998; see below). Massive extracellular glutamate accumulation as that resulting from an associated neuronal lysis may instead favor necrosis (Figure 1).

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Figure l. The intensity of insult may determine the shape of neuronal death. In the case of peroxynitrite

(ONO0) exposure to low concentrations may lead to neuronal changes that can be compensated by cellular defense mechanisms. Increasing concentrations would lead to apoptosis, whereas a further increase of concentration or exposure time would result in neuronal necrosis. Notably, apoptosis and necrosis may involve different execution mechanisms, when different concentrations of toxins are used. Under excitotoxic conditions, the involvement of caspases, and thus, prevention of neuronal death by protease inhibitors is most effective at low intensities of insult. Part of the problem in deciding the relevance of apoptosis in neurodegeneration has originated from the assumption that inhibitors of protein synthesis would protect cells from the apoptotic, but not necrotic, demise. However, cell death with typical apoptotic morphology may occur in the presence of inhibitors of protein synthesis (Leist et al., 1995; Weil et el., 1996;), and neuronal apoptosis triggered by autocrine excitotoxicity, NO donors, or Hg 2+ is not modified by agents that inhibit protein synthesis (Leist et al., 1997a; Rossi et al., 1997). In vivo, this is further complicated by the possibility that delayed cell death following brain ischemia may be at least in part due to induction of death receptors (Martin-Villaba et al., 1998). Thus, neurons in the core ischemic area die by necrosis directly after the excitotoxic insult (CharriautMarlangue et el., 1996). Delayed apoptotic cell death in the penumbra may initially progress regardless of new protein synthesis, while at a later stage, expression of death signaling molecules may further contribute to extend cell demise.

T h e T u n i n g o f Cell Death: R e c r u i t m e n t o f Different S u b r o u t i n e s

Contrasting interpretations of the mode of excitotoxic death have also originated from the significance attributed to single apoptosis-linked alterations in dying cells. However, different apoptotic features may be displayed in different situations. For instance, glutamate elicits chromatin condensation and high molecular weight DNA-fragmentation

Neuronal Apoptosis and Excitotoxicity

327

in CGC, i.e. typical indications of apoptosis; oligonucleosomal DNA-laddering and nuclear fragmentation, which are also indicative of apoptosis are less apparent (Ankarcrona et al., 1995). In cortical neurons, DNA-fragmentation has been reported to occur without nuclear condensation at low glutamate concentrations (Sohn et al., 1998), while necrosis occurs at high NMDA concentrations (Bonfoco et al., 1995). CGC, exposed to other apoptotic stimuli (e.g. staurosporine or 4-hydroxynonenal), exhibit the typical DNA laddering, chromatin condensation, and nuclear fragmentation (Kruman et al., 1997; Koh et al., 1995). Because not all the indications of apoptosis are necessarily present simultaneously, excitotoxic neuronal death has often been described as non-apoptotic. DNA laddering has received much attention as an elective marker of apoptosis. While it is undisputed that typical DNA laddering is a feature of many apoptosis models, it is also clear that apoptosis can occur without oligonucleosomal DNA fragmentation. The lack of DNA laddering has indeed no effect on the rate of death (Cohen et al., 1992). Oligonucleosomal DNA-fragmentation seems to depend on proteins activated by caspase-3 and should therefore not be expected in forms of apoptosis where this caspase isoenzyme is not active. Caspase-3 has a dominant role in developmental neuronal death (Kuida et al., 1996) and it is also induced in stroke-like conditions. However, caspase-3 independent apoptosis and programmed cell death are well documented in neurons (Stefanis et al., 1996; Leist et al., 1997c, 1998; Kuida et al., 1996; Miller et al., 1997) and other cells (Kuida et al., 1996; J~inicke et al., 1998). The same applies to caspase-9, a key enzyme in the cytochrome c-dependent caspase-3 activation pathway (Kuida et al., 1998; Hakem et al., 1998). Various intracellular changes triggered by death stimuli may decide on the subset of apoptotic routines activated. For instance, membrane changes (phosphatidylserine flip) have been shown to be uncoupled from other apoptotic changes (DNA-fragmentation, caspase activation) by agents that modify mitochondrial function (Zhuang et al., 1998). In the same vein, the activity of PAK2, a kinase activated by caspases, may have a bearing on the shape-determining subroutines of cell death (Rudel et al., 1997). Moreover, in excitotoxic conditions, the caspase-3 processing, and the subroutines depending on this protease, may be impaired by ion influx into the cell (Hampton et al., 1998). Accordingly, caspase inhibitors prevent NO triggered autocrine excitotoxicity in CGC at low intensities of insult. When the NO concentration is increased, caspase inhibitors become ineffective (Leist et al., 1997c), although the morphological features of apoptosis (i.e., exposure of phosphatidylserine on the outer leaflet of the plasma membrane and chromatin condensation) are still retained by dying neurons. With even stronger insults, the degradative processes typical of apoptosis are prevented, and neurons die by necrosis (Bonfoco et al., 1995) (Figure 1). Such necrosis, triggered by a potentially apoptotic signal may be due to rapid energy dissipation (see below)

Therapeutic Implications of a Branched Death Program Stimulation of self-feeding loops, which maintain both the activation of executioners and the neutralization of anti-apoptotic defense systems, seems to be necessary for

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the completion of most death programs. Cells are equipped with defense systems that compensate damage at multiple levels. It seems likely that accumulation of damage incompatible with cell survival would require disruption of several vital functions. Once such a threshold is trespassed multiple positive feed-back loops would ensure the progression of the death program to the end, and the safe disposal of the injured cell. This also implies that the morphological appearance of cell death (apoptosis or necrosis) is not linked to a single commitment point, but rather is the result of a more or less complete execution of subroutines deciding on the shape of dying cells. Notably, apoptosis can still be blocked downstream of putative commitment points (i.e. caspase 3 activation) (J~itt~il~i~i et al., 1998), which emphasizes the concept that even the degree of activation of intracellular subroutines of the death program may be relevant to decide death or survival. This may be very important when considering apoptosis-related targets for possible therapeutic approaches. Observations in stroke models suggest that apoptosis occurs mainly in the border regions (penumbra), while necrosis dominates in the more severely stressed areas of the ischemic core (Charriaut-Marlangue et al., 1996). Apoptosis of penumbral neurons may be due both to a mild direct excitotoxic-ischemic insult, but also to secondary mediators such as oxygen radicals, cytokines and lipid peroxidation products from the necrotic core (Kruman et al., 1997; Mattson, 1998; Lipton and Rosenberg, 1994). Intervention, a few hours after the ischemic insults, is normally aimed to reduce spreading of the lesion and to inhibit delayed cell death in the border areas (Figure 2A). Assuming that the level of injury decides the activation of different pathways for the execution of cell death, it is apparent that caspase inhibitors, for example, may be most effective in areas where the intensity of the excitotoxic insult is low, and positive feedback loops between different execution subroutines are not fully established. In the regions where the stress is more intense, inhibition of caspases alone may not prevent cell death (Green and Kroemer, 1998). Thus, strategies that combine agents to reduce the overall intensity of the insult and the overall lesion size (i.e. N-methyl-D-aspartate (NMDA)antagonists and other ion channel blockers or selective bNOS inhibitors), with agents that block execution of apoptosis (caspase inhibitors) has been proven more successful than individual treatments (Ma et al., 1998; Schulz et al., 1998) (Figure 2B).

The Tuning of Cell Death by Energy Metabolism: ATP and the Shape of Neuronal Demise Mitochondria and energy metabolism play an important role in the decision on the rate (Kroemer, 1997) and shape (Nicotera and Leist, 1997) of cell death. A mitochondrial function frequently relevant to the shape of death is the production of ATP. Circumstantial evidence for this comes from experiments with inhibitors of the respiratory chain, which induce apoptosis at low concentrations (where they only partially deplete ATP) and necrosis at higher concentrations (when ATP is entirely lost) (Hartley et al., 1994). Additional evidence comes from excitotoxicity experiments, where neurons with severely reduced energy charge died by necrosis, whereas those recovering ATP died by apoptosis (Ankarcrona et al., 1995).

329

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Figure 2B.

Effectiveness of protease inhibitors, depending on the shape of cell death and the

intensity of insult In vivo, protease inhibitors may protect neurons from low level excitotoxic insult or NO exposure and, in addition, from apoptosis triggered by oxygen radicals, lipid peroxidation products and other endogenous mediators generated under ischemic conditions. Receptor antagonists, nitric oxide synthase inhibitors and radical scavengers may strongly lower the intensity of the primary excitotoxic insult. Under those conditions, inhibitors of caspases or other proteases, may be more effective, and combined treatment may lead to increased neuroprotection.

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M. Leist and P. Nicotera

To examine more directly the role of ATP for the mode of cell death, intracellular ATP levels have been manipulated in a controlled way. With this approach it has been possible to show that when the intracellular ATP concentration was reduced, typical apoptotic stimuli caused instead necrosis (Leist et al., 1997b). If ATP concentrations are markedly reduced, activation of downstream caspases and all most typical apoptotic changes are blocked. More recent work suggests that one of the ATP requiring steps may be at the level of the formation of the protein complex between Apaf-1, cyt-c and procaspases (Li et al., 1997), blocking the resulting downstream degradative processes including caspase-3 activation, poly-(ADP-ribose)-polymerase cleavage, lamin cleavage, and exposure of PS on the outer membrane delayed cell death, but other (caspase-independent) subroutines finally results in cell demise by necrosis (Leist et al., 1997b; Eguchi et al., 1997).

Indirect Excitotoxicity -- Apoptosis by Energy Depletion Mitochondria play multiple roles in neurodegeneration (Figure 3). They prevent excessive intracellular Ca 2÷ accumulation and generate ATP thus: (i) energy failure as that elicited by genetic defects or by mitochondrial toxins sensitizes neurons to excitotoxicity (Novelli et al., 1988), and fosters the release of excitotoxic mediators. (ii) Mitochondria represent themselves a target of excitotoxic mechanisms (Nicotera and Leist, 1997; Schinder et al., 1996; Isaev et al., 1996; Leist et al., 1998), and (iii) mitochondrial damage can aggravate the initial damage by releasing Ca 2+, ROS and factors essential for apoptotic protease activation. As briefly discussed above, mitochondrial dysfunction is amongst the general causes favouring the development of neurodegenerative diseases (Beal, 1996; Wallace et al. 1995). For example, Huntington's disease (HD) is modelled by exposing specific neuronal subpopulations to mitochondrial toxins (Zeevalk et al., 1995a,b; Ferrante et al. 1997; Pang and Geddes, 1997), and ischemic damage can be examined in vitro following mitochondrial impairment due to oxygen-glucose deprivation (Choi and Rothman, 1990). In all these models, a close relationship has been established between mitochondrial impairment and excitotoxicity. In fact, it has been suggested that in pathological situations glutamate receptor stimulation is not necessarily the triggering event of excitotoxicity, but rather a consequence of energy failure. Accordingly, the "energy-linked excitotoxicity hypothesis" (Henneberry et al., 1989; Zeevalk and Nicklas, 1990; Albin and Greenamyre, 1992; Beal et al., 1993) proposes that energy failure would lead to partial hypopolarization of neurons, thereby releasing the voltage-dependent Mg 2+ block of the N-methyl-Daspartate receptor (NMDA-R)/channel. Such a release would make the receptor/channel hypersensitive to glutamate stimulation. NMDA-R-mediated influx of Na + and Ca z+ would increase the energy demand, enhance depolarization, trigger further [Ca2÷]i increase, and stimulate glutamate release (Figure 4). Indeed, secondary excitotoxic mechanisms contribute to neuronal cell death triggered by mitochondrial poisons (Pang and Geddes, 1997; Zeevalk et al., 1995a,b; Schulz et al., 1996; Beal et al., 1993; Ferrante et al., 1997; Turski et al. 1991; Leist et al., 1998).

Neuronal Apoptosis and Excitotoxicity

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functions, by taking up Ca 2÷ from the cytosol and providing the cell with ATP. Several triggers of cell death require an impairment of the counteractive mitochondrial mechanisms to become effective. (middle) Frequently, mitochondria have a modulatory or accelerating role in cell death, one their proper function has been impaired. Their failure may switch apoptosis to necrosis, and they may actively contribute to cellular demise by releasing Ca 2÷ and reactive oxygen species (ROS). (right) in may forms of programmed cell death mitochondria act as a key switch deciding on life or death of the cell. The release of apoptogenic factors (e.g. cytochrome c) is prevented by Bcl-2 and triggered by Bax, and the balance of these proteins may determine whether programmed cell death occurs.

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inhibitors, including the endogenous mediator NO, can impair mitochondrial respiration and deplete cellular ATP levels. Loss of ATP impairs ion pumps and eventually results in plasma membrane hypopolarization. This triggers the opening of voltage-dependent ion channels and a hypersensitization of N-methyl-D-aspartate receptors (NMDA-R). In addition, increased cellular [Ca2+]i may favor exocytosis of excitotoxins, which further trigger the NMDA-R. The resultant massive and uncompensated influx of extracellular Ca 2+ leads to a complete mitochondrial failure, associated with rupture of the outer membrane and release of cytochrome c.

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Autocrine release of NMDA-R agonists, which trigger excitotoxicity, may occur by different mechanisms (Szatkowski and Attwell, 1994; Nicholls and Attwell, 1990) (Figure 5). First, release may occur by exocytosis. This process may be relevant, when ATP levels are still high enough to promote the fusion of neurotransmitter vesicles with the presynaptic membrane, or when the pool of vesicles that has already been primed for exocytosis is released. At early stages, ATP production may be maintained by residual mitochondrial production together with the very potent glycolytic capacity of neurons (Budd and Nicholls, 1996). The relevance of this release mechanism for secondary excitotoxicity has been demonstrated in models of oxygen-glucose deprivation (Monyer et al., 1992), NO-triggered excitotoxicity (Bonfoco et al., 1996; Leist et al., 1997a), or autocrine excitotoxic MPP ÷ toxicity in CGC cultures (Leist et al., 1998). Second, glutamate may be released by the reversal of the glutamate transporter. This mechanism does not require ATP, but is rather triggered by intracellular and extracellular ionic changes, which occur under conditions of ATP loss.

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Figure 5. Different mechanisms of glutamate release in exeitotoxicity. The glutamate concentrations in the synaptic cleft (0-10 pM), the cytoplasm (1-10 mM) and in vesicles (10-100 mM) differ by several orders of magnitude. ATP is required to sequester glutamate into secretory vesicles and for the movement and fusion of secretory vesicles. (left) Normally, exocytosis requires the availability of ATP and a trigger by Ca 2÷. (middle) In various cells a pool of pre-docked vesicles exists, that can be triggered for exocytosis by Ca 2+, independent of ATP. (right) under conditions of severe ATP-depletion, glutamate can be released from neurons by non-exocytotic mechanisms. After disturbance of normal ionic gradients across the plasma membrane, the glutamate transporter may be reversed and cytosolic glutamate would be released into the synaptic cleft. In contrast to the former two mechanisms, this form of glutamate release cannot be inhibited by botulinum toxins.

A well-examined example of a mitochondrial poison involved in neurodegeneration is the Parkinson syndrome inducing neurotoxin 1-methyl-4-phenypyridinium (MPP+). Binding to complex I in vivo (Kilbourn et al., 1997) seems to be the primary biochemical effect of MPP +. Thus, excitotoxic mechanisms should be secondary to the initial mitochondrial dysfunction. In animals, removal of glutamatergic inputs (decortication), blockers of glutamate release, or NMDA-R antagonists reduce MPTP- and MPP +induced striatal damage and dopamine depletion (Srivastava et al., 1993; Brouillet and

Neuronal Apoptosis and Excitotoxicity

333

Beal, 1993; Schulz et al., 1996) or loss of dopaminergic neurons in the substantia nigra (Turski et al., 1991). In CGC cultures, MPP + triggered apoptosis was prevented by N M D A - R blockers (Leist et al., 1998). Thus, the reciprocal interaction of mitochondrial function and glutamate receptor activation seems to be a fundamental concept of excitotoxic cell death. Recent findings show that, in neurons undergoing excitotoxicity, an ATP-independent and caspase-3 independent form of apoptosis may be triggered by mitochondrial inhibition itself. For example, direct modification of respiratory chain complex-I with a subsequent decline in ATP generation may be responsible for the apoptosis elicited by the neurotoxin methylphenylpyridinium (MPP +) in neurons establishing glutamatergic synapses (Leist et al., 1998). In this system, proteolytic cascades involving both Ca2+-activated proteases (calpains) and caspases other than caspase-3, seem to execute apoptosis (Figure 6). While it is still unclear whether calpain activation is linked to that of caspases by a causal relationship, or whether it represents an alternative, independent lethal effect of the Ca 2÷ overload (Wang and Yuen, 1997), it is apparent that distinct proteolytic systems participate in the execution of neuronal apoptosis (Jordan et al., 1997; Leist et al., 1998). It may be speculated that the balance of caspases and alternative execution systems may be determined here by the type of stimulus, e.g. the extent of Ca 2+ influx in excitotoxicity. An initial decline of ATP may be required to facilitate glutamate release and autocrine stimulation, and to prevent the buffering of cytosolic Ca z÷ overload. A similar series of events has been described for mitochondrial inhibitors in vivo (Blandini et al., 1997). C a 2+

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death can be facilitated/triggered by mitochondrial failure and ATP-depletion without caspase-3 activation: functioning mitochondria normally protect cells from Ca2÷overload, by sequestering the ion in a membrane potential (AW)-dependent manner and by providing ATP for its sequestration across the plasma membrane or into the endoplasmic reticulum (Ca2+-pumps). Inhibition of mitochondrial respiration and ATP-depletion would facilitate/aggravate [Ca2+]iincrease (bold arrows) and activationof Ca2+-dependentproteolyticsystems. Calpains may activate caspases and caspases may cleave the calpains inhibitory protein calpastatin (Wang et al., 1998). Both classes of proteins may cause mitochondrial permeability transition and inactivation of Ca2÷ sequestering transport systems. This would further increase [Ca2+]i,and Ca2+-dependentprotease activity in a vicious loop eventually leading to cell death.

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Nitric O x i d e as a M e d i a t o r o f N e u r o n a l Death

In the central nervous system, nitric oxide is a pleiotropic messenger molecule involved in the regulation of blood flow and cellular signalling (Beckmann and Koppenol, 1996; Garthwaite and Boulton, 1995; Zhang and Snyder, 1995.). However, experiments using inhibitors of NOS, studies performed in gene-targeted mice which lack different isoforms of NOS, and measurement of NO metabolites suggest that NO production is also involved in various neuropathological processes (Schulz et al., 1996; Huang et al., 1994; Huang and Fishman, 1996; Sandani et al., 1997; Snyder, 1996; Schulz et al., 1995 (JN); Galpern et al., 1996; Crow and Beckman, 1995; Gross and Wollin, 1995). NO exerts physiologic effects by regulating guanylate cyclase activity or possibly by mild, partially-reversible, covalent protein modifications, such as S-nitrosylation (Stamler et al., 1992; Stamler, 1994). E.g. hemoglobin-nitrosylation has a role in the fine-tuning of cerebral oxygen supply (Stamler et al., 1997), and NO may trigger Ca2÷-independent neurotransmitter release by S-nitrosylation of synaptic proteins (Meffert et al. 1994; Meffert et al., 1996). The targets and mechanisms of adverse NO-effects are commonly thought to be different from those involved in its physiological actions. They may include DNA damage or irreversible protein modifications such as tyrosine nitration or thiol oxidation. Enzymatically produced NO may undergo a large variety of non-enzymatic reactions (Figure 7) e.g. with cellular thiols, metals or with superoxide (02.). Products formed from these secondary reactions may have differing biological activities that have been elucidated only in part (Garthwaite and Boulton, 1995; Zhang and Snyder, 1995; Stamler, 1994; Lipton et al., 1993). To explain the apparent dichotomy between physiological and pathological effects of NO, it has been proposed that its different reaction products may have contrasting effects (Stamler, 1994; Lipton et al., 1993; Beckmann and Koppenol, 1996; Sandani et al., 1997). Peroxynitrite (ONOO-), formed by the reaction of NO' with O2, has been implicated in cortical neuronal apoptosis (Bonfoco et al., 1995), and as inducer of apoptosis in various cell lines (Est6vez et al., 1995; Lin et al., 1995; Troy et al., 1996). ONOO can nitrate or hydroxylate protein tyrosine residues (Crow and Beckman, 1995; Beckman et al., 1994; Ischiropoulos et al., 1992), it oxidizes thiols (Stamler et al., 1992; Radi et al., 1991), or decomposes into OH' and NO 2" and finally forms NO2/NO 3- at neutral pH (Crow and Beckman, 1995; Beckman et al., 1990; Beckman et al., 1995). In neurons, the targets of ONOO- have been identified only partially. Possible cytotoxic mechanisms involve DNA damage and activation of poly-(ADP-ribose) synthase with subsequent depletion of NAD ÷ and ATP (Zhang et al., 1994), or mitochondrial damage (Bolanos et al., 1995). An entirely different paradigm of NO-toxicity is based on its presynaptic actions. NO is known to stimulate neurotransmitter release (Meffert et al., 1994; O'Dell et al., 1991; Hirsch et al., 1993; West and Galloway, 1997). Recently, it has been shown that NO stimulates exocytosis of neurotransmitters from synaptic vesicles probably by S-nitrosylating proteins responsible for the fusion of neurosecretory vesicles with the plasma membrane (Meffert et al., 1996).

335

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oxygen. Different stimuli either induce the inducible isoform (iNOS) via transcription factors such as NF-KB or the constitutive neuronal nitric oxide synthase (bNOS) via an increase of [Ca2+]v Nitric oxide may be converted by reaction with molecular oxygen or with oxy-hemoglobin (Oxy-Hb) to nitrite or nitrate. Alternatively, it may react in the presence of thiols, oxygen and transition metals (Me(")+/Me("+~)+) with thiols and metals in proteins and other cellular structural elements (nitrosylation; reversible). A pathway with particular relevance to NO toxicity may be the reaction of.NO with.O 2- to form ONOO-. ONOO- may nitrate or hydroxylate protein tyrosines (Tyr) and oxidise or nitrate protein thiols and thereby cause hardly reversible alterations in cellular structures.

Possibly NO may also trigger neurotransmitter release by energy depletion in synapses, since NO inhibits different complexes of the mitochondrial respiratory chain and glyceraldehyde-3-phosphate dehydrogenase (Gross and Wollin, 1995). E.g. it has been shown that NO is involved in the physiological control of respiration and in the control of insulin secretion via its influences on mitochondrial Ca 2+ release (Schweizer and Richter, 1996; Giulivi, 1998). Recently, an isoform of NOS has also been shown to be located in the inner mitochondrial membrane and respiration and mitochondrial potential were controlled by its activity (Ghafourifar and Richter, 1997; Giulivi, 1998). NO-effects on neurosecretion may explain why in NO-induced apoptosis of CGC cytotoxicity seems to result from excessive stimulation of neurotransmitter release (Bonfoco et al., 1996; Leist et al., 1997a). In this model of neuronal apoptosis elicited by NO/ONOO the activation of the NMDA-receptor and a receptor-dependent increase of the intracellular free calcium concentration ([Ca2+]i) are key mechanisms (i.e. when NMDA-R are blocked, NO does not induce any significant increase in CGC

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apoptosis) (Bonfoco et al., 1996; Leist et al., 1997a). A presynaptic action of NO (i.e. triggering of neurotransmitter-release) would imply that the mechanisms underlying physiologic and toxic NO-actions on neurons may involve, at least in part, similar pathways (e.g. neurotransmitter release by controlled S-nitrosylation of fusion proteins). In this case, the final outcome would be determined by the intensity of the insult or by the metabolic condition of the cell.

Nitric Oxide: Dr. Jekyll or Mr. Hyde? The existence of a complex death program with differentially modulated subroutines and counter-regulatory mechanisms implies that pleiotropic mediators such as NO can either trigger or prevent apoptosis, or switch apoptosis to necrosis, The diverse and often contrasting effects elicited by NO in different cell-types may in part be due to its actions at several steps and hierarchies of the death program. In some systems, NO donors or NO metabolites cause apoptosis (Albina et al., 1993; Messmer and Brtine, 1995; Bonfoco et al., 1995; Bonfoco et al., 1996; Leist et al., 1997a), which eventually involves the activation of caspases (Chlichlia et al., 1998; Mohr et al., 1997, 1998; Brockhaus and Brfine, 1998). On the other hand, NO can also prevent cell death induced by a variety of stimuli in cells as diverse as hepatocytes (Harbrecht et al., 1994; Bohlinger et al., 1995), neurons (Wink et al., 1993, Tenneti et al., 1997) or leukocytes (Mannick et al., 1994, 1997; Beauvais et al., 1995; Genaro et al., 1995). However, in some cases, inhibition of cell death by NO may be only ephemeral and recent findings have suggested that NO would rather delay cell death and change its shape from apoptosis to necrosis (Melino et al., 1997). Such a change in the prevalent shape of cell death may be relevant in several pathological conditions. Extensive necrosis would lead to uncontrolled release of lipid peroxidation products or other cytotoxins from dying cells, which would further increase cellular losses, favor/sustain inflammation, and ultimately prevent an efficient tissue reorganizsation and restoration (Ren and Savill, 1998). NO-dependent prevention of cell death, as well as NO-mediated conversion of apoptosis into necrosis have been often attributed to inhibition of caspase activity (Melino et al., 1997; Dimmeler et al., 1997; Kim et al., 1997; Mohr et al., 1997). Caspases have multiple roles in cell death (Green and Kroemer, 1998). Some members of this protease family may have a prevailing signalling function. In this case, their inhibition could completely prevent cell death (Longthorne and Williams, 1997). Other members of the family, primarily caspases-3,6,7,9 are required for the execution of apoptosis. In this case, caspase activation is relevant for the apoptotic morphology, but not always for the decision on cell death (Lemaire et al., 1998; Hirsch et al., 1997; Green and Kroemer, 1998), which may be triggered by alternative execution pathways (Adjei et al., 1996; McCarthy et al., 1997). Accordingly, a general inhibition of all caspase activity by NO should reduce the rate of cell death, whereas inhibition of downstream caspases may simply change the mode of cell death. In some systems, direct S-nitrosylation of caspases has been shown to interfere with the execution of apoptosis (Dimmeler et al., 1997; Kim et al., 1997; Melino et al., 1997). However, caspases are only partially nitrosylated in intact cells (Kim et al., 1997;

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Mohr et al., 1997), and evidence in other experimental paradigms suggests that NO can inhibit caspase activation by mechanisms different from S-nitrosylation (Kim et al., 1997), e.g. by NO-dependent formation of cGMP that could interfere with the cell death signal upstream of caspase activation (Mannick et al., 1994; Kim et al., 1997; Hebestreit et al., 1998). Since a well documented action of NO is the inhibition of the mitochondrial respiratory chain (Stadler et al., 1991; Cleeter et al., 1994; Schweizer and Richter, 1994; Cassina and Radi, 1996; Clementi et al., 1998), it seems conceivable that the resulting ATP-depletion might be relevant for the effects of NO on cell death. In fact, recent results in our laboratory have shown that NO prevents caspase activation by inhibiting mitochondrial respiration, thereby lowering intracellular ATP levels. By this mechanism, NO prevented DNA-fragmentation, chromatin condensation and the translocation of phosphatidylserine, which is a relevant recognition parameter for phagocytosis (Ren and Savill, 1998). However, as shown previously (Melino et al., 1997), the prevention of cell death was only ephemeral. Cell death was delayed and doomed cells died eventually by necrosis. When non-mitochondrial, glycolytic ATP generation was supported via glucose supplementation to the culture medium, death restored its apoptotic appearance. In vivo, a halt of the apoptotic program would have two possible implications: first, cells protected by NO via a stop of the apoptotic execution cascade would have time to recover from a transient or mild insult, and thus survive; second, cells hit by a lethal, normally apoptotic insult would eventually lyse without having been removed by phagocytosis. Thus, depending on the situation, endogenous mediators such as NO, may either prevent cell demise, or may convert apoptosis into necrosis. In the latter case, the release of factors from dead cells and the ensuing inflammation would further aggravate tissue damage (Figure 8).

Conclusions and Outlook

An initially simple program of cell death has evolved into a variety of subroutines, and multiple parallel pathways of death execution may have developed to ensure removal of injured and unwanted cells. This safeguard mechanism implies that therapeutic targeting of a single execution pathway or subroutine may not always prevent cell death. The different subroutines may be interconnected by positive feedback loops to prevent the persistence of "undead" cells. However, it may be speculated that in complex and large cells, such as neurons or oligodendrocytes, different death routines and counter-regulatory mechanisms may be activated within a single cell (e.g. in axon and soma) (Sagot et al., 1995, Martinou et al., 1994). Under pathologic situations, the ordered activation of subroutines of the death program may be inhibited to a different degree in individual cells of a tissue. Then, apoptotic and non-apoptotic cell death occur simultaneously, and neuronal necrosis may represent the shape of death resulting from a failure of cells to execute the full apoptotic program. This suggests a continuum of possible shapes of cell demise, where different morphologies do not reflect a fundamental mechanistic difference, but rather the subset of death routines activated in a particular cell.

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cytokines hypoglycemia/ischemia lipid peroxidation viruses 4 excitotoxins bacterial toxins xenobiotics reactive oxygen species . , , . . , , ,

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Implications of a switch of apoptosis to necrosis. Different stimuli may trigger programmed

cell death. In the case of caspase-3 dependent apoptosis, the progression of the program is controlled at the level of the Apaf complex by the availability of ATP. If ATP levels are depleted, death takes the shape of necrosis. The ensuing inflammation produces new cell death triggers which perpetuate tissue damage. Increased NO production under inflammatory conditions impairs ATP synthesis and further switches the mode of death to necrosis.

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Summary Programmed cell death is a vital process in the development of the mammalian nervous system. However, mechanisms and subroutines of this program are maintained throughout adult life, and operate in neuronal killing when activated inappropriately. For instance, the proteins p53, Bcl-2, Bax, and several members of the caspase family of proteases are implicated in animal models of stroke, and in excitotoxic neuronal death in vitro. In particular the involvement of execution caspases in models of neurodegeneration, such as neuronal injury by excitotoxins, has suggested new strategies for possible therapeutic interventions. For example, caspase inhibitors protect neurons from glutamate and nitric oxide, two key mediators involved in the pathology of stroke. Protection, however, is most effective at low levels of injury. At high intensity of insult other subroutines of the death program may become active. Thus, caspase involvement, and the recruitment of caspase-independent subroutines of the death program may depend on the neuronal subpopulation, the type and intensity of the noxious agent, and the metabolic situation of the cell. For instance, ATP-depletion may prevent the activation of caspases-3 and -9. Also, exposure to high concentrations of nitric oxide or glutamate may preclude the activation of caspase-3/9 and favour the activation of different sets of proteases (e.g. calpains or other caspases). Under such conditions, the shape of neuronal death may switch from apoptosis to necrosis. Thus, neuronal apoptosis and necrosis may share initial triggers and signals, whereas divergence of downstream execution subroutines results in different shapes of cell death.

References Adjei, P.N., Kaufmann, S.H., Leung, W.-Y., Mao, F. & Gores, G.J. (1996). Selective induction of apoptosis in Hep 3B cells by toposiomerase I inhibitors: evidence for a protease-dependent pathway that does not activate cysteine protease P32. J. Clin. Invest. 98, 2588-2596. Aguilar, H.I., Botla, R., Arora, A.S., Bronk, S.F. & Gores, G.J. (1996). Induction of the mitochondrial permeability transition by protease activity in rats: a mechanism of hepatocyte necrosis. Gastroenterol. 110, 558-566. Albin, R.L. & Greenamyre, J.T. (1992). Alternative excitotoxic hypotheses. Neurology 42, 733-738. Albina, J.E., Cui, S., Mateo, R.B. & Reichner, J.S. (1993). Nitric oxide-mediated apoptosis in murine peritoneal macrophages. J. Immunol. 150, 5080-5085. Ankarcrona, M., Dypbukt, J.M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S.A. & Nicotera, P. (1995). Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15,961-973. Beal, M.F. (1996). Mitochondria, free radicals, and neurodegeneration. Curr. Opin. Neurobiol. 6:661-666. Beal, M.F., Hyman, B.T. & Koroshetz, W. (1993). Do defects in mitochondrial energy metabolism underlie the pathology of neurodegenerative diseases? Trends Neurosci. 16, 125-131. Beauvais, F., Michel, L. & Dubertret, L. (1995). The nitric oxide donors, azide and hydroxylamine, inhibit the programmed cell death of cytokine-deprived human eosinophils. FEBS Lett. 361,229-232. Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., & Freeman, B.A. (1990). Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc.

340

M. Leist and P. Nicotera

Natl. Acad. Sci. USA 87, 1620-1624. Beckman, J.S., Chen, J., Ischiropoulos, H., & Crow, J.P. (1995). Oxidative chemistry of peroxynitrite. Meth. Enyzmol. 233,229-240. Beckman, J.S. & Koppenol, W. H. (1996). Nitric oxide, superoxide, and peroxynitrite: the goood, the bad, and the ugly. Am. J. Physiol. 271, C1424-C1437. Beckmann, J.S., Ye, Z.Y., Anderson, P.G., Chen, J., Accavitti, M.A., Tarpey, M.M. & White, C.R. (1994) Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol. Chem. Hoppe-Seyler 375, 81-88. Blandini, F., Higgins, D.S., Jr., Greene, J.G. & Greenamyre, J.T. (1997), Glutamate and mitochondrial defects in neurodegeneration. In: Mitochondria and free radicals in neurodegenerative diseases. (Beal, M. F., Howell, N. & Bodis-Wollner, I., eds.) Wiley-Liss. Inc., pp. 145-158. Bohlinger, I., Leist, M., Barsig, J., Uhlig, S., Tiegs, G. & Wendel, A. (1995). Interleukin-1 and nitric oxide protect against tumor necrosis-factor alpha-induced liver injury through distinct pathways. Hepatology 22, 1829-1837. Bolanos, J.P., Heales, S.J.R., Land, J.M. & Clark, J. B. (1995). Effect of peroxynitrite on the mitochondrial respiratory chain: differential susceptibility of neurones and astrocytes in primary culture. J. Neurochem. 64, 1965-1972. Bonetti, B. & Raine, C.S. (1997). Multiple sclerosis: oligodendrocytes display cell death-related molecules in situ but do not undergo apoptosis. Ann. Neurol. 42, 74-84. Bonfoco, E., Krainc, D., Ankarcrona, M., Nicotera, P. & Lipton, S.A. (1995). Apoptosis and necrosis: two distinct events induced respectively by mild and intense insults with NMDA or nitric oxide/superoxide in cortical cell cultures. Proc. Natl. Acad. Sci. USA 92, 72162-72166. Bonfoco, E., Leist, M., Zhivotovsky, B., Orrenius, S., Lipton, S.A. & Nicotera, P. (1996). Cytoskeletal breakdown and apoptosis elicited by NO-donors in cerebellar granule cells require NMDA-receptor activation. J. Neurochem. 67, 2484-2493. Brockhaus, F. & Brtine, B. (1998). U937 apoptotic cell death by nitric oxide: bcl-2 downregulation and caspase activation. Exp. Cell Res. 238, 33-41. Brouillet, E. & Beal, M.F. (1993). NMDA antagonists partially protect against MPTP induced neurotoxicity in mice. Neuroreport 4, 387-390. Budd, S.L. & Nicholls, D.G. (1996). Mitochondria, calcium regulation, and acute glutamate excitotoxicity in cultured cerebellar granule cells. J. Neurochem. 67, 2282-2291. Cassina, A. & Radi, R. (1996). Differential inhibitory action of nitric oxide and peroxynitrite on mitochondrial electron transport. Arch. Biochem. Biophys. 328, 309-316. Charriaut-Marlangue, C., Aggoun-Zouaoui, D., Represa, A. & Ben-Ari, Y. (1996). Apoptotic features of selective neuronal death in ischemia, epilepsy and gpl20 toxicity. Trends Neurosci. 19, 109-114. Chlichlia, K., Peter, M.E., Rocha, M., Scaffidi, C., Bucur, M., Krammer, P.H., Schirrmacher, V. & Umansky, V. (1998). Caspase activation is required for nitric oxide-mediated, CD95 (APO-lXfas)dependent and independent apoptosis in human neoplastic lymphoid cells. Blood 91,4311-4320. Choi, D.W. (1988). Glutamate neurotoxicity and diseases of the nervous system. Neuron 1,623-634. Choi, D. W. & Rothman, S. M. (1990). The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Annu. Rev. Neurosci. 13, 171-182. Cleeter, M.W.J., Cooper, J.M., Darley-Usmar, V.M., Moncada, S. & Schapira, A.H.V. (1994). Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative disease. FEBS Lett. 345, 50-54. Clementi, E., Brown, G.C., Feelisch, M. & Moncada, S. (1998). Persistent inhibition of cell respiration

Neuronal Apoptosis and Excitotoxicity

341

by nitric oxide: crucial role of S-nitrosylation of mitochondria complex I and protective action of glutathione. Proc. Natl. Acad. Sci. USA 95,7631-7636. Cohen, G.M. (1997). Caspases: the executioners of apoptosis. Biochem. J. 326, 1-16. Cohen, G.M., Sun, X.-M., Snowden, R. T., Dinsdale, D. & Skilleter, D.N. (1992). Key morphological features of apoptosis may occur in the absence of internucleosomal DNA fragmentation. Biochem. J. 286, 331-334. Crow, J.P. & Beckman, J.S. (1995). Reactions between nitric oxide, superoxide, and peroxynitrite: footprints of peroxynitrite in vivo. Adv. Pharmacol. 34, 17-43. Dimmeler, S., Haendeler, J., Nehls, M. & Zeiher, A.M. (1997). Suppression of apoptosis by nitric oxide via inhibition of interleukin- lbeta-converting enzyme (ICE)dike and cysteine protease protein (CPP)-32-1ike proteases. J. Exp. Med. 185,601-607. Du, Y., Bales, K.R., Dodel, R.C., Hamilton-Byrd, E., Horn, J.W., Czilli, D.L., Simmons, L.K., Ni, B. & Paul, S.M. (1997). Activation of a caspase 3-related cysteine protease is required for glutamate-mediated apoptosis of cultured cerebellar granule neurons. Proc. Natl. Acad. Sci. USA 94, 11657-11662. Eguchi, Y., Shimizu, S. & Tsujimoto, Y. (1997). Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res. 57, 1835-1840. Est6vez, A.G., Radi, R., Barbeito, L., Shin, J.T., Thompson, J.A. & Beckman, J.S. (1995). Peroxynitriteinduced cytotoxicity in PC12 cells: evidence for an apoptotic mechanism differentially modulated by neurotrophic factors. J. Neurochem. 65, 1543-1550. Ferrante, R.J., Brouillet, E., Palfi, S., Kowall, N.W., Hantraye, P. & Beal, M.F. (1997). Mitochondrial dysfunction as a model for huntington's disease using the electron transport chain inhibitor 3-nitropropionic acid. In: Mitochondria and free radicals in neurodegenerative diseases. (Beal, M. F., Howell, N. & Bodis-Wollner, I., eds.) New York: Wiley-Liss., pp. 229-244. Galpern, W.R., Matthews, R.T., Beal, F.M. & Isacson, O. (1996). NGF attenuates 3-nitrotyrosine formation in a 3-NP model of Huntington's disease. NeuroReport 7, 2639-2642. Garthwaite, J. & Boulton, C.L. (1995). Nitric oxide signaling in the central nervous system. Annu. Rev. Physiol. 57,683-706. Genaro, A.M., Hortelano, S., Alvarez, A., Martfnez-A., C. & Bosc~i, L. (1995). Splenic B lymphocyte programmed cell death is prevented by nitric oxide release through mechanisms involving sustained Bcl-2 levels. J. Clin. Invest. 95, 1884-1890. Ghafourifar, P. & Richter, C. (1997). Nitric oxide synthase activity in mitochondria. FEBS Lett. 418, 291-296. Green, D. & Kroemer, G. (1998). The central executioners of apoptosis: caspases or mitochondria? Trends Cell Biol. 8, 267-271. Gross, S.S. & Wolin, M.S. (1995). Nitric oxide: pathophysiological mechanisms. Annu. Rev. Physiol. 57, 737-769. Giulivi, C. (1998). Functional implications of nitric oxide produced by mitochondria in mitochondrial metabolism. Biochem. J. 332, 376-379. Gwag, B.J., Lobner, D., Koh, J.Y., Wie, M.B. & Choi, D.W. (1995). Blockade of glutamate receptors unmasks neuronal apoptosis after oxygen-glucose deprivation in vitro. Neurosci. 68,615-619. Hakem, R., Hakem, A., Duncan, G.S., Henderson, J.T., Woo, M., Soengas, M.S., Ella, A., de la Pompa, J.L., Kagi, D., Khoo, W., Potter, J., Yoshida, R., Kaufman, S.A., Lowe, S.W., Penninger, J.M. & Mak, T.W. (1998). Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94, 339-352. Hampton, M.B., Zhivotovsky, B., Slater, A.F.G., Burgess, D.H. & Orrenius, S. (1998). Importance of the redox state of cytochrome c during caspase activation in cytosolic extracts. Biochem. J. 329, 95-99.

342

M. Leist and P. Nicotera

Hara, H., Friedlander, R. M., Gagliardini, V., Ayata, C., Fink, K., Huang, Z., Shimizu-Sasamata, M., Yuan, J. & Moskowitz, M. A. (1997). Inhibition of interleukin 1beta converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage. Proc. Natl. Acad. Sci. USA 94, 2007-2012. Harbrecht, B.G., Stadler, J., Demetris, A.J., Simmons, R.L. & Billiar, T. R. (1994). Nitric oxide and prostaglandins interact to prevent hapatic damage during murine endotoxemia. Am. J. Physiol. 266, G1004-G1010. Hartley, A., Stone, J.M., Heron, C., Cooper, J.M. & Schapira, A.H.V. (1994). Complex I Inhibitors induce dose-dependent apoptosis in PC12 cells: relevance to Parkinson's disease. J. Neurochem. 63, 1987-1990. Hebestreit, H., Dibbert, B., Balatti, I., Braun, D., Schapowal, A., Blaser, K. & Simon, H.-U. (1998). Disruption of fas receptor signaling by nitric oxide in eosinophils. J. Exp. Med. 187,415-425. Henneberry, R.C., Novelli, A., Cox, J.A. & Lysko, P.G. (1989). Neurotoxicity at the N-methyl-D-aspartate receptor in energy-compromised neurons. An hypothesis for cell death in aging and disease. Ann. NY Acad. Sci. 568,225-233. Hirsch, D.B., Steiner, J.P., Dawson, T.M., Mammen, A., Hayek, E. & Snyder, S.H. (1993). Neurotransmitter release regulated by nitric oxide in PC-12 cells and brain synaptosomes. Curr. Biol. 3,749-754. Hirsch, T., Marchetti, P., Susin, S.A., Dallaporta, B., Zamzami, N., Marzo, I., Geuskens, M. & Kroemer, G. (1997). The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene 15, 1573-1581. Huang, P.L. & Fishman, M.C. 0996). Genetic analysis of nitric oxide synthase isoforms: targeted mutation in mice. J. Mol. Med. 74, 415-421. Huang, Z., Huang, P.L., Panahian, N., Dalkara, T., Fishman, M.C. & Moskowitz, M.A. (1994). Effects of Cerebral ischemia in mice deficient in neuronal nitric oxide synthase. Science 265, 1883-1885. Isaev, N.K., Zorov, D.B., Stelmashook, E.V., Uzbekov, R.E., Kozhemyakin, M.B. & Victorov, I.V. (1996). Neurotoxic glutamate treatment of cultured cerebellar granule cells induces Ca2*-dependent collapse of mitochondrial membrane potential and ultrastructural alterations of mitochondria. FEBS Lett. 392, 143-147. Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M., Martin, J.C., Smith, C.D. & Beckman, J.S. (1992). Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. Arch. Biochem. Biophys. 298, 431-437. J~i~ittel~i,M., Wissing, D., Kokholm, K., Kallunki, T. & Egeblad, M. (1998). Hsp70 exerts its anti-apoptotic function downstream of caspase-3-1ike proteases. EMBO J. 17, in press J~inicke, R.U., Sprengart, M.L., Wati, M.R. & Porter, A.G. (1998). Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J. Biol. Chem. 273, 9357-9360. Jord~in, J., Galindo, M.F. & Miller, R.J. (1997). Role of calpain- and intedeukin-lg converting enzyme-like proteases in the B-amyloid-induced death of rat hippocampal neurons in culture. J. Neurochem. 68, 1612-1621. Kerr, J.F., Wyllie, A.H. & Currie, A.R. (1972). Apoptosis: A basic biological phenomenon with wide ranging implications in tissue kinetics. Br. J. Cancer 26, 239-247. Kilbourn, M.R., Charalambous, A., Frey, K.A., Sherman, P., Higgins, D.S.J. & Greenamyre, J.T. (1997). Intrastriatal neurotoxin injections reduce in vitro and in vivo binding of radiolabeled rotenoids to mitochondrial complex I.J. Cereb. Blood. Flow Metab. 17, 265-272. Kim, Y.-M., Talanian, R. V. & Billiar, T. R. (1997). Nitric oxide inhibits apoptosis by preventing increases in caspase-3-1ike activity via two distinct mechanisms. J. Biol. Chem., 272, 31138-31148. Koh, J.-Y., Wie, M.B., Gwag, B.J., Sensi, S.L., Canzoniero, L.M.T., Demaro, J., Csernansky, C. & Choi,

Neuronal Apoptosis and Excitotoxicity

343

D.W. (1995). Staurosporine-induced neuronal apoptosis. Exp. Neurol. 135, 153-159. Kroemer, G. (1997). The proto-oncogene bcl-2 and its role in regulating apoptosis. Nat. Med. 3,614-620. Kroemer, G., Zamzami, N. & Susin, S.A. (1997). Mitochondrial control of apoptosis. Immunol. Today 18, 44-51. Kruman, I., Bruce-Keller, A.J., Bredesen, D., Waeg, G. & Mattson, M.P. (1997). Evidence that 4-hydroxynonenal mediates oxidative stress-induced neuronal apoptosis. J. Neurosci. 17, 5089-5100. Kuida, K., Zheng, T.S., Na, S., Kuan, C.-y., Yang, D., Kamsuyama, H., Rakic, P. & Flavell, R.A. (1996). Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 384, 368-372. Kuida, K., Haydar, T.F., Kuan, C. Y., Gu, Y., Taya, C., Karasuyama, H., Su, M.S., Rakic, P.& Flavell, R.A. (1998). Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 94, 325-337. Leist, M. & Nicotera, P. (1997). Calcium and neural death. Rev. Physiol. Pharmacol. Biochem. 132, 79-125. Leist, M., Gantner, F., Bohlinger, I., Tiegs, G., Germann, P.G. & Wendel, A. (1995). Tumor necrosis factor-induced hepatocyte apoptosis precedes liver failure in experimental murine shock models. Am J. Pathol., 146, 1220-1234. Leist, M., Fava, E., Montecucco, C. & Nicotera, P. (1997a). Peroxynitrite and NO-donors induce neuronal apoptosis by eliciting autocrine excitotoxicity. Eur. J. Neurosci. 9, 1488-1498. Leist, M., Single, B., Castoldi, A.F., K0hnle, S. & Nicotem, P. (1997b). Intracellular ATP concentration: a switch deciding between apoptosis & necrosis. J. Exp. Med. 185, 1481-1486. Leist, M., Volbracht, C., Kiihnle, S., Fava, E., Ferrando-May, E. & Nicotera, P. (1997c). Caspase-mediated apoptosis in neuronal excitotoxicity triggered by nitric oxide. Mol. Med. 3,750-764. Leist, M., Volbracht, C., Fava, E. & Nicotera, P. (1998). Excitotoxic mechanisms in 1-methyl-4phenylpyridinium (MPP+)-induced neuronal apoptosis. Mol. Pharmacol. in press. Lemaire, C., Andrrau, K., Souvannavong, V. & Adam, A. (1998). Inhibition of caspase activity induces a switch from apoptosis to necrosis. FEBS Lett. 425,266-270. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S. & Wang, X. (1997). Cytochrome c and dATP-dependent formation of Apaf-1/Caspase-9 complex initiates an apoptotic protease cascade. Cell 91,479-489. Lin, K.-T., Xue, J.-Y., Nomen, M., Spur, B. & Wong, P.Y.-K. (1995). Peroxynitrite-induced apoptosis in HL-60 cells. J. Biol. Chem. 270, 16487-16490. Lipton, S. A. & Rosenberg, P. A. (1994). Excitatory amino acids as a final common pathway for neurologic disorders. New Engl. J. Med. 330, 613-622. Lipton, S.A., Choi, Y.-B., Pan, Z.-H., Lei, S.Z., Chen, H.-S.V., Sucher, N.J., Loscalzo, J., Singel, D.J. & Stamler, J. S. (1993). A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature 364, 626-632. Loddick, S.A., MacKenzie, A. & Rothwell, N.J. (1996). An ICE inhibitor, z-VAD-DCB attenuates ischaemic brain damage in the rat. NeuroReport 7, 1465-1468. Longthorne, V.L. & Williams, G.T. (1997). Caspase activity is required for commitment to fas-mediated apoptosis. EMBO J. 16, 3805-3812. Luo, X., Budihardjo, I., Zou, H., Slaughter, C. & Wang, X. (1998). Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94, 481-490. Ma, J., Endres, M. & Moskowitz, M. A. (1998). Synergistic effects of caspase inhibitors and MK-801 in brain injury after transient focal cerebral ischaemia in mice. Br. J. Pharmacol. 124, 756-762. Mannick, J.B., Asano, K., Izumi, K., Kleff, E. & Stamler, J.S. (1994). Nitric oxide produced by human B

344

M. Leist and P. Nicotera

lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation. Cell 79, 1137-1146. Mannick, J.B., Miao, X. Q. & Stamler, J. S. (1997) Nitric oxide inhibits Fas-induced apoptosis. J. Biol. Chem. 272, 24125-24128. Martinou, J.C., Dubois-Dauphin, M., Staple, J.K., Rodriguez, I., Frankowski, H., Missotten, M., Albertini, P., Talabot, D., Catsicas, S., Pietra, C. & Huarte, J. (1994). Overexpression of bcl-2 in transgenic mice protects neurons from naturally occuring cell death and experimental ischemia. Neuron 13, 1017-1030. Martin-Villalba, A., Herr, I., Jeremias, I., Vogel, J., Brandt, R. Winter, C., Herdegen, T., Debatin, K-M., Schenkel, & J. Zimmerman, M. (1998) Protection against brain ischemia induced apoptosismediated by the CD95 ligand system in lpr mice 28m Annual meeting of the Society for Neuroscience, Volume 24 Abstract 112.9 pag 275, 1998 Mattson, M.P. (1998). Modification of ion homeostasis by lipid peroxidation: roles in neuronal degeneration and adaptive plasticity. Trends Neurosci. 21, 53-57. McCarthy, N.J., Whyte, M.K.B., Gilbert, C. S. & Evan, G. I. (1997). Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the bcl-2 homologue bak. J. Cell Biol. 136, 215-227. Meffert, M.K., Calakos, N.C., Scheller, R. H. & Schulman, H. (1996). Nitric oxide modulates synaptic vesicle docking/fusion reactions. Neuron 16, 1229-1236. Meffert, M.K., Premack, B.A. & Schulman, H. (1994). Nitric oxide stimulates calcium-independent synaptic vesicle release. Neuron 12, 1235-1244. Meldrum, B. & Garthwaite, J. (1990). Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol. Sci. 11,379-387. Melino, G., Bernassola, F., Knight, R.A., Corasaniti, M. T., Nistico, G, & Finazzi-Agro, A. (1997). S-nitrosylation regulates apoptosis. Nature 388, 432-433. Messmer, U.K. & B~ne, B. (1995). Nitric oxide-induced apoptosis: p53-dependent and p53-independent signalling pathways. Biochem. J. 319, 299-305. Miller, T.M., Moulder, K.L., Knudson, C.M., Creedon, D.J., Deshmukh, M., Korsmeyer, S.J. & Johnson, E.M., Jr. (1997). Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death. J. Cell Biol. 139, 205-217. Mohr, S., McCormick, T.S. & Lapetina, E.G. (1998). Macrophages resistant to endogenously generated nitric oxide-mediated apoptosis are hyposensitve to exogenously added nitric oxide donors: dichotomous apoptotic response independent of caspase-3 and reversal by the mitogen-activated protein kinase (MEK) inhibitor PD 098059. Proc. Natl. Acad. Sci. USA 95, 5045-5050. Mohr, S., Zech, B., Lapetina, E.G. & Briane, B. (1997). Inhibition of caspase-3 by S-nitrosation and oxidation caused by nitric oxide. Biochem. Biophys. Res. Commun. 238, 387-391. Monyer, H., Giffard, R.G., Hartley, D.M., Dugan, L.L., Goldberg, M.P. & Choi, D.W. (1992). Oxygen or glucose deprivation-induced neuronal injury in cortical cell cultures is reduced by tetanus toxin. Neuron 8, 967-973. Nicholls, D. & Attwell, D. (1990). The release & uptake of excitatory amino acids. Trends Pharmacol. Sci. 11,462-468. Nicotera, P. & Leist, M. (1997). Energy supply and the shape of death in neurons and lymphoid cells. Cell Death Differ. 4, 435-442. Nicotera, P., Hartzell, P., Baldi, C., Svensson, S.-A., Bellomo, G. & Orrenius, S. (1986). Cystamine induces toxicity in hepatocytes through the elevation of cytosolic Ca2÷ and the stimulation of a nonlysosomal proteolytic system. J. Biol. Chem. 261, 14628-14635. Novelli, A., Reilly, J.A., Lysko, P.G. & Henneberry, R.C. (1988). Glutamate becomes neurotoxic via the

Neuronal Apoptosis and Excitotoxicity

345

N-methyl-D-aspartate receptor when intracellular energy levels are reduced. Brain Res. 45 I, 205-212. O'DelI, T.J., Hawkins, R.D., Kandel, E.R. & Arancio, O. (1991). Tests of the roles of two diffusible substances in long-term potentiation: evidence for nitric oxide as a possible early retrograde messenger. Proc. Natl. Acad. Sci. USA 88, 11285-11289. Oppenheim, R.W. (1991). Cell death during development of the nervous system. Annu. Rev. Neurosci. 14, 453-501. Pang, Z. & Geddes, J.W. (1997). Mechanisms of cell death induced by the mitochondrial toxin 3-nitropropionic acid: acute excitotoxic necrosis and delayed apoptosis. J. Neurosci. 17, 3064-3073. Portera-Cailliau, C., Price, D.L. & Martin, L. J. (1997). Excitotoxic neuronal death in the immature brain is an apoptosis-necrosis morphological continuum. J. Comp. Neurol. 378, 88-104. Radi, R., Beckmann, J.S., Bush, K.M. & Freeman, B.A. (1991). Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. J. Biol. Chem. 266, 42444250. Raff, M.C., Barres, B.A., Burne, J.F., Coles, H.S., Ishizaki, Y. & Jacobson, M.D. (1993). Programmed cell death and the control of cell survival: lessons from the nervous system. Science 262, 695-700. Ren, Y. & Savill, J. (1998). Apoptosis: the importance of being eaten. Cell Death Differ. 5,563-568. Rossi, A.D., Viviani, B., Zhivotovsky, B., Manzo, L., Orrenius, S., Vahter, M. & Nicotera, P. (1997). Inorganic mercury modifies calcium -z* signalling, triggers apoptosis and potentiates NMDA toxicity in neural cells. Cell Death Differ. 4, 317-324. Rudel, T. & Bokoch, G. M. (1997). Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. Science 276, 1571-1574. Sagot, Y., Dubois-Dauphin, M., Tan, S.A., de Bilbao, F., Aebischer, P., Martinou, J.C. & Kato, A.C. (1995). Bcl-2 overexpression prevents motoneuron cell body loss but not axonal degeneration in a mouse model of a neurodegenerative disease. J. Neurosci. 15, 7727-7733. Sandani, A.F., Dawson, T.M. & Dawson, V.L. (1997). Nitric oxide synthase in models of focal ischemia. Stroke 28, 1283-1288. Schinder, A.F., Olson, E.C., Spitzer, N.C. & Montal, M. (1996). Mitochondrial dysfunction is a primary event in glutamate neurotoxicity. J. Neurosci. 16, 6125-6133. Schulz, J.B., Huang, P.L., Matthews, R.T., Passov, D., Fishman, M.C. & Beal, M.F. (1996). Striatal malonate lesions are attenuated in neuronal nitric oxide synthase knockout mice. J. Neurochem. 67,430-433. Schulz, J.B., Matthews, R.T., Jenkins, B.G., Ferrante, R.J., Siwek, D., Henshaw, D.R., Cipolloni, P.B., Mecocci, P., Kowall, N.W., Rosen, B.R. & Beal, M. F. (1995). Blockade of neuronal nitric oxide synthase protects against excitotoxicity in vivo. J. Neurosci. 15, 8419-8429. Schulz, J.B, Weller, M., Matthews, R.T., Heneka, M.T., Groscurth, P., Martinou J-C, Lommatzsch, J., von Coelln, R., Wtillner, U., L6schmann, P-A., Beal, M.F., Dichgans, J. & Klockgether. T. (1998). Extended therapeutic window for caspase inhibition and synergy with MK-801 in the treatment of cerebral histotoxic hypoxia Cell Death Differ. 5, 847-857. Schwartz, L.M., Smith, S.W., Jones, M.E.E. & Osborne, B.A. (1993). Do all programmed cell deaths occur via apoptosis? Proc. Natl. Acad. Sci. USA 90, 980-984. Schweizer, M. & Richter, C. (1994). Nitric oxide potently and reversibly deenergizes mitochondria at low oxygen tension. Biochem. Biophys. Res. Commun. 204, 169-175. Schweizer, M. & Richter, C. (1996) Peroxynitrite stimulates the pyridine nucleotide-linked Ca 2÷ release from intact rat liver mitochondria. Biochem. 35, 4524-4528. Shimizu, S., Eguchi, Y., Kamiike, W., Waguri, S., Uchiyama, Y, Matsuda, H. & Tsujimoto, Y. (1996). Retardation of chemical hypoxia-induced necrotic cell death by Bcl-2 and ICE inhibitors: possible involvement of common mediators in apoptotic and necrotic signal transductions. Oncogene 12,

346

M. Leist and P. Nicotera

2045-2050. Snyder, S.H. (1996). No NO prevents parkinsonism. Nature Med. 2, 965-966. Sohn, S., Kim, E.Y. & Gwag, B.J. (1998). Glutamate neurotoxicity in mouse cortical neurons: atypical necrosis with DNA ladders and chromatin condensation. Neurosci. Lett. 240, 147-150. Srivastava, R., Brouillet, E., Beal, M. F., Storey, E. & Hyman, B.T. (1993). Blockade of 1-methyl-4phenylpyridinium ion (MPP*) nigml toxicity in the rat by prior decortication or MK-801 treatment: a stereological estimate of neuronal loss. Neurobiol. Aging 14, 295-301. Stadler, J., Curran, R.D., Ochoa, J.B., Harbrecht, B.G., Hoffman, R.A., Simmons, R.L. & Billiar, T.R. (1991). Effect of endogenous nitric oxide on mitochondrial respiration of rat hepatocytes in vitro and in vivo. Arch. Surg. 126, 186-191. Stamler, J.S. (1994). Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell 78,931-936. Stamler, J.S., Jia, L., Eu, J.P., McMahon, T.J., Demchenko, I.T., Bonaventura, J., Gernert, K. & Piantadosi, C.A. (1997). Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient. Science 276, 2034-2037. Stamler, J.S., Simon, D.I., Osborne, J.A., Mullins, M.E., Jaraki, O., Michel, T., Singel, D.J. & Loscalzo, J. (1992). S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc. Natl. Acad. Sci. USA 89, 444-448. Stefanis, L., Park, D.S., Yan, C.Y.I., Farinelli, S.E., Troy, C.M., Shelanski, M.L. & Greene, L.A. (1996). Induction of CPP32-1ike activity in PC12 cells by withdrawal of trophic support. Dissociation from apoptosis. J. Biol. Chem. 271, 30663-30671. Szatkowski, M. & Attwell, D. (1994). Triggering and execution of neuronal death in brain ischaemia: two phases of glutamate release by different mechanisms. Trends Neurosci. 17, 359-365. Tenneti, L., D'Emilia, D.M. & Lipton, S.A. (1997). Suppression of neuronal apoptosis by S-nitrosylation of caspases. Neurosci. Lett. 236, 139-142. Tenneti, L., D'Emilia, D.M., Troy, C.M. & Lipton, S,A. (1998). Role of caspases in N-methyl-D-aspartateinduced apoptosis in cerebrocortical neurons. J. Neurochem. 71,946-959. Troy, C.M., Derossi, D., Prochiantz, A., Greene, L.A. & Shelanski, M.L. (1996). Downregulation of Cu/Zn superoxide dismutase leads to cell death via the nitric oxide-peroxynitrite pathway. J. Neurosci. 18, 253-261. Turski, L., Bressler, K., Rettig, K.-J., L~schmann, P.-A. & Wachtel, H. (1991). Protection of substantia uigra from MPP+ neurotoxicity by N-methyl-D-aspartate antagoists. Nature 349, 414-418. Vercammen, D., Beyaert. R., Denecker, G., Goossens, V., Van Loo, G., Declercq, W., Grooten, J., Fiers, W. & Vandenabeele, P. (1998). Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor. J. Exp. Med. 187, 1477-1485. Wallace, D.C., Shoffner, J.M., Trounce, I., Brown, M.D., Ballinger, S.W., Corral-Debrinski, M., Horton, T., Jun, A. S. & Lott, M.T. (1995). Mitochondrial DNA mutations in human degenerative diseases and aging. Biochim. Biophys. Acta. 1271, 141-151. Wang, K.K.W. & Yuen, P.-W. (1997). Development and therapeutic potential of calpain inhibitors. Adv. Pharmacol. 37, 117-153. Wang, K.K.W., Posmantur, R., Nadimpalli, R., Nath, R., Mohan, P., Nixon, R.A., Talanian, R.V., Keegan, M., Herzog, L. & Allen, H. (1998). Caspase-mediated fragmentation of calpain inhibitor protein calpastatin during apoptosis. Arch. Biochem. Biophys. 356, 187-196. Weil, M., Jacobson, M.D., Coles, H. S. R., Davies, T.J., Gardner, R.L., Raft, K.D. & Raft, M.C. (1996). Constitutive expression of the machinery for programmed cell death. J. Cell. Biol. 133, 1053-1059.

Neuronal Apoptosis and Excitotoxicity

347

West, A.R. & Galloway, M.P. (1997). Endogenous nitric oxide facilitates striatal dopamine and glutamate efflux in vivo: role of ionotropic glutamate receptor-dependent mechanisms. Neuropharmacol. 36, 1571-1581. White, R.J. & Reynolds, I.J. (1996). Mitochondiral depolarization in glutamate-stimulated neurons: an early signal specific to excitotoxin exposure. J. Neurosci. 16, 5688-5697. Wink, D.A., Hanbauer, I., Krishna, M.C., DeGraff, W., Gamson, J. & Mitchell, J.B. (1993). Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species. Proc. Natl. Acad. Sci. U.S.A. 90, 9813-9817. Xiang, H., Kinoshita, Y., Knudson, C.M., Korsmeyer, S.J., Schwartzkroin, P.A. & Morrison, R.S. (1998). Bax involvement in p53-mediated neuronal cell death. J. Neurosci. 18, 1363-1373. Zeevalk, G.D. & Nicklas, W.J. (1990). Chemically induced hypoglycemia and anoxia: relationship to glutamate receptor-mediated toxicity in retina. J. Pharmacol. Exp. Themp. 253, 1285-1292. Zeevalk, G.D., Derr-Yellin, E. & Nicklas, W.J. (1995a). NMDA receptor involvement in toxicity to dopamine neurons in vitro caused by the succinate dehydrogenase inhibitor 3-nitropropionic acid. J. Neurochem. 64, 455-458. Zeevalk, G.D., Derr-Yellin, E. & Nicklas, W.J. (1995b). Relative vulnerability of dopamine and GABA neurons in mesencephalic culture to inhibition of succinate dehydrogenase by malonate and 3-nitropropionic acid and protection by NMDA receptor blockade. J. Pharmacol. Exp. Ther. 275, 1124-1130.

Zhang, J. & Snyder, S.H. (1995). Nitric oxide in the nervous system. Annu. Rev. Pharmacol. Toxicol. 35,213-233. Zhang, J., Dawson, V.L., Dawson, T.M. & Snyder, S.H. (1994). Nitric oxide activation of poly(ADP-ribose) synthetase in neurotoxicity. Science 263,687-689. Zhong, L.-T., Sarafian, T., Kane, D.J., Charles, A.C., Mah, S.P., Edwards, R.H. & Bredesen, D.E. (1993) bcl-2 inhibits death of central neural cells induced by multiple agents. Proc. Natl. Acad. Sci. U.S.A 90, 4533-4537. Zhuang, J., Ren, Y., Snowden, R.T., Zhu, H., Gogvadze, V., Savill, J.S. & Cohen, G.M. (1998). Dissociation of phagocyte recognition of cells undergoing apoptosis from other features of the apoptotic program. J. Biol. Chem. 273, 15628-15632. Zoratti, M. & Szabo, I. (1995). The mitochondrial permeability transition. Biochim. Biophys. Acta. 1241, 139-176.

CONTRIBUTOR ADDRESSES

Volume I: Programmed Cell Death: Cellular and Molecular Mechanisms Edited by Mark P. Mattson, Steven Estus and Vikek M. Rangnekar

BENJAMIN O. ANDERSON, University of Washington, Department of Surgery, Box 356410, Seattle, WA 98195-6410 Phone: (206) 543-6352, Fax: (206) 543-8136, e-mail: [email protected] ANNADORA J. BRUCE-KELLER, PhD., Department of Anatomy and Neurobiology, Mn 210 Chandler Medical Center, University of Kentucky, Lexington, KY 40536-0298 Phone: (859) 323-4601 , Fax: (859) 323-5946, e-mail: [email protected] SOPHIE CAMILLERI-BROET, Service d'Anatomie Pathologique, Hotel Dieu, 1 place du parvis Notre Dame, 75004 Paris Phone: (01) 42348709, Fax: (01) 42348641 SIC L. CHAN, Sanders-Brown Center on Aging, 211 Sanders-Brown Center, University of Kentucky, Lexington, KY 40536-0230 Phone: (606) 257-1412, ext. 244, Fax: (606) 323-2866 QUIN L DEVERAUX, Genomics Institute of the Novartis Research Fondation STEVEN ESTUS, PhD., Sanders-Brown Center on Aging, Room 332 Sanders-Brown Building., University of Kentucky, Lexington, KY 40536-0230 Phone: (859) 323-3985, ext. 264, Fax: (859) 323-2866, e-mail: sestus @aging.coa.uky.edu Q1NG GUO, PhD., Assistant Professor of Neurobiology, Department of Neurobiology and Pharmacology, Northeastern Ohio Universities College of Medicine (NEOUCOM), 4209 State Route 44, P.O. Box 95, Rootstown, Ohio 44272-0095, U.S.A. Phone: (330) 325-6655 (Office), (330) 325-6644 (Labl), (330) 325-6488 (Lab2), Fax: (330) 325-5916, e-mail: [email protected] DAVID M. HOCKENBERY, MD., Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue. N., Mailstop: C3-168, Seattle, Washington 98195 Phone: (206) 667-4611 or (206) 667-6524, e-mail: [email protected] INNA KRUMAN. PhD., Sanders-Brown Center on Aging, 211 Sanders-Brown Center, University of Kentucky, Lexington, KY 40536-0230 Phone: (606) 257-1412, ext. 244, Fax: (606) 323-2866

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GARY E. LANDRETH, PhD., Alzheimer Research Laboratory, RmE504, Case Western Reserve University Medical School, 10900 Euclid Avenue, Cleveland, OH 44106 Phone: (216) 368-6101, Fax: (216) 368-3079, e-mail: [email protected] MARCEL LEIST, University of Konstanz, Box X911, D-78457 Konstanz, Germany Phone: (+49) 7531-884035, Fax: (+49) 7531-884033 DAN L. LONGO, MD., National Institute on Aging, 5600 Nathan Shock Drive, Baltimore, MD 21224 Phone: (410) 558-8110, Fax: (410) 558-8137, e-mail: [email protected] MARIANGELA MANCINI, Institute di Urologia, University of Padova, Via Giustiniani 2, 35128 Padova Phone: (39)049 8212724, Fax: (39)049 8212721, e-mail: [email protected] MARK P. MATTSON, Chief, Laboratory of Neurosciences, National Institute on Aging. Professor, Department of Neurosciences, Johns Hopkins University. NIA Gerontology Research Center - 4F02, 5600 Nathan Shock Drive, Baltimore, MD 21224 Phone: (410) 558-8463, Fax: (410) 558-8465, Email: [email protected] BERNARD MIGNOTTE, Universite De Versailles/Saint-Quentin, CNRS UPRESA 8087, Equipe Apoptose, Batiment Fermat, 45 avenue des Etats Unis, 78035 Versailles Cedex FRANCE Phone: (33) 1 39 25 36 50, Fax: (33) 1 39 25 36 55, e-mail: [email protected], Web: http://genome.genetique.uvsq.fr/apoptose/ JASON MILLS, MD., PhD., Department of Molecular Biology and Pharmacology, Washington University School of Medicine, Box 8103, 660 S. Euclid Avenue, St. Louis, MO 63110 Phone: (314) 362-5443, Fax: (314) 362-7058, e-mail: [email protected] / [email protected] Prof. PIERLUIGI NICOTERA, Chair of Molecular Toxicology, University of Konstanz, Germany Phone: (+49) 7531-884035, Fax: (+49) 7531-884033, e-mail: Pierluigi.Nicotera@ uni-konstanz.de VIVEK M. RANGNEKAR, Departments of Radiation Medicine, and Microbiology/ Immunology, University of Kentucky, Combs Cancer Research Building, Room 303, 800 Rose Street, Lexington, KY 40536 Phone: (606) 257-2677; Fax: (606) 257-9608, e-mail: [email protected]

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JOHN C. REED, MD., PhD., The Burnham Institute, Program on Apoptosis and Cell Death Research, 10901 N. Torrey Pines Rd., La Jolla, CA 92037-1062 Phone: (619) 646--3140, Fax: (619) 646-3194, e-mail: [email protected] RAKESH K. SRIVASTAVA, Laboratory of Immunology, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Box 28, Baltimore, MD 21224-6825 Phone: (410) 558-8480, Fax: (410) 558-8284, e-mail: [email protected] STEVEN P. TAMMARIELLO, Assistant Professor, Department of Biological Sciences, Binghamton University, Science III, Room 142, Binghamton, NY 13902-6000 Phone: (607) 777-2008, ext. 244, Fax: (607) 777-6521, e-mail: [email protected] CAROL M. TROY, MD., PhD., Department of Pathology, College of Physicians & Surgeons, Columbia University, 630 W. 168th St, New York, NY 10032 Phone: (212) 305-6371, Fax: (212) 305-5498, e-mail: [email protected] JEAN-LUC VAYSSIERE, Universite De Versailles/Saint-Quentin, CNRS UPRESA 8087, Equipe Apoptose, Batiment Fermat, 45 avenue des Etats Unis, 78035 Versailles Cedex FRANCE Phone: (33) 1 39 25 36 50, Fax: (33) 1 39 25 36 55, e-mail: mignotte @genetique.uvsq.fr