Advances in Cancer Research Volume 71

Advances in

CANCER RESEARCH Volume 71

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Advances in

CANCER RESEARCH Volume 71

Edited by

George F. Vande Woude ABL-Basic Research Program National Cancer Institute Frederick Cancer Research and Development Center Frederick, Maryland

George Klein Microbiology and Tumor Biology Center Karolinska Institutet Stockholm, Sweden

ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto

This book is printed on acid-free paper.

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Copyright 0 1997 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1997 chapters are as shown on the title pages, if no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-230X/97 $25.00

Academic Press a division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.apnet.com Academic Press Limited 24-28 Oval Road, London NWl 7DX, UK http://www.hbuk.co.uk/ap/ International Standard Book Number: 0- 12-006671-8

P ” W D IN THE UNITEDSTATES OF AMERlCA 97 9 8 9 9 00 01 0 2 B B 9 8 7 6

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Contents

Contributors to Volume 71 ix

FOUNDATIONS IN CANCER RESEARCH p 5 3 and ATM: Cell Cycle, Cell Death, a n d Cancer Susan E. Morgan and Michael B. Kastan I. Introduction 1 11. The p53 Tumor Suppressor Gene 2 111. ATM 9 IV. Cell Cycle Control 16 References 19

Deletions of the Short Arm of Chromosome 3 in Solid Tumors and t h e Search for Suppressor G e n e s Klaas Kok, Susan L. Naylor, and Charles H. C. M. Buys I. Tumor Suppressor Genes: The Concept 28 30 111. Chromosome 3 Losses in Different Types of Tumors 35 IV. Functional Assays of Tumor Suppression on Chromosome 3 54 V. (Presumed) Tumor Suppressor Genes on the Short Arm of Chromosome 3 58 VI. Evolutionary Aspects of Human Chromosome 3 73 VII. Concluding Remarks 75 References 77 11. Methods of Localizing Tumor Suppressor Genes

Mutations Predisposing to Hereditary Nonpolyposis Colorectal Cancer Paivi Peltomaki and Albert de la Chapelle I. Introduction 94 11. The HNPCC Syndrome 95 111. HNPCC and DNA MMR 97

IV Mutations Predisposing to HNPCC 101 V. Phenotypic Effects of MMR Gene Mutations

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VI. Implications of Mutation Findings 114 References 115

Functional Aspects of Apoptosis in Hematopoiesis and Consequences of Failure Sharon L. McKenna and Thomas G. Cotter I. Introduction 122 11. Morphological and Biochemical Features of Apoptosis 123 III. Molecular Mechanisms in Apoptosis 125 IV. Functional Aspects of Apoptosis in the Hematopoietic System 131 V. Disruption of Apoptosis in Hematopoiesis 141 VI. Future Perspectives 152 References 154

Cyclin-Dependent Kinase Regulation during G 1 Phase and Cell Cycle Regulation by TGF-f3 Michael 1. Ravitz a n d Charles E. Wenner I. Introduction 166 II. Cyclins and Cyclin-DependentKinases 168 III. Conclusions 198 References 199

The Natural Somatic Mutation Frequency and Human Carcinogenesis Andrew 1. G . Simpson I. Introduction 210 11. Somatic Mutation of Microsatellite Sequences 211 111. Somatic Mutations of Minisatellite Sequences 217 IV. Somatic Mutation of the HPRT Gene 217 V. The Frequency of Somatic Mutation in Solid Tissues Can Account for Multistep Carcinogenesis 221 VI. Cellular Proliferation as a Risk Factor for Cancer 222 VII. Germline Mutations 224 VIII. The Mutation Rate as the Fundamental Biological Pacemaker 231 IX. The Importance of Measuring Somatic Mutation Rates 233 X. The Mutational Clock and Cancer Prevention 234 References 235

CD44: Structure, Function, and Association with the Malignant Process David Naor, Ronit Vogt Sionov, a n d Dvorah Ish-Shalom I. Introduction

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11. CD44 Nomenclature 245 111. CD44 Biochemical Structure 246 IV. CD44 Expression on Normal Cells 253 V. Hyaluronic Acid Is the Principal Ligand of CD44 259 VI. Non-HA Ligands of CD44 272 VII. Soluble CD44 274 VIII. Genetic Control of CD44 Expression 275 IX. CD44 Functions 275 X. Involvement of CD44 in Physiological and Pathological Cell Activities 285 XI. CD44 Association with the Malignant Process in Experimental Models 287 XII. CD44 Expression in Human Neoplasms and Its Correlation with the Malignant Status 291 XIII. CD44 Association with Malignancy: Some Practical Comments 304 XIV. Conclusions 305 References 307

Human Papillomaviruses and Cervical Cancer Luisa Lina Villa I. 11. 111. IV. V.

Introduction 321 Biology of Papillomaviruses 322 Taxonomy and Genomic Variability of Papillomaviruses Epidemiological Aspects 327 HPV Interaction with Cofactors 330 VI. Viral Persistence and Disease Progression 331 VII. Viral Burden and Cervical Disease 333 VIII. HFV in Cervical Screening Programs 333 References 335

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HER-2heu Protein: A Target for Antigen-Specific lmmunotherapy of Human Cancer Mary L. D i s k a n d Martin A. Cheever I. Introduction

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11. HER-2heu Vaccines for Cancer Therapy

346 111. Potential Pitfalls Associated with HER-2/neu-Specific Imrnunotherapy IV. HER-2heu Specific Antibodies for Cancer Therapy 360 V. Conclusion 366 References 367

Index

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Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Charles H. C. M. Buys, Department of Medical Genetics, University of Groningen, 9713 AW Groningen, The Netherlands (27) Albert de la Chapelle, Department of Medical Genetics, Haartman Institute, University of Helsinki, 00290, Finland (93) Martin A. Cheever, Division of Oncology, University of Washington, Seattle, Washington 98195 (343) Thomas G. Cotter, Tumour Biology Laboratory, Department of Biochemistry, University College, Cork, Ireland (121) Mary L. Disis, Division of Oncology, University of Washington, Seattle, Washington 98195 (343) Dvorah Ish-Shalom, The Lautenberg Center for General and Tumor Immunology, The Hebrew University Hadassah Medical School, Jerusalem 91120, Israel (241) Michael B. Kastan, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205 (1) Klaas Kok, Department of Medical Genetics, University of Groningen, 9713 AW Groningen, The Netherlands (27) Sharon L. McKenna, Tumour Biology Laboratory, Department of Biochemistry, University College, Cork, Ireland (121) Susan E. Morgan, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205 (1) David Naor, The Lautenberg Center for General and Tumor Immunology, The Hebrew University Hadassah Medical School, Jerusalem 91120, Israel (241) Susan L. Naylor, Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284 (27) Paivi Peltomaki, Department of Medical Genetics, Haartman Institute, University of Helsinki, 00290 Helsinki, Finland (93) Michael J. Ravitz, Department of Biochemistry, Roswell Park Cancer Institute, hffalo, New York 14263 (165) ix

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Contributors

Andrew J. G. Simpson, Laboratory of Cancer Genetics, Ludwig Institute for Cancer Research, 01509-010, S5o Paulo, SPYBrazil (209) Ronit Vogt Sionov, The Lautenberg Center for General and Tumor Immunology, The Hebrew University Hadassah Medical School, Jerusalem 91120, Israel (241) Luisa Lina Villa, Ludwig Institute for Cancer Research, 01509-010, Sio Paulo, SP, Brazil (321) Charles E. Wenner, Department of Biochemistry, Roswell Park Cancer Institute, Buffalo, New York 14263 (165)

FOUNDATIONS IN CANCER RESEARCH p53 and ATM: Cell Cycle, Cell Death, and Cancer Susan E. Morgan and Michael B. Kastan Thelohns Hopkins Oncology Center, Baltimore, Maryland 21 205

I. Introduction 11. The p53 Tumor Suppressor Gene A. p53 Mediates a G1 Arrest in Response to DNA Damage B. p53 and Genetic Instability C. pS3 and Programmed Cell Death 111. ATM A. ATM and the p53-Dependent Signal Transduction Pathway B. Structural and Phenotypic Homologs of ATM: Implications for ATM Function N. Cell Cycle Control A. Implications for Cancer Development B. Implications for Cancer Therapy References

The development of a normal cell into a tumor cell appears to depend in part on mutations in genes that normally control cell cycle and cell death, thereby resulting in inappropriate cellular survival and tumorigenesis. ATM (“mutated in ataxia-telangiectasia”)and pS3 are two gene products that are believed to play a major role in maintaining the integrity of the genome such that alterations in these gene products may contribute to increased incidence of genomic changes such as deletions, translocations, and amplifications, which are common during oncogenesis. p53 is a critical participant in a signal transduction pathway that mediates either a G1 arrest or apoptosis in response to DNA damage. In addition, p53 is believed to be involved in the mitotic spindle checkpoint and in the regulation of centrosome function. Following certain cytotoxic stresses, normal ATM function is required for pS3-mediated G1 arrest. ATM is also involved in other cellular processes such as S phase and G2-M phase arrest and in radiosensitivity. The understanding of the roles that both p53 and ATM play in cell cycle progression and cell death in response to DNA damage may provide new insights into the molecular mechanisms of cellular transformation and may help identify potential targets for improved cancer therapies.

I. INTRODUCTION Cellular responses to DNA-damaging agents are believed to be critical determinants of human tumorigenesis. Cell cycle arrests and DNA repair folAdvances in CANCER RESEARCH 0065-23OW97 $25.00

Copyright Q 1997 by Academic Press. All rights of reproduction in any form reserved.

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Susan E. Morgan and Michael B. Kastan

lowing DNA damage require the coordination of multiple gene products that, as a whole, serve to maintain the integrity of the genome. Within the cell cycle, both G1-S and G2-M phase transitions are under constant surveillance by checkpoint genes for the protection of cells from either exogenous DNA-damaging agents (environmental carcinogens), endogenous agents such as free oxygen radicals, or intrinsic cellular processes such as abnormal gene rearrangments during immunoglobulin development. Both the p53 tumor suppressor gene and the gene product ATM (“mutated in ataxia-telangiectasia”) mediate cell cycle perturbations in response to DNA damage, and play a role in cell death, genetic stability, and cancer susceptibility. p53 is a critical participant in a signal transduction pathway that mediates either a G1 arrest or programmed cell death in response to DNA damage. In addition, p53 has been implicated to be an active component of a mitotic spindle checkpoint and a regulator of centrosome function. Inactivation of normal p53 function could thus result in inappropriate replication of damaged DNA, inappropriate cellular survival after cellular stresses, or abnormal segregation of chromosomes during mitosis, thus contributing to the malignant transformation of cells. After certain cytotoxic stresses, normal ATM gene function is required for optimal signaling to p53 and the subsequent G1 checkpoint. ATM is also involved in the p53-independent S and G2-M checkpoints, suggesting that the ATM signal transduction pathway may send multiple signals to distinct effector molecules, other than p53, for each cell cycle transition. This article focuses on the current level of understanding of the roles both p53 and ATM play in cell cycle control in response to DNA damage and how alterations in these genes may contribute to tumorigenesis.

11. THE p 5 3 TUMOR SUPPRESSOR GENE

A. p 5 3 Mediates a GI Arrest in Response

to DNA Damage Overexpression of the p.53 phosphoprotein inhibits cell growth by mediating an arrest in the cell cycle prior to or near the restriction point in G1 following exposure to certain DNA-damaging agents (Baker et al., 1990; Diller et al., 1990; Martinez et al., 1991; Ullrich et al., 1992; Vogelstein and Kinzler, 1992; Zambetti and Levine, 1993).The concept that this growth arrest function of p53 was utilized to mediate a G1 arrest after DNA damage arose from the observation that wild-type p.53 protein levels are transiently induced in response to ionizing radiation (IR) in temporal correlation with an arrest of cells in the G1 phase of the cell cycle (Kastan et al., 1991; Kuer-

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bitz et al., 1992). This functional role of p53 was further supported by studies demonstrating that either loss of expression of wild-type p53 or overexpression of mutant p53 resulted in the lack of a G1 cell cycle arrest following damage (Kessis et al., 1993; Kuerbitz et al., 1992). Furthermore, embryonic fibroblasts from p53-null mice completely lose the G1 arrest in response to IR (Kastan et al., 1992). The induction of p53 in response to DNA damage results in p53-mediated transcriptional activation of genes involved in the G1 arrest. The induction of wild-type p53 protein levels in response to DNA damage is believed to be regulated by a posttranscriptional mechanism, with metabolic stabilization of the p53 protein being, in part, an important feature of this process (Kasran et al., 1991; Maltzman and Czyzyk, 1984; Tishler et al., 1993). DNA strand breaks are believed to be the primary signal leading to the induction of p53 protein levels following DNA damage (Nelson and Kastan, 1.994; Zhan et al., 1994). This was supported by studies using nuclear microinjection of defined DNA substrates (Huang et al., 1996). These studies suggested that double-strand breaks and single-strand gaps of greater than 30 nucleotides are sufficient to induce a p53-dependent G1 arrest and, furthermore, that as few as one double-strand break may be adequate for p53 induction. p53 is a phosphorylated protein, and in vitro experiments have demonstrated that phosphorylation of p53 can modulate its sequence-specific DNA-binding activity (Meek, 1994). Protein kinases that may be involved in p53 phosphorylation include cyclin-dependent kinases (cdks), casein kinase I and 11, protein kinase C, DNA-dependent protein kinase (DNA-PK) (Lees-Miller et al., 1992), mitogen-activated protein kinase (Milne et al., 1994),jun amino-terminal kinase (Milne et al., 1995),and rafkinase (Jamal and Ziff, 1995). Whether such protein kinases potentiate p53-dependent transcriptional transactivation specifically in response to IR, however, remains unclear. Elucidation of the mechanism by which p53 mediates a G1 cell cycle arrest was facilitated by the identification of several growth arrest-related gene products that are transcriptionally activated by p53. The induction of p53 in response to IR activates the expression of several genes, including gadd45 (growth arrest and DNA damage-inducible), p21 (WAF-1, cipl, SDIl), and mdm2 (Fig. 1).The gadd45 gene was the first p53-regulated gene identified whose induction following IR is dependent on normal p53 function (Kastan et al., 1992; Papathanasiou et al., 1991; Zhan et al., 1994).The presence of a consensus p53 binding site located within intron 3 of the gadd45 gene suggested that gadd45 transcriptional enhancement following IR is mediated by direct binding of p53 to this gadd4.5 intronic sequence (Kastan et al., 1992). Gadd45 overexpression results in the inhibition of both colony formation and progression of cells into S phase (Smith et al., 1994; Zhan et al., 1994),

Susan E. Morgan and Michael B. Kastan

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Ionizin Radiation

f

(DNA strand breaks)

U1 (ATM)

Fig. 1 Schematic diagram of irradiation-induced, pS3-dependent G1 checkpoint, which results in either a cell cycle arrest or apoptosis. Ionizing radiation causes DNA strand breaks that lead to a posttranscriptional increase in p53 protein levels. Optimal induction of p53 after irradiation appears to require a normal ATM gene product. Increased p53 protein levels lead to the transcriptional transactivation of several genes, including gudd45, mdm2, p21, bux, and cyclin G. It is not yet known whether the p53-responsive genes IGF-BP3 and TSP-1 can be induced specifically in response to IR. An increase in p21 protein levels appears to contribute to the arrest of cells in the G1 phase of the cell cycle through inhibition of cyclin-cdk activation, thus preventing the phosphorylation of a number of protein targets, including Rb, that are required for the G1-to-S transition. One mechanism by which Rb functions appears to be by binding to a family of E2F transcription factors, thereby preventing transcriptional activation of genes required for progression into the next phase of the cell cycle; consequently,cells arrest in G1. The roles of cyclin G and gadd45 in this p53-dependent pathway are not yet known. Increased box expression may contribute to apoptosis in certain cell types.

consistent with a role for Gadd45 in inhibition of cell cycle progression. Although the mechanism by which Gadd45 induces a cell cycle arrest is unknown, recent studies have suggested an association of Gadd45 with proliferating cell nuclear antigen (PCNA), a DNA polymerase processivity factor involved in both DNA repair and DNA replication (Smith et al., 1994).Transcriptional activation of Gadd45 by p53, in response to DNA damage, may serve to play a role in the DNA repair process by Gadd45 facilitating the interaction of PCNA with DNA repair complexes. It has been hypothesized

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that some forms of damage-activated DNA repair may be p53-dependent since loss of p53 function correlated with a decrease in DNA excision repair in vitro (Ford and Hanawalt, 1995; Wang et al., 1995).In light of these studies, the role p53 may play in damage-activated DNA repair may be mediated either by direct interaction with repair complexes or through activation of Gadd45, which in turn, may bind to PCNA to stimulate DNA repair (Smith et al., 1994). Experiments that directly assess the contribution of Gadd45 to the repair process, however, have been difficult to document, suggesting that, at most, Gadd4S has only subtle effects on the DNA repair process (Kazantsev and Sancar, 1995). The definitive mechanisms by which Gadd45 functions remain under investigation. The discovery of a second p53-responsive gene, the G1 cdk inhibitor p21 (El-Deiry et al., 1993; Harper et al., 1993; Noda et al., 1994; Xiong et al., 1993a,b), directly associated p53 with cell cycle control. Cdks exist in protein complexes with a cell cycle stage-specific protein called a cyclin (Grana and Reddy, 1995; Pines, 1995; Sherr, 1994; Sherr and Roberts, 1995). The cdk component becomes activated by a cdk-activating kinase (Fisher and Morgan, 1994; Kato et al., 1994). The sequential association with G1 cyclins and activation of their appropriate cdk partners are necessary for the timely progression of cells into the next phase of the cell cycle. There are several families of G1 cdk inhibitors that directly bind and inhibit cyclin-cdk complexes by preventing cdk activation or inhibiting the kinase activity. Thus far, p21 is the only cdk inhibitor known to be regulated by p53 in response to DNA damage. Whether p21 is the only mediator of p53-mediated G1 arrest remains to be elucidated. One study suggested that a human tumor cell line that is ~ 2 1 ~ ’was - completely defective in a G1 arrest (Waldman et al., 1995), whereas studies of fibroblasts that were homozygous null for p21 appeared to be only partially defective in their ability to undergo a G1 arrest in response to IR (Brugarolas et al., 1995; Deng et al., 1995). The latter result led to the suggestion that other p53-mediated growth arrest gene products may also be required for a complete G1 arrest response. The apparent discrepancies between these studies may be a function of differences in either cell types (tumor cell versus normal fibroblast) or species (human versus mouse) and thus require further investigation. p21-mediated inhibition of cyclin E- and cyclin D-associated cdk2 complexes prevents the phosphorylation of a number of protein targets that are required for the timely progression of cells into the next phase of the cell cycle (Matsushime et al., 1992; Nevins, 1992). One of the downstream targets of cyclin-cdks is the retinoblastoma protein (pRb). One mechanism by which pRb functions appears to be by binding of hypophosphorylated pRb to a family of S-phase-promoting E2F transcription factors and prevention of transcriptional activation of gene products required for the G1-to-S transition (Nevins, 1992) (Fig. 1).Some of these gene products activated by E2F

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Susan E. Morgan and Michael B. Kastan

complexes include DNA polymerase CY and dihydrofolate reductase (Nevins, 1994). The importance of E2F activity in cell cycle progression has been demonstrated by the fact that overexpression of E2F-1 can overcome both IR-induced G1 arrest and p21-mediated inhibition of cyclin-cdk activity (DeGregori et al., 1995a,b; Johnson et al., 1993). A role for pRb and/or its related molecules, p107 and p130, in p53-mediated growth arrest has been suggested by the observation that overexpression of the human papillomavirus (HPV)E7 protein, which binds pRb, inhibited radiation-induced G1 arrest despite the induction of p21 by p53 (Demers et al., 1994; Slebos et al., 1994). In addition to interacting with cyclin-cdk complexes, p21 also has been found to bind to PCNA-replication factor C-DNA polymerase 6 complexes in vitro (Flores-Rozas et al., 1994; Li et al., 1994; Waga et al., 1994). PCNA is a subunit of the DNA polymerase 6 enzyme complex and is involved in both DNA replication and DNA excision repair. Studies using an in vitro system have shown that p21, while inhibiting similar virus 40 DNA replication, does not appear to interfere with PCNA-dependent nucleotide excision repair, even though repair and replication are carried out by the same set of proteins. The model proposed by Waga et al. (1994)suggests that p21 prevents long primer-extension DNA replication by preventing the PCNA-enzyme complex from sliding along the DNA, but does not inhibit the shorter gap-filling DNA synthesis that occurs as a result of DNA repair. The precise mechanism, however, by which p21 selectively affects the role of PCNA in repair versus replication is currently unclear. Following irradiation, wild-type p53 also transcriptionally activates a 90kDa negative regulator called mdm2 (Barak et al., 1993; Chen et al., 1994; Juven et al., 1993; Otto and Deppert, 1993). Unlike gadd45 or p21, IRinduced p53 transactivation of mdm2 appears to block p53-mediated transcriptional activity (Barak et al., 1993; Chen et al., 1994; Momand et al., 1992; Oliner et al., 1993). The downregulation of p53 function by mdm2 is thought to serve as a negative feedback loop with p53 whereby mdm2 may serve to limit the extent of a G1 arrest after DNA damage in order to allow cells to enter into the next phase of the cell cycle (Barak et al., 1993; Chen et al., 1994; Perry et al., 1993; Wu et al., 1993). The exact mechanism of mdm2-mediated abrogation of p53 transactivational activity, however, remains to be elucidated. Observations of mice with disrupted mdm2 genes supported the suggestion that there exists a delicate balance between the levels of p53 and mdm2 in mediating cell cycle progression. While the mdm2null embryos did not survive past day 5.5 of gestation, mice in which both p53 and mdm2 were disrupted survived. This suggested that even normal levels of p53 protein, unopposed by mdm2, can arrest cell growth (Jones et al., 1995; Montes de Oca Luna et al., 1995). Other p53-responsive genes have been reported, including cyclin G, the

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cell death-promoting gene bax, the insulin-like growth factor binding protein 3 (IGF-BP3), and the angiogenesis inhibitor thrombospondin-1 (TSP-1) (Fig. 1).In response to DNA damage, cyclin G was found to be induced in a p53-dependent manner (Okamota and Beach, 1994; Zauberman et al., 1995). The functional role of cyclin G has not yet been elucidated. IGF-BP3 can also be induced in response to genotoxic stress, as demonstrated in EB1 colon carcinoma cells containing an inducible wild-type, but not mutant, p53 transgene (Buckbinder et al., 1995). The enhanced secretion of an active form of IGF-BP3 by p53 can inhibit insulin-like growth factor-1 mitogenic signaling, thereby representing another potential mechanism by which p53 could mediate growth arrest. Bax induction by IR has been reported to be p53-dependent, but this induction may be cell type dependent (Miyashita et al., 1994; Selvakumaran et al., 1994).Whether Bax induction by p53 contributes to p53-mediated apoptosis is not yet clear. Finally, p53 has also been suggested to inhibit angiogenesis through regulation of TSP-1 synthesis. Tumor cells are potently angiogenic as a result of decreased production of inhibitors and an increased secretion of factors that stimulate new vessel growth. Fibroblasts derived from Li-Fraumeni patients displayed an angiogenic phenotype in vitro and in vivo coincident with the loss of wild-type p53 and reduced expression of TSP-1 (Dameron et al., 1994). Reintroduction of wild-type p53 into Li-Fraumeni fibroblasts restored both TSP-1 mRNA levels and the antiangiogenic phenotype. p53 may thus play a role in preventing tumor development by mediating TSP-1 antineoplastic activity. Further identification of p53-regulated genes is likely to continue, which will further facilitate identifying potential mechanisms by which p53 could mediate cell growth or cell death.

B. p53 and Genetic Instability Considerable experimental data support the view that loss of p53 function can lead to genomic instability and inappropriate survival of genetically damaged cells, leading to the evolution of a cancer cell. Mutation or inactivation of p53 has been linked to a wide variety of human cancers in which aneuploidy and gene amplification are common occurrences (Livingstone et al., 1992; Shuefer et al., 1993; Yin et al., 1992). The link between mutant p.53 and aneuploidy was further supported by results from studies implicating p53 as an active component of a mitotic spindle checkpoint (Cross et al., 1995) and as a regulator of centrosome function (Fukasawa et al., 1996). In yeast, several genes have been identified as components of a checkpoint that causes a mitotic arrest in response to spindle aberrations (Hoyt et al., 1991; Li and Murray, 1991). Mutations in these genes result in bypassing of this checkpoint, permitting premature rounds of DNA replication and ultimate-

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Susan E. Morgan a n d Michael B. Kastan

ly resulting in polyploidy. Studies examining the role p53 may play in the mitotic checkpoint assessed the ability of fibroblasts from p53+‘+ and p53-l- mouse embryos to arrest in mitosis after exposure to spindle inhibitors (Cross et al., 1995). In contrast to wild-type p53 fibroblasts, which arrest in response to spindle inhibitors, p53-l- mouse embryonic fibroblasts did not exhibit an arrest in the presence of mitotic inhibitors (Cross et al., 1995). Instead, these p53-null cells underwent multiple rounds of DNA synthesis without chromosome segregation, resulting in aneuploidy. These studies thus suggest a mechanism for the development of tetraploid cell populations that have a mutant p53. Specifically, mouse cells defective in the p53-dependent spindle checkpoint can undergo subsequent cell cycle events, such as DNA replication and reduplication of centrosomes and centrioles, without completing chromosomal segregation. This reduplication of centrosomes could lead to the formation of tetraploid cell populations that harbor an abnormal number of mitotic poles, resulting in aberrant chromosomal segregation and aneuploidy.

C. p53 and Programmed Cell Death In addition to the p53-mediated growth arrest in the G1 phase of the cell cycle, p53 also plays a critical role in the induction of apoptosis following DNA damage. p53-mediated apoptosis can result from a variety of stimuli, such as IR (Clarke et al., 1993; Lotem and Sachs, 1993; Lowe et al., 1993), hypoxia exposure (Graeber et al., 1996), and overexpression of transforming oncogenes such as adenovirus E1A and c-myc (Debbas and White, 1993; Lowe and Ruley, 1993; Wagner et al., 1994). The apoptosis-promoting ability of p53 was first observed following restoration of wild-type p53 activity in several different tumor cell lines that lack endogenous p53 (Levy et al., 1993; Ramqvist et al., 1993; Ryan et al., 1993; Shaw et al., 1992; Yonish-Rouach et al., 1991). Studies have shown that overexpression of a temperature-sensitive (ts) p53 in murine leukemia cells, which lack p53, resulted in apoptotic cell death at the permissive temperature, in which p53 assumes wild-type conformation and activity. Overexpression of ts p53 in other cell types such as murine fibroblasts, however, resulted in a G1 arrest upon shift to the permissive temperature without any loss of cell viability (Levy et al., 1993; Michalovitz et al., 1990; YonishRouach et al., 1991). It has been suggested that these two cellular decisions, growth arrest versus apoptosis, are distinctive endpoints of p53 induction and that which endpoint is reached may be dependent upon cell type or “cellular context” (Han et al., 1996; Slichenmeyer et al., 1993). It has been well established that cytokines and growth factors can serve as survival factors by protecting cells from apoptosis (Sachsand Lotem, 1995),

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and, in fact, many different cytokines have been shown to protect cells from p53-mediated apoptosis (Lin and Benchimol, 1995). Studies have revealed that exposure of an interleukin-3 (IL-3)-dependentmouse hematopoietic cell line (Baf-3 cells) to irradiation in the absence of IL-3 results in rapid apoptosis, whereas little apoptotic cell death is apparent following IR in the presence of IL-3 (Collins et al., 1992). These initial observations were developed into a model system in which Baf-3 cells, following exposure to IR in the presence of IL-3, undergo a transient p53-dependent G1 arrest, whereas irradiation in the absence of growth factor results in rapid p53-mediated apoptotic cell death (Canman et al., 1995). Clonogenic survival studies demonstrated that the IL-3 growth factor provides a survival signal for irradiated Baf-3 cells. Interestingly, overexpression of activated oncogenetic kinases, cRaf and v-Src, also protected irradiated Baf-3 cells from apoptotic cell death, suggesting that proto-oncogenes that are involved in growth factor signaling pathways may play a critical role in protecting tumor cells from p53-mediated apoptosis (Canman et al., 1995). Thus growth factors also serve as survival signals in response to cytotoxic stresses. To date, the cell death-promoting gene, bax, appears to be the only member of a family of apoptotic genes that is upregulated by p53 in response to IR (Miyashita et al., 1994; Selvakumaran et al., 1994). bax contains homology to the antiapoptotic gene bcl-2; bcl-2 enhances cell survival, whereas bax accelerates programmed cell death. The two proteins can form heterodimers with each other in which the ratio of Bcl-2 to Bax determines cell survival or death following an apoptotic stimulus, suggesting that this family of proteins play a critical role in the regulation of the apoptotic process (Oltvai et al., 1993). It is unknown, however, whether Bax specifically plays a role in p53-mediated apoptosis. The regulation of p53-mediated apoptosis, however, is undoubtedly complex, and the relative contributions of apoptosis-related gene products may be tissue or cell type specific.

111. ATM

A. ATM and the p53-Dependent Signal Transduction Pathway Ataxia-telangiectasia (AT) is a rare autosomal recessive disease characterized by a combination of progressive cerebellar ataxia, cellular and humoral immune dysfunction, lymphoreticular malignancies, growth retardation, and premature aging (Harnden, 1994; Shiloh, 1995). Cells derived from AT patients are hypersensitive to IR and radiomimetic drugs, exhibit chromosomal instability and abnormalities in genetic recombination, have higher re-

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Susan E. Morgan and Michael B. Kastan

quirements for serum growth factors, and exhibit cell arrest defects (Gatti et al., 1991; McKinnon, 1987; Nagasawa et al., 1985). AT cells have elevated frequencies of spontaneous and induced chromosomal aberrations, including defects in immune gene rearrangements and abnormally high spontaneous rates of intrachromosomal recombinations (Kobayashi et al., 1991; Lipkowitz et al., 1990; Meyn, 1993). Dysfunctional G1, S, and G2 cell cycle checkpoints have all been documented in AT cells (Beamish et al, 1994; Painter, 1985). An inability to arrest the cell cycle at one or more of these stages in response to DNA damage may contribute to chromosomal breaks and gaps that promote the formation of acentric chromosomes, generation of aneuploidy, and abnormal chromosomal translocations. In light of the pleiotropic manifestations of the disease, the nature of the AT defect has been the subject of much speculation. For years, researchers have focused their attention on studying the radiosensitivity of AT cells, addressing what cellular defects could be primarily responsible for such abnormalities. Several investigators have suggested that the radiation sensitivity of AT cells was due to either DNA repair deficits, abnormalities in chromatin structure, or defects in genetic recombination (Cox et al., 1984; Kojis et al., 1991; Paterson et al., 1976), but concrete evidence for such defects is still lacking. Alternatively, investigators have proposed that the insufficient time to repair DNA, due to the lack of cell cycle arrest in response to DNA damage in AT cells, may be the contributing factor toward radiosensitivity. However, reversible arrest of AT cells prior to irradiation, thus theoretically allowing time for DNA repair, demonstrated no significant improvement in survival after irradiation (Arlett and Priestly, 1983; Little and Nagasawa, 1985). This suggests that radiosensitivity in AT is not a simple function of abnormal cell cycle checkpoint function. The ATM gene product has been linked to the signal transduction pathway that utilizes p53 in causing a G1 arrest following DNA damage. Several studies have revealed that cells derived from AT patients exhibit defective/delayed increases in p53 protein following IR, suggesting that normal ATM function is required for optimal transduction of the signal from the initial DNA damage induced by IR to the modulators of this increase in p53 protein levels (Canman et al., 1994; Kastan et al., 1992; Khanna and Lavin, 1993). Further characterization of the genes in this pathway led to the observation that AT cells are also defective in their ability to induce downstream genes, including GADD45, p21, and M D M 2 (Canman et al., 1994) (Fig. 2). The fact that the IR induction of all of these genes is dependent on wild-type p53 provides further evidence that normal ATM gene function is required to optimally induce this p53-dependent pathway and subsequent G1 arrest following DNA damage. The suboptimal induction of p53 and lack of a G1 arrest is only one manifestation of the AT defect, since AT cells are also defective in the p53-

p53 and ATM

Ionizing Radiation

=:

0

DNA damage signals

0 n V

gATM 1- n

rI

fpZ1 fgadd45 fmdm2

d l

cyclidcdk

Fig. 2 Schematic illustration of IR-induced cell cycle arrest or cell death pathways that may involve the ATM protein product. Ionizing radiation causes breaks in the DNA, and it is this DNA damage signal that is believed to require an intact ATM for optimal p.53 induction and subsequent G1 arrest. ATM is also required for effective induction of the p53-responsive genes p21, gadd4.5, and mdm2 (see Fig. 1 for details) in response to IR. ATM is also believed to play a role in the p53-dependent S and G2-M checkpoints of the cell cycle in response to DNA damage. The biochemical modulators leading to these arrests have not been identified. The role ATM may play in p53-dependent or -independent programmed cell death pathways has not yet been clarified.

independent S and G2-M checkpoints of the cell cycle (Fig. 2). Cells defective in the ATM gene fail to arrest DNA synthesis in S-phase upon irradiation (known as “radioresistant DNA synthesis”), thereby increasing the potential for genomic instability (Painter and Young, 1993). Evidence of the G2-M defect was provided by observations of a lack of a normal delay at G2 following IR in nonsynchronized AT cells. This abnormal block in AT cells following damage, however, is more complex since AT cells irradiated while in S-phase appear to pile up in G2, suggesting that they are arresting at this checkpoint, whereas AT cells irradiated in G2 bypass the G2 checkpoint and progress into mitosis (Beamish et al., 1994). This variability may reflect differences in the response of ATM to specific types of DNA strand breaks. Alternatively, a delay of G2 of AT cells irradiated at earlier stages of

/

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Susan E. Morgan and Michael B. Kastan

the cell cycle may reflect accumulation of cells harboring severe unrepaired DNA damage, which does not allow progression through the cell cycle (Paules et al., 1995). The role of the ATM gene product in modulating programmed cell death has not been clearly demonstrated, although many in vitro and in vzvo studies support such a role. Earlier studies suggested that increased apoptosis observed in Purkinje cells accounted for their enhanced cell death in AT homozygotes (Agamanolis and Greenstein, 1979; Amromin et al., 1979). In addition, Meyn et al. (1994) reported that AT cells appeared to exhibit enhanced apoptotic cell death in culture following low-dose exposures to radiation and streptonigrin. They suggested that this low threshold for triggering programmed cell death was mediated by p53, since a functionally inactivated p53 (by transfection of a dominant negative p53 or a HPV E6 protein) in AT cells resulted in a loss of radiosensitivity to IR. An alternative mechanism for heightened triggering of apoptotic cell death in AT cells, however, may include defective detoxification of reactive oxygen intermediates, since apoptosis is believed to be triggered by oxidative stress (Buttke and Sandstrom, 1994).

B. Structural and Phenotypic Homologs of ATM: Implications for ATM Function Given the variety of phenotypic abnormalities in AT cells, the ATM protein product is believed to play a major role in multiple cellular signaling pathways such that elucidation of ATM function will undoubtably provide critical insights into the fundamentals of cell cycle and cell growth control. The recent cloning of the ATM gene by Yosef Shiloh and colleagues (Savitsky et al., 1995a,b) should help to unravel the role that ATM plays in p53dependent signal transduction pathways, including apoptosis, and in other cellular processes such as S phase and G2-M phase arrest and in radiosensitivity. The ATM gene occupies approximately 150 kb of genomic DNA and encodes a 13-kb mRNA transcript that is expressed in a wide variety of tissues. The open reading frame of the ATM transcript encodes a protein of 3056 amino acids with a predicted molecular mass of 350 kDa (Savitsky et al., 1995a,b). The ATM protein belongs to an expanding family of large eukaryotic proteins involved in cell cycle control, DNA repair, and DNA recombination. This family of proteins have been grouped together with ATM primarily for the strong homology of their carboxyl termini to the 100-kDa catalytic subunit of the mammalian signal transduction mediator phosphatidylinositol-3 (PI-3) kinase. PI-3 kinases have been widely studied and appear to partici-

13

p53 and ATM

pate in many cellular processes, including insulin-dependent glucose transport, growth factor responses, and cellular differentiation (Dhand et al., 1994; Freund et al., 1995; Hanks et al., 1988; Hiles et al., 1992; Tanti et al., 1994). This family of PI-3 kinase-like proteins can be subdivided into several groups, all defined by sequence and functional similarities (Table I). One subgroup includes the yeast TOR1 and TOR2 proteins as well as the mouse and human homologs, mTOR (RAFT1) and FRAP, respectively. This latter group of proteins are all involved in the G1-S cell cycle progression and were identified as the targets of the immunosuppressant rapamycin (Brown et al., 1994; Heitman et al., 1991; Helliwell et al., 1994; Kunz et al., 1993; Sabatini et al., 1994; Sabers et al., 1995; Stan et al., 1994). Another group of proteins that may be functionally more closely related to ATM include the cell cycle checkpoint-DNA recombination and repair proteins Meclp (Esrl/Sad3), Mei-41, and Rad3. The Saccharomyces cerevisiae Meclp encodes an essential protein involved in the G1, S, and G2 cell cycle checkpoints as well as DNA repair and meiotic recombination (Allen et al., 1994; Kato and Ogawa, 1994; Kato et al., 1994; Weinert, 1992; Weinert et al., 1994). The Drosophila melanogaster Mei-41 is involved in mitotic and meiotic recombination and double-strand break repair (Baker et al., 1978; Banga et al., 1986; Hari et al., 1995), and the Saccharomyces pombe Rad3 is required for both S and G2-M checkpoints as well as DNA repair (Al-Khodairyand Carr, 1992; Al-Khodairy et al., 1994; Enoch and Norbury, 1995; Jimenez et al., 1992; Seaton et al., 1992). Like ATM homozygotes, mei-41, m e c l , and rad3 mutants exhibit defects in cell cycle arrest and increased sensitivity to ionizing radiation (Hari et al., 1995; Jimenez et al., Table I Properties of a Family of PI-3 Kinase-like Proteins’ Protein

Species

ATM

Vertebrate

DNA-PK

Vertebrate

Mei-41

Drosphila

Rad3

S. pombe

Tor ll Tor2

S . cerevisiae

Tell Mecl

S. cerevisiae S. cerevisiae

Function

Plays a role in a variety of cellular responses to ionizing radiation Protein kinase involved in V(D)Jrecombination and double-strand break (and DSB) repair Involved in meiotic and mitotic recombination and DSB repair Required for S and G2-M checkpoints; involved in DNA repair Involved in GI-S cell cycle progression; binds rapamycid FKBPl2 Involved in maintenance of telomere length Involved in G1, S, and G2 cell cycle checkpoints; DNA repair and meiotic recombination

aSee text for details comparing functional similarities between ATM and related proteins listed here.

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Susan E. Morgan and Michael B. Kastan

1992; Weinert et al., 1994), and the mei-41 and rad3 homozygotes exhibit high spontaneous rates of mitotic recombination and increased chromosomal instability (Hari et al., 1995; Phipps et al., 1985). ATM shows the highest sequence similarity to the S. cerevisiae Tell protein, which is involved in maintenance of telomere length (Greenwell et al., 1995; Lustig and Petes, 1986; Morrow et al., 1995) rather than cell cycle control. It is interesting to note that AT cells, like the tell yeast mutants, appear to exhibit shorter telomeres, telomere-telomere end associations, and increased frequencies of aberrant chromosomal segregation (Greenwell et al., 1995; Kojis et al., 1991; Pandita et al., 1995). It has been suggested that loss of telomeric sequences is associated with senescing of human cells and that shortened telomeres may activate a checkpoint pathway that inhibits cellular proliferation (Hastie et al., 1990; Levy et al., 1992). These characteristics may thus explain the reduced life span and premature senescence seen in AT cell lines that may be defective in telomere length maintenance. Tell and the functionally related yeast protein, Mecl, are suggested to have related but not identical functions, since (1)the radiation sensitivity and lethality of mecl mutants can be suppressed by overexpression of Tell, and (2) the double tell lmecl mutants are synergistically sensitive to hydroxyurea, ultraviolet radiation, and IR than the mecl mutant alone (Morrow et al., 1995; Sanchez et al., 1996). Interestingly, like AT cells, the tell/mecl double mutants exhibit sensitivity to radiomimetic and DNA-damaging drugs such as streptonigrin and bleomycin, whereas neither mutant alone is sensitive (Morrow et al., 1995). These studies suggest that the human ATM may be a multifunctional protein involved in both cell cycle control and telomere maintenance, two functions that may be controlled separately in yeast. Studies by Sanchez et al. (1996) have further elucidated a DNA damage response pathway that involves Rad53 as a downstream target of the ATM homologs Mecl and Tell. Rad53 is believed to be one of several proteins necessary for regulating the onset of mitosis and the rate of progression through S phase in response to DNA damage (Sanchez et al., 1996; Sun et al., 1996). In these studies, the phosphorylation status of Rad53 was found to be controlled by Mecl and Tell. Furthermore, overexpression of Rad53 restored checkpoint function of mecl and tell mutants. The suggestion that Rad53 may serve as a downstream target to the “ATM-like” proteins Mecl and Tell may aid in the elucidation of human homologs that may function as critical cellular targets of ATM in the DNA damage response pathway. The catalytic subunit of the human DNA-PK, which contains the PI-3 kinase-like domain at its carboxyl terminus, is another member of this family of “ATM-like” proteins. DNA-PK is a serine-threonine protein kinase that is activated by DNA double-strand breaks and is suggested to play a role in DNA damage detection and/or DNA repair and V(D)Jrecombination (Hart-

p53 and ATM

15

ley et al., 1995; Gottlieb and Jackson, 1993; Taccioli et al., 1994; Tuteja et al., 1994). DNA-PK is a heteromultimer consisting of a 350-kDa catalytic subunit and a dimer of 70- and 80-kDa subunits known as the Ku antigen (Anderson, 1993; Gottlieb and Jackson, 1993). The Ku antigen is suggested to serve as a sensor of DNA strand breaks and activates the serine-threonine protein kinase activity of the catalytic subunit, which subsequently phosphorylates DNA-bound substrates. Like DNA-PK, ATM may also be recruited to the sites of DNA damage by other protein subunits to form an active rnultiprotein complex. Severe combined immunodeficiency mice, which are defective in DNA-PK, confer hypersensitivity to IR and exhibit deficiencies in both DNA double-strand break repair and V(D)J recombination, phenotypic defects that parallel those seen in cell lines derived from AT patients (Biedermann et al., 1991; Blunt et al., 1995; Lieber e t al., 1988). In light of similarities of ATM with the PI-3 kinase catalytic domain, the possibility exists that ATM may possess intrinsic lipid kinase activity, although no member of the family of ATM-related proteins discussed previously has, thus far, been shown to exhibit such activity. Alternatively, this family of PI-3 kinase-containing proteins may possess intrinsic protein kinase activity as exhibited by the DNA-PK catalytic subunit. Regardless of whether this family of proteins contain lipid or protein kinase activity, PI-3 kinases in general are believed to play an important role in growth factor signaling, mediated through direct interaction with regulatory subunits. Downstream targets of PI-3 kinases that have been identified include serinethreonine protein kinase B, the protein kinase C family, and the ribosomal protein S6 kinase (p70s6k), which is activated in response to a wide variety of mitogens (Burgering and Coffer, 1995; Downward, 1994; Franke et al., 1995; Toker et al., 1994). The hypersensitivity of AT cells to ionizing radiation and radiomimetic drugs could be due in part to a defective response to growth factor-induced signaling pathways mediated by PI-3 kinases. Interestingly, PI-3 kinase was shown to be required for the inhibition of apoptotic cell death in a rat pheochromocytoma cell line by nerve growth factor (Yao et al., 1995). This may help explain why AT cells, which may have a defective PI-3 kinase, exhibit heightened nerve cell death. Consistent with this model of PI-3 kinases serving as “survival factors,” growth factor withdrawal in cells of hematopoietic origin leads to an increase in cell death in response to IR (Canman et al., 1995; Collins et al., 1992; Lin and Benchimol., 1995). The essential role the PI-3 kinase domain may serve in ATM-mediated growth factor signaling is further strengthened by the following observations: (1)that a single amino acid deletion within the PI-3 kinase signature sequence results in ATM dysfunction (as suggested by an AT homozygote who, harboring this point mutation, manifests the full AT phenotype); and (2) the fact that mutations within the PI-3 kinase domain of the yeast homolog Tell result in its

16

Susan E.Morgan and Michael B. Kastan

characteristic phenotype of shortened telomeres (Greenwell et al., 1995; Savitsky et ul., 1995a). Both the structural and phenotypic similarities between ATM and the gene products discussed previously strongly implicate involvement of ATM in signal transduction, cellular responses to DNA damage, and cell cycle control. Given the large nature of this protein and the pleiotropic nature of the disease, ATM may very well be multifunctional, constituting an essential “link” between several critical physiological processes of the cell that ultimately work in concert with each other to maintain the integrity of the genome.

IV. CELL CYCLE CONTROL A. Implications for Cancer Development The transformation of a normal cell to a tumor cell appears to depend in part on mutations in genes that normally control the cell cycle and cell death, thereby resulting in inappropriate cellular survival and tumorigenesis. Cell cycle checkpoints are believed to play a major role in maintaining the integrity of the genome, such that defects at these control points may contribute to increased incidence of genomic changes such as deletions, translocations, and amplifications, which are common during the evolution of a normal cell to a cancer cell. The fact that p53 is the most commonly mutated gene in a wide variety of human cancers (Hollstein et al., 1991) and the observation that mice with disrupted p53 genes have an increased rate of tumor formation (Donehower et al., 1992, 1995a,b) demonstrate the importance of disabling of p53dependent pathways in order to achieve cellular transformation. Loss of p.53 function may contribute to enhanced tumorigenesis either by loss of p53-mediated checkpoint control, with resultant enhanced genetic instability (Livingstone et al., 1992), or by failure to induce cell death in inappropriate physiological situations, or both (Symonds et al., 1994). Mechanisms of p53 inactivation in tumor cells include p53 gene mutations, gene deletions, overexpression of the negative regulator mdm2, or infection with a DNA tumor virus that encodes protein products that inhibit p53 function (Oliner et al., 1992; Scheffner et al., 1990). In addition to loss of function of tumor suppressor genes such as p53, a defective ATM gene that results in spontaneous chromosomal loss and genetic instability may also turn out to be another event contributing to tumor progression. If one of the major functions of ATM involves activating multiple cell cycle checkpoints in response to DNA damage to protect the cell from genomic instability, then one would expect that, during the process of

p53 and ATM

17

tumorigenesis, the cell would incur mutations within the ATM locus (1lq23). Multiple studies have in fact suggested that loss of heterozygosity at 1lq22-q23 loci may be a frequent event in breast, cervical, ovarian, and colon cancers (Carter et al., 1994; Hampton et al., 1994; Keldysh et al., 1993; Pejovic, 1995). However, it has been suggested that ATM is not the critical tumor suppressor gene in this region (Vorechovsky et al., 1996). In addition, loss of ATM function would be expected to decrease tumor cell survival and thus might not be selected for during tumorigenesis. Loss of ATM function, however, has been linked to tumorigenesis by patient studies. Cancer predisposition in AT heterozygotes has been reported to be three- to fivefold that of the general population, with a relative risk for breast cancer approximately fivefold that of normal women (Swift et al., 1987, 1991). AT homozygotes have an approximately 250- to 700-fold increased risk of developing leukemia and lymphoblastic lymphomas (Hecht and Hecht, 1990; Swift et al., 1987), tumors that harbor gene rearrangements of immunoglobulin and T-cell receptor gene families (Kojis et d., 1991). During immunoglobulin and T-cell receptor gene rearrangements, in which naturally occurring breaks within the DNA take place, cell cycle progression is presumably normally inhibited. Cell lines derived from AT patients, however, appear to incur enhanced errors in gene rearrangements, generating chromosomal instability and activation of cellular oncogenes and ultimately leading to the development of immune system tumors such as lymphomas and leukemias. The specific increase in lymphoid tumors in AT homozygotes suggests that the abnormal production of DNA strand breaks and chromosomal rearrangements, in conjunction with lack of repair, may in fact be a rate-limiting step in lymphoid tumorigenesis.

B. Implications for Cancer Therapy Since many of the anticancer agents in use today include DNA-damaging compounds, increasing understanding of cell cycle control in response to DNA damage may help us to better manipulate selective tumor cell death. Tumor types that respond to treatment (e.g., lymphomas and germ cell cancers) may have a better tendency to undergo DNA damage-induced apoptosis, whereas relatively resistant tumor types (e.g., colon, breast, lung carcinomas) may be more likely to survive following exposure to DNA-damaging chemotherapeutic agents or radiation therapy since chemotherapy/radiation therapy does not induce rapid apoptosis as effectively in these cells. A better understanding of the biochemical pathways controlling rapid DNA damageinduced apoptosis may enable us to better therapeutically manipulate a re-. sistant tumor cell to undergo a rapid apoptosis response on exposure to chemotherapy or radiotherapy. Manipulating the tendency of tumor cells to

18

Susan E. Morgan and Michael B. Kastan

undergo cell death could also be achieved by selective inhibition or activation of growth factor or hormone pathways (Canman et al., 1995). Alternatively, apoptotic cell death may be induced through overexpression of adenovirus protein products E1A or HPV E7, which have been suggested to sensitize cells to p53-mediated apoptosis (Fufiwara et al., 1994; Lowe et al., 1994; Lowe and Ruley, 1993; Rao et al., 1992; White et al., 1992). Perhaps a more technically difficult approach to inducing a rapid apoptosis response of tumor cells on exposure to chemotherapy or radiotherapy involves the biochemical manipulation of mutant p53 back to the wild-type conformation or gene therapy techniques. Restoring p53 function by either retroviral- or adenoviral-mediated transfer of wild-type p53 has resulted in the induction of apoptosis in a variety of cell lines harboring a mutant p53, such as in the non-small-cell lung cancer and squamous carcinoma cell lines (Debbas and White, 1993; Fufiwara et al., 1994). Alternatively, inactivation of Mdm2 protein, an inhibitor of p53 function, may provide another potential target for enhancing p.53 function. In addition, since many tumors express viral gene products that inactivate p53 and affect cell cycle checkpoint function, new pharmacological agents could theoretically be developed that interfere with viral p53-associating proteins, such as HPV E6, thus restoring the wild-type p53 status. HPV infection has been tightly linked to the development of certain human cancers, primarily cervical and anal carcinoma (Brachman et al., 1992; Field, 1992; Sheffner et al., 1990; Zaki et al., 1992; zur Hausen, 1989). Tumor cell lines defective in ATM may be more susceptible to DNA damage-inducible apoptotic cell death since cells derived from AT patients are extremely sensitive to the cytotoxic effects of irradiation. Therefore, a mutation within the ATM gene could possibly be exploited to render cells more sensitive to DNA-damaging agents such as topoisomerase inhibitors and radiomimetic chemicals. In terms of tumor treatment, ATM could be a target for drug design that transiently targets and inhibits ATM protein function at the time of chemotherapyhadiotherapy exposure, thereby making a resistant tumor more susceptible to cell death. Alternative approaches to enhancing therapy based on knowledge of checkpoint controls and DNA damage responses include abrogation of both the G1 and G2 checkpoints of tumor cells. Although loss of the G1 checkpoint in tumor cells by itself does not render the cell more susceptible to irradiation, various combinations of DNA-damaging agents may be able to selectively kill tumor cells that continue through the cell cycle after treatment with the initial anticancer agent while normal cells, which normally arrest, are presumably protected. Data suggest that tumor cells devoid of both a G1 and G2 checkpoint may be more sensitive to DNA-damaging agents than tumor cells lacking only a G1 arrest (Fan et al., 1995; Powell et al., 1995; Russell et al., 1995).

p53 and ATM

19

Continued investigations into the molecular controls of cell cycle progression and cell death may eventually provide new insights into the molecular differences between tumor cells and normal cells. This new understanding may dictate the choice and schedule of agents to be used in therapy. Characterizations of new compounds are also likely to be developed that take advantage of differences between cell cycle control in normal versus cancer cells to enhance therapeutic efficacy.

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Deletions of the Short Arm of Chromosome 3 in Solid Tumors and the Search for Suppressor Genes Klaas Kok,' Susan L. Naylor,2 and Charles H. C. M. Buys' 'Department of Medical Genetics, University of Groningen, 9713 AW Groningen, The Netherlands; and 2Department of Cellular and Structural Biology, The University of Texas Health Science Center at Sun Antonio, San Antonio, Texas 78284-7762

I. Tumor Suppressor Genes: The Concept 11. Methods of Localizing Tumor Suppressor Genes A. Karyotyping of Tumor Cells B. Analysis of Allelic Losses C. Comparative Genome and RNA Analysis D. Cell Fusion and Transfection Studies 111. Chromosome 3 Losses in Different Types of Tumors A. Lung Cancer B. Renal Cell Carcinoma C. Head and Neck Squamous Cell Carcinoma D. Gastrointestinal Tumors E. Breast Cancer F. Female Genital Tract Tumors G. Other Types of Cancer H. Loss of 3p Sequences in Experimental Cell Systems IV. Functional Assays of Tumor Suppression on Chromosome 3 A. Transfer of Chromosome 3 into Renal Cell Carcinoma Cell Lines B. Transfer of Chromosome 3 into Other Human Tumor Cell Lines C. Chromosome Transfer and Mismatch Repair D. Human Chromosome 3 in Rodent Lines V. (Presumed) Tumor Suppressor Genes on the Short Arm of Chromosome 3 A. The Von Hippel-Lindau Disease Gene B. The hMLHl Gene C. The TGF-f3 Receptor Type I1 Gene D. THRB and RARB E. Candidate Genes from 3p21 F. Candidate Genes from 3p13-pl4 VI. Evolutionary Aspects of Human Chromosome 3 VII. Concluding Remarks References

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The concept that cells can become malignant upon the elimination of parts of chromosomes inhibiting cell division dates back to Boveri in 1914. Deletions occurring in tumor cells are therefore considered a first indication of possible locations of tumor suppressor genes. Approaches used to localize and identify the paradigm of tumor suppressors, RB1, have also been applied to localize tumor suppressor genes on 3p, the short arm of chromosome 3. This review discusses the methodological advantages and limitations of the various approaches. From a review of the literature on losses of 3p in different types of solid tumors it appears that some tumor types show involvement of the same region, while between others the regions involved clearly differ. Also discussed are results of functional assays of tumor suppression by transfer of part of chromosome 3 into tumor cell lines. The likelihood that a common region of deletions would contain a tumor suppressor is strongly enhanced by coincidence of that region with a chromosome fragment suppressing tumorigenicity upon introduction in tumor cells. Such a situation exists for a region in 3p21.3 as well as for one or more in 3p12-pl4. The former region is considered the location of a lung cancer suppressor. The same gene or a different one in the same region may also play a role in the development of other cancers including renal cell cancer. In the latter cancer, there may be additional roles of the VHL region and/or a 3p12-pl4 region. The breakpoint region of a t(3;8) originally found to be constitutively present in a family with hereditary renal cell cancer now seems to be excluded from such a role. Specific genes on 3p have been suggested to act as suppressor genes based on either their location in a common deletion region, a markedly reduced expression or presence of aberrant transcripts, their capacity to suppress tumorigenicity upon transfection into tumor cells, the presumed function of the gene product, or a combination of several of these criteria. A number of genes are evaluated for their possible role as a tumor suppressor according to these criteria. General agreement on such a role seems to exist only for VHL. Though hMLHl plays an obvious role in the development of specific mismatch repair-deficient cancers, it cannot revert the tumor phenotype and therefore cannot be considered a proper tumor suppressor. The involvement of VHL and MLHl also in some specific hereditary cancers allowed to successfully apply linkage analysis for their localization. TGFBR2 might well have a tumor suppressor function. It does reduce tumorigenicity upon transfection. Other 3p genes coding for receptor proteins, THRB and RARB, are unlikely candidates for tumor suppression. Present observations on a possible association of FHIT with tumor development leave a number of questions unanswered, so that provisionally it cannot be considered a tumor suppressor. Regions that have been identified as crucial in solid tumor development appear to be at the edge of synteny blocks that have been rearranged through the chromosome evolution which led to the formation of human chromosome 3. Although this may merely represent a chance occurrence, it might also reflect areas of genomic instability.

1. TUMOR SUPPRESSOR GENES: THE CONCEPT Tumor development and progression are generally considered to be the result of multiple mutations. Activating mutations of proto-oncogenes and inactivating mutations of tumor suppressor genes play a role in this process. In fact, this concept had already been formulated by Boveri in 1914 (English translation published in 1929), who suggested that cells could become malignant either from a predominance of chromosomes that promote division

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or upon the elimination of inhibiting chromosomes. Epidemiological studies on retinoblastoma by Knudson (1971) led to the idea that in hereditary cases of retinoblastoma a predisposition to tumor development would exist as a germline mutation of one allele of the gene involved and that a tumor would arise as a consequence of deletion or inactivation of the other allele by some somatic event. In nonhereditary cases, both alleles would need to become inactivated by somatic events in the same cell. Microscopically visible deletions consistently involving band q14 of chromosome 13 in retinoblastoma patients indicated where the gene was located (Yunis and Ramsey, 1978; Turleau et al., 1985). Cavenee et al. (1983)used DNA markers in a comparison of tumor and constitutive tissues to analyze the chromosomal events involved in tumor initiation. When a marker showed loss of heterozygosity in the tumor, this was taken as an indication of deletion of the marker region from one of the chromosome 13 homologs. From results obtained with proximal and distal markers, several underlying chromosomal mechanisms could be inferred: loss of a whole homolog, loss followed by duplication of the remaining homolog, and mitotic recombination. In familial retinoblastoma, the homolog retained in the tumor could be traced to the affected parent (Caveneeet al., 1985).Thus, epidemiological studies by Knudson (1971) combined with cytogenetic observations and with DNA analysis by Cavenee et al. (1983, 1985) made the retinoblastoma gene, the paradigm of tumor suppressors even before its cloning by Friend et al. (1986). A clear-cut distinction between hereditary and nonhereditary cases of cancer does not always exist or cannot always readily be made (e.g., when there is a high incidence of the cancer). Still, in such cases, the occurrence of chromosomal deletions or loss of heterozygosity in a tumor is generally taken as an indication of inactivation by elimination of one allele of a tumor suppressor gene involved in the development of the cancer. It has also been suggested that loss of heterozygosity is a general and rather stochastic process in tumor progression, due to loss of DNA replication fidelity (Chigira et al., 1993). Loss of heterozygosity might thus be the result of carcinogenesis instead of its cause. This has been phrased in even more general terms by Prehn (1994),who suggests that it may be more correct to say that cancers beget mutations than it is to say that mutations beget cancers. Although most likely this is partially true, it cannot be denied that the observation of consistent allelic losses has led to the successful identification of tumor suppressor genes in several types of cancer. Although allelic losses may only reflect an inherent genomic instability, some cellular growth advantage may result if the lost region contains a tumor suppressor locus. A kind of sequential order of involvement of chromosome regions in allelic losses seems apparent in several types of tumor and might be related to differential genomic instability (Buys, 1991). A tumor suppressor gene whose (functional) elimination may be essential

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for the development of a certain type of tumor may also play a role in the progression of other types of tumor. This is well illustrated by RB1, which shows ubiquitous expression in a variety of tissues, is deleted or mutated in many different tumor types (Horowitz et a/., 1990), but seems to have an essential initiating role only in the development of retinoblastoma. Specific developmental stage and tissue characteristics, such as presence of redundant regulatory mechanisms, may determine a tumor suppressor’s role in either tumor initiation or tumor progression. This, combined with the fact that many of the tumor suppressors identified to date show a wide diversity in structure and function (Levine, 1993), makes it difficult to find common denominators. Nevertheless, starting from the retinoblastoma story, some common strategies of identification of tumor suppressors have been developed and are here discussed further in relation to tumor suppressor loci on 3p.

11. METHODS OF LOCALIZING TUMOR SUPPRESSORGENES The two approaches routinely used to detect deletions in tumor cells or tumor-derived cell lines as a possible indication of the location of a tumor suppressor gene are karyotyping and analysis of loss of heterozygosity. Considerable technical improvements have been obtained with the implementation of fluorescence in situ hybridization (FISH) techniques, including comparative genomic hybridization, of polymerase chain reaction (PCR)-mediatedmicrosatellite analysis, and of representational difference analysis. Furthermore, several techniques have been developed that bypass the genome analysis and directly identify genes differentiallyexpressed in tumor cells compared to normal cells. The differential display technique may serve as an example. An alternative approach to localizing tumor suppressor genes is to transfer DNA presumed to contain a suppressor gene. A DNA segment ranging from a cosmid to a complete chromosome is transfected into a tumorigenic cell line, after which the changes in tumorigenicity of the cell line can be tested. Each of the previously mentioned approaches is briefly discussed in the next sections. Beyond the scope of this paragraph, but representing a direct way to immediately identify genes possibly involved in tumorigenesis, are the identification and analysis of human homologs of genes known to play a role in proliferation and differentiation in other organisms. It should also be mentioned that, in familial situations where germline mutations of a tumor suppressor gene predispose family members to the same type of cancer that occurs sporadically due to somatic mutations, genetic linkage analysis is an

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alternative method to localize such a tumor suppressor gene (see Sections V.A and V.B). A. Karyotyping of

Tumor Cells

Individual tumor cells may contain both structural and numerical chromosome abnormalities. They may vary from cell to cell, even within a single tumor specimen. Such heterogeneous changes are probably due to the increased genomic instability of cancer cells compared to normal somatic cells and have no direct relation to tumor initiation or progression. Therefore, analysis of a large number of cells from a single culture is warranted to identify clonal abnormalities of a certain tumor or tumor cell line. When a high percentage of specimens, or derived cell lines, from the same type of tumor have a specific clonal aberration in common, such an aberration is usually regarded as a primary event, related to tumor initiation. In contrast, clonal events observed in smaller percentages of tumors are considered secondary events, related to progression or metastasis. Unfortunately, many tumor specimens are difficult to grow in vitro. Therefore, microscopic analysis has often been carried out on metaphase preparations from established tumor cell lines. Despite a continuous changing of tumor cell karyotypes over time, early deletions that are possibly necessary for tumor development will never be reversed. Consequently, they represent a consistent feature of such cell lines. Present tissue preparation and cell culturing conditions allow successful short-term culture of most tumor types. Consistent chromosome abnormalities may provide a clue to loci involved in tumor development. In the case of deletions, involvement of suppressor loci may be supposed. An advantage of microscopic analysis on a cell-by-cell basis is that abnormalities occurring in only a minority of tumor cells may well be detected. On the other hand, reduction of genomic regions to homozygosity or hemizygosity by mechanisms such as mitotic recombination or chromosome loss followed by duplication of the remaining copy will remain unnoticed. Still, these are fairly common mechanisms of suppressor gene inactivation. Although karyotyping has revealed consistent abnormalities, including deletions, in many types of tumor, the quality of tumor metaphases does not always allow a good definition of breakpoints. Moreover, parts lost from one chromosome can be translocated, sometimes fragmented, to other chromosomes, where they may escape detection. FISH with whole chromosome libraries or with specific clones originating from the presumed deleted region should therefore routinely be used for verification. In situ hybridization protocols have been developed that are based on combinatorial labeling strategies using several different fluorochromes. Speicher et a]. (1996) were able

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to give each chromosome a different color in a single in situ hybridization using combinations of five different fluorochromes.

B. Analysis of Allelic Losses The use of polymorphic probes to detect allelic losses in tumor material compared with constitutive tissue was introduced by Cavenee et al. (1983). This technique offers a number of advantages over microscopic analysis. Mitotic cells, sometimes difficult to obtain from tumor specimens, are no longer required. Deletion analysis by DNA markers also excludes misinterpretation caused by complex chromosome rearrangements and has a resolution that is much higher than that of microscopic analysis of mitotic chromosomes. It is dependent only on the density of accurately mapped polymorphic probes, including the highly informative microsatellite sequences (Weber and May, 1989). Since high numbers of cells are included in a single analysis, detection of aberrations is limited to those that are clonal. On the other hand, clonal aberrations that occur in only a minority of cells may well be missed. In part, this problem can be circumvented by microscopic selection of specific areas of a tumor (Zhuang et d.,1995). A major pitfall in loss of heterozygosity studies is the presence in tumors of infiltrating stromal tissue, resulting in an apparently partial loss of heterozygosity. This necessitates densitometric analysis of the resulting banding patterns or phosphoimaging of gels, but perhaps even more a careful selection of tumor material. Again, microscopic examination should be used, both to select areas with limited amounts of infiltrating tissue and to roughly estimate the proportion of normal cells in a given tumor sample, causing the residual signal for the “lost” allele. The residual band may, however, also reflect heterogeneity of the tumor for a particular allele loss (Chen et al., 1992) or different copy numbers of the homologs of the chromosome under study. Only those tumors that show allelic loss for a subset of markers, and thus retention of heterozygosity for other markers on the same chromosome, may be expected to show a genuine loss of heterozygosity. The inclusion in each tumor analysis of a number of markers on the same chromosome and quantification of allele intensities should therefore be a requirement in studies of loss of heterozygosity. Unfortunately, many reports in the literature fail to provide criteria for loss of heterozygosity and may well contain ambiguous results. The present availability of dense sets of microsatellite markers for all chromosomes has greatly increased the resolution of analysis of loss of heterozygosity and allowed the detection of small regions that are homozygously lost in tumors and tumor-derived cell lines. Homozygous deletions are often regarded as pinpointing regions that contain tumor suppressor genes; that is, they are considered to represent cases in which both alleles of a tumor

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suppressor gene are lost by a deletion. Therefore, tumors are now being systematically screened for homozygous deletions by sophisticated techniques such as representational difference analysis (see Section 1I.C). Indeed, this approach has led to the identification of a new tumor suppressor gene on the long arm of chromosome 18 (Hahn et al., 1996). However, the possibility that some homozygous deletions have nothing to do with loss of tumor suppression cannot be excluded. With the increased power to detect small homozygous deletions, this might turn out to hold true in more and more cases.

C. Comparative Genome and RNA Analysis A comparison of related genomes to identify deletions can be made by different techniques. A microscopic technique making use of metaphase chromosome preparations is comparative genomic hybridization (Kallioniemi et al., 1992). Equal amounts of tumor DNA and normal DNA, each labeled with a different fluorochrome, are admixed and used as a probe for in situ hybridization on a normal (human) metaphase spread. Variations in the ratio of the intensities of the two fluorochromes on a chromosome segment are determined solely by the relative abundance of this segment in the tumor DNA. Although most suitable for determining regions of amplification, this technique can in principle also be used to determine deletions. An advantage of this technique is the possibility of obtaining semicytogenetic data without the need for high-quality metaphase spreads from the tumor cells. There are, however, some limitations of the technique. Since it is based upon relative changes in DNA sequence copy number, the sensitivity of deletion detection is critically dependent on the number of homologs present in a tumor cell. Moreover, the relative change is an average of the relative changes of all the cells in the tumor sample under analysis. Consequently, heterogeneity in deletions may be missed. The size of a deletion also has an effect. Deletions smaller than 3-5 Mb may readily escape detection. A comprehensive discussion of the power and limitations of this technique can be found in Kallioniemi et al. (1994). A promising direction in which comparative genomic hybridization techniques are developing is the hybridization of the previously described probes on gridded genomic and/or cDNA libraries so that sequences that are over- or underrepresented in the tumor can be directly identified without morphological analysis of chromosomes. Another method for analyzing differences between complex genomes is called representational difference analysis (Lisitsyn et al., 1993). Starting with two DNA samples, called the driver and the tester, respectively, a series of experimental steps leads to the identification of DNA fragments that are present in the tester but are absent from the driver. Thus, when tumor DNA

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is used as the driver, one may identify homozygous deletions in the tumor. In principle, allelic losses can also be determined, since loss of one allele of a two-allele restriction fragment length polymorphism (RFLP) will also result in the complete loss of a restriction fragment from the tumor DNA. With this method, identification of deletions in fact precedes their localization. As even low amounts of normal DNA in the driver will prevent enrichment of those fragments that are deleted from the tumor, successful application of this method depends critically on the ability to obtain high-quality tumor DNA with very little or no admixture of stromal tissue. Lisitsyn et al. (1995) identified by representational difference analysis seven genomic loci that were homozygously lost in different tumor types. Two of these loci mapped to the short arm of chromosome 3, including one that coincided with the FRA3B region (discussed in Section V.F.). A number of techniques have been developed to identify genes that are differentially expressed in tumor cells and normal cells. These techniques are generally based on the construction of subtraction libraries (e.g., Schrampl et al., 1993), the differential display method (Liang and Pardee, 1992; Liang et al., 1995; reviewed by McClelland et al., 1995), the previously described method of representational difference analysis (Hubank and Schatz, 1994), or a combination of these methods (Suzuki et al., 1996). Identification of differences in levels of expression precedes the localization of the genes involved. It is beyond the scope of this review to give a detailed description of the procedures. All these methods aim at the identification of mRNAs differentially expressed in tumor and normal cells. Even with the availability of control RNA from the genuine precursor cells of the tumor cells to be analyzed-in fact a rare situation-it may be expected that many genes will be found differentially expressed between the two mRNA pools because of secondary effects of tumor development, such as an increased growth rate.

D. Cell Fusion and Transfection Studies Suppression of tumor growth was first demonstrated in somatic cell hybrids. Early hybrid studies indicated that fusion of a tumor cell to a “nonmalignant” cell resulted in tumorigenic cells and thus that malignancy (i.e., the ability of tumor cells to grow progressively and kill their host) was dominant (see Harris et al., 1969, for references). Harris and his co-workers (Harris et a/., 1969; Harris, 1971), however, used several different combinations of malignant and nonmalignant cells and found the opposite to be true-that is, that malignancy is recessive. The difference between the two observations is due to the choice of “nonmalignant” parents and to the number of chromosomes retained in the hybrids. Harris (1971) and other investigators (Kaelbling and Klinger, 1986) found that the malignant phenotype

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was suppressed in cells containing a complete complement of chromosomes, but that malignant subclones could be isolated from later passages after chromosome loss had occurred. Other studies indicated that malignant x malignant crosses of two different tumor types resulted in the loss of malignancy. These early studies conclusively showed tumor suppression, but since whole cell hybrids were used, it was difficult to define the location of the suppressor gene. The advent of microcell hybrids (Fournier and Ruddle, 1977) allowed the transfer of single chromosomes (or chromosomal fragments) into tumor cells. Basically, the microcell method (Fournier and Ruddle, 1977; Killary and Fournier, 1995) consists of exposing cells for prolonged periods to colcemid. During this time, the nuclear envelope forms around one to a few chromosomes, creating micronuclei. These nuclei are isolated as microcells by centrifugation in the presence of cytochalasin B. The microcells are then fused to a recipient cell using polyethylene glycol. In chromosome 3 studies, two methods have been used to select the hybrid containing the transferred chromosome: (1)the chromosome under study is transferred to a chromosome containing a selectable marker (e.g., the X chromosome, with hypoxanthine-guanine phosphoribosyltransferase) (Saxon et al., 1986; Shimizu et al., 1990); or (2) a selectable marker such as the neomycin resistance gene is randomly integrated on a “normal” copy of the chromosome (Rimessi et al., 1994; Sanchez et al., 1994; Shimizu et al., 1990).The resulting microcell hybrid clones are then analyzed for the integrity of the chromosome, as this procedure itself can result in breakage of chromosomes. The tumorigenicity of individual clones is tested by injecting lo5 to l o 7 cells into nude mice.

Ill. CHROMOSOME 3 LOSSES IN DIFFERENT TYPES OF TUMORS A. Lung Cancer 1. MICROSCOPIC CHROMOSOME ANALYSIS

Shortly after the first reports of successful culturing of small-cell lung cancer (SCLC)-derivedtumor cells, the first karyotypes of lung cancer cells from SCLC cell lines, short-term cultures of primary SCLC tumors, and an SCLC bone marrow preparation were published (Whang-Peng et al., 1982a,b). They revealed the presence of multiple chromosomal aberrations per cell with a deletion always occurring in at least one chromosome 3. The major2 3 the shortity of these deletions appeared to be interstitial, 3 ~ 1 6 ~ being est region of overlap. Despite some initial dispute about the consistency of

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this 3p involvement (Wurster-Hill et al., 1984; Zech et al., 1985; Morstyn et al., 1987; De Fusco et al., 1989), a frequent if not consistent deletion of the short arm of chromosome 3 could be confirmed and further delimited by cytogenetic analysis of established cell lines (Buys et al., 1984; Waters et al., 1988). Deletions of 3p were also confirmed in direct preparations of tumor material (Falor et al., 1985; De Fusco et al., 1989). Most authors agree on a part or the whole of 3p14-p23 as the region involved. The variation in the reported minimal region of overlap of the del(3p) can be attributed to difficulties in obtaining good chromosome preparations from lung tumors and to difficulties in accurately determining the breakpoints involved in the structural changes, due to the complexity of the rearrangements. Tumors that do not carry microscopically detectable deletions on 3p may have lost a whole chromosome 3 and reduplicated the remaining homolog, resulting in hemizygosity without any visible cytogenetic alteration. In addition, 3p marker chromosomes may be lost during long-term culture of tumor cells (Zech et al., 1985). This might explain the occurrence of multiple apparently normal copies of chromosome 3 that occasionally have been reported. It may now be concluded that cytogenetic aberrations of 3p occur in most if not all SCLCS. For non-SCLC, karyotypes can also be extremely complex and thus difficult to interpret. This might explain the discrepancy in the various reports with respect to the loci most frequently involved. For instance, whereas one report describes 9p alterations in 9 of 10 cases (Lukeis et al., 1990), this frequent involvement of chromosome 9 was not found by others (Miura et al., 1990; Whang-Peng etal., 1991).Visible aberrations of the short arm of chromosome 3 occur in about 60-75% of cases (Miura et al., 1990; Lukeis et al., 1990; Whang-Peng et al., 1991; Testa and Siegfried, 1992; Matturri and Lavezzi, 1994). It has been reported (Testa et al., 1994) that the frequency of 3p loss was significantly higher in squamous cell carcinomas (94%) than in adenocarcinomas (60%).Breakpoints have been determined to occur most frequently in band 3p14, which may in general imply loss of the distal region. In a series of 63 non-SCLCs, the smallest region of overlap of 3p losses was at 3p21 (Testa et al., 1994). Thus, according to cytogenetic analysis, deletion of the region 3p14-p23 appears to be a common abnormality in non-SCLC too. Efforts have been made to distinguish different regions of deletion on 3p by microscopic analysis (Whang-Peng et al., 1991). FISH analysis using chromosome 3 libraries as a probe could, however, clearly demonstrate that in some cell lines parts of chromosome 3, including minute fragments, were distributed over several other chromosomes (Van der Veen et al., 1992; Kok et al., 1994). At a microscopic level, a correct interpretation of classic karyotypes therefore hardly seems possible without supplementary FISH data. Comparative genomic hybridization (Kallioniemiet al., 1992) has also been

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applied to SCLC cell lines and could reveal some new frequent changes (Levin et al., 1994). However, comparative genomic hybridization cannot be considered very helpful in precisely identifying the size of heterozygous deletions. The boundaries of the deletions can most accurately be determined by analysis of loss of heterozygosity. Nevertheless, microscopic analysis of lung cancer did provide the first clue to the genomic region in which a primary event in the somatic-genetic etiology of this cancer would take place. 2. ANALYSIS OF LOSS OF HETEROZYGOSITY Pairwise analyses of lung tumors and normal tissue with RFLP markers have demonstrated that all small-cell lung tumors suffer from allelic losses of markers of the short arm of chromosome 3 (Brauch et al., 1987; Kok et al., 1987; Naylor etal, 1987; Yokota et al., 1987; Johnson et al., 1989; Mori et al., 1989; Kok et al., 1989). The vast majority of SCLC tumors appear to have lost the entire short arm distal to D3S3 at 3p12. This finding may, however, be biased by the limited availability of polymorphic markers in earlier analyses. The determination of a smallest region of overlap of the deletions thus had to be based on only a few cases that showed retention of heterozygosity at loci distal to 3p12. Retention of heterozygosity for the thyroid hormone receptor p gene (THRB) has been described in a few reports, also in combination with loss of heterozygosity for more proximal loci (Drabkin et al., 1988; Leduc et al., 1989; Ganley et al., 1992). Likewise, a few tumors have been described that retained heterozygosity at D3S2 or D3S3 in combination with allelic losses at one or more distal loci (Johnson et al., 1989; Naylor et al., 1989). One SCLC tumor with retention of heterozygosity at D3F15S2 in combination with loss of heterozygosity for 3q has been described (Johnson et al., 1989). Although further 3p markers were not informative in this case, most likely the whole of chromosome 3 proximal to D3F15S2 was deleted. For a map of the markers mentioned here and throughout the rest of this review, the reader is referred to Figure 1. The introduction of microsatellite markers allowed more refined analyses. One such study reported one tumor with retention of heterozygosity at D3S686, which maps just telomeric to D3F15S2, and two with retention of heterozygosity distal to this locus (Hibi et al., 1992). Several groups have screened large numbers of SCLC-derived cell lines for the presence of heterozygous 3p loci (Sithanandam et al., 1989; Brauch et al., 1990a; Daly et al., 1993). Heterozygosity was detected only rarely, but in these studies never for D3F15S2, nor for D2S1235 mapping just proximal to D3F15S2. One tumor (SC3)has been described that was heterozygous for D3S2 and, based on densitometric analysis, also retained both D3F15S2 alleles (Daly et al., 1991). This tumor did show loss of heterozygosity for a more centromeric region identified by D3S30, D3S3, and D3S4. With the exception of this single case,

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D3S1304 D3S18 D3S601 D3S1038 D3S1250 RAF D3S656 D3S1110 D3S1255 D3S266

m

ARp

FHIT PTPRG

IC B

I

I

21.1

I -I

8 8 8

D3S647 D3S1611 D3S1298 D3S1260 D3S1029 D3S966 D3S32 D3S643 D3F1SS2 D3S2968 D3S1235 D3S1573 D3S1227 D3S1289 D3S2 D3S1295 D3S1766 D3S1313 D3S1234 D3S1300 D3S1481 D3S1480 D3S1312 D3S1287 D3S1228 D3S1233 D3S1217 D3S1210 D3S30 D3S1284 D3S2623 D3S1511 D3S1274 D3S3 D3S1254 D3S1276 D3S1251 D3S1101

l-

IMLHl

I

D3S686

I

D3S1478

ARP

FHIT

IPTF'RG IE4,%560 D3S714

I

D3S642 D3S659

Fig. 1 Location of genes, markers, deletions, and fragments that suppress tumorigenicity on chromosome 3. The hatched bars indicate areas of homozygous deletions. (A) At 3p22 is the deletion described by Murata et al. (1994). (B) At 3p21.3 are the three overlapping deletions (Daly et al., 1993; Kok et al., 1994; Roche et al., 1996) that overlap with a fragment of chromosome 3 (upper dotted bar) that suppresses tumorigenicity in mouse A9 cells (Killary et al., 1992). (C) At 3p21.1 is the homozygous deletion observed in a breast cancer (Buchhagen et al., 1994). (D) At the location of the fragile site and the FHlT gene, a number of homozygous deletions occur (see text). (E-F) At 3p12-pl4 are three large deletions found in breast cancer (E) (Chen, L.-C., et al., 1994), cervical cancer (F) (Aburatani et al., 1994), and SCLC (G) (U2020) (Rabbitts et al., 1990) that overlap with a chromosomal fragment (lower dotted bar) that suppresses tumorigenicity of a renal cell carcinoma line (Sanchez et al., 1994). Bars have not been drawn to scale. Details are given in the text.

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which might be explained by an unequal crossing-over event in the D3F15S2 region, a smallest region of overlap can be defined as distal to D3S2 and proximal to THRB, or perhaps to D3F15S2 (i.e., band 3 ~ 2 1 ) . Non-SCLC tumors often show partial loss of one of the two alleles. As discussed, this need not always imply a genuine loss of heterozygosity. Since many reports fail to provide criteria for loss of heterozygosity, it is understandable that the literature shows some disagreement with respect to the percentage of non-SCLC tumors with loss of heterozygosity at 3p. Reported frequencies vary from 50 to 100% (Yokota et al., 1987; Weston et al., 1989; Tsuchiya et al., 1992; Yokoyama et al., 1992; Rabbitts et al., 1989; Kok et al., 19-89).Methods of selection of tumor material, choice of probes, and differences in interpretation of the resulting data all contribute to this variation. The lowest percentage for loss of heterozygosity has been reported for adenocarcinoma of the lung (Tsuchiya et al., 1992; Yokoyama et al., 1992), the subtype known to have the highest amount of infiltrating normal tissue. Thiberville et al. (1995b) collected their tumor samples using a very precise microdissection technique and detected loss of heterozygosity for 3p21 in 87% of squamous cell carcinomas and in 84% of large-cell undifferentiated carcinomas. Most authors do agree upon the importance of 3p deletions in non-SCLC. Only a few non-SCLC tumors have been described that show loss of heterozygosity at a subset of 3p markers. Three tumors have been reported to show retention of heterozygosity for either D3S3 (Rabbitts et al., 1989) or D3S2 (Kok et al., 1987) in combination with allelic losses at more distal loci. Several tumors have shown retention of heterozygosity for THRB in combination with loss at D3F15S2 (Leduc et al., 1989).In a large study using 19 RFLP markers from 3p, two smallest regions of overlap of deletions were defined for adenocarcinoma of the lung, namely, 3p21.3 and 3p14.1-p21.1 (Yokoyama et d.,1992). The 3p21.3 region, flanked by the markers D3S686 and D3S643, was the most frequently occurring common deletion region. In a similar large study, an almost identical smallest region of overlap of deletions was defined for both adenocarcinoma and squamous cell carcinoma of the lung (Hibi et al., 1992). All these data are in support of a smallest region of overlap for non-SCLC tumors identical to that for SCLC tumors.

3. LOSS OF HETEROZYCOSITY IN PRENEOPLASTIC LESIONS The high frequency of allelic losses at the short arm of chromosome 3 in all histological types of lung cancer suggested that an allelic loss at 3p represents an early event in lung cancer pathogenesis. Its prior detection in preinvasive lesions of the bronchial epithelium (Sundaresan et al., 1992; Gazdar et al., 1994; Sozzi et al., 1995; Chung et al., 1995) strongly supports this conclusion. Despite absence of an agreed-on classification of preinvasive bronchial lesions, which complicates a comparison of the different results,

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there are common findings. Allelic losses at 3p are detected in a broad range of preneoplastic lesions, from mild hyperplasia to severe dysplasia. The frequency of allelic losses and the size of the deletions seem to increase as the tissue progresses toward a more neoplastic phenotype (Hung et al., 1995; Thiberville et al., 1995a). In mild hyperplasia, loss of heterozygosity was not detected by all groups. Deletions of 3p in preneoplastic lesions are mostly interstitial and confined to a small region, whereas carcinomas of the same patients invariably show extensive allelic losses that mostly comprise the whole p arm of chromosome 3 (Chung et al., 1995; Hung et al., 1995). Two dysplastic samples obtained with a 9-month interval from the same patient and the same anatomical site showed an increase of the region of allelic losses at 3p (Chung et al., 1995). A similar correlation of the size of 3p deletions with the stage of the disease has also been observed in a comparative study of renal cell adenoma and carcinoma (Van den Berg et al., 1996a). This might indicate that, as tumor development progresses, distinct genes located at 3p become sequentially inactivated, although not necessarily always in the same order. As cytogenetic studies (Whang-Peng et al., 1982a,b) have shown that most if not all lung tumors contain at least one apparently normal copy of chromosome 3 (i.e., one not containing microscopically visible deletions), the progressive loss of chromosome 3 material is probably confined to one of the homologs. Chung et al. (1995) proposed that an initial random genetic loss destabilizes the chromosomal integrity and predisposes that homolog to further genetic damage. A more simple explanation might be that, following the initial loss of a large region of 3p at one homolog, a second large deletion occurring at the other homolog would result in a large homozygous deletion lethal to the cell. Allelic losses at 3p have been detected in preneoplastic lesions from patients with all types of lung carcinoma (Sundaresan et d., 1992; Chung et al., 1995; Hung et al., 1995) and irrespective of the site from which the sample was taken. The preneoplastic tissue samples that have been investigated were taken not only at relatively close distances from the actual tumor (Sundaresan et al., 1992; Chung et al., 1996), but also from various sites of the respiratory tree remote from the tumor site (Hung et al., 1995),and from individuals without any lung carcinoma at the time of sampling (Sundaresan et al., 1992; Thiberville et al., 1995a). The analyses of Hung et al. (1995) showed deletions to be present in multiple lesions throughout the respiratory epithelium, including bronchi, bronchioles, and alveoli. Moreover, various preneoplastic lesions taken from the same patient had lost different regions of 3p. Such data are difficult to explain by a clonal origin of the preneoplastic lesions. Instead, they seem to support the “field cancerization” theory, which suggests that the entire upper aerodigestive tract has been mutagenized and is at risk for the development of multiple cancers (Strong et al., 1984; Sozzi et al., 1995; Smith et al., 1996). In this process of field can-

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cerization of the epithelium of the aerodigestive tract, which is presumably the result of exposure to carcinogens, loss of 3p sequences appears to be one of the initial events. On the other hand, the data of Sundaresan et al. (1992), who investigated bronchial neoplasias that were distinct from, but situated close to, the primary lesion, are in agreement with a clonal relationship between adjacent lesions of different grades in the same patient. Analyses of TP53 mutations in their specimens confirmed this (Chung et al., 1996). The fact that cells from the respiratory epithelium have or acquire some kind of mobility may provide an explanation for this situation (Habuchi et al., 1993b). In their analysis of multiple lesions from various sites throughout the respiratory tract, Hung et al(1995) found that in 89% of the lesions the same chromosome 3 homolog was affected in all samples from the same patient. Such homolog-specific losses have also been observed in preneoplastic breast lesions (O’Connell et al., 1994) and in renal cell adenomas (Van den Berg et al., 1996a).Although a difference in the susceptibility to genetic damage between maternal and paternal homologs has been suggested (Chung et d., 1995; Hung et al., 1995), this phenomenon still awaits a satisfactory explanation. 4. HOMOZYGOUS DELETIONS

The increasing number of markers available to search for allelic losses has led to the detection of several homozygous deletions in lung tumors and derived cell lines. Rabbitts et al. (1990) were the first to report a homozygous deletion at the locus D3S3 in an SCLC cell line, namely, U2020. Detailed mapping later indicated that it is located in 3p12 and is about 8 Mb in size (Drabkin et al., 1992; Latif et al., 1992). In an effort to search for genes in this region, a YAC contig has been constructed that almost completely spans the deleted region flanked by the markers D3S1276 and D3S2623 (Todd et al., 1995). The U2020 homozygous deletion is far from the smallest region of overlap of heterozygous deletions in SCLC, and overlaps with a region within 3p12-pl4 that mediates rapid cell death of renal cell carcinoma (RCC) in vivo (Sanchez et al., 1994; see also Section IV). A single cosmid from 3p21 has been reported to detect homozygous deletions in 5 of 36 lung cancer-derived cell lines (Yamakawa et al., 1993). One of these homozygous deletions has been determined to be nearly 800 kb in length (Murata et al., 1994). Bicolor FISH analysis using CHEPH YAC 936C1 overlapping this 800-kb deletion (Murata et al., 1994)and YACs that flank a 3p21.3 deletion, described later, demonstrated that the 800-kb deletion lies distal to the latter deletion, probably in 3p22 (Van den Berg et al., 1 9 9 6 ~ )Using . a DNA segment from this YAC, Roche et al. (1996)identified homozygous deletions in two additional lung cancer specimens. The hMLHl

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gene is not included in this homozygous deletion as it hybridizes with YACs that lie telomeric to 936C1, and not with 936C1 itself (Roche et al., 1996). Three overlapping homozygous deletions have been identified in three different SCLC-derived cell lines (Daly et al., 1993; Kok et al., 1994; Roche et al., 1996). They map in 3p21.3 directly centromeric to D3F15S2 and have a DNA segment of about 400 kb in common. The homozygously deleted DNA segments are wholly or partly contained within a 2-Mb DNA fragment able to suppress the tumorigenicity of a mouse fibrosarcoma cell line (Killary et al., 1992). This strongly suggests that this DNA region of 3p21 contains a tumor suppressor gene involved in the development of lung cancer. A partial YAC contig of the region (Kok et al., 1994), a P1 contig (Xiang et al., 1996), and a 600-kb cosmid contig (Wei et al., 1996) have been constructed. The cosmids defined by Wei et al. (1996) are in the process of being sequenced by the Washington University Sequencing Center and the Sanger Centre. A P1 phage containing some 80 kb of this region has been reported to suppress the tumorigenicity of the mouse A9 fibrosarcoma cell line Todd et al., 1996). Although a number of genes have already been identified in this apparently gene-rich region (see Section V.D), tumor suppressor gene candidacy has yet to be demonstrated for any of them. It should be noted that this region contains a DNA segment that has proven very unstable when cloned (Kok et al., 1994; Timmer et al., 1996), so some DNA sequences may still be missing from the contigs. A homozygous deletion in a different region, namely, in the FRA3B region, has recently been found in a lung adenocarcinoma cell line (Van den Berg et al., 1995a). The possible significance of this deletion is further discussed in Section V.F.

B. Renal Cell Carcinoma 1 . MICROSCOPIC CHROMOSOME ANALYSIS The first chromosome analysis of an RCC tumor was published by Pathak et al. (1982) and revealed an apparently balanced t(3;ll) with the chromosome 3 breakpoint in 3p13. Although the patient belonged to a family with several members suffering from RCC, the translocation was only observed in tumor cells. Many RCC tumors, mainly sporadic, have been karyotyped since then. Deletions or other structural aberrations in 3pl3-pter appeared to be the most common chromosome abnormalities in sporadic RCC (Yoshida et al., 1986; Kovacs et al., 1987; De Jong et al., 1988; Kovacs and Frisch, 1989; Presti et al., 1991a; Maloney et al., 1991; Meloni et al., 1992; Van den Berg et al., 1993). In most cases of familial RCC constitutive chromosome abnormalities could not be found (Li et al., 1982; Pathak and Goodacre,

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1986). A family reported by Cohen et al. (1979), however had a constitutive t(3;8) with the chromosome 3 breakpoint in 3p14.2 (Wang and Perkins, 1984). Kovacs et al. (1989)described a constitutive t(3;6) with a breakpoint microscopically defined between 3p13 and 3 ~ 1 4 . 1 ,occurring in three generations in a family in which the oldest translocation carrier had developed bilateral RCC. The other translocation carriers were too young to reveal whether they also have a predisposition for RCC. Using YACs from a 6-Mb contig containing the t(3;8) breakpoint (Boldog et al., 1994)in a FISH analysis of t(3;8) and t(3;6) cells, flanking YACs could be identified for each of the two chromosome 3 breakpoints (Van den Berg et al., 1995b). From the order of the YACs it could be clearly confirmed that the t(3;6) breakpoint is located more proximal than the t(3;s) breakpoint. Moreover, inclusion in the FISH analysis of YACs close to the distal boundary of the 3p12 homozygous deletion in the SCLC cell line U2020, described by Rabbitts et al. (1990),indicated that the t(3;6) breakpoint mapped distal to that deletion. Thus, in a model assuming that the constitutive translocations would disrupt and thereby inactivate one of the alleles of a tumor suppressor gene, at least two different suppressor genes would have to be hypothesized because of the different location of the breakpoints. The t(3;8) breakpoint has been cloned and a cDNA clone identified by exon trapping has been reported to lie immediately adjacent to it (Boldog et al., 1993). However, as yet no data have been provided suggesting this gene to be the RCC gene. Recently, another gene, called FHIT, has been found overlapping the breakpoint (Ohta et al., 1996). A possible role of this gene in the development of different types of tumors is discussed in Section V.F. No evidence has yet been presented, however, of a role of FHIT in the development of RCC. Cytogenetic analysis of tumors from the t(3;6) patient and from t(3;8) patients revealed that in all cases the derivative chromosome containing the distal part of chromosome 3 was lost, whereas the morphologically normal homolog was retained (Kovacs et al., 1989; Li et al., 1993). In the disruption model, one would expect loss of the normal chromosome 3 homolog. Therefore, as an alternative, the constitutive translocations may be considered to be random, independent eventsincreasing, however, the probability of chromosome loss from any cell. A preferential loss of translocation products would then represent the hereditary predisposition for RCC in these cases. As has been suggested earlier (Kovacs and Kung, 1991; Kovacs, 1993), the random loss of the derivative chromosomes during embryonic development or regeneration may yield a proportion of renal cells retaining only one copy of the 3p segment distal to the breakpoint. Tumor formation would result from subsequent mutation of the remaining allele of an RCC gene in this distal segment. The finding of a von Hippel-Lindau (VHL) gene mutation in a tumor from a patient of the t(3;8) family is in agreement with this suggestion (Gnarra et al., 1994). RCC is also one of the tumors for which patients with VHL disease, a

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hereditary cancer syndrome, are at risk. RCC occurs in 3 0 4 0 % of these patients, who also have a predisposition to develop a.0. retinal angioma, cerebellar hemangioblastoma, and pheochromocytoma. Constitutive chromosome abnormalities have not been described (Neumann etal., 1988).In RCC tumors from VHL patients, loss of 3p13-pter was found in all cases studied (Kovacs and Kung, 1991), indicating 3p as a possible site for the VHL gene. An answer to the question of whether the genes involved in sporadic RCC, in the translocation families, and in VHL are identical could only come from additional data that are discussed in Section V.A. 2. ANALYSIS OF LOSS OF HETEROZYGOSITY The involvement of 3p in the development of RCC as suggested by the results of chromosome analysis could readily be further evaluated by direct DNA analysis of matched pairs of tumor and constitutive tissue (Zbar et al., 1987; Kovacs et al., 1988; van der Hout et al., 1988; Anglard et al., 1991). Loss of heterozygosity at the short arm of chromosome 3 in RCC correlates with the cytological tumor type. It is predominantly detected in clear celltype nonpapillary RCC, and not in papillary RCC tumors or in nonpapillary oncocytomas (Brauch et al., 1990b; Ogawa et al., 1991; Presti et al., 1991a, 1993; van der Hout et al., 1993). These investigators detected loss of heterozygosity in some 60% of clear cell RCC by using only a limited number of 3p markers. This frequency of heterozygous loss by far exceeds the frequencies detected for other chromosome arms (Morita et al., 1991; Brauch et al., 1994; Thrash-Bingham et al., 1995a). Analysis of loss of heterozygosity of a great number of RCCs resulted in the identification of different smallest regions of overlap of 3p deletions, namely, one in 3p12 (Lubinski et al., 1994), one in 3p13-pl4 (Yamakawa et al., 1991; Lubinski et al., 1994), one in 3p21 (Anglard et al., 1991; van der Hout et al., 1991; Yamakawa et al., 1991; Foster et al., 1994a),and one in 3p2.5-pter (Brauch et al., 1994; Foster et al., 1994a). The increase in density of polymorphic markers applied led to the detection in a number of tumors of multiple deletions separated by regions of retention of heterozygosity (Yamakawa et al., 1991; Foster et al., 1994a; Lubinski et al., 1994). In a recent analysis of sporadic RCCs, it turned out that, in all cases where 3p deletions were detected, the deletion always included the 3p21 region (Van den Berg et al., 1996b). In cases of multiple deletions, loss of the 3p21 region occurred either in combination with deletion of the 3p25-p26 region or with deletion of the 3p12-pl4 region (Van den Berg et al., 1996b). This seems to point to a major role for the 3p21 region in the development of RCC. The smallest region of deletion overlap in 3p21 was between D3S643 and D3S123.5, that is, very close to if not overlapping with the 3p21.3 region most likely involved in the development of lung cancer (see Fig. 1).An

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involvement of one of at least two other 3p loci has been indicated by several studies. One is the VHL locus, which shows loss of heterozygosity in 45-96% of sporadic RCC (Brauch et al., 1994; Foster et al., 1994a; Van den Berg et al., 1996b) and mutations in 31-57% of nonfamilial clear cell RCC (Foster et al., 1994b; Gnarra et al., 1994; Shuin et al., 1994; Whaley et al., 1994).The other locus is in the region 3pcen-p14. Lubinsky et al. (1994)defined a smallest region of overlap between the markers D3S1285 in 3p14.1 and D3S1295 in 3 ~ 2 1 . 1 ,a region that included the t(3;8) translocation breakpoint and the FRA3B site. Later the region was further delineated to a common region in 3 ~ 1 4 . 2flanked by the markers D3S1481 and D3S1312 (Druck et al., 1995). D3S1481 maps between FRA3B and the t(3;8) breakpoint; D3S1312 is a PTPRG intragenic marker. However, some of the RCC samples analyzed by Van den Berg et al. (1996b) had retained heterozygosity for two markers, D3S1481 and D3S1480, that immediately flank the t(3;8) translocation breakpoint, whereas allelic losses were detected for a region more proximal to the breakpoint. This observation seems to support exclusion of the t(3;8) breakpoint region from a role in the development of sporadic RCC. Most publications describe deletions of only part of 3p. In contrast, Wilhelm et al. (1995) report terminal deletions comprising the whole p arm distal to the breakpoint in all 41 RCC tumors that they had analyzed. This conflicting finding may, however, be explained by their choice of microsatellite markers that only included two markers distal to 3 ~ 1 4 . 2 . They could show that all tumors had breakpoints proximal to D3S1300 in 3~14.2.Lubinski et al. (1994) identified a second minimal region of allelic loss in 3p12-pl3, flanked by the markers D3S1274 and D3S1254, which both map within the homozygously deleted region in the cell line U2020. Frequent loss of heterozygosity of this region has also been described by others (Wilhelm et al., 1995; Van den Berg et al., 1996b). These data, in combination with the frequent finding of homozygous deletions and suppression of tumorigenicity of an RCC cell line by the segment 3p12-pl4 (Sanchez et al., 1994), indicate the presence in the proximal part of 3p of at least one further gene involved in RCC development.

C. Head and N e c k Squamous Cell Carcinoma Head and neck squamous cell carcinomas (HNSCC) constitute a heterogeneous group of tumors encompassing several subtypes, including nasopharyngeal carcinoma, laryngeal carcinoma, and carcinoma of the oral cavity. Abnormalities of 3p have been revealed by cytogenetic analysis, but results from different studies seem to vary. Heo et a/. (1989) and Van Dyke et al. (1994) report microscopically detectable deletions of the 3p13-pl4 region as a common finding in primary cultures and cell lines from HNSCC.

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Jin et al. (1993), however, found loss of 3p material in only 7 of 50 tumors with clonal abnormalities. In laryngeal carcinoma, Allegra et al. (1992) described deletion of 3p21-p23 as the most frequent chromosomal abnormality. Underrepresentation of the short arm of chromosome 3 for this type of tumor has been confirmed by comparative genomic hybridization (Brzoska et al., 1995; Speicher et al., 1995). From loss of heterozygosity studies of HNSCC, it could be concluded that 3p was one of the chromosome arms most frequently involved (Ah-See et al., 1994; Nawroz et al., 1994). Loss of heterozygosity is detected in 50-80% of the cases (Maestro et al., 1993; Ah-See et al., 1994; Lo et al., 1994; Nawroz et al., 1994; Ishwad et al., 1996). A number of publications have tried to define a minimal region of allelic loss at 3p (Huang et al., 1990; Choi et al., 1993; El-Nagger et al., 1993; Maestro et al., 1993; Li et al., 1994; Lo et al., 1994; Scholnick et al., 1994). It is not easy to compare the results from the different studies, since in many cases only a limited number of often different markers has been used. From a few larger studies that applied a greater number of markers, there appear to be three regions of 3p with relatively high frequencies of allelic losses separated by regions with retention of heterozygosity (Maestro et al., 1993; Lo et al., 1994; Wu et al., 1994; Partridge et al., 1996). The pattern of allelic loss resembles that of RCC, as the regions involved are 3p12-p14.3, flanked by D3S30 and D3S1228; 3 ~ 2 1 . 3 flanked , by D3S966 and D3S647; and 3p25, distal to THRB. Despite the frequent loss of heterozygosity for 3p24-p25, VHL mutations have not been found in this type of tumor (Wager et al., 1996). Waber et al. (1996) also did not detect loss of heterozygosity for a polymorphic marker in exon 1 of the VHL gene for any of 18 informative tumors. In one study that specifically focused on loss of heterozygosity in the distal region of 3p, a smallest region of overlap was defined as the interval 3p24-p25.1 bordered by the markers D3S1293 and D3S6.56, which both map proximal to VHL (Rowley et al., 1996). These studies suggest the presence of another gene involved in cancer development in 3p24-p25. Allelic losses at multiple chromosome arms appear to correlate with a poor prognosis in HNSCC (Li et al., 1994; El-Nagger et al., 1995; Field et al., 1995). In oral squamous cell carcinoma, Partridge et al. (1996) found a significant correlation between the number of deleted regions at 3p and tumor stage. Since they analyzed only loss of heterozygosity of 3p, the possibility that this correlation applied to the number of genomic losses in general cannot be excluded. As in dysplastic regions, 9p deletions occurred at a higher frequency than 3p deletions, El-Naggar et al. (1995) concluded that the latter may occur at a later stage of tumor development. Roz et al. (1996) detected allelic losses of 3p13-p21.1 and 3p25-pter in oral dysplastic lesions at frequencies comparable to those in invasive oral carcinoma. They therefore concluded that these deletions are probably early events in cancer development. In the dysplastic lesions, the

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frequency of allelic loss at the central region of 3p was lower than that in the oral squamous carcinomas.

D. Gastrointestinal Tumors A total of 45 adenocarcinomas of the stomach and lower esophagus have been cytogenetically analyzed in several studies. Structural anomalies of chromosome 3 occurred in 28 cases (Rodriguez et al., 1990; Whang-Peng et al., 1990; Xiao et al., 1992; Barletta et al., 1993; Seruca et al., 1993). In 4 of 9 cases Rodriguez et al. (1990) detected a clustering of breakpoints in 3p21. Whang-Peng et al. (1990) observed breakpoints in 3p13, resulting in loss of the telomeric part of 3p, in 9 of 14 esophageal carcinoma cell lines. In two DNA marker analyses of esophageal carcinoma (Aoki et al., 1994; Shibagaki et al., 1994), 3p allelic losses were detected in 3 5 4 0 % of informative cases, with loss at several other chromosome arms scoring equal or higher percentages. According to the data of Ogasawara et al. (1995), loss of heterozygosity was most frequent for D3S1255 at 3p21.3 (33%) and for C13-1169 (D3S1110) at 3p25 (35%). In an analysis of 78 specimens of gastric adenocarcinoma, D3S1478 at 3p21 showed loss of heterozygosity in 36% of informative cases (Schneider et al., 1995). Again, markers at several other chromosome arms showed equal or higher frequencies of allelic losses. The same study indicated that band 3 ~ 2 1 . 3 flanked , by the markers D3S1227 and D3S1029, was the common region of deletion of these tumors. According to Mori et al. (1994),3p deletions occur in all histological grades of esophageal carcinoma. They can already be detected in low-grade esophageal dysplasia, implying that they represent an early event. Shimada et al. (1996) analyzed 106 premalignant lesions of the esophagus selected from 32 patients with esophageal squamous cell carcinoma and also detected a high frequency of allelic loss at 3p in mildly dysplastic lesions. The authors concluded that a gene on 3p may be responsible for the dysplastic changes in esophageal epithelium, possibly changing normal stratified squamous epithelial cells into dysplastic cells. In colorectal carcinoma, loss of heterozygosity of 3p markers appeared to be a very infrequent event (Vogelstein et al., 1989; Devilee et al., 1991; Barletta et d., 1993). One of the two 3p clones recently isolated by representational difference analysis, D3S3 155, appeared to be homozygously deleted in four colon cancer cell lines (Lisitsyn et al., 1995). The clone turned out to originate from the region of FRA3B at 3p14.2 (Ohta et al., 1996). The implications of this finding are discussed in Section V.F. Kastury et al. (1996) detected frequent loss of heterozygosity for markers from this region, flanked by D3S1234 and D3S1481, in about 50% of uncultured stomach carcinomas and colon carcinomas.

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A few anal canal cancer tumors have been karyotyped. Deletions of the short arm of chromosome 3 were observed in five of eight cases analyzed (Muleris et al., 1987).

E. Breast Cancer Cytogenetic analyses of primary breast tumors have revealed relatively few cases with structural abnormalities involving chromosome 3 (Geleick et al., 1990; Dutrillaux et al., 1990). Some cases have been described, however, in which a deletion of 3p was the only clonal abnormality. Zang et al. (1989) defined such a deletion in a primary tumor as de1(3)(p14-p21). Pandis et al. (1993) reported three such cases and defined the minimal deletion as de1(3)(p13-p14). These findings might indicate that loss of 3p sequences can be an early event in a subset of breast tumors, a conclusion substantiated by Dietrich et al. (1995),who found a de1(3)(p12-p14) in 3 of 16 benign lesions of the breast (two atypical epithelial hyperplasias and one papilloma). Loss of heterozygosity at 3p was detected in 2 of 28 cases of ductal carcinoma in situ of the breast, a preinvasive form of breast cancer (Radford et al., 1995). In general, allelic losses of 3p have been detected in 30-56'70 of informative breast tumors (Ali et al., 1989; Devilee et al., 1989, 1991; Chen et al., 1992).In these studies only one or a few polymorphic markers had been used for the short arm of chromosome 3. Other chromosome arms, however, showed even higher frequencies of loss of heterozygosity (Devilee et al., 1989, 1991; Sat0 et al., 1991a; Chen et al., 1992). Sat0 et al. (1991a) used 12 markers from 3p, mostly mapping proximal to 3p22, to define a smallest region of overlap of allelic losses and found this to be in 3p13-p14.3, flanked by D3S660 and D3S714. Two separate regions of loss at 3p, namely, 3p13-pl4 and 3p24-p26, were defined by Chen et al. (1994). They also found a higher frequency of loss of heterozygosity at 3p21, but always in combination with allelic losses of one of the other two regions. Although they concluded that allelic losses at 3p21 may therefore not represent a crucial event, the situation may be comparable to that in RCC (see Section III.B.2). A frequent involvement of the telomeric region in allelic losses had already been demonstrated (Ali et al., 1989; Chen et al., 1992). Eiriksdottir et al. (1995) observed allelic imbalance for 3p markers in 48 of 140 (34%) of the breast tumors they analyzed. The highest frequencies of allelic loss were detected with the probe pH3H2, defining loss at D3FlSS2 in 3p21.3 (46%) and at D3S1233 in 3p14 (33'70),whereas markers at 3p21.1 showed much lower frequencies of allelic loss. The allelic losses appeared to be correlated with DNA nondiploidy, with a high S-phase fraction, and with an increased mortality rate. Bergthorsson et al. (199.5) detected allelic imbalance for at least one marker in 26 of 35 paired normal and breast tumor samples

Chromosome 3 Suppressor G e n e s

49

from seven breast cancer kindreds. The highest frequencies of allelic losses were detected at D3S1217 in 3p14.2 (68%) and at D3S1029 in 3~21.3p21.2 (42%). The high frequency of allelic imbalance for D3S1217 is noteworthy, since in sporadic breast carcinoma Eiriksdottir et al. (1995)detected allelic imbalance with this marker in only 28% of the cases. Bergthorsson et al. (1995) found no evidence for linkage to 3p in any of the seven breast cancer kindreds they analyzed. By combining their loss of heterozygosity data with the results of a FISH analysis of the same tumor specimens using cosmids originating from chromosome 3, Chen et al. (1994) showed that in the majority of cases loss of heterozygosity was caused by physical deletion of part of the short arm of chromosome 3. In about 10% of the cases it was due to loss of a complete chromosome. A homozygous deletion in a breast cancer specimen was detected with a probe defining D3S2 and thus mapped to the region 3p14.3-p21.1 (Buchhagen et al., 1994).In addition, rearrangements at D3S2 were found in a few breast cancer cell lines (Buchhagen et al., 1994). Another homozygous deletion was detected with D3S642 at 3p13 (Chen et al., 1994). It did not overlap with the homozygous deletion of the U2020 lung cancer cell line (Geil et al., 1994).

F. Female Genital Tract Tumors The female genital tract tumors can be divided in three main types: endometrial, ovarian, and cervical carcinoma. The highest frequency of loss of heterozygosity for 3p is found for cervical carcinoma. Although viral infection with certain types of human papillomavirus (HPV) is recognized as an important factor in the development of this cancer, the infection in itself appears to be insufficient to initiate tumor development (Zur Hausen, 1991). Loss of 3p sequences has been detected in both HPV-positive and HPVnegative cervical lesions (Yokota et al., 1989; Chung et al., 1992; Mitra et al., 1994) in all stages of the disease (Yokota et al., 1989), including all stages of intraepithelial neoplasia (Chung et al., 1992), with the highest frequencies of allelic losses found in advanced stages (Kohno et af., 1993; Mullokandov et al., 1996). High frequencies of allelic losses have been reported for several markers: 90% for D3S3 at 3p14 and 83% for cRAFl at 3p25 (Chung et al., 1992); 75% for THRB at 3p24 (Karlsen et al., 1994); 46% for D3S32 at 3p21.3 (Mitra et al., 1994); 100% for D3S2 at 3p21.1 (Yokota et al., 1989);and 86% for D3S659 at 3p13 (Jones et al., 1994).A detailed analysis of 47 tumor specimens using 24 markers for 3p showed loss of heterozygosity for at least one locus in 21 of the tumors (Kohno et al., 1993). Only 4 tumors showed loss and retention of heterozygosity for different

50

Klaas Kok et al.

parts of 3p. Based on these four cases, 3p13-p21.1 was defined as a common region of deletion (Kohno et al., 1993).This region includes the markers D3S2, D3S659, and D3S3, which is in agreement with the findings of Yokota et al. (1989), Chung et al. (1992),Jones and Nakamura (1992),and Karlsen et al. (1994). A homozygous deletion in the region 3p12-pl3 was reported for one cervical carcinoma by Aburatani et al. (1994).Although 3p has been shown to be one of the most frequently deleted chromosome arms in cervical carcinoma, loss of heterozygosity has also frequently been observed for several other chromosome arms (Jones et al., 1994; Mitra et al., 1994; Mullokandov et al., 1996). In endometrial carcinoma, loss of heterozygosity occurs less frequently (Jones and Nakamura, 1992; Jones et al., 1994), but frequencies for different loci, including several at 3p, vary considerably between different studies (Fujino et al., 1994; Jones et al., 1994). Jones and Nakamura (1992) suggested 3p13-p21.1 as a common region of deletion in endometrial carcinoma but D3S32 at 3p21.3 is also deleted at a high frequency (Jones et al., 1994). In ovarian carcinoma, Van der Riet-Fox et al. (1979) reported microscopically visible deletions of 3p in two of five tumors. In a much larger study by Whang-Peng et al. (1984),chromosome 3 turned out to be the chromosome second most frequently involved in structural abnormalities. This conclusion was confirmed by Bello and Rey (1990). It was, however, the long arm of chromosome 3 that was most often involved in structural rearrangements. Pejovic et al. (1992) karyotyped 52 short-term cultures of primary ovarian carcinomas and found that 12 had either deletions of a segment distal to 3p12-pl4 or unbalanced translocations, with breakpoints clustering in 3p12-pl3. Results from several studies on allelic losses showed that 3p was involved in only a minority of cases. Cliby et al. (1993) did not find a single case among 23 informative cases. Most studies with 3p markers gave frequencies of loss of heterozygosity between 15 and 27% (Sato et al., 1991b; Zheng et al., 1991; Dodson et al., 1993; Yang-Feng et al., 1993). Many other chromosome arms showed allelic losses at equal or higher frequencies. Deletions of 3p have been reported to occur predominantly in high-grade tumors (Zheng et al., 1991), although Dodson et al. (1993) also found loss of heterozygosity for 3p in a subset of low-grade tumors showing allelic losses at several chromosome arms. None of the analyses of ovarian carcinoma clearly defined a smallest region of overlap of allelic losses, but loss of heterozygosity appeared to be frequent at the THRB locus in 3p24 (Zheng et al., 1991; Yang-Feng et al., 1993; Foster et al., 1995). A single study (Worsham et al., 1991) has been published on squamous cell carcinoma of the vulva. Karyotyping showed loss of the region 3pcen-p14 in five of six cases, making this one of the most consistent structural changes.

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G. Other Types of Cancer For several types of cancer, including malignant astrocytoma (Fults et al., 1990),hepatocellular carcinoma (Fujimori et al., 1991),prostate cancer (Kunimi et al., 1991; Latil et al., 1994),meningioma (G. Schneider et al., 1992), and acoustic neuroma (Irving et al., 1993), no substantial allelic losses of chromosome 3 sequences have been found. It should be noted, however, that many of these tumors have not yet been studied extensively. Moreover, it might have been the choice of the markers used that caused the low percentages of allelic losses. Also, 3p deletions have not been found in the relatively slow-growing and rarely metastasizing basal cell carcinomas of the skin (Quinn et al., 1994), although these tumors show a specific chromosomal pattern of infrequent allelic losses. Squamous carcinomas of the skin, which grow faster and have a distinct metastatic potential, however, are characterized by much higher frequencies of allelic losses. Loss for 3p was detected in 23% of the cases, but several chromosome arms showed allelic losses at higher frequencies, indicating that 3p deletion may not be a primary event in these tumors (Quinn et al., 1994). Cytogenetic analyses of some 16 cases of Merkel cell carcinoma, also called small-cell carcinoma of the skin, did not reveal any alterations of the short arm of chromosome 3 (Leonard et al., 1993; references in Leonard et al., 1996).However, in a molecular genetic analysis by Leonard et al. (1996), loss of heterozygosity for at least one 3p marker was detected in 18 of 26 tumors. The smallest region of overlap of the heterozygous deletions was determined as 3p13-p21.1, centered around D3S2. Malignant mesothelioma is a highly malignant type of tumor derived from the lung mesoderm and closely associated with asbestos exposure. Involvement of chromosome 3 in malignant mesothelioma was first found by cytogenetic analysis of primary tumors and tumor-derived cell lines (Gibas et al., 1986). Losses of the region 3p13-p21 were detected in 7 of 7 cases with abnormal karyotypes by Popescu et al. (1988)in 7 of 25 such cases by Tiainen et al. (1989), and in 1 3 of 20 cases by Taguchi et al. (1993). Zeiger et al. (1994) reported allelic losses of 3p markers in 1 0 of 24 mesotheliomas, with a possible common deletion region flanked by D3F15S2 and D3S30. In a comparable analysis, Y. Y. Lu et al. (1994)detected allelic losses of 3p markers in 15 of 24 informative cases, with a smallest region of overlap flanked by D3S2 and THRB. The smallest regions of common deletion reported in these two publications overlap the region between D3S2 and DNFlSS2, a region coinciding with that of lung cancer. For testicular germ cell tumors, reported frequencies of allelic losses at 3p vary considerably, from 10% (Al-Jehani et al., 1995) to 28% (Lothe et al., 1989) to as much as 54% (Foster et al., 1995). Losses appear largely confined to seminomas.

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In follicular thyroid carcinoma, Herrmann et af. (1991) detected loss of heterozygosity for the short arm of chromosome 3 in 6 of 6 cases. No such losses were found in papillary thyroid carcinoma and follicular adenoma, of which 12 and 3 cases have been analyzed, respectively. These data are in agreement with earlier cytogenetic analyses of these types of tumors (Jenkins et af., 1990). Medullary thyroid carcinoma and pheochromocytoma are part of multiple endocrine neoplasia type 2, a familial cancer syndrome. Both types of tumors also occur sporadically. Allelic losses of chromosome 3 are almost exclusively found in pheochromocytoma (Mulligan et af., 1993)and appear to be concentrated on the short arm. Pheochromocytoma is also a feature of another cancer syndrome, VHL disease. VHL-associated pheochromocytomas also show allelic losses of 3p (Zeiger et al., 1995). In pleomorphic adenomas of the salivary glands, abnormalities of 3p21 have been found in 30% of cases with clonal chromosome abnormalities. The most commonly detected rearrangement is a reciprocal translocation t(3;8)(p21;q12) (Sandros et al., 1990; Stern et af., 1990). In one case a de1(3)(p21)was reported as the sole anomaly (Stern et af., 1990). An RFLP analysis of 35 pleomorphic adenomas with four different markers from the region 3p21-p25 detected loss of heterozygosity in four cases, all of which also had t(3;8)(p21;q12) (Sahlin et af., 1994). Cytogenetic analysis of seven lacrimal gland neoplasms revealed a aberrant chromosome 3 with a breakpoint in p21 in one case (Hrynchak et al., 1994). In malignant melanoma, structural abnormalities with breakpoints in 3pll-p25 were detected in 4 of 21 cases (Ozisik et al., 1994). In primary cutaneous melanomas they occurred in 6 of 3 1 informative cases (Healy et d., 1996), but in benign melanocytic nevi they occurred in none of the 28 informative cases (Healy et af., 1996). Uveal melanoma is characterized by a high frequency of monosomy of chromosome 3, often in combination with trisomy of 8q. Monosomy for chromosome 3 is detected in some 50% of the tumors both by conventional cytogenetics (Horsthemke et al., 1992, and references therein; Horsman and White, 1993) and by comparative genomic hybridization (Speicher et al., 1994; Gordon et al., 1994). Monosomy of chromosome 3, together with trisomy 8q, appears to be specific for one of two subgroups of uveal melanoma, the ciliary body type (Wiltshire et af., 1993; Sisley et d.,1990 Prescher et af., 1995). In bladder cancer, loss of 3p sequences appears to be correlated with tumor progression (Presti et al., 1991b; Habuchi et al., 1993a; Dalbagni et al., 1993). It is found predominantly in late stage and high-grade (invasive)tumors. In superficial papillary tumors confined to the mucosa, loss of heterozygosity of 3p has not been observed (Presti et af., 1991b; Dalbagni et al., 1993a), in contrast to the frequent loss of heterozygosity of 9q (Dalbagni et

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al., 1993). Presti et al. (1991b) and Dalbagni et al. (1993) detected allelic losses at 3p in about 45% and Habuchi et al. (1993a) in 20% of informative cases. Knowles et al. (1994) found loss of heterozygosity of 3p in only 5 of 64 informative cases, despite the inclusion of more than 20 high-grade and/or late-stage specimens. The variation in the frequency of allelic loss may very well be caused by the choice of markers. The common region of allelic losses at 3p appeared to be 3p13-p21.1. These studies indicate that loss of this region in bladder cancer is a late event, correlated with the transition from a superficial papillary tumor stage to an invasive disease (Presti et al., 1991b; Dalbagni et al., 1993).

H. Loss of 3p Sequences in Experimental Cell Systems The data obtained from the analysis of loss of 3p sequences in bladder cancer as described previously are in agreement with results from a multistep in vitro transformation system of human uroepithelial cells (Wu et al., 1991; Klingelhutz et al., 1991). Nontumorigenic simian virus 40 (SV40)-immortalized uroepithelial cells were transformed in vitro, either by exposing them to carcinogens or by transfection with EJ/ras, and subsequently inoculated into athymic nude mice to test the tumorigenicity of the cells. In 8 of 17 resulting carcinomas, karyotyping showed 3p losses (Wu et al., 1991). Using DNA marker analysis, loss of heterozygosity of 3p was detected solely in 9 of 11grade 111 tumors and in none of the 5 lower grade tumors (Klingelhutz et al., 1991).This clearly indicates an association of loss of 3p sequences with the transition to a high-grade malignant phenotype. The critical region of loss was narrowed down to the region 3p13-p14.2, flanked by D3S30 and D3S2, thus coinciding with the proximal RCC critical region (Klingelhutz et al., 1991; Kao et al., 1993). Hybrids resulting from a nontumorigenic SV40-immortalized human uroepithelial cell line and an isogenic tumorigenic line that had lost 3p contain normal chromosome 3 copies and are nontumorigenic. A tumorigenic revertant from such a hybrid cell line appeared no longer to contain normal copies and again showed deletion of 3p13-p21.2, suggesting this region as the location of a bladder cancer suppressor gene (Klingelhutz et al., 1992). Human epithelial cells can also be immortalized by transformation with the E6E7 genes from HPV. Again, the resulting lines only become tumorigenic after many passages in culture or exposure to chemical carcinogens, and at that time show chromosome alterations. Reznikoff et al. (1994) used both the HPV16 E6 gene alone and the E7 gene alone to immortalize human uroepithelial cells. The E7-immortalized lines showed minimal genotypic alterations, even after 35 passages, but contained amplification of 20q sequences. The E6-immortalized cell lines lacked amplification of 20q and in-

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stead contained multiple chromosome aberrations, with deletions of 3p in four of five cell lines. HPV E6 protein is known to bind and thereby to functionally inactivate TP53 (Scheffneret al., 1993). Thus, in this model system, loss of 3p appears to work in synergy with p.53 loss. Klingelhutz et al. (1996) demonstrated that HPV E6 protein activates telomerase in human keratinocytes and mammary epithelial cells at very early passages, before the cells become immortal. The use of various E6 mutants indicated that telomerase activation was independent of HPV E6 promoted TP53 degradation. Instability of chromosome 3 has also been observed after treatment of HPV-immortalized human bronchial epithelial cells with ionizing radiation (Willey et al., 1993), in HPV-immortalized and tumorigenic human keratinocytes (Montgomery et al., 1995), and in radiation-induced neoplastic transformation of human prostate epithelial cells (Kuettel et al., 1996). A tumorigenic subline spontaneously developing from a SV40-immortalized human bronchial epithelial cell line also showed loss of chromosome 3 sequences (Reddel et al., 1993). A similar result was obtained by Lebeau et al. (1995), who studied SV4O-transformed human mammary epithelial cells. Again, the acquisition of tumorigenicity appeared to be associated with loss of 3p. In most cases, the chromosome 3 losses have not yet been analyzed in detail. Still, these experimental systems may constitute a valuable addition to the armory for analyzing the association of specific losses with tumor development.

IV. FUNCTIONAL ASSAYS OF TUMOR SUPPRESSION ON CHROMOSOME 3 A. Transfer of Chromosome 3 into Renal Cell Carcinoma Cell Lines Shimizu et al. (1990) transferred the short arm of chromosome 3 into a RCC line (YCR) that, when inoculated into nude mice, resulted in the complete absence of tumor growth or a vast reduction in tumor volume. However, hybrids containing chromosome 7, l l, or X resulted in tumors that developed at the same rate as the parental RCC line. At least three different sources of normal chromosome 3, including an X;3 translocation and independently isolated pSV2neo-marked chromosomes 3, have been used in transfection studies (Shimizuet al., 1990, Rimessi et al., 1994, Killary et al., 1992). The tumor lines used varied in their tumorigenicity and morphology, but all showed changes upon transfer of chromosome 3. Some subclones of the YCR line, whether containing a rearranged neo-marked chromosome 3 (neo3t) or an X;3 translocation, were no longer tumorigenic (Shimizu et al.,

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1990). Although another RCC cell line did not form tumors, the addition of chromosome 3 decreased its growth rate and saturation density and also changed its morphology (Yoshida et al., 1994). The line RCC23 (Yoshida et al., 1994) has been reported to change organization of actin cables after the addition of chromosome 3. Sanchez et al. (1994) used a RCC line containing a 3;s translocation (SN12C.19) to transfer fragments of chromosome 3. These experiments delineated the region of chromosome 3 that suppressed the tumor phenotype to 3p12-pl4. After extended culture of the RCC23 cells containing chromosome 3, Ohmura et al. (1995) found that the cells senescenced. The loss of growth was associated with repression of telomerase function and shortening of telomeres.

B. Transfer of Chromosome 3 into Other Human Tumor Cell Lines Chromosome neo3t has also been transferred to a lung adenocarcinoma, A549 (Satoh et al., 1993). The resulting microcell hybrids did not form tumors in nude mice or denuded trachea, unlike hybrids containing transferred copies of chromosomes 7 or 11. Preliminary observations have been made that chromosome 3 inhibits the formation of tumors by small-cell lung cancer lines (Naylor et al., unpublished data). An ovarian cancer cell line, HEY, has been used as a recipient for chromosome 3 in a microcell-mediated gene transfer experiment. Rimessi et al. (1994) found that a normal chromosome 3 suppressed the tumorigenicity of this ovarian cell line. One clone that had lost three regions of chromosome 3 (two at 3p24.2-p25 and one at 3p21.1-p21.2) was not suppressed. Their data suggest that chromosome 3 has a key gene involved in ovarian tumors. Oral squamous cell carcinoma, as discussed earlier, has been associated with a very high loss of heterozygosity of chromosome 3. Uzawa et al. (1995) have transferred the neo3t into three different oral squamous cell carcinoma lines (HSC-2, HSC-3, and HSC-4). All three of the lines showed a significant decrease in growth in vitro and morphological changes upon introduction of 3p. All the microcell hybrids failed to form tumors in nude mice even after 6 months in the animal. Chromosome 7 did not affect in vitro growth of the cells, although it suppressed the tumorigenicity of one of them, namely, HSC-3.

C. Chromosome Transfer and Mismatch Repair In a different type of study, the colon cancer line HCTll6, which has been shown to have a mutation in the MLHl mismatch repair gene, was the re-

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cipient of chromosome 3. Koi et al. (1994) found that the addition of chromosome 3 not only restored the repair of G.G mismatch in an m13 heteroduplex assay, but also corrected microsatellite instability as a CA repeat was no longer found in altered forms after examining 200 clones. These effects were not seen with a transfected chromosome 2, the site of another repair gene, MSH2. The addition of chromosome 3 also increased sensitivity tolerance, which is asof the cells to N-methyl-N’-nitro-N-nitrosoguanidine sociated with poor mismatch repair (Koi et al., 1994). Hawn et al. (1995) continued work on these cell lines and found that HCTl16 with chromosome 3 was growth-arrested in G2 by the addition of 6-thioguanine to the medium. Their data suggest that the mismatch repair system is necessary to traverse the G2 checkpoint. Although chromosome 3 appears to have an effect on several tumor types, with the exception of the colon cancer line HCT116, it is not clear which or how many loci are acting to suppress tumor growth. Suppression of tumorigenicity by transfer of chromosome 3 is not a universal change, as a prostate cancer line (DU 145), which is not associated with loss of heterozygosity of chromosome 3, is not suppressed for tumorigenicity. For this cell line suppression could be achieved by the addition of chromosome 12p (Btrubk et al., 1994).

D. Human Chromosome 3 in Rodent Lines Although chromosome 3 has been demonstrated as the location of a tumor suppressor locus in several tumor types, these experiments have not narrowed the location of the genes involved any further than approximately 50 Mb. An observation made by Killary et al. (1992) has provided a means to isolate the tumor suppressor gene lying at 3 ~ 2 1 . 3 Introduction . of a complete human chromosome 3 into the mouse fibrosarcoma cell line A9 resulted in microcell hybrids with significantly reduced tumorigenicity in nude mice. Human chromosome 2 and the X chromosome did not affect the tumorigenicity, nor did the long arm of chromosome 3. Using derivatives of this cell line, a hybrid containing a fragment of chromosome 3 was found which greatly reduced capacity to form tumors in nude mice. This hybrid cell line, HA(3)BB9F7contained 2 Mb of human DNA as its only human material. This DNA was derived from 3p21-p22 and also from q21. As hybrids containing the long arm of chromosome 3 did not show a reduction of tumorigenicity, the effect of HA(3)BBSF could be attributed to 3p21-p22. InterAlu PCR was used to isolate human-specific sequences from this 2-Mb region. PCR primers made to some of these sequences detected a homozygous deletion in an SCLC cell line (Daly et al., 1993). Two strategies have been undertaken to further define the region of chro-

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mosome 3 which suppresses tumorigenicity of A9. One is to assess clones that contain fragments of chromosome 3 for their capacity to form tumors. Using this method, Imreh et al. (1994) have delineated the region between 3p21 and p25 that suppresses tumorigenicity. Todd et al. (1996) have used a P1 contig of the region contained in HA(3)BBSF.Individual P1 clones were cotransfected with a selectable marker and the resulting transfectants assayed for tumorigenicity. One of the P1 clones near the GNAI2 locus (Fig. 2) greatly suppressed the formation of fibrosarcomas by A9. This assay will presumably lead to the identification of a human gene that suppresses tumorigenicity of a mouse cell line. The gene will then need to be evaluated for its role in the genesis of human tumors.

V. (PRESUMED) TUMOR SUPPRESSOR GENES ON THE SHORT ARM OF CHROMOSOME 3 A number of genes assigned to the short arm of chromosome 3 have been suggested to act as tumor suppressors in cancer development. Such suggestions have been based on (1)location in a common deletion region; (2) a markedly reduced expression, possibly in combination with the presence of transcripts of an aberrant size; (3)the capacity to suppress tumorigenicity of tumor cells upon transfection; and (4) the function of the gene product as derived from the gene sequence or as analyzed. Only one or two of the genes detected on 3p so far can be considered as tumor suppressors; VHL and possibly TGFBR2 (see later). A few more genes show some feature(s) of tumor suppression but lack others or cannot yet be considered as proven examples. Finally, there are a number of genes that are poor candidates in terms of criteria but that nevertheless have been suggested at some time as possible tumor suppressors. The following discussion is meant as an evaluation of all these 3p genes.

A. The Von Hippel-Lindau Disease Gene The most frequently occurring tumors in the dominantly inherited cancer syndrome VHL disease are retinal angiomatosis, cerebellar and spinal hemangioblastomas, and renal cell cancer (Maher et al., 1990). Pheochromocytoma occurs in less than 10% of the patients. Two types of VHL families can be distinguished dependent on the absence or presence of pheochromocytomas. The usual age of onset is in the second and third decades, but onset may also occur in infancy or in old age (Maher et al., 1990; Ridley et al.,

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1986). The most common cause of death in VHL disease is RCC (Maher et al., 1990). The gene for VHL disease was mapped to band p25 of chromosome 3 by linkage analysis (Seizinger et al., 1988, 1991; Hosoe et al., 1990; Maher et al., 1991). Subsequent genetic mapping narrowed down the region to a segment of about 4 cM between markers D3S1250 and D3S18 in one study (Richards et al., 1993) and between D3S1038 and D3S18 in another study (Crossey et al., 1993), both including the tightly linked marker D3S601. A YAC contig and a partial cosmid contig covering the VHL critical region were constructed (Latif et al., 1993a) and a gene homologous to the rat PMCA isoform 2 cDNA and encoding a plasma membrane Ca2+-transporting ATPase isoform 2 protein was identified. This human PMCA-2 gene was not included in the smallest nested deletion defined by gross rearrangements affecting the VHL region in three unrelated VHL patients (Yao et al., 1993). Therefore, PMCA-2 could not be considered the VHL gene. Two more cDNAs were identified, one of which, when used in southern analysis, revealed aberrant bands in 12% of the VHL patients. Moreover, a few small mutations were demonstrated by SSCP analysis (Latif et al., 1993b). Mutations were also detected in four of five RCC cell lines. This gene was therefore called VHL. Gross germline rearrangements of the VHL gene occur in a minority (12-19%) of patients (Latif et al., 1993b; Richards etal., 1994). Most of the VHL families (39-75%) were shown to carry small germline (point) mutations within the gene (Crossey et al., 1994b; Whaley et al., 1994; Chen et al., 1995b). Different types of mutations appear to occur in VHL families with and without pheochromocytoma. VHL families with pheochromocytomas almost exclusively have missense mutations (Crossey et al., 1994b; Chen et al., 1995b; Zbar, 1995). This suggests that a full-length mutant VHL protein is required to produce pheochromocytomas. About half of the mutations affect codon 23 8, leading to a substitution of either tryptophan or glutamine for arginine. Large deletions, microdeletions-insertions, and nonsense mutations are specific for VHL families without pheochromocytoma and are found in 56% of such cases. The constitutive mutations in the VHL families appear to cluster in the coding regions of exons 1 and 3 (Chen et al., 1995b). If the VHL gene functions as a tumor suppressor gene and constitutive mutations of the gene predispose to cancer development, one should expect that in VHL-related tumors the remaining VHL allele is inactivated. This is indeed the case. Tory et al. (1989) and Crossey et al. (1994a) both detected loss of the remaining allele in a large proportion of VHL-associated tumors. As may be expected, the allele lost had been inherited from the unaffected parent in all (seven) cases that could be analyzed (Crossey et al., 1994a). Since loss of the remaining allele was not detected in all VHL-associated tu-

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mors, other mechanisms of inactivation may also occur. Herman et al. (1994) analyzed the methylation pattern of the CpG island in the 5’ region of the VHL gene and found it to be hypermethylated in 5 of 26 cases. None of these 5 tumors expressed VHL. Thus, hypermethylation may explain the inactivation of VHL in these cases. Sporadic RCCs (Foster et al., 1994b; Shuin et al., 1994; Whaley et al., 1994) and renal cancer-derived cell lines (Gnarra et al., 1994; Whaley et al., 1994) have been screened extensively for VHL mutations. The combined data reveal mutations in 123 of 271 tumors (45%),with most mutations occurring in exon 2, despite its absence in one of the two mRNA isoforms. A strong correlation with loss of heterozygosity at the VHL locus emerged from the analysis by Foster et al. (1994b). In 23 of 27 informative sporadic RCC tumors with a somatic VHL mutation, concomitant allelic loss for the region 3p25 was detected, demonstrating functional loss of both copies of the VHL gene in 85% of the cases. VHL mutations were confined to the clear cell-type RCCs. The VHL gene was not mutated in 28 non-clear-cell RCCs (Gnarra et al., 1994; Foster et al., 1994b). Allelic loss of the VHL region is also seldom found in this group of tumors. These data are in agreement with the finding that renal lesions in VHL patients are always of the clear cell type (Poston et al., 1995; Walther et al., 1995). Somatic VHL mutations were detected in one other type of sporadic carcinoma, namely, hemangioblastoma. This benign tumor of the central nervous system also occurs in association with VHL disease. VHL mutations were detected in 7 of 13 sporadic tumors (Kanno et al., 1994). Several other types of tumor, including those that show high frequencies of allelic losses at 3825, such as lung cancer and HNSCC, only rarely carry VHL mutations (Gnarra et d., 1994; Sekido et al., 1994; Whaley et al., 1994; Foster et al., 1994; Sun et al., 1995; Waber et al., 1996). An analysis of nasopharyngeal carcinoma did show frequent loss of heterozygosity of the region 3p24-pter, but none of 18 informative cases showed allelic loss for a VHL intragenic marker (Waber et al., 1996). This suggests that the 3p24-pter region might contain another gene that is targeted by the heterozygous deletions. Kovacs and Kung (1991) performed a cytogenetic analysis on 28 RCC tumors from VHL patients and found that the most frequent abnormality was an unbalanced translocation resulting in monosomy of chromosome 3p13-pter. They suggested that VHL mutations were responsible for the development of renal cysts but not for the initiation of RCC. In a study by Lott et al. (1994), loss of heterozygosity for the entire short arm of chromosome 3 was detected in the pancreatic tumors of three sisters affected with VHL. They hypothesized, too, that the VHL gene may be required for benign hyperplastic growth. In fact, most of the tumors associated with VHL are indeed benign. Loss of additional loci on chromosome 3 may be a prerequisite

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for malignant conversion in VHL. A molecular genetic analysis of RCC tumors from VHL patients by Crossey et al. (1994a) also revealed extensive allelic losses all over 3p, but always in combination with loss of heterozygosity of the VHL locus. Rather than suggesting inactivation of another tumor suppressor gene, the authors explained these extensive losses as a reflection of the mechanism by which the second allele of VHL is lost. Introduction of wild-type VHL, but not of mutant VHL, into a RCC cell line inhibited its ability to form tumors in nude mice, although in vitro growth of the cell line was not affected (Iliopoulos et al., 1995).In contrast, in vitro growth of two other renal cell cancer-derived cell lines was strongly suppressed upon transfection with wild-type VHL (Chen et al., 1995a). In the latter study, however, tumorigenicity in nude mice had not been tested. The VHL amino acid sequence does not show any significant homology to known proteins but contains eight copies of an acidic tandemly repeated pentamer (Gly-X-Glu-Glu-X), which suggests a function in signal transduction (Latif et al., 1993b). VHL expression is ubiquitous, with high levels in the urogenital system, brain, spinal cord, and bronchial epithelium (Richards et al., 1996). The VHL gene encodes two widely expressed transcripts of approximately 6 and 6.5 kb that probably reflect alternative splicing resulting in absence or presence of the second exon. The mouse and rat homologs of the VHL gene have also been cloned (Gao et al., 1995; Kikuchi et al., 1995). The derived amino acid sequences show a high degree of identity with the human protein, but both lack the eight copies of the tandemly repeated pentamer Gly-X-Glu-Glu-X. Rat VHL contains signals both for a nuclear and for a cytosolic localization of the protein (Lee et al., 1996). No VHL mutations were detected in spontaneous and chemically induced rat RCCs (Gao et al., 1995; Kikuchi et al., 1995). New insights into a possible function of the VHL gene have been offered by the identification of two proteins that interact with the VHL protein (Kibe1 et d., 1995; Duan et d., 1995). The VHL protein appears to compete with elongin A in binding to the transcription elongation factors elongin B and elongin C. In vitro, the transcription elongation activity of DNAdependent RNA polymerase I1 is inefficient in the absence of elongin SIII, a complex of elongin A, elongin B, and elongin C (Aso et a/., 1995). Elongin A appears to be the transcriptionally active subunit in this complex, whereas elongin B and elongin C are positive regulatory subunits. The VHL protein decreases the elongation rate by competing with elongin A for the binding of elongin B and elongin C, and thus inhibiting elongin SIII activity. Duan et al. (1995) showed in addition that the VHL protein could be located either in the nucleus, in the cytosol, or in both. Lee et al. (1996) demonstrated a tightly regulated, cell density-dependent transport of VHL protein into and/or out of the nucleus. In densely grown cells (i.e., in situations where there is much intracellular contact), the VHL protein has a predominantly

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cytoplasmic location, whereas in sparse cultures most of the protein is in the nucleus. The results suggest that in a normal cell both elongin complexes, the one with elongin A and the one with VHL, are present and that the ratio of these complexes might determine the transcription elongation activity of DNA-dependent RNA polymerase 11. Loss of the VHL protein would result in a constitutive high elongation rate and gene expression. Thus, VHL does not seem to be directly involved in cell cycle or DNA damage pathways, but rather in cell density-dependent regulation of transcription elongation efficiency.

B. The hMLH1 Gene In line with the argument that “the spontaneous mutation rate in somatic cells is not sufficient to account for the multiple mutations often seen in solid tumors” and that supposedly “an early step in tumor progression is one that induces a mutator phenotype” (Loeb, 1991), mutations in a number of DNA mismatch repair genes have been identified as underlying the phenomenon of frequent mutations in simple repeat or microsatellite sequences. This was first observed in sporadic colorectal tumors (Peinado et al., 1992; Ionov et al., 1993; Thibodeau et al., 1993) and later in a variety of tumors developing in hereditary nonpolyposis colorectal cancer (HNPCC) patients (Aaltonen et al., 1993,1994; Ionov etal., 1993). Tumors exhibiting this phenotype are called replication error positive (RER’), as microsatellite instability was suggested to result from defects in the replication machinery. One of the genes involved is located at 3p21-p23, as demonstrated by Lindblom et al. (1993) by linkage analysis in three HNPCC families. A few months after the genetic localization of this gene, its cloning was reported independently by two groups (Bronner et al., 1994; Papadopoulos et al., 1994) that both made use of comparative gene analysis. Bronner et al. (1994) used degenerate oligonucleotides targeted at two of the most conserved regions of the MutL family of DNA repair genes from yeast and bacteria. Papadopoulos et ul. (1994)screened a data base of human expressed sequences for sequences homologous to the bacterial and yeast mutator sequences. In accord with its homology to the yeast and bacterial genes, the human gene was named hMLH1. Papadopoulos et al. (1994) also defined the marker D3S1611 as intragenic. Four more DNA mismatch repair genes have been identified, MSH2, PMS1, PMS2, and GTBP (Fishel et al., 1993; Leach et al., 1993; Horii et al., 1994a; Nicolaides et al., 1994; Papadopoulos et al., 1994, 1995; Palombo et al., 1995). It is estimated that in some 40% of HNPCC families cancer susceptibility cosegregates with a germline mutation in MLHl on chromosome 3 (Papadopoulos et al., 1994; Bronner et al., 1994; Nystrom-Lahti et

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al., 1994, 1996; Han et al., 1995; Wijnen et al., 1996). In the Finnish population this frequency is over 80% due to the presence of two founder mutations (Nystrom-Lahti et al., 1995, 1996). Cells from HNPCC patients that are heterozygous for a mutation of a mismatch repair gene are not DNA mismatch repair deficient (Parsons et al., 1993). A defect in mismatch repair requires functional loss of both copies of a responsible gene. In a carrier of a germline mutation, inactivation of the remaining wild-type allele by a somatic mutation or deletion will result in a repair-deficient cell. Such a cell will easily accumulate additional mutations, resulting in an increased chance to become tumorigenic. Indeed, loss of heterozygosity at 3p is a common finding in tumors from families in which the cancer susceptibility was due to mutations in hMLHl (Hemminki et al., 1994). However, one HNPCC patient with a hMLHl mutation appeared to have widespread mutations of simple repeat sequences in nonneoplastic cells that still contain a wild-type allele of the mismatch repair gene (Parsons et al., 1995a).This suggests that specific hMLHl mutations may cause a dominant negative effect. It also shows that in isolated cases mismatch repair deficiency can be compatible with normal development. Reports on the identification of the underlying mutations that cause the microsatellite instability in sporadic colorectal tumors are scarce (Boyer et al., 1995; Liu et al., 1995). By complementation analysis using extracts of cell lines containing previously identified mutations in either hMSH2 or hMLH1, two of four colon cancer-derived cell lines were shown to be defective in hMLHl (Boyer et al., 1995). An analysis of fibroblasts from 10 patients with RER+ sporadic colorectal cancer revealed an hMLHl germline mutation in one of them (Liu et al., 1995). In an analysis of seven cell lines derived from RER+ sporadic colorectal tumors, Liu et al. (1995) could not detect any hMLHl messenger in two, while one contained a truncated hMLHl protein product. In a survey of 31 RER+ tumors from patients with sporadic colon carcinoma, somatic mutations of MLHl accounted for 26% of cases, while possible germline mutations were detected in two cases (Wu et al., 1997). Boyer et al. (1995) also detected defective hMLHl in one endometrial carcinoma-derived cell line, one ovarian carcinoma-derived cell line, and one prostatic carcinoma-derived cell line among five cell lines with microsatellite instability. Although they analyzed all four mismatch repair genes, Katabuchi et al. (1995)identified the underlying mutation in only two of nine endometrial carcinomas with microsatellite instability. Microsatellite instability has been reported for a variety of other sporadic tumors (Peltomaki et al., 1993; Horii et al., 1994b), including gastric carcinoma (Chong et al., 1994; Rhyu et al., 1994; Strickles et al., 1994),head and neck carcinoma (Ma0 et al., 1994),oral carcinoma (Ishwad et al., 1995),testicular germ cell tumors (Huddart et al., 1995), endometrial carcinoma (Risinger et al., 1993; Burks et al., 1994; Duggan et al., 1994; Kobayashi et

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al., 1995), ovarian cancer (Orth et al., 1994), esophageal adenocarcinoma (Meltzer et al., 1994), breast carcinoma (Yee et al., 1994), all types of lung cancer (Merlo et al., 1994; Shridhar et al., 1994b), and RCC (Uchida et al., 1995). Typically, some 15-30% of the analyzed tumors show microsatellite instability, which often appears to be correlated with a poorly differentiated phenotype and a poor prognosis. In some reports it is argued that the occurrence of mismatch repair deficiency is an early event. However, for certain types of tumors a low frequency or even absence of microsatellite instability has been reported as well (Peltomaki et al., 1993; Thrash-Bingham et al., 1995b). Kobayashi et al. (1995) detected microsatellite instability in only 2 of 68 ovarian carcinomas, and Fong et al. (1995) in 7 of 108 nonsmall-cell lung cancers. Whereas Meltzer et al. (1994) detected microsatellite instability in only 2% of squamous cell carcinomas of the esophagus, Ogasawara et al. (1995) found it in 60% of them. As the hMLHl gene is located at 3p22, a region frequently showing allelic losses, many tumors will have one allele inactivated. hMLHl might be a target gene for complete inactivation in sporadic tumors. However, until now a strict correlation of microsatellite instability and loss of heterozygosity of 3p22 has been reported for none of the sporadic tumor types mentioned previously. Probably, many sporadic tumors with microsatellite instability have alterations in genes other than the five now known to participate in mismatch repair (Liu et al., 1995; Katabuchi et al., 1995). Although their role in cancer development is obvious, strictly speaking mismatch repair genes cannot be regarded as tumor suppressors. The gene products do not directly influence progression through the cell cycle, as does, for instance, the retinoblastoma gene. Loss of the mismatch repair function will render a cell prone to accumulation of mutations that may affect genes that are important for the regulation of cell growth. Transfection of a wildtype copy of the hMLHl gene into a colon cell line known to have a homozygous defect of the hMLHl gene corrected the mismatch repair deficiency (Koi et al., 1994), but, unlike what has been described for (or is expected from) tumor suppressors, restoration of the function of a mismatch repair gene in a deficient tumor cell is not likely to revert its tumorigenicity.

C. The TGF-f3 Receptor Type I1 Gene A gene for the transforming growth factor-p (TGF-P) type I1 receptor has been mapped to chromosome 3p22 (Mathew et al., 1994). Its product is the type I1 serine-threonine kinase receptor, which, together with the type I serine-threonine kinase receptor, encoded by a gene on 9q33-q34.1, forms a receptor complex to which the TGF-P binds. TGF-P is a potent inhibitor of epithelial cell growth and plays a central role in cell cycle control (Fynan and

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Reiss, 1993). It is capable of suppressing the growth of certain cancers and cell lines. The various roles of TGF-P in regulating cell proliferation and differentiation are mediated through the type I and type I1 receptors. In addition, the type I1 receptor is required for the inhibition of pRB phosphorylation (Chen et al., 1993). The majority of epithelial tumors are insensitive to TGF-P. Gastric cancer cell lines resistant to the growth-inhibitory effect of TGF-P expressed either truncated or no detectable TGF-P type I1 receptor mRNA, whereas TGF-f3 type I receptor mRNA expression was normal (Park et al., 1994). This implies that an altered type I1 receptor gene is the primary cause of insensitivity toward TGF-P. A similar result was obtained by Markowitz et a1 (1995) in their analysis of the expression of the two TGF-P receptors in human colon cancer cell lines and xenografted human colon cancers. They found strongly reduced or no expression of the type I1 receptor gene in 12 of 14 cell lines and xenografted tumors that showed microsatellite instability, but in only 5 of 52 cell lines and xenografted tumors that did not show microsatellite instability. A subsequent mutation analysis of 7 of the former group of cell lines with a reduced type 11 expression revealed a change in length of a sequence of 10 adenines with one or two nucleotides, causing frameshifts in every case. Such a sequence is a typical mutation hotspot in mismatch repair-deficient cells. Identical mutations were detected in 100 of 111 primary and cultured RER+ colon tumors (Parsons et al., 1995b), and in 17 of 24 RER+ tumors from HNPCC patients (Lu et al., 1995).This type of mutation was again not found in a total of 136 RER- tumors (Lu et al., 1995; Parsons et al., 1995b), although one of them had a missense mutation in codon 537 (Lu et al., 1995). Mutations affecting the length of the adenine stretch in TGFBR2 are also frequent in RER+ gastric cancers (Myeroff et al., 1995),but less so (17%)in RER+ endometrial cancers (Myeroff et al., 1995; Lu et al., 1995). These findings have linked DNA repair defects to a specific pathologic event, as loss of both copies of the TGFBR2 gene will cause unresponsiveness toward TGF-P-mediated growth inhibition of several cell types. Although the presence of an adenine stretch in TGFBR2 makes this gene an obvious target in mismatch repair-deficient cells, a few mutations have been detected in non-RER tumors. In these cases, the mutations occurred not in the adenine stretch but at various sites in the gene (Markowitz et al., 1995; Park et al., 1994; Garrigue-Antar et al., 1995; Lu et al., 1995). The occurrence of mutations of TGFBR2 in combination with a location in a region showing frequent allelic loss in several types of epithelial tumors may well lead to inactivation of both copies of the gene and thus constitute tumor progression. Restoration of wild-type TGFBR2 expression by gene transfection restored TGF-P responsiveness and strongly reduced the cloning efficiency in

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soft agar of the recipient breast cancer MCF-7 cells (Sun et al., 1994). Xenograft formation in nude mice was delayed and not completely suppressed. However, no expression of the transfected TGFB2 gene could be detected in the resulting xenografts. Similar results have been obtained when the RER+ colorectal carcinoma cell line H C T l l 6 was transfected with wildtype TGFBR2 (Wang et al., 1995). The fact that transfection of just this one gene into a cell line that will have accumulated many mutations due to its RER+ phenotype suppresses its tumorigenicity underscores the key function of this gene in determining the malignancy in RER+ colon cancers (Sun et al., 1994; Wang et al., 1995).In these specific situations, TGFBR2 shows features of a tumor suppressor gene.

D. THRBandRARB Two further genes coding for receptor proteins-namely, THRB (Dobrovic et al., 1988), encoding the thyroid hormone receptor p and RARB (Mattei et al., 1988), encoding the retinoic acid receptor P-have been mapped distal to the common region in 3p21 of heterozygous and homozygous deletions in a number of tumor types. Heterozygous loss of a receptor gene might have a direct phenotypic effect, possibly contributing to differences in tumor differentiation (Leduc et al., 1989; Kok et al., 1989). With the possible exception of ovarian carcinoma (see Section IILF), loss of heterozygosity is not a general phenomenon for these genes. Indeed, for THRB, the most proximal of the two genes, retention of heterozygosity has been shown in lung cancer (Drabkin et al., 1988) as well as in other tumors. This makes THRB an unlikely candidate for a tumor suppressor gene. The RARB gene is a member of a family of retinoic acid receptor genes. Its expression is strongly inducible by retinoic acids. In lung cancer cell lines, RARB expression varies considerably (Gerbert et al., 1991). Lack of expression has been suggested to correlate with epidermoid differentiation (Houle et al., 1991). Upon Southern analysis, DNA rearrangements were seen in 3 of 48 epidermoid lung tumor DNA samples, but in each case they appeared to involve only one allele, leaving the other copy of the gene intact (Gerbert et al., 1991; Houle et al., 1991). Houle et al. (1993) transfected two epidermoid lung tumor-derived cell lines with a RARB gene construct. The resulting cell lines had an increased RARB expression and an increased doubling time and were less tumorigenic in nude mice. As retinoic acid affects many developmental and differentiation processes, especially in bronchial epithelium, it is not surprising that changes in the level of RARB expression affect cellular growth characteristics. It should also be noted that the cell lines used by Houle et al. (1993) did show a weak RARB expression even before transfection (Gerbert et al., 1991). As inactivation of RARB in tumors by two somatic events has

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also never been reported to occur at any significant frequency, a tumor suppressor-like function for this receptor seems unlikely.

E. Candidate Genes from 3p21 The early detection of high frequencies of allelic loss in several tumor types with probe H3H2 from the D3F15S2 locus in 3p21.3 has focused many mapping efforts on the 3p21 region. This has resulted in detailed physical maps (see the Genome Data Base [GDB]), in various YAC and cosmid contigs, and in the isolation of a number of genes (Fig. 2). In total, well over 50 genes have been assigned to 3p21, including several so called zinc finger genes (Lanfrancone et al., 1992; Hoovers et al., 1992; Calabro et al., 1995). A complete listing of all 3p21 genes can be found in the GDB. Several of them have been analyzed in light of a possible relation to tumor development, but thus far none of them has “passed the test.” It turned out that D3F15S2 was itself part of a gene. Sequence analysis revealed a putative amino acid sequence with a domain structure identical to that of the hepatocyte growth factor (Han et al., 1991).A study by Yoshimura et al. (1993) indicated that the gene (MST1) encodes a human macrophage-stimulating protein predominantly expressed in liver. A gene that maps only a few kilobase pairs from the locus D3F15S2 has been identified independently by several groups (Naylor et a/., 1989; Erlandsson et al., 1990; Harbour et al., 1990). The gene (APEH) encodes the human acylpeptide hydrolase, as concluded from its high degree of homology with the rat and porcine genes (Erlandsson et al., 1991; W. A. Jones et al., 1991). It has been stated that a majority of primary RCCs expressed the gene at a level of less than 20% that in normal kidneys (Erlandsson etal., 1990),but this could not be confirmed by others (Van der Hout, 1992). In the majority of SCLCderived cell lines the gene is expressed at a level similar to that in normal lung tissue (Naylor et al., 1989; Carritt et al., 1992), and neither genomic rearrangements nor transcripts of an aberrant size could be detected in lung cancer cells (Naylor et al., 1989). The APEH product catalyzes the hydrolysis of the terminal acetylated amino acid in small acetylated peptides. Hydrolysis of the resulting acetylated amino acids is then catalyzed by the enzyme aminoacid acylase. The gene coding for this enzyme (ACY1) has been assigned to 3p21.1 (Miller et al., 1990), and maps close to D3S2 at a considerable distance centromeric from D3F15S2. ACYl is expressed uniformly in a variety of human tissues, tumors, and tumor-derived cell lines, including non-SCLC cell lines. For SCLC cell lines, reduced enzyme activity has been demonstrated for about half of the cell lines analyzed (Miller et al., 1989). Although the loss of one copy of the gene may provide a potential explanation for the reduced activity, it cannot explain the four cases for which

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complete absence of enzyme activity was reported. Genomic rearrangements have not been detected in any SCLC cell line investigated. In normal lung tissue the gene is expressed at relatively low levels. Although its expression as assessed by Northern analysis was reduced in 9 of 12 SCLC cell 1'ines, complete absence of expression or transcripts of aberrant sizes were never seen (Cook et al., 1993). It has been suggested that severely reduced activity of both the APEH and ACY 1 gene products could lead to accumulation of an acetylated peptide growth factor, which might contribute to the development of tumors (Jones et al., 1991), but experimental evidence for this suggestion has yet to be shown. The assembly and analysis of two cosmid contigs in the close vicinity of ACYl resulted in the identification of five new transcripts (Shridhar et al., 1994a), none of which appeared to be altered in lung cancers. One of these genes, called APR, maps approximately 600 kb telomeric to the ACYl locus and encodes a ubiquitously expressed, extremely basic, arginine-rich protein (Shidhar et al., 1996). The gene contains an imperfect ATG trinucleotide repeat that codes for a stretch of 15 arginines interrupted by 3 other amino acids. Thirteen of 21 RCC tumor samples were shown to contain a mutation in this repeat, 10 of which affected the same ATG codon. However, there is no evidence as yet on a functional role this gene might have in the development of cancer. At about 150 kb centromeric to D3F15S2, a gene was identified whose product has a 45% identity with the human ubiquitin-activating enzyme E l (Kok et al., 1993a).The gene, ubiquitously expressed in human tissues (Carritt et d., 1992), was named UBElL. As expected from its location, one allele was lost in all lung cancer specimens and lung cancer-derived cell lines analyzed (Carritt et al., 1992). Despite the presence of one functionally intact allele of the UBElL gene, Northern analysis failed to detect any expression in lung cancer-derived cell lines. Upon reverse transcriptase (RT)-PCR analysis some expression was detectable (Kok et al., 1993b). Quantitative PCR showed that the level of this expression was 2 4 % of that in normal lung. Such a consistent dramatically decreased expression in lung cancer has not been described for any other gene in 3p21. Single-strand conformation polymorphism (SSCP) analysis of 15 SCLC-derived cell lines, however, did not reveal any mutations in the remaining allele (Kok et al., 1995). This excludes a primary tumor suppressor role for UBElL. The virtual absence of expression may be due to a regulatory block caused by mutation or absence of yet unknown transcription factors. The human homolog of murine UNP, a gene putatively involved in the cleavage of ubiquitin tags from substrates targeted for proteolytic degradation, has also been assigned to 3p21.3 (Gray et al., 1995). Murine UNP encodes a ubiquitous nuclear protein whose overexpression leads to oncogenic transformation of NIH 3T3 cells (Gupta et al., 1994). The human gene, UNPH, turned out to be consistently overexpressed

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in small-cell lung tumors and in adenocarcinoma of the lung as compared to normal lung tissue (Gray et al., 1995). The human p-catenin gene (CTNNB1) is located at chromosomal region 3p21.3-p22 (Kraus et al., 1994; van Hengel et al., 1995). p-Catenin plays a central role in the E-cadherin-catenin cell-cell adhesion complex of epithelial cells. It also binds to the tumor suppressor gene product APC, and may act as a central regulator of cell adhesion and tumor progression (Hulsken et al., 1994). From its location, it is obvious that many types of tumor show frequent loss of heterozygosity at the CTNNBl locus. Reduced p-catenin expression has been detected in more than 50% of esophageal, gastric, and colorectal cancers (Takayama et al., 1996). It is suggested that loss of function of p-catenin might promote anchorage-independent growth of (tumor) cells. However, clear evidence for a possible role as a tumor suppressor is missing. The two regions of homozygous deletions reported to be in 3p21 have been examined extensively for candidate genes. The deletions described by Yamakawa et al. (1993) in fact appeared to be distal to 3p21. Murata et al. (1994) have defined a YAC clone and a cosmid contig that encompass this entire region. The region has been sequenced as part of the Japanese genome project in the laboratory of Yusuke Nakamura. To date, no candidate from this region has been proven to be a tumor suppressor gene. One gene, the integrin a subunit gene (aRLC), has, inconsistent with the tumor suppressor gene concept, been shown to be expressed at higher levels in lung cancer than in lung (Hibi et al., 1994). The region defined by three overlapping homozygous deletions in small-cell lung cancer lines is in 3p21.3 and overlaps with the tumor suppressor activity defined by Killary et al. (1992). Th'is region, depicted in Figure 2, has also been extensively mapped in YACs, cosmids, and P1 clones (Kok et al., 1994; Wei et al., 1996; Roche et al., 1996; Xiang et al., 1996) and has been shown to be very gene-rich. To detect transcribed sequences, exon trapping and hybridization selection have been applied to the entire region corresponding to the NCI H740 deletion (We; et al., 1996). Transcripts identified (see Fig. 2) include those for two guanine nucleotide-binding proteins previously known to map in this region, namely, the retinal specific a-transducing polypeptide 1 (GNAT1) (Blatt et al., 1988) and the a-transducing polypeptide 2 (GNAI2) (Magovcevic et al., 1992). The gene for 3pK, a mitogen-activated protein kinase-activated protein kinase, is located within the homozygous deletions of NCI H740 and NCI H1450 (Sithanandam et al., 1996), but it is not deleted in the SCLC line GLC20 and it is not contained in the fragment that suppresses tumor formation. Also contained within this region are SEMA-IIW and SEMA-A, two semaphorin genes (Xiang et al., 1996; Roche et al., 1996; Sekido et al., 1996) belonging to a family of genes involved in signal transduction and cell-cell

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communication. Although variable levels of one of the semaphorins (IIW, also named IV) have been noted (Xiang et al., 1996; Roche et al., 1996), a thorough screening (Xiang et al., 1996, Sekido et al., 1996) did not reveal mutations that might be considered causative in lung cancer. The cosmids defined by Wei et al. (1996) have been sequenced by the Washington University Sequencing Center and the Sanger Centre. As stated, to date none of the genes, including those newly identified, has proven to be a tumor suppressor gene. However, the transcription map from this region is far from complete yet, and several newly identified transcripts from this region are still being analyzed.

F. Candidate Genes from 3p 13-p 14 Band p14 of chromosome 3 contains two regions that have attracted broad interest: the previously discussed t(3;s) breakpoint associated with hereditary RCC (see Section 1II.B) and the fragile site FRA3B (see Fig. 3). Boldog et al. (1993), who cloned the t(3;S) breakpoint of the family with hereditary RCC described by Cohen et al. (1979),claimed the identification of at least one gene in the breakpoint region. However, no further information has been published to date. The establishment and analysis of an integrated YAC contig confirmed the short physical distance between the FRA3B region and the t(3;S) breakpoint (Wilke et al., 1994; Boldog et al., 1994) but also made clear that they do not coincide (Wilke et a]., 1996). Fragile sites are chromosomal regions that easily show elongation, gaps, and breaks in metaphase preparations, either spontaneously or after exposure to specific culture conditions (Sutherland, 1991). FRA3B at 3p14.2 is

-3'

FHIT

500 kb

5'

5'

300 kb

PTPRG

3'

750 kb

Fig. 3 Map of part of band p14.2 of chromosome 3. Positions of markers and the F H F and PTPRG genes are shown relative to the t(3;S) breakpoint and the FRA3B region. The hatched bar indicates the approximate position of homozygous deletions in the FRA3B region. FHIT exons are indicated by small vertical bars.

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one of the most common fragile sites in humans. Upon chromosome analysis of peripheral blood lymphocytes, breakage of chromosome 3 at band p14 can be observed for every individual, with frequencies varying from 2 to 4% of the cells (Smeets et al., 1986). This frequency increases strongly if the cells are treated with aphidicolin (Glover et al., 1984). FRA3B appears to comprise a region of some 200 kb (Boldog et al., 1994). As yet, no specific sequences have been reported that could be responsible for the fragility. There have been early suggestions that fragile sites may be preferentially involved in chromosome translocations and/or deletions, and thus would play a mechanistic role in cancer development (Yunis and Soreng, 1984; Glover and Stein, 1988).With respect to FRA3B, this idea has recently been revitalized. Two findings account for this. First, the FRA3B region has been shown to be the integration site of HPV16 in a primary cervical carcinoma (Wilke et al., 1996). It is noteworthy that cervical cancers are often HPV16 associated (Bosch et al., 1995) and that the 3p14.2 region lies within the smallest commonly deleted region in this cancer (Kohno et al., 1993). Second, when Lisitsyn et a2. (1995) used representational difference analysis to isolate new markers that were homozygously lost in different tumors, one of their new markers appeared to map within the FRA3B region (Van den Berg et al., 1995a; Ohta et al., 1996). This very marker, D3S3155, detected homozygous deletions in several colon cancer- and RCC-derived cell lines (Lisitsyn et al., 1995) and also in a bladder cancer cell line and a breast cancer cell line (Lisitsyn et al., 1995), a lung adenocarcinoma cell line (Van den Berg et al., 1995a), and a gastric carcinoma cell line and two nasopharyngeal carcinoma cell lines (Kastury et al., 1996). As far as they have been analyzed, these homozygous deletions ranged in size from 50 to 300 kb. They map between D3S1234 and D3S1481, the common deletion region of stomach and colon cancers (Kastury et al., 1996). A high frequency of loss of heterozygosity for this region has also been reported for RCC and nasopharyngeal carcinoma (see Sections 1II.B and 1II.C).The deletion breakpoints were scattered (Ohta et al., 1996), which favors the notion of a fragile region rather than a fragile site. A cosmid contig has been assembled that covers the homozygous deletions and, by exon trapping, Ohta et al. (1996)identified a novel gene in this region. The deduced amino acid sequence showed a significant homology to the group of so-called HIT (for histidine triad motif) proteins. The gene was accordingly designated FHIT, for fragile histidine triad gene. It has 10 relatively small exons that code for an mRNA of about 1.1kb. However, its nine introns span a region of at least 500 kb. The FRA3B region coincides with the fifth exon and the large fifth intron. The t(3;8) breakpoint, which occurs constitutively in the hereditary RCC family described by Cohen et al. (1979), maps in the third intron. Based on these mapping data, the gene has been suggested as a strong candidate for involvement in the development of different types of sporadic tu-

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mors, as well as in the initiation of familial RCC. The previously mentioned retention of a normal copy of chromosome 3 in RCC tumors of members of the t(3;S) family (Li et al., 1993), however, strongly argues against the latter suggestion. With respect to the former suggestion, the following observations must be taken into account. A low level of expression was detected for all human tissues (Ohta et al., 1996). Tumor-derived cell lines, including those with a homozygous deletion, had levels of FHIT mRNA varying from almost undetectable to normal. At least some of the homozygous deletions will therefore probably be restricted to intron sequences. This has been demonstrated for the lung adenocarcinoma cell line described by Draaijers et al. (1996). In cancer-derived cell lines (Ohta et al., 1996), lung tumors (Sozzi et al., 1996b), and Merkel cell carcinomas (Sozzi et al., 1996c), a variety of aberrant bands have been detected by RT-PCR analysis. In many cases the aberrant bands occur in combination with a band of normal size. In the majority of cases, the aberrant bands corresponded to transcripts that completely lack one or more exons and could thus be the result of aberrant processing of the pre-mRNA. Upon Southern analysis with a full-length FHIT cDNA probe, normal hybridization patterns were detected in 11 of 15 lung cancer cell lines, although most of these cell lines had shown aberrant bands by RT-PCR analysis. Another observation that awaits explanation is the finding that some of the previously mentioned lung cancer cell lines had more than one aberrant transcript, although they were all hemizygous for the FHIT region. FHIT transcripts of aberrant size are also frequently detected in nontumorigenic, nonimmortal cell lines (Van den Berg et al., 1997). What the present data have shown is that beyond any doubt the FRA3B region is an unstable region and that tumors show homozygous deletions of this region at relatively high frequencies. Sequences responsible for this instability will also be transcribed into the FHIT pre-mRNA and may cause irregular splicing-processing, leading to aberrant-sized rnRNAs. This might explain why aberrant bands are not solely detected in tumor cells, but also occur in normal cells. Whether the FRA3B region plays a role in tumor development or not fully depends on the possible presence of (a) gene(s) with a tumor suppression function. Presence of a gene per se does not answer the question. Establishment of a possible role of FHIT in the process of tumorigenesis must await its functional analysis. PTPRG, a gene coding for receptor protein-tyrosine phosphatase y (PTPy) (Kaplan et al., 1990), originally assigned to 3p21 (LaForgia et al., 1991), has been located at 3p14.2 (Tory et al., 1992), 200 kb centromeric to the t(3;s) breakpoint. Protein-tyrosine phosphatases are enzymes that dephosphorylate protein-tyrosine residues. Since they thus reverse the effect of protein-tyrosine kinases, many of which are oncogenes, they can conceivably act as tumor suppressors, and the PTPRG gene has been suggested to be a candidate tumor suppressor. Densitometric analysis has indicated reduction

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to hemizygosity for this gene in roughly 5 of 10 lung cancers and three of four RCCs (LaForgia et al., 1991). In normal lung tissue, PTPy expression is relatively high (Tsukamoto et al., 1992). In 9 of 31 lung cancer cell lines, expression as assessed by Northern analysis was low or undetectable. However, SSCP analysis of the two PTPase-like domains of PTPy in 3 l lung cancer cell lines did not reveal any mutation (Tsukamoto et al., 1992). Thus, there is in fact no evidence in support of a tumor suppressor function of this gene.

VI. EVOLUTIONARY ASPECTS OF HUMAN CHROMOSOME 3 Chromosome 3 is homologous to several mouse chromosomes: 3,6,9,14, and 16 (Fig. 4). The regions that have been identified as key in cancer-3~25, 3p21.3,3p14.2, and 3p12-are also at the edge of synteny blocks. This may reflect areas of genomic instability or merely a chance occurrence. For example, the region of 3 ~ 2 1 . 3 the , location of the genes GNAT1 and GNAI1, in mouse borders the regions corresponding to genes mapping to 3q21. Also, there is a scrambling of markers in the region of the fragile site as well as the region involved in the U2020 deletion. The changes resulting in chromosome 3 of humans are rather recent events in evolution. Comparative banding patterns of human and several primate species have revealed several recent changes (Dutrillaux, 1979; Yunis and Prakash, 1982). A series of papers compared the banding patterns of several species, including humans, chimpanzees, gorillas, and orangutans. There have been very few changes in the chromosomal banding pattern of these primates with the exception of chromosome 2 being homologous to two chromosomes in each of the other primate species. Chimpanzees and gorillas vary only in minor ways from the human chromosome 3 homolog. However, in orangutans and all organisms earlier in evolution, the chromosome 3 is quite different. There have been three inversions between orangutans and humans. One of the inversions has a breakpoint at 3p21. A more recent study by Jauch et al. (1992) utilized chromosome-specific libraries to follow the rearrangements among primate species. Using FISH analysis with libraries from flow-sorted chromosomes, they found that the human chromosome 3 library hybridized to the single chromosomes that had the same banding pattern: PTR2 (chimpanzee), G G 0 2 (gorilla), and PPY2 (orangutan). However, the more distant gibbon had four chromosomes and five regions that hybridized to the chromosome 3 library. In contrast, some chromosomes, such as human chromosome 11, are relatively intact. Thus, chromosome 3 has undergone a great deal of rearrangement, even in primate evolution.

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RAFl lL5RA RARB CTNNBl GLBl APEH GNAT1 GNAl2 PTPRG

MlTF ITIH3

GAP43 DRD3

RHO RPN TF RYK MME EVI FIM3 SST

3 Fig. 4 A comparison of human chromosome 3 to mouse chromosomes. Chromosome 3 is syntenic to mouse chromosomes 3, 6 , 9, 14, and 16. The short arm of chromosome 3 shows scattered blocks of syntenic groups primarily homologous to mouse chromosomes 6 and 9. Definite rearrangement has occurred at the sites of tumor suppressor genes.

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VII. CONCLUDING REMARKS From a number of approaches discussed in this review, the picture emerging with respect to the involvement of chromosome 3 in tumor suppression is one of multiple loci on the short arm of the chromosome, each or several together presumably playing a major role in the development of a large variety of solid tumors. It is hard to escape the impression that chromosome 3 has been attributed by nature a larger role in this than other chromosomes. The fact that during mammalian evolution more changes, including recent ones, have occurred in the formation of chromosome 3 than in the formation of other chromosomes might just be a random correlation, although one wonders whether that might have contributed to a greater genomic instability reflected in a proneness to deletions. Frequencies of allelic losses also vary on 3p. If there is a high frequency of loss of heterozygosity at a certain locus, deletions at that locus presumably represent a primary event. Deletions in 31321.3 in lung cancer are a good example. Their presence in preneoplastic lesions confirms that they occur early. If a certain locus shows a low frequency of loss of heterozygosity, then different explanations are possible. The deletions occurring at that locus may represent a secondary event. They will usually occur later during progression and thus be associated with more advanced tumor stages. The deletions occurring in 3p13-3p21.1 in bladder cancer may serve as an example. Alternatively, a low frequency of allelic losses may be due to unrecognized heterogeneity. Although the frequency of loss of heterozygosity in RCC in general is not very high, introduction of a cytohistological classification immediately shows that 3p allelic losses do occur at a high frequency in clear cell RCC. The number of presumed suppressor loci on 3p is hard to estimate. Different tumor types show involvement of similar (identical?)or different 3p regions, for example, 3p13-3~21.1in both bladder cancer and cervical cancer and 3p21.3 in lung cancer and mesothelioma. In some tumor types multiple loci on 3p seem to be involved. Three main regions of allelic losses have been defined in RCC,namely 3p25,3p21.3, and 3p12-pl4. The situation in HNSCC looks very similar. This is possibly also true for breast cancer. The constitutive occurrence of a t(3;8) in an RCC family is still used as a rationale for an analysis of the significance of the breakpoint region in the development of tumors, in particular RCC.The retention of the normal copy of chromosome 3, but loss of the derivative containing the distal 3p, from tumors of t(3;S) carriers in the family and the retention of the very breakpoint region in sporadic RCC with 3p allelic losses argue against a role of this region in RCC development. The role of 3p in tumor suppression is confirmed by observations that spon-

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taneous tumorigenic mutants in nontumorigenic immortalized cell lines show loss of parts of 3p. Such cellular models offer an alternative approach to define the 3p regions involved in some specific tumors. This is also and more generally true for the transfer of chromosome fragments suppressing the tumorigenicity of transfected tumor cell lines. The possible involvement of a common region of deletions in tumor development is strongly corroborated by coincidence with a chromosome fragment suppressing tumorigenicity. Such a situation exists for the 3p21.3 region, which by analysis of overlapping homozygous deletions in lung cancer cell lines has been reduced to 400 kb and possibly even further by a P1 transfer experiment, and for a much less delimited region in 3p12-pl4 defined by analysis of loss of heterozygosity in RCC and HNSCC and by transfer of part of 3p in an RCC cell line. In several types of tumor (lung, RCC, HNSCC) an increase in the size of 3p deletions with the stage of disease has been observed. This might mean that additional suppressor loci become involved during tumor progression. Searches for genes are in progress for some of the more precisely defined smaller regions with presumed tumor suppressor activity on 3p. Other regions are still far too large to start a search for genes. The only gene on 3p generally considered as a tumor suppressor, VHL, has actually been identified because of its involvement in a hereditary cancer syndrome, allowing the collection of families and the application of linkage analysis to pinpoint the gene. Although loss of heterozygosity does occur and has been well documented in sporadic clear cell-type RCC, other tumors, in particular nasopharyngeal carcinoma, show a high frequency of allelic losses of the VHL region while still having an unmutated wild-type VHL allele on the remaining chromosome homolog. Thus, inactivation of VHL cannot be held responsible for all types of tumors that show a high frequency of loss of heterozygosity in 3~2.5,and also this distal part of 3p might still harbor an unidentified tumor suppressor. The most promising region in terms of possible identification of a new tumor suppressor(s) is undoubtedly 3 ~ 2 1 . 3 The . materials and tools seem to be available. In view of the involvement of deletions of this region in a variety of tumor types, either a cluster of tumor suppressor genes or, perhaps more likely, a kind of a broad-spectrum suppressor may be present.

ACKNOWLEDGMENTS The authors thank Anke van den Berg for her assistance in compiling the literature and in preparing the figures. They acknowledge all other researchers who have spent so long elucidating the role of chromosome 3 in the development of tumors. They also gratefully acknowledge support from the Dutch Cancer society (GUKC86-9 89-5 and 90-15, RUG93-509 and 94-831) and from the National Institutes of Health (CA56626, CA54174, and HG00470).

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Mutations Predisposing to Hereditary Nonpolyposis Colorectal Cancer Paivi Peltomaikt and Albert d e la Chapelle Department of Medical Genetics, Haartman Institute, 00290 University of Helsinki, Finland

I. Introduction 11. The HNPCC Syndrome A. CRC Associated with HNPCC B. Extracolonic Cancer C. Occurrence of Colorectal Adenomas 111. HNPCC and DNA MMR A. Identification of Human MMR Genes B. Biochemical and Functional Properties of the MMR System C. Microsatellite Instability as an Indicator of MMR Deficiency IV.Mutations Predisposingto HNPCC A. Structure of HNPCC-Related MMR Genes B. Proportion of HNPCC Attributable to Different Genes C. Types and Sites of MMR Gene Mutations D. Founding Mutations V. Phenotypic Effects of MMR Gene Mutations A. “Mutator Phenotype” and Tumorigenesis B. Basis of HNPCC Tumor Spectrum C. Clinical Correlations of HNPCC Mutations VI. Implications of Mutation Findings References

Since 1993 four genes have been identified that, when mutated, confer predisposition to a form of hereditary colon cancer (hereditarynonpolyposiscolorectalcancer [HNPCC]). These genes belong to the Mut-related family of DNA mismatch repair genes whose protein products are responsible for the recognition and correction of errors that arise during DNA replication. Mutational inactivation of both copies of a DNA mismatch repair gene results in a profound repair defect demonstrable by biochemical assays, and in vivo this defect is presumed to lead to progressive accumulation of secondary mutations throughout the genome, some of which affect important growth-regulatory genes and, hence, give rise to cancer. To date, more than 70 different germline mutations have been detected in DNA mismatch repair genes and shown to be associated with HNPCC. Current evidence suggeststhat two genes, MSH2 and MLHI, account for roughly equal proportions of HNPCC kindreds, together being responsible for a majority of these families, but striking interethnic differences occur. Most mutations lead to truncated protein Advances in CANCER RESEARCH 0065-230W97 $25.00

Copyright 0 1997 by Academic Press. All rights of reproduction in any form reserved.

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products. Mutation screening is quite demanding in HNPCC since, with a few exceptions, the predisposing mutations typically vary from kindred to kindred and individual mutations are scattered throughout the genes. Knowledge of the predisposingmutations allows genotype-phenotype correlations and forms the basis for further studies clarifying the pathogenesis of this disorder. In at-risk individuals, it allows predictive testing for cancer susceptibility and, consequently, appropriate clinical management of mutation carriers and noncarriers.

1. INTRODUCTION Colorectal cancer (CRC) is one of the most common forms of neoplasia in industrial countries (Dunlop, 1992). It is rare before age 40, but after that age the incidence starts to rise, reaching a peak at 75-80 years. Epidemiological risk factors for CRC have been extensively studied. Studies on migrant populations have shown that within one or two generations the incidence rates in immigrants approach those of the host country, suggesting that the disease is sensitive to changes in environmental factors (Haenszel and Kurihara, 1967). Dietary intake is implicated in the etiology of CRC, so diets high in meat and fat and low in fiber and cereal grains are thought to increase the risk of the disease (Bruce, 1987). A family history of CRC is recognized as a risk factor, and the relative risk of CRC has been estimated to be approximately two- to three-fold in individuals with one affected first-degree relative and three- to eight-fold in individuals with two affected first-degree relatives, as compared to those without a family history of the disease (Houlston et al., 1990; Fuchs et al., 1994). Although family history generally represents both shared genes and shared environment, kindred studies suggest that familial clustering of common (apparently sporadic) CRC often occurs as a result of a partially penetrant inherited susceptibility (CannonAlbright et al., 1988). Approximately 5 % of CRC cases represent monogenic Mendelian disorders with autosomal dominant transmission and penetrance close to 100% (Lynch et al., 1993). The main forms of hereditary CRC are familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC). While FAP is recognized by the appearance of hundreds or even thousands of polyps in the large intestine by the teens, HNPCC is not associated with polyposis or other specific stigmata, making its diagnosis impossible on clinical grounds. Until recently, the diagnosis of HNPCC was mainly based on family history. HNPCC kindreds are commonly defined as those in which at least three relatives in two generations have CRC, with one of the relatives being a first-degree relative to the other two and, furthermore, one relative being diagnosed at less than 50 years of age (Vasen et al., 1991).

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These so-called Amsterdam criteria are relatively strict and serve to eliminate most cases of chance clustering of CRC. A definitive diagnosis of HNPCC is based on gene analyses. Since 1993 four genes have been identified that, when mutated, confer predisposition to this cancer syndrome. The normal function of the proteins encoded by these genes is to participate in DNA mismatch repair (MMR). In this review, we describe recent advances in the study of MMR genes, with the emphasis on mutations predisposing to HNPCC, their phenotypic effects, and scientific and clinical implications of these findings.

11.

THE HNPCC SYNDROME

A. CRC Associated with HNPCC The first HNPCC kindred, family G, was described by Warthin in 1913. As a clinical entity, the syndrome is characterized by the development of CRC and certain other adenocarcinomas at an early age (mean 44 years) (Lynch et al., 1993). While most sporadic CRCs occur in the distal part of the large intestine or in the rectum, up to 70% of CRCs in HNPCC are proximal, occurring proximal to the splenic flexure. There is an excess of synchronous and metachronous colonic cancers. Histologically, the carcinomas are often poorly differentiated and of mucinous type (Mecklin et al., 1986). Despite aggressive histological features, the survival has been reported to be better than in sporadic CRC (Albano et al., 1982; Love, 1985). This may in part be related to tumor stage, implying a decreased tendency to metastasize. For instance, Lynch et al. (1993) found that 74% of HNPCC patients presented with local disease (Dukes stage A or B) as compared to 44% of other CRC patients. Further characteristics of HNPCC cancers in accordance with indolent behavior include a heavy peritumoral lymphocyte infiltration, indicating a marked host lymphocytic response, and diploid DNA content as evaluated by flow cytometry (Kouri et al., 1990; Lothe et al., 1993). The previously described histopathological and prognostic characteristics have been shown to be common features of colorectal tumors with replication errors, no matter whether hereditary or sporadic (see later).

B. Extracolonic Cancer The HNPCC syndrome is sometimes divided into Lynch syndromes I and 11, based on the absence or presence, respectively, of extracolonic cancer.

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Clinical studies, however, argue against the view that there are two variants, one in which only the colorectum is affected and another in which the colorectum as well as other organs are affected (Watson and Lynch, 1993). Of all cancers diagnosed in HNPCC patients, approximately 60% are colorectal while 40% occur at extracolonic sites (Mecklin and Jarvinen, 1991; Watson and Lynch, 1993). Apart from CRC, at least cancers of the endometrium, stomach, biliopancreatic system, urinary tract, small bowel, and ovaries occur in excess in HNPCC kindreds as compared to the general population, and are therefore considered to be part of the “HNPCC tumor spectrum.” Interestingly, changes in the cancer phenotype have been reported to have occurred over time in some HNPCC kindreds, resembling trends observed in the general population and possibly reflecting changes in lifestyle or other environmental factors. For example, the original description of family G shows an excess of gastric cancer, whereas the general trend of a decrease of gastric cancer and an increase in CRC is observed in later generations of this family (Warthin, 1913; Lynch and Krush, 1971). Two syndromes have been identified that have a shared genetic basis with HNF’CC based on the involvement of DNA MMR genes. One is the Muir-Torre syndrome, which is characterized by the occurrence of sebaceous gland tumors and skin cancers in addition to a tumor spectrum similar to that found in the Lynch I1 syndrome (Lynch et al., 1981). Additionally, in some families with Turcot syndrome, characterized by the presence of a primary brain tumor and multiple colorectal adenomas (Turcot et al., 1959), the condition is attributable to mutations in DNA MMR genes, although in the majority of kindreds with this syndrome it appears to be due to mutations in the APC gene (Hamilton et al., 1995).

C. Occurrence of Colorectal Adenomas CRC in HNPCC, as in sporadic cases, is believed to develop via a precancerous growth, adenoma. In HNPCC, the occurrence of adenomas is not significantly higher than in the general population (Lanspa et al., 1990; Jass and Stewart, 1992), but the adenomas develop at a young age, reach a large size, and are often villous, suggesting that they are more prone to malignant transformation than sporadic adenomas or individual adenomas occurring in FAP (Jass and Stewart, 1992). Results from a 10-year follow-up study by Jarvinen et al. (1999, showing that CRC can effectively be prevented in HNPCC patients by colonoscopic screening and removal of adenomas, provide indirect evidence of an important role of adenomas as precursor lesions for cancer in HNPCC.

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111. HNPCC AND DNA MMR A. Identification of

Human MMR Genes

A systematic search through the entire genome using highly informative short tandem repeat (microsatellite) markers resulted in the identification of the first HNPCC susceptibility locus on chromosome 2p by linkage analysis (Peltomaki et al., 1993). In studies that followed the mapping of this locus, tumors from HNPCC patients were found to display length variation at multiple random microsatellite sequences throughout the genome (Aaltonen et al., 1993). A similar phenotype had previously been observed in bacterial and yeast strains with DNA MMR gene mutations (Levinson and Gutman, 1987; Strand et al., 1993). These data together provided a functional clue that soon led to the identification of human homologs of bacterial and yeast MMR genes (Fig. 1).Proof that human MMR genes caused susceptibility to E. coli

S. cerevisiae

MutL

MLHl

PMSl

H. sapiens

E. coli

S. cerevisiae

H.sapiens Fig. 1 MutS and MutL families of DNA mismatch repair genes in bacteria, yeast, and humans. Chromosomal locations are shown in parentheses. Genes that, when mutated, cause susceptibility to HNPCC are underlined and those shown to be associated with microsatellite instability in human cancer are indicated with an asterisk,

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HNPCC was based on the subsequent demonstration of germline mutations in these genes that segregated with the disease phenotype in HNPCC kindreds (Leach et d., 1993; Papadopoulos et al., 1994; Bronner et d., 1994; Nicolaides et al., 1994).

B. Biochemical and Functional Properties of the MMR System In Escherichia coli, mutS and mutL proteins participate in two main repair pathways, the methyl-directed long patch and the very short patch pathways (Modrich, 1991, 1994). The methyl-directed pathway functions by correcting base-base mispairs, small insertions and deletions mainly resulting from errors in DNA replication. The specific function of the very short patch pathway is to correct G T mispairs in nonreplicating DNA that result from deamination of S-methylcytosineresidues. Methyl-directed MMR in E. coli depends on 10 activities. Repair is initiated by binding of the MutS protein to the mismatch, followed by the addition of MutL. This complex activates MutH, an endonuclease, which makes a nick at a GATC site with unmethylated adenine, located 1-2 kb away on either side of the mismatch. Subsequently, the portion containing the mismatch is excised by a 3 ’ 4 ’ or 5’-3’ exonuclease and replaced by a new tract synthesized by DNA polymerase. In yeast, after the mismatch has been recognized by the MutS homolog 2 (MSH2) protein, a heterodimer is formed by the MutL homolog 1 (MLH1) and postmeiotic segregation increased 1 (PMS1)proteins, followed by a ternary complex formation by MLH1, PMS1, and MSHl (Prolla et al., 1994). This complex then recruits additional proteins that accomplish the repair as in the bacterial system. The human MMR system is believed to operate in more or less the same general fashion. Biochemical analyses in human cells have demonstrated that repair is strand specific and is directed by a nick located 5’ or 3’ to the mismatch (Parsons et al., 1993; Umar et al., 1994b).The mismatch binding factor in humans consists of two distinct proteins, the 100-kDa MSH2, encoded by the 2p gene (Leach et al., 1993; Fishel et al., 1993), and a 160-kDa polypeptide, G/T binding protein (GTBP), encoded by an adjacent gene on 2p (Palombo et al., 1995). A third MutS-related gene, MSH3, has been identified in humans (Fujii and Shimada, 1989; Watanabe et al., 1996), but its role in MMR remains to be clarified. While E. coli has a single mutL gene, human cells have at least 16 genes that specify MutL-like proteins, including the MLHl gene on 3p (Papadopoulos et al., 1994; Bronner et al., 1994), the PMSl gene on 2q (Nicolaides et al., 1994), the PMS2 gene on 7p (Nicolaides et al., 1994), and the PMS3-8 (Horii et al., 1994) as well as PMSR1-7 (for PMS2-related genes; Nicolaides et al., 1995a) genes on chromosome 7.

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Whether or not these different MutL-like proteins have different functions with respect to each other is not known. Studies have revealed some differences between MMR proteins in their capacity to repair specific types of biochemical defects. While MSH2 and MLHl seem to be equally important in single-base MMR, MSH2 plays a more prominent role in the repair of loops containing five or more unpaired bases (Umar et al., 1994a). Human cells may be capable of repairing loops of up to 14 nucleotides (Fishel et al., 1994), which is important since human DNA contains numerous microsatellites that may generate large loops as a consequence of strand slippage during replication. Furthermore, it is suggested that GTBP is necessary for the correction of base-base mispairs and one- (or two)-nucleotide loops but may not be absolutely required in the correction of larger loops (Drummond et al., 1995; Papadopoulos et al., 1995b; Marsischky et al., 1996). Correction of biosynthetic errors in newly synthesized DNA is not the only function of the MMR system. This system is also presumed to recognize other types of lesions-for example, those caused by alkylating mutagens-and is implicated in the process that causes growth arrest in G2 in response to DNA damage by these agents (Hawn et al., 1995). In E . coli, there is a connection between two major DNA repair systems, MMR and nucleotide excision repair, since mutations in MutS and MutL have been shown to abolish transcription-coupled repair of photoproducts induced by ultraviolet light (Mellon and Champe, 1996). A similar connection has been shown to exist in humans (Mellon et al., 1996).The previously described findings provide examples of mechanisms by which environmental carcinogens could contribute to the etiology of tumors associated with genetic defects in MMR. Finally, the MMR system also prevents recombination between nonidentical (“homeologous”) sequences (Selva et al., 1995).

C. Microsatellite Instability as an Indicator of MMR Deficiency Instability at short tandem repeat sequences (microsatellites) reflects malfunction in the replication or repair of DNA. For this reason, it is also referred to as the replication error (RER) phenomenon (Aaltonen et al., 1993). Although the length of microsatellite sequences varies among the homologous chromosomes of a single individual and among different individuals (polymorphism), such variation should not occur among different cells of any single individual. In the HNPCC and some sporadic tumors, microsatellite DNA varies in length from that of normal cells from the same patient, indicating that during tumor formation the sequences have gained or lost one or several repeat units (e.g., CA dinucleotides in a (CA), repeat). Approxi-

100

Paivi Peltornaki and Albert de la Chapelle

mately 1O0,OOO microsatellite repeats are scattered throughout the human genome (Weber and May, 1989), and a majority of these loci are apparently unstable in tumors from HNPCC patients (Aaltonen et al., 1993). Thus, in HNPCC tumors, the total number of mutations at microsatellite loci alone could be as high as 100,000 per cell, and microsatellite instability serves as a useful marker of the “mutator” phenotype characteristic of these tumors. Biochemical studies by Parsons et al. (1993) provided a link between microsatellite instability and defective MMR. In CRC cells with microsatellite instability, the mutation rate of (CA), repeats was at least 100-fold that in tumors without instability, and, by in vitro assay, the increased mutability was associated with a profound defect in strand-specific MMR. While most tumors developing in HNPCC patients and belonging to the HNPCC tumor spectrum (see earlier) contain mutations in microsatellite sequences (Aaltonen et al., 1993, 1994a), such variability is usually not observed in normal tissue from the same individuals; in keeping with this, lymphoblasts from an HNPCC patient were found to be repair proficient (Parsons et al., 1993). Later studies have shown that, in cells with deficient DNA MMR, both copies of a given MMR gene are usually inactive, leaving the cells with no functional protein (Liu et al., 1995b; Leach et al., 1996). Hereditary defects in one of four MMR genes-MSH2, MLH1, PMS1, or PHS2-were detected in 70% of HNPCC kindreds with microsatellite instability, suggesting that, in HNPCC, this abnormality mainly results from inactivation of these genes (Liu et al., 1996). Apart from HNPCC, approximately 15% of apparently sporadic CRCs and a variable proportion of other cancers show the RER phenotype (see de la Chapelle and Peltomaki, 1995, for a review). Although HNPCC and sporadic CRCs with microsatellite instability share several clinical and pathological features, including proximal tumor location, diploid DNA content, and indolent behavior (Aaltonen et al., 1993; Ionov et al., 1993; Thibodeau et al., 1993; Kim et al., 1994), the genetic background of the RER abnormality may not be the same in these two groups. While inactivating mutations in one of four HNPCC-related MMR genes are demonstrable in a proportion of sporadic colorectal and endometrial tumors as well (Liu et d., 1995b; Katabuchi et al., 1995; Bsrresen et al., 1995), a significant fraction probably arise by other mechanisms. These include mutations in other MMR genes, such as GTBP, whose defects mainly cause instability in mononucleotide tracts (Papadopoulos et al., 1995b) or MSH3, whose defects preferentially result in deletions in microsatellite sequences (Strand et al., 1995). Other genes involved in DNA replication or repair, such as DNA polymerase 6, may underlie microsatellite instability in some cases (da Costa et al., 1995). Even exogenous damage has been implicated (Canzian et al., 1994). In sporadic tumors that do not belong to the HNPCC spectrum, the pattern

Mutations Predisposingto HNPCC

101

of instability may be different from that typically associated with HNPCC and MMR deficiency (Ma0 et al., 1994; Wooster et al., 1994). Based on current experience, a majority of MMR gene mutations detected in unselected RER+ colorectal tumors are acquired (Liu et al., 1995b; Bsrresen et al., 1995). However, microsatellite instability is exceptionally common (with an incidence of 58%) in colorectal tumors from very young patients (<35 years), and in these cases it often indicates inherited susceptibility even in the absence of any family history (Liu et al., 1995a). Furthermore, microsatellite instability in a colorectal adenoma may indicate HNPCC, since this abnormality is often observed in adenomas from HNPCC patients but is rare in apparently sporadic colorectal adenomas (Aaltonen et al., 1994a).

IV. MUTATIONS PREDISPOSING TO HNPCC

A. Structure of HNPCC-Related MMR G e n e s So far, four MMR genes-MSH2, MLH1, PMS1, and PMS2-have been demonstrated to cause predisposition to HNPCC based on germline mutations segregating with the disease phenotype in HNPCC kindreds. Some essential characteristics of these genes are shown in Table I. As is evident, the length of coding DNA and the number of exons display little variation between different genes, while the estimates of the genomic size of these genes range from 16 to 100 kb, implying significant variation in the lengths of intron sequences. To date, the promoter regions of MSH2 (Schereret al., 1996) and PMS2 (Nicolaides et al., 1995b) have been isolated and characterized. The human and yeast MMR proteins show 3 0 4 0 % similarity in amino acid sequences (Chung and Rustgi, 1995), and mutations affecting the conserved regions are expected to significantly impair DNA MMR. The amino acid sequences of the human and yeast MutS homologs are highly similar in the carboxyl-terminal region (Palombo et al., 1995). The greatest homology (77%) between the human and yeast MSH2 protein is observed between codons 615 and 788, encompassing the helix-turn-helix domain thought to be responsible for the binding of the protein to DNA (Leach et al., 1993). In contrast, the different MutL-related proteins are highly homologous to each other at their amino-terminal portion (Nicolaides et al., 1994). Human and yeast MLHl proteins display 55 % identity at codons 11-3 17; additionally, the last 13 amino acids are identical between these two proteins (Bronner et al., 1994). Comparison of the yeast and human PMS proteins shows 3 0 4 2 % identity between codons 40 and 390; in addition, they have anoth-

L

0 N

Table 1 Characteristics of Four HNPCC-Related Human DNA MMR Genes

Gene

Chromosomal location

Length of cDNA

(kb)

Number of exons

Genomic sizeu (kb)

MSH2

2p16

2.8

16

73

MLHl

3p21-p23

2.3

19

58-100

PMSl PMS2

2q31-q33 7p22

2.8 2.6

Not known 15

Not known 16

‘Without the promoter region.

References for structure Leach et al. (1993), Fishel et al. (1993), Kolodner et al. (1994) Papadopoulos et al. (1994),Bronner et al. (1994), Kolodner et al. (1995),Han et al. (1995) Nicolaides et al. (1994) Nicolaides et al. (1994, 1995a)

Mutations Predisposing to HNPCC

I03

er region of significant homology (22%) at the carboxyl terminus (Nicolaides et al., 1994).

B. Proportion of HNPCC Attributable to Different Genes To date, more than 70 different germline mutations in MMR genes have been published and shown to be associated with HNPCC. Results from a few comprehensive investigations are shown in Table 11. When interpreting the results, one should pay attention to which genes were studied, which method was used for mutation screening, and which ethnic and geographic group the kindreds originated from. In a study by Liu et al. (1996), five genes (MSH2, MLH1, PMS1, PMS2, and GTBP) were screened for mutations in a series of 48 HNPCC kindreds that had been shown to manifest microsatellite instability. Germline mutations were identified in the first four genes, accounting for 70% of the kindreds. A three-step strategy was used for mutation detection. Messenger RNA alterations produced by gross deletions or insertions or splicing defects were first screened for by reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR products were then transcribed and translated in vitro by the in vitro synthesized protein (IVSP) assay, to detect primarily truncations caused by various nonsense or frameshift mutations. If no alterations were observed by the previously described approach, the entire coding regions of the genes were sequenced to detect possible nontruncating (missense) mutations. In contrast to the comprehensive study of Liu et al. (1996), in the remaining investigations described in Table I1 the overall proportion of kindreds with a detectable MMR gene mutation was generally lower, probably because fewer genes were studied, usually with a single technique such as denaturating gradient gel electrophoresis, single-strand conformation polymorphism analysis, or direct sequencing. When used alone, these techniques lead to lower detection rates. Several investigations suggest that two MMR genes, MSH2 and MLH1, together account for a majority (approximately 70%) of HNPCC kindreds fulfilling the international diagnostic criteria for the disorder (see references in Table I1 and Nystrom-Lahti et al., 1994a). The combined proportion of PMSl and PMS2 genes appears to be around 5% (Liu et al., 1996). In most studies cited in Table 11, MSH2 and MLHl account for roughly equal proportions of HNPCC. However, ethnic differences clearly occur. A striking example is provided by Finnish kindreds, in which the presence of two widespread founding mutations has resulted in the proportion of 83% for MLHl mutations, compared to only 3% for mutations in MSH2, as discussed in greater detail later.

L

0 P

Table I1 Results of Mutation Analyses in HNPCC Kindreds Meeting the International Diagnostic Criteria Country of origin of the kindreds

Gene

Proportion of kindreds with a mutation

MSH2 MLHl PMS 1 PMS2 GTBP

15/48 (31%) 16/48 (33%) 1/48 (2%) 2/48 (4%) 0/48 (0%)

1)RER analysis 2) RT-PCR + IVSP + sequencing of cDNA

North America, Europe, New Zealand

Liu et al. (1996)

MSH2 MLHl

3/10 (30%) 3/10 (30%)

Sequencing of genomic DNA

Switzerland

Buerstedde et al. (1995)

MSH2 MLHl

3/7 (43%) 117 (14%)

Sequencing of genomic DNA

Russia, Moldavia

Maliaka et al. (1996)

MSH2 MLHl

1/35 (3%) 29/35 (83%)

RT-PCR + 2-dimensional DNA electrophoresis

Finland

Nystrom-Lahti et al. (1996)

MSH2 MLHl

7/34 (21%) 12/34 (35%)

DGGE

The Netherlands, Italy, Denmark

Wijnen et al. (1995,1996)

MLHl

5/21 (24%)

DGGE

Sweden

Tannergird et al. (1995a)

MLHl

8/34 (24%)

SSCP

Japan, Korea, United States

Han et al. (1995)

Screening method'

Reference

s

a DGGE, denaturing gradient gel electrophoresis; SSCP, single-strand conformation polymorphism.

3:

9n R

n

c

Mutations Predisposing to HNPCC

I05

What underlies HNPCC in those approximately 30% of kindreds in which no germline mutation is found in the four HNPCC-related MMR genes? A proportion are likely to result from mutations that have escaped detection by the present techniques (an example is given in Papadopoulos et al., 1995a). As nearly all tumors from HNPCC patients show microsatellite instability (Aaltonen et al., 1994a), other genes affecting DNA stability would be good candidates, including the numerous new members of the families of mutS- and mutL-related genes that have been discovered in humans but not yet systematically tested (see earlier). Moreover, mutations in the proofreading domain of DNA polymerase 6 could be involved, since in at least one case such a mutation was detected in the germline of a colon cancer patient with microsatellite instability (da Costa et al., 1995). Preliminary evidence suggests that, among kindreds not fulfilling the international criteria, the proportion with no detectable mutation in the known HNPCC genes may be even larger (Table 111). In such kindreds, additional explanations that must be considered include a non-Mendelian (e.g., multifactorial) genetic predisposition and chance clustering of cancer.

C. Types and Sites of MMR Gene Mutations The types of published mutations in MSH2 and MLHl are shown in Figure 2. All but missense mutations, which only substitute an amino acid, are expected to alter the size of the protein product, most commonly leading to truncation. That is why Liu et al. (1996)were able to detect as many as 70% of the mutations (Table 11) by the IVSP assay. A majority (53%)of MSH2 mutations cause frameshifts (i.e., changes the reading frame) as a consequence of insertions or deletions. Twenty-nine percent are nonsense mutations, creating stop codons. In comparison with MSH2, and MLHl gene shows a high proportion of missense mutations, which are almost as frequent as frameshift mutations in this gene. Furthermore, splice site defects are relatively common as specific causes of frameshifts or in-frame deletions in MLHl (Fig. 3B). In both MSH2 and MLH1, single-base substitution mutations seem to involve CpG dinucleotides more often than expected based on the calculated CpG content of these genes, a phenomenon probably related to methylation and spontaneous deamination of cytosine residues (Maliaka et al., 1996). The frequent finding of missense mutations in MLHl is problematic from the clinical point of view because it is difficult to evaluate the biological effect and the relative risk associated with these mutations. Such a mutation is likely to be pathogenic if the substituted amino acid has different polarity, if the involved codon is evolutionarily conserved, if the mutation cosegregates with the disease (which is a necessary but not sufficient condition),

Table 111 Results of Mutation Analyses in Kindreds Not Meeting Formal Diagnostic Criteria Gene

Proportion of kindreds with a mutation

Screening method

Country of origin of the kindreds

MSH2 MLHl

1/4 (25%) 1/4 (25%)

Sequencing of genomic DNA

Russia, Moldavia

Maliaka et al. (1996)

MSH2 MLHl

1/20 (5%) 5/20 (25%)

RT-PCR + 2-dimensional DNA electrophoresis

Fin1and

Nystrom-Lahti et al. (1996)

MLHl

3/15 (20%)

DGGE

Sweden

Tannergard et al. (1995a)

Reference

g s. 3 ; ; s

B 5

Mutations Predisposing to HNPCC

No. 25

MSH2

107 MLHl

1 missense nonSenSe

frameshift

in-frame deletion

Fig. 2 Relative proportions of different types of germline mutations reported in MSH2 ( n = 28) and MLHl ( n = 48). The numbers of the mutations are indicated by the scale on the left and the respective percentages are given in parentheses. The category “in-frame deletion” includes genomic deletions that do not alter the reading frame as well as splice site mutations that result in an in-frame deletion of one or several exons from the transcript.

and, finally, if the mutation is absent in the normal population. Pathogenicity is further supported if an amino acid substitution at the equivalent position results in a nonfunctional protein in yeast (Tannergird et al., 1995a). MSH2 and MLHl mutations are scattered throughout the genes, as demonstrated by Figure 3. This implies that, in mutation searches, as a rule the entire genes must be screened. A few potential hot spots for mutations may be emerging, comprising exons 5 , 7 , and 12 of MSH2 and exons 2, 16, and 19 of MLH1. It is noteworthy that several of these potential hot spot regions affect evolutionarily conserved portions (Fig. 3 ) . >

Fig. 3. Sites of germline mutations within the MSH2 gene, with 16 exons (A),and the MLHl

gene, with 19 exons (B). Black bars below the schematic diagrams of the genes indicate areas of coding DNA with the highest homology to the respective yeast genes. Nontruncating (missense, M) mutations are shown in the upper part and truncating mutations (F, frameshift; N, nonsense; I, in-frame deletion) in the lower part of the figures. Splice site mutations (S) resulting in frameshifts or in-frame deletions are indicated separately. The numbers in circles refer to published reports as follows: 1, Buerstedde et al. (1995); 2, Liu et al. (1995a);3, Lazar et al. (1994);4, Wijnen et al. (1995);5, Liu et al. (1996); 6, Froggatt et al. 1995; 7, Miliaka et al. (1996); 8, Nystrom-Lahti et al. (1996); 9, Mary et al. (1994); 10, Bsrresen et al. (1995); 11, Kolodner et al. (1994);12, Tannergird et al. (1995b); 13, Tannergird et al. (1995a); 14, Bronneretal. (1994);15,LiuetaL (1995b);16,LuceetaL (1995);17,Hanetal. (1995); 18,Kolodner et al. (1995);and 19, Wijnen et al. (1996).Illustration on following page.

MSH2

0 M

0 M

7 171

6

H

7122

123215

216- 265- 315264 314 359

oolg F

100 bp Of coding DNA 10 kb of intron DNA

359426

13

426462

463- 504- 554- 587504554 587 669

669737

H

14 737820

H

15

H 16 1

820878

879- ,.,dons 934

0

MLHl

M

1-

39

3969

70102

103- 127-152- 182-197- 226- 264- 295127 151 182 196226 264 295 346

347470

470520

F

H

100 bp of coding DNA 10 kb of intron DNA

Fig. 3 Legend on page 107.

520- 556556 577

578- 633-664- 702632 663701 756

F

Iq I

I

S(F)

I

I

@ F

c.ons

110

Paivi Peltomaki and Albert d e la Chapelle

D. Founding Mutations As a rule, the predisposing mutation varies from kindred to kindred. The situation is different in the case of so-called founding mutations, which have arisen once in a common ancestor and thereafter been transmitted through a variable number of generations to present-day members of HNPCC kindreds. Three likely or possible founding mutations have already been described in HNPCC, two of which, both affecting the MLHl gene, occur in Finnish HNPCC kindreds, while the third one, affecting the MSH2 gene, occurs in European and North American kindreds. Kindreds with either of the two Finnish mutations are characterized by common ancestry and/or geographical origin as well as conservation of a large disease haplotype around MLH1, establishing the ancestral nature of the mutations (Nystrom-Lahti et al., 1994b, 1995; Moisio et al., 1996). The enrichment of these mutations among the Finns is believed to result from genetic drift in an isolated population (Nystrom-Lahti et al., 1994b). Together, they account for as many as 68% (24/35) of kindreds fulfilling the Amsterdam criteria and 51% (28/55) of all kindreds with verified or putative HNPCC in Finland. The most prevalent mutation consists of a large genomic deletion affecting exon 16 and its flanking introns and leads to an in-frame deletion of this exon from the transcript (Nystrom-Lahti et al., 1995, and Fig. 3B). It seems to have arisen through Alu-mediated recombination. The other mutation consists of a single base change at the splice acceptor site of exon 6 and results in an out-of-frame deletion of this exon (Nystrom-Lahti et al., 1995, and Fig. 3B). Results from genealogical studies and haplotype analyses suggest that the former mutation is likely to be at least 500 years old, whereas the latter mutation is probably younger (Nystrom-Lahti et al., 1994b; Moisio et al., 1996).If the presence of these mutations is suspectedfor example, based on observed deletions of exons 6 or 16 from the MLHl transcript-simple DNA-based tests are available for exclusion or verification (Nystrom-Lahti et al., 1995). In contrast to the two mutations described previously, the ancestral nature of the MSH2 mutation is somewhat controversial and needs further clarification. This mutation consists of a single base change at the splice donor site of exon 5, leading to an in-frame deletion of this exon (Leach et al., 1993, and Fig. 3A). It creates an MseI restriction site, which is useful in the detection of this mutation. The mutation was reported to occur in 12% (4/33) of English kindreds and was the most frequent single mutation, with a proportion of 8%, in the series of 48 kindreds from Europe and North America studied by Liu et al. (1996). However, in the former study the authors state having excluded a common disease haplotype (markers not specified) in two kindreds informative for haplotype, and, in the latter study, “no obvious geographic or ethnic relationship was shared” among kindreds with the mu-

Mutations Predisposing to HNPCC

111

tation. Therefore, it is unclear if the mutation observed in several families has a common origin or arises de novo with appreciable frequency.

V. PHENOTYPIC EFFECTS OF MMR GENE MUTATIONS

A. “Mutator Phenotype” and Tumorigenesis Statistically, it has been estimated that colorectal tumors require four to seven mutations to develop (Renan, 1993). Sequential accumulation of mutations in the APC, m s , DCC, and p53 genes is associated with increased cell proliferation in the colonic epithelium, resulting first in benign growths called polyps, which may then gradually progress to carcinomas (Vogelstein et al., 1988; Boland et al., 1995). The presence of APC mutations in polyps is closely correlated with dysplastic histology (“adenomatous polyp”), implying a tendency to malignant transformation (Jen et al., 1994). HNPCC tumors are believed to arise by a classic tumor suppressor mechanism involving two inactivating mutations, one inherited and one somatic, in a MMR gene (Hemminki et al., 1994; Liu et al., 1995b). Loss of a critical MMR activity in a tumor precursor cell probably leads to genetic destabilization and initiates a cascade of secondary mutations throughout the genome, some of which affect important growth-regulatory genes and, hence, give rise to cancer (Loeb, 1994). In support of this hypothesis, Lazar et al. (1994) found that tumors from two HNPCC patients showed multiple somatic mutations in the APC and p53 genes (up to six mutations per gene). Interestingly, a polyp from a patient with an inherited MSH2 mutation displayed only a single mutation (in APC), whereas CRC from the same patient showed 10 mutations (in APC and p53), suggesting that inactivation of the MSH2 gene was a source of progressive accumulation of mutations in critical genes during colorectal tumorigenesis (Lazar et al., 1994). Genes with small repeated sequences as part of their coding DNA would be particularly susceptible to structural alterations in tumors with defective DNA mismatch repair. In keeping with this, colon cancer cells with microsatellite instability were found to show frequent inactivation of the transforming growth factor-p receptor I1 (TGF-PRII) gene, which was due to frameshift mutations targeted on two repeat sequences, an (A)lo and (to a lesser extent) a (GT), repeat, both contained in the coding region of the gene (Markowitz et al., 1995; Parsons et al., 1995b). Mutations of the RII gene result in the absence of cell surface RII receptors and consequent escape from TGF-P-mediated growth control. These mutations thus link DNA repair defects with a specific pathway of tumor progression. Another gene implicat-

112

Paivi Peltomaki and Albert d e la Chapelle

ed in colon cancer that also has a repeat sequence [(CT),] as a potential target for mutations is the p2-microglobulin (P2M) gene (Bicknell et al., 1994; Branch et al., 1995). The product of this gene is a subunit of the human leukocyte antigen (HLA) class I molecule that is necessary for the presentation of peptides derived from endogenous proteins to cytotoxic T cells. All four “mutator” colorectal cell lines studied by Branch et al. (1995) showed loss of P2M expression due to inactivating mutations in both alleles of the gene, as compared to none of 33 CRC cell lines without microsatellite instability. Close correlation between the loss of P2M/HLA expression and the mutator phenotype may indicate that mutator tumor cells have undergone selection to evade T-cell surveillance (Branch et al., 1995). Prior to successful selection, frequent mutations in the P2M gene and other genes affecting HLA expression are likely to provoke a strong immune response against tumor cells, which may explain the favorable prognosis associated with tumors manifesting the mutator phenotype (Bodmer et al., 1994; see earlier). Although inactivation of both alleles of a MMR gene is usually necessary for a phenotypic effect, three HNPCC patients were found to display widespread mutations not only in their tumors, but also in their nonneoplastic cells presumed to be heterozygous for the mutation (Parsons et al., 1995a). One patient had a MLHl gene mutation, while PMS2 was involved in the other two. The authors suggested a dominant negative mechanism based on the assumption that mutated MLHl and PMS2 proteins might be able to interact with MSH2 but inable to recruit the enzymes carrying out the actual repair. Despite elevated mutation rates in all tissues examined, these patients had surprisingly few tumors and had otherwise normal development (Parsons et al., 1995a). That DNA MMR deficiency is compatible with normal development is further supported by knockout mouse experiments. Mice homozygous for a MSH2 or PMS2 mutation are viable and display no major abnormalities, but some are infertile (PMS2-deficient males) and many develop tumors, primarily lymphomas and sarcomas, starting at 2 months of age (Reitmair et al., 1995; de Wind et al., 1995; Baker et al., 1995). It will be interesting to see whether these animals develop other cancers with time.

B. Basis of HNPCC Tumor Spectrum The finding that MSH2 and PMS2 knockout mice are mainly prone to lymphoid tumors and not colon cancer or other cancers typical of HNPCC is somewhat surprising and related to the broader question of the genetic basis of the HNPCC tumor spectrum. Several explanations have been proposed for the apparent tissue-specific differences in cancer predisposition in HNPCC. First, the development of various cancers may be regulated by genes that differ in their vulnerability to replication errors. An example is provided by

Mutations Predisposing to HNPCC

I I3

TGF-PRII, which is frequently mutated within its polyadenine tract in colorectal and gastric cancers with microsatellite instability (90 and 71%, respectively), but rarely in RER+ endometrial cancers (17%),suggesting a different pathogenetic route for the latter tumors (Markowitz et al., 1995; Parsons et al., 1995b; Myeroff et al., 1995). Second, there may be tissue-specific differences in MMR gene expression. Although Northern blot and RT-PCR analyses of various human tissues have shown that DNA MMR genes are widely expressed, as expected from their presumed housekeeping function (Leach et al., 1993; Papadopoulos et al., 1994), immunohistochemical studies have revealed cell-specific patterns of expression (Wilson et al., 1995; Leach et al., 1996). Using antibodies specific for MSH2, the highest expression was observed in esophageal and intestinal epithelia, where expression was limited to the replicating compartment (Leach et al., 1996). Third, there may be redundancy among members of the Mut family, which could prevent MMR deficiency and microsatellite instability in some tissues but not in others (Varlet et al., 1994). Evidence of redundancy was recently demonstrated in Saccharomyces cerevisiae, which was found to have two pathways of MSH2-dependent MMR: one that recognizes single-base mismatches and requires MSHG (homologous to GTBP in humans) and another that recognizes insertioddeletion mismatches and requires either MSHG or MSH3, in addition to MSH2 (Marsischky et al., 1996). Fourth, different rates of exposure to exogenous carcinogens might play a role. Cells deficient in MMR have been shown to be tolerant to alkylating agents (Aquilina et al., 1993), which may confer selective advantage in these cells. Alkylating agents are probably more common in the lumen of the colon than in many other organs, which would contribute to the high frequency of colon cancer in HNPCC. Finally, our current concept of the HNPCC tumor spectrum, which depends on the comparison of cancer frequencies in HNPCC kindreds and the normal population, may change substantially upon reevaluation based on confirmed mutation carriers (see later).

C. Clinical Correlations of HNPCC Mutations Mutation studies published so far, most of which focus on Lynch I1 kindreds fulfilling the Amsterdam criteria, have failed to demonstrate any clear relationship between the clinical phenotype and the gene involved or the site or type of mutation. Vasen et al. (1996) reported a higher rate of extracolonic cancer in MSH2 mutation carriers as compared to MLHl mutation carriers (61 vs. 42%), but the difference was not statistically significant. The Muir-Torre syndrome may be predominantly associated with MSH2 mutations, based on published reports of five kindreds all of which have shown the involvement of this gene (Kolodner et al., 1994; Liu et al., 1994; Nys-

114

Paivi Peltomaki and Albert de la Chapelle

trom-Lahti et al., 1994a). Germline mutations in MLHl and PMS2 were found in two patients with Turcot syndrome, both of whom manifested deficient MMR, in normal cells as well as tumor cells (see earlier and Hamilton et al., 1995). Interestingly, glioblastomas from these patients were RER+ and were associated with unusually long survival, suggesting pathogenetic mechanisms similar to those in typical HNPCC. A potent modifying locus of intestinal tumorigenesis, encoding secretory phospholipase A,, was identified in mice with a germline APC mutation (MacPhee et al., 1995). Kindreds sharing an identical predisposing MMR gene mutation provide the opportunity to study the possible role of such modifying genes in HNPCC. Phenotypic diversity observed in kindreds with the same genetic predisposition suggests that other genetic or environmental factors are involved. For example, the mean age of onset varies from 36.5 to 54.8 years in Finnish kindreds with the MLHl exon 16 founding mutation (Moisio et al., unpublished data). Similarly, of four kindreds with the same mutation involving MSH2 exon 5 , one showed skin tumors characteristic of the Muir-Torre syndrome while the remaining three had no skin tumors (Liu et al., 1996).

VI. IMPLICATIONS OF MUTATION FINDINGS Knowledge of the mutations predisposing to HNPCC forms the basis for further investigations to clarify different aspects of the disease. As described earlier, mutation information allows genotype-phenotype correlations and studies on different factors contributing to the phenotype. The ability to distinguish mutation carriers from phenocopies among affected individuals from HNPCC kindreds makes it possible to refine or redefine the HNPCC tumor spectrum (see Kolodner et al., 1994,1995, for examples). Due to the lack of reliable diagnostic criteria, current estimates of the incidence of the disease show large variation (Houlston et al., 1992; Aaltonen et al., 1994b); genetic studies will for the first time allow accurate evaluation of the frequency of HNPCC in different populations. Clinically, it is now possible to determine who has and who has not inherited the mutation segregating in a particular family and use the mutation information to offer predictive testing for asymptomatic at-risk individuals. Such testing and counseling has already started (Van de Water et al., 1994). Guided by studies on expected cancer risk (Aarnio et al., 1995; Vasen et al., 1996), careful surveillance by different methods, mainly colonoscopy (Vasen et al., 1993),is offered to mutation carriers with the aim of early cancer detection and cure, while individuals without the mutation can be exempted from lifelong screening pro-

Mutations Predisposing to HNPCC

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cedures and excessive fear of cancer. Future studies are necessary to assess the psychosocial impact and ethical issues of genetic testing in HNPCC kindreds.

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Functional Aspects of Apoptosis in Hematopoiesis and Consequences of Failure Sharon L. McKenna and Thomas G. Cotter Tumour Biology Laboratory, Department of Biochemistry, University College, Cork, Ireland

I. Introduction 11. Morphological and Biochemical Features of Apoptosis III. Molecular Mechanisms in Apoptosis A. The BCL-2 Gene Family B. p53 C. MYC D. RASGenes E. Abl Tyrosine Kinases F. FasIApo-1 G. ICE-Related Cysteine Proteases H. Lessons from Viruses IV. Functional Aspects of Apoptosis in the Hematopoietic System A. Survival Factors and Population Control B. Survival Factors and Stem Cell Viability C. Survival Factors and Myeloid Differentiation D. T-cell Differentiation E. Cytotoxic T Lymphocytes F. B-Cell Differentiation G. Affinity Maturation H. Downregulation of an Immune Response V. Disruption of Apoptosis in Hematopoiesis A. Inflammation B. Autoimmunity C. Acquired Immunodeficiency Syndrome D. Apoptosis in the Acquisition of Malignancy VI. Future Perspectives References

Apoptosis is an internally directed, physiological method of cell destruction. Cellular components are dismantled within the confines of an intact cell membrane, and rapid ingestion by phagocytic cells prevents local idammation. A variety of genes have now been identified as positive or negative regulators of apoptosis. Transfection experiments and studies of gene cooperation in viral transformation suggest that full cellular trans-

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formation requires not only the deregulation of proliferation, but also the inhibition of concomitant apoptosis programs. The regulation of apoptosis is fundamental to hematopoietic homeostasis. Stem cell renewal is continuously counterbalanced by apoptosis in functionally inactive or terminally differentiated cells. Extensive cell death in developing lymphocyte populations ensures that only cells recognizing non-self antigens are released into the periphery, and the finite lifespan of terminally differentiated cells enables the extensive cell turnover demanded by functional aspects of the hematopoietic system. The requirement of each hematopoietic sub-population for a specific sub-set of survival factors, provides a flexible mechanism for dictating the cellular composition of the mature population and for controlling population size. Surplus cell production and apoptosis are therefore normal features of hematopoiesis. The consequences of deregulated apoptosis are severe. Excessive apoptosis in lymphocyte populations plays a major role in the pathogenesis of acquired immunodeficiency syndrome (AIDS), whereas ineffective apoptosis has been associated with the development of inflammation, autoimmunity and hematological malignancies. The identification of various genetic abnormalities which influence apoptosis in leukaemic cells (e.g., mutant pS3, Bcr-Abl and over-expression of Bcl-2), suggests that the acquisition of an anti-apoptotic lesions is an important event in the multi-step evolution of hematological malignancies. In addition, the nature of some leukaemias particularly the chronic leukemias, in which the leukemic cells are nonproliferative and long lived, suggests that anti-apoptotic lesions are early events in the pathogenesis of these diseases. It is likely that the utilization of mechanisms to evade apoptosis would facilitate disease progression in all leukemias and contribute to the development of multi-drug resistance. A better understanding of apoptosis mechanisms in hematopoietic cells, and their exploitation by leukemic cells should be useful in the development of improved cytotoxic regimes.

I. INTRODUCTION It is now apparent that the regulation of apoptotic cell death is fundamental to the development and homeostatic maintenance of cell populations. This method of cell death has been described in a wide range of eukaryotes, including unicellular organisms, simple invertebrates, and complex mammalian cell systems (Ameisen et al., 1995b; Vaux et al., 1994). The extent of the evolutionary conservation in structural, biochemical, and molecular events underscores the physiological significance of the process. In mammals, one of the best studied systems with regard to apoptotic cell death is the hematopoietic system. This system has recruited a number of mechanisms whereby it selectively induces apoptosis in undesirable cells, and nurtures only the most functionally competent effectors. Despite the large turnover of cells, the boundaries of each specialized population are impeccably maintained and do not infringe upon other populations occupying the same environment. The size of each hematopoietic subpopulation is controlled by continually counterbalancing stem cell renewal and differentiation with apoptotic cell death.

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The importance of apoptosis in maintaining hematopoietic homeostasis is evident from the disastrous consequences of its deregulation. The inappropriate induction of apoptosis has been associated with the pathogenesis of acquired immunodeficiency syndrome, whereas ineffective apoptosis has been associated with autoimmunity, inflammation, and the development of hematological neoplasia. This article initially reviews the main morphological, biochemical, and molecular events that have been described in apoptotic cells. The functions and failures of apoptosis in the hematopoietic system are then discussed, with particular emphasis on the impact of deregulated apoptosis on the development of hematological malignancies. In addition, some attempt is made to explore those general concepts that may be central to the evolution of a variety of neoplastic diseases.

11. MORPHOLOGICALAND BIOCHEMICAL FEATURES OF APOPTOSIS Apoptosis is a morphologically defined, internally directed method of cell death. Apoptotic cells coordinate the dismantling of their own cellular machinery, and provide all of the necessary energy and enzymes for the process. The entire process takes place within the confines of an intact plasma membrane. The dying cells frequently appear shrunken, with condensed and marginalized chromatin, and undergo a characteristic convolution or “blebbing” of the plasma membrane. Although subtle differences in the destructive process have been described in various cell types (Clark, 1990; Zakeri et al., 1995), apoptosis is readily distinguishable from necrosis, which is a pathological method of cell death. Membrane integrity is lost in necrosis, resulting in the release of cellular contents, and thus is often accompanied in vivo by the development of inflammation. The destructive nature of necrosis makes it unlikely to be a significant player in physiological processes of cell deletion. Numerous biochemical alterations underlie the visible changes in apoptotic cells. Alterations in ion fluxes are thought to be significant in cell shrinkage (Arends and Wyllie, 1991), and a major reorganization of the cytoskeleton contributes to the changes in cell shape and budding of membrane-bound apoptotic bodies (Cotter et al., 1992). The activation of tissue transglutaminase results in protein cross-linking, and thus minimizes any leakage of soluble proteins from the dying cell (Fesus et al., 1987). Energy metabolism breaks down, and macromolecules are destroyed by a variety of proteases and nucleases. The most characteristic biochemical event in most apoptotic cells is DNA fragmentation into multiples of nucleosome-

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sized units (180-200 bp). Analysis of higher molecular weight DNA from apoptotic cells has indicated that the formation of large DNA fragments (50 and 300 kb) precedes oligonucleosomal DNA fragmentation. The sizes of these fragments have been suggested to arise from the release of loops (50 kb) or rosettes (300 kb) of chromatin as they become detached from the nuclear scaffold (Filipski et al., 1990). High-molecular-weight fragments may be present in apoptotic cells in the absence of the low-molecular-weight oligonucleosomal-sized fragments (Oberhammer et al., 1993). The identity of the endonuclease(s) responsible for DNA degradation in apoptosis remains controversial. A Ca2+-Mg2+-dependent endonuclease was reported to be involved in DNA degradation in apoptotic thymocytes (Cohen and Duke, 1984; Wyllie et al., 1992).A later study has suggested that this endonuclease is DNase I (Peitsh et al., 1993). Other reports have suggested that DNase I1 is the predominant enzyme in apoptotic Chinese hamster ovary cells (Barry and Eastman, 1993) and apoptotic human neutrophils (Gottlieb et al., 1995). Several novel endonucleases have also been reported in apoptotic thymocytes (Tanuma and Shiokawa, 1994; Montague et al., 1994; Nikonova et al., 1993) and cytotoxic T lymphocytes (Deng and Podack, 1995). In addition, Anzai et al. (1995) found that completely different types of endonucleases were activated in apoptotic bone marrow pro-genitor cells and their granulocytic descendants. These studies suggest that DNA digestion in apoptotic cells is an ordered, multistep process that may involve the concerted action of a number of endonucleases, and that the cell type and differentiation status may influence the nature of the endonucleases activated. Apoptotic cells display alterations in surface molecules that are recognized by neighboring cells or macrophages, resulting in their rapid ingestion (Hall et al., 1994; Dini et al., 1995).Apoptotic cell death does not, therefore, elicit an inflammatory response in vivo. The nature of the cell surface changes enabling phagocytic recognition has been the subject of active investigation. Lectin-like receptors have been suggested to mediate macrophage recognition of changes in surface carbohydrates on apoptotic cells (Dini et al., 1992; Hall et al., 1994). The vitronectin receptor (a&) and CD36 macrophage surface receptor have also been shown to cooperate in the recognition of apoptotic human neutrophils (Savill et al., 1990, 1992), and loss of phospholipid asymmetry of the plasma membrane, resulting in the inappropriate externalization of phosphatidylserine, can influence the recognition of apoptotic lymphocytes by macrophages (Martin et al., 1995; Fadok et al., 1992). In addition, a 75-kDa polypeptide (61D3 antigen) on monocyte-derived macrophages has been shown to be important for the recognition of apoptotic lymphocytes and neutrophils (Flora and Gregory, 1994). It is conceivable that the mechanism used by macrophages for recognition of apoptotic cells may be dependent upon the subpopulation of macrophages and the presence or absence of specific cytokines in their cellular environment.

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111. MOLECULAR MECHANISMS IN APOPTOSIS As the interest in apoptosis has expanded exponentially in the past decade, so has the list of molecular players in the process. Many new genes have been identified that can either positively or negatively influence the ability of a cell to undergo apoptosis (see Table I). In addition, a number of genes that have been previously associated with proliferation or cell cycle regulation (e.g., MYC, RAS, and p53) have been shown to mediate pathways to cell death. Emerging evidence suggests that many key players in cell proliferation are inextricably linked to cell death, and that this necessitates the acquisition of multiple lesions for full cellular transformation. A.

The B C L 2 Gene Family

Bcl-2 was the first protein found to exclusively maintain cell viability without promoting cell proliferation. The gene was originally cloned from the Table I Genetic Regulators of Apoptosis Positive regulators

Negative regulators

MYC P53 Ras * Bax B~1-x~ Bad Bak*

Bcl-2 Bcl-X, A1 Mcl-1 BHRFl

ICE-like proteases

CrmA P35 IAP

FasIFasL Bcr-Abl v-Abl Adenovirus E1A

ElB 55K ElB 19K

HPV E7

E6

"Genes that have been reported to positively or negatively influence apoptosis depending on the cell system, or presence of other genetic regulators.

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t(14:18) translocation breakpoint associated with follicular lymphoma.

Overexpression of BCL-2 has been shown to protect mammalian cell lines from apoptosis induced by a broad range of signals, including growth factor deprivation, ultraviolet irradiation, cytotoxic lymphokines (e.g., tumor necrosis factor), and heat shock (reviewed in Reed, 1995). Bcl-2 is an integral membrane protein associated with the mitochondria, endoplasmic reticulum, and nuclear envelope. It has been shown to protect cells from apoptosis in the absence of a nucleus, indicating that its effects are mediated in the cytoplasm (Jacobson 1994). A large BCL-2 gene family has now been identified on the basis of sequence similarity or physical interactions with other family members. The family includes both positive and negative regulators of apoptosis. Bax is a Bcl-2-related protein that has been shown to promote cell death. Bax and Bcl-2 are capable of forming both homodimers and heterodimers (Oltvai et al., 1993). Bax homodimers render a cell more susceptible to apoptosis, whereas binding of Bcl-2 to Bax neutralizes its activity and thus protects cells from apoptosis. It has been proposed that Bcl-2 negatively regulates apoptosis by sequestering the positive effector Bax (Yin et al., 1994). Another BCL-2 gene family member, BCL-x, produces two products by alternative splicing. The long product, Bcl-xL, is a negative regulator of apoptosis, whereas the short product, Bcl-xs, is a positive regulator of apoptosis. Like Bcl-2, Bcl-x, can inhibit cell death induced by growth factor withdrawal from an interleukin (IL)-3-dependentcell line (Boiseet al., 1993). Bclx, can also heterodimerize with Bax, but this interaction is not essential for its death-repressing activity (Cheng et al., 1996). Bcl-xs can antagonize cell death inhibition by both Bcl-2 and Bcl-x, (Boise et al., 1993). The positive regulator Bad can dimerize with both Bcl-2 and (more strongly) Bcl-x,. Bad can promote apoptosis by displacing Bax from Bcl-x, in vivo (Yang et al., 1995).Bak is another Bcl-2 homolog that can interact with both Bcl-2 and Bcl-x,. Bak can accelerate apoptosis following IL-3 withdrawal (Chittenden et al., 1995), but can also inhibit apoptosis in an Epstein-Barr virus-transformed cell line (Kiefer et al., 1995). A1 and Mcl-1 are Bcl-2-related proteins that are expressed during myeloid differentiation (Lin et al., 1993; Kozopas 1993). Mcl-1 can inhibit apoptosis induced by Myc overexpression (Reynolds et al., 1994) and can heterodimerize with Bax (Bodrug et al., 1995). A complex interplay of BCL-2 family members appears to be involved in the regulation of susceptibility to apoptosis. The evolution of such a large gene family in multicellular organisms suggests that there may be many regulatory pathways to cell death involving BCL-2 family members, perhaps related to the cell type or the nature of specific internal-external stimuli.

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B. p53 The p53 tumor suppressor gene directs cell cycle arrest following sublethal DNA damage, allowing DNA repair to take place prior to further replication. In the event of excessive DNA damage, apoptosis will follow cell cycle arrest (see reviews by Canman and Kastan, 1995; Cox and Lane, 1995).p53 has also been implicated in mediating the physiological response of hematopoietic cells to survival factors. Ectopic expression of wild-type p53 in a myeloid leukemia cell line, M1, induced apoptosis that could be inhibited by IL-6 (Yonish-Rouach et al., 1991). In the myeloid 32D cell line, endogenous p53 expression was shown to significantly accelerate apoptosis upon withdrawal of IL-3 (Blandino et al., 1995),and antisense p53 oligonucleotides reduced the level of apoptosis in factor-dependent leukemia cells after withdrawal of growth factor (Zhu et al., 1994). Reporter gene assays have suggested that p53 can influence the expression of BCL-2 family members. A p53 negative response element has been mapped in the BCL-2 gene, and a p53 binding site in the BAX gene can transactivate the BAX promoter (Miyashita et al., 1994; Miyashita and Reed, 1995). Thus p53 may promote cell death by increasing the Bax:Bcl-2 ratio. p53 has also been reported to induce Fas expression, which can increase cell susceptibility to apoptosis (Owen-Schaub et al., 1995). p53 is not required for glucocorticoid-mediated apoptosis of thymocytes (Clark et al., 1993), and cell lines that do not express p53 are still capable of undergoing apoptosis. Apoptosis can therefore proceed by both p53-dependent and p53-independent pathways.

C. MYC Expression of the c-MYC proto-oncogene is an immediate early response to mitogenic stimulation. MYC expression is maintained throughout the cell cycle and is rapidly downregulated following mitogen withdrawal, leading to cell cycle arrest in G1 (Marcu et al., 1992). Constitutive unregulated expression of c-MYC abolishes cell cycle arrest in fibroblasts following serum deprivation and results in concomitant proliferation and apoptosis (Evan et al., 1992). MYC-dependent apoptosis has also been demonstrated in factordependent myeloid progenitor cells. Failure to downregulate MYC prevented these cells from arresting in G1 after removal of IL-3, and accelerated the induction of apoptosis (Askew et al., 1991). Myc-dependent apoptosis requires dimerization with Max, yet apoptosis can proceed in the absence of protein synthesis in fibroblasts. This suggested that Myc must be continuously implementing the molecular machinery

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required for apoptosis, but that the execution of apoptosis is dependent upon other factors. It was subsequently shown that the insulin-like growth factors and platelet-derived growth factor could inhibit Myc-induced apoptosis in low serum (Harrington et al., 1994). Other studies have shown that Bcl-2, v-Abl, and mutant PS3 can inhibit apoptotic cell death induced by Myc and cooperate in cellular transformation (Bissonnette et al., 1992; Fanidi et al., 1992; Vogt et al., 1987; Lotem and Sachs, 1995a). Thus “survival signals” such as antiapoptotic cytokines or overexpression of a negative regulator of apoptosis are necessary to inhibit a default apoptosis program induced by Myc. The combination of deregulated Myc and survival signals promotes cell proliferation in the absence of apoptosis, and may explain the requirement for oncogene cooperation in cellular transformation by MYC (Green et al., 1994).

D. RAS Genes Proteins encoded by RAS gene family members have also been associated with both proliferation and apoptosis. These GTP-binding proteins play a critical role in signal transduction and the mediation of mitogenic responses. RAS genes can become oncogenic when activated by mutation, and have been shown to cooperate with other genes such as pS3, MYC, ElA, and SV40 in cellular transformation (Barbacid, 1987). The transcription factor interferon regulatory factor-1 (IRF-1) has also been found to cooperate in RAS transformation. Embryonic fibroblasts lacking the IRF-1 gene can be transformed by activated H-RAS. In the presence of IRF-1 cDNA, cells with activated RAS undergo apoptosis. These results suggest that IRF-1 may act like a tumor suppressor gene as it can mediate the death of an oncogene-activated cell (Tanaka et al., 1994). Another study has suggested that H-Ras can direct signals toward cell growth or apoptosis depending on the activity of protein kinase C. When protein kinase C activity is suppressed, expression of activated RAS induces apoptosis in a T-lymphoblastoid cell line. Apoptosis induced by Ras in this system could be blocked by Bcl-2 (Chen and Faller, 1995). Other groups have reported that activated forms of H-RAS, or overexpression of RAS, can have a protective effect on apoptosis (Lin et al., 1995; Arends et al., 1993; Moore et al., 1993). The effects of H-RAS may therefore be largely dependent on the presence of other proteins. R-Ras is a Ras-related protein that was isolated in a screen for BCL-2-interacting proteins (Fernandez-Sarabia and Bischoff, 1994). R-Ras has been shown to promote apoptosis in growth factor-deprived cells by a mechanism that is suppressible by Bcl-2 (Wang, H.-G., et al., 1995).

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E. Abl Tyrosine Kinases Several lines of evidence suggest that the Abl tyrosine kinase is a negative regulator of apoptosis (reviewed in Samali et al., 1996). Both v-Abl and Bcr-Abl can rescue growth factor-dependent cell lines from apoptosis induced by factor withdrawal (Rovera etal., 1987; Daley and Baltimore, 1988; Hariharan et al., 1988). Both kinases have also been shown to protect myeloid cells from apoptosis induced by a variety of cytotoxic drugs (McGahon et al., 1994; Bedi et al., 1994) and Fas-mediated apoptosis (McGahon et al., 1995b).Bcr-Abl has been reported to prevent apoptosis in factordependent murine hematopoietic cells by inducing a Bcl-2 expression pathway. Bcr-Abl-expressing cells reverted to factor dependence and nontumorigenicity after Bcl-2 expression was suppressed (Sanchez-Garcia, 1995).Another study has suggested that the inhibition of apoptosis by v-Abl is independent of both Bcl-2 and Bax expression (McGahon et al., 1995a). Expression of Bcr-Abl in myeloid cells was found to induce the accumulation of GTP-bound Ras (Mandanas et al., 1993). The inhibition of Ras function with the Gap carboxyl terminus or dominant negative Ras impaired the transforming activity of Bcr-Abl and v-Abl, suggesting that the deregulation of Ras is required for the oncogenic activity of Abl (Sawyers et al., 1995). Bcr-Abl has been shown to physically interact with a number of proteins known to influence Ras activity, including GAP, Grb-2, and Shc (Druker et al., 1992; Tauchi et al., 1994; Matsuguchi et al., 1994; Pendergast et al., 1993).

F. FadApo-1 The Fas antigen (also known as Apo-1 and CD95) is a 48-kDa cell surface protein that belongs to the tumor necrosis factor (TNF) and nerve growth factor family of surface receptors (Smith et al., 1994). Stimulation of the Fas receptor by its natural ligand (FasL), or with anti-Fas antibodies, induces apoptosis. The oligomerization of an 80-amino-acid cytoplasmic “death domain” in Fas and TNF-R1 receptors is essential for receptor function. A number of novel proteins, including FADD, MORT1, FAF-1, and RIP, have been shown to associate with death domains and are thought to transmit death-inducing signals to intracellular signaling pathways (reviewed by Peter et al., 1996). Biochemical events that have been implicated in Fas signaling include activation of phosphatidylcholine-specific phospholipase C (Cifone et al., 1995), production of ceramides by an acidic sphingomyelinase (Cifone et al., 1994), and activation of IL-1 converting enzyme (ICE)-like protease (Enari et al., 1995; Los et al., 1995).

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Fas is widely expressed on normal and malignant hematopoietic cells, and on cells outside the immune system. Its function on nonhematopoietic cells remains to be established as the consequences of Fas deficiency are primarily manifested in the immune system (see later sections).

G. ICE-Related Cysteine Proteases Two genes were found to be essential for the 131 deaths that occur during normal development of Caenorhabditis elegans: CED-3 and CED-4. Numerous homologs of CED-3 have now been identified in mammalian cells, including ICE, Nedd2, CPP32, ICErel,,/TX/Ich-2, ICE,,, and Mch2 (reviewed in Kumar, 1995; Pate1 et al., 1996; White, 1996). These proteins are all cysteine proteases, which are initially produced as inactive precursors and are activated by proteolytic cleavage. Overexpression of these proteases has been shown to induce apoptosis in a variety of cell types (see reviews and references therein). Substrates for ICE-like proteases include pro-interleukin 1 (cleaved by ICE), U1-70kDa small ribonucleoprotein (cleaved by CPP32), and poly(ADP) ribose polymerase (PARP; cleaved by CPP32). The proteolytic cleavage of PARP may be required to prevent its DNA repair activity in apoptotic cells, although evidence from PARP-deficient mice suggests that this activity is dispensable for apoptosis (Wang, Z.-Q., et al., 1995).The key effector substrates for these enzymes remain to be identified.

,,,,

H. Lessons from Wruses A number of viruses have evolved mechanisms of circumventing their host cells’ apoptotic machinery. The DNA tumor virus, adenovirus, produces an oncoprotein (ElA) that can by itself initiate foci formation; however, most of the cells generated subsequently die by apoptosis (Rao et al., 1992). The ability of E1A to induce apoptosis has been shown to be heavily dependent on p53. Cells transformed with E1A and a temperature-sensitive p.53 proliferate at the restrictive temperature when p53 is inactive, and undergo apoptosis at a permissive temperature when p53 is predominately wild type (Debbas and White, 1993). E1A has been shown to bind to the retinoblastoma susceptibility gene product Rb, resulting in the release of the transcription factor E2F (Nevins, 1992). E2F will activate the transcription of genes required for proliferation, including MYC, at the appropriate time in the cell cycle (Oswald et al., 1994; Johnson et al., 1993). The ability of E1A to direct apoptosis is thought to be related to its ability to cause the release of E2F. Enforced overexpression of E2F in the presence of wild-type p53 has

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been shown to induce both DNA synthesis and apoptosis (Qin et al., 1994; Wu and Levine, 1994). Adenovirus also produces two additional proteins that inhibit p53-mediated apoptosis. E1B 55K directly associates with p53 (Yew and Berk, 1992), whereas E1B 19K blocks apoptosis downstream of p53 activation. E1B 19K can interact with Bax and is thought to inhibit apoptosis by a mechanism similar to that of Bcl-2 (Han et al., 1996) (see Fig. 1). Human papillomavirus (HPV) encodes a protein product, E7, that also promotes cellular proliferation through its interaction with Rb. An additional HPV protein, E6, inhibits apoptosis by promoting the intracellular degradation of p53 (White et al., 1994). Other antiapoptotic genes encoded by viruses include the Epstein-Barr virus BHRFl protein, which is a Bcl-2 homolog (Henderson et al., 1993); the CrmA protein from cowpox virus, which inactivates the ICE protease (Ray et al., 1992); and the p35 and IAP proteins from baculoviruses, which are also thought to inhibit the activity of proteases (Clem et al., 1996). The existence of antiapoptotic genes in viruses implies that their host cells are not easy victims, and that it is undoubtedly preferable to the organism as a whole if they become martyrs. This propensity to undergo apoptosis in response to cellular corruption is facilitated by the close association between proliferation and apoptosis pathways. It is possible that physiological mitogenic signals may also feed into at least two pathways to induce proliferation. For example, activation of Ras in combination with active protein kinase C will allow proliferation, whereas activation of Ras alone will induce apoptosis (see Section 1.D). Alternatively, the antiapoptotic signal may be provided by independent “survival factors” as demonstrated for Myc (see Section 1.C). The requirement for accompanying survival signals may provide a cellular protection mechanism against oncogene deregulation.

IV. FUNCTIONAL ASPECTS OF APOPTOSIS IN THE HEMATOPOIETIC SYSTEM The extensive cell turnover in the mammalian hematopoietic system requires stringent physiological control of cell death. The expansion and differentiation of new cell populations is continually counterbalanced by apoptotic cell death. Various functional aspects demand selectivity in the generation of cell populations, and a considerable degree of flexibility in population size control in the event of an immune response. The life span of all hematopoietic cells is regulated by a combination of internal genetic programs and external survival factors. The molecular mechanisms that propel

I Proliferation I

m

I

W

N

Mutant

Gene transcription Myc Cell cycle deregulation

IIIIIIIIIIIIIIII

'*n Wild type

v) W

z

a-

r

nl

a

Fig. 1 Cell cycle corruption by adenovirus. Cell transformation by adenovirus depends on both the promotion of proliferation and the inhibition of apoptosis. The product of the E1A gene binds to the retinoblastoma susceptibility gene product Rb, resulting in the release of the transcription factor E2F. Heterodimerization of E2F with DP-1 generates an active transcription complex that will activate genes required for proliferation. In the presence of wild-type p53, cell cycle deregulation will induce apoptosis. Adenovirus produces two additional proteins that inhibit p53-mediated apoptosis. E1B 55K directly associates with p53, whereas ElB 19K interacts with Bax and blocks apoptosis downstream of p53 activation.

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a terminally differentiating cell toward death, or those that translate receptor signals into viability or death, are largely unknown. Transfection studies with apoptosis regulating genes and studies with transgenic animals have helped to define some important molecular players.

A. Survival Factors and Population Control An increasingly popular concept with regard to population size regulation is the “social control hypothesis,” which suggests that all cells express a default apoptosis program and will undergo cell death unless directed not to do so by survival signals (Raff, 1992). This mechanism would confine specific cell types to the tissue producing the required set of survival factors, and would limit the population size according to the availability of survival factors. Evidence suggests that this mechanism may be important in the homeostatic regulation of hematopoietic cell populations. Cytokines that have been shown to promote the viability of differentiated myeloid cells include granulocyte colony-stimulating factor (G-CSF),granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (1FN)y, TNF, IL-1, and IL-2 (Lopez et al., 1986; Mangan and Wahl, 1991; Hogquist et al., 1991; Lotem and Sachs, 1995b). Many of these cytokinesfor example, the colony-stimulating factors (CSFs)-are also capable of inducing proliferation; however, several studies have suggested that growth and viability are separately regulated processes. IL-1, IL-6, and Steel factor can promote viability in immature myeloid cells without inducing proliferation (Lotem and Sachs, 1990; Caceres-Cortes et al., 1994), and viability of mature neutrophils can be promoted by CSFs in the absence of proliferation (Begley et al., 1986). If the expansion of each of the various subpopulations of hematopoietic cells is dependent upon a specific subset of viability factors, then the concentrations of these factors will dictate the composition of the mature hematopoietic population (see Fig. 2). The requirement for survival signals may also provide a flexible mechanism for the expansion and retraction of cell populations during and after an immune response. For example, several proinflammatory cytokines, including IL-lp, TNF-a, GM-CSF, and IFN-y, were shown to prevent monocyte apoptosis when sufficient levels of these cytokines were maintained in culture. Monocytes will also produce IL-1 and TNF-a when activated, suggesting that both autocrine and paracrine mechanisms may influence cell survival. A local decrease in cytokines, as would occur during the resolution of inflammation, could then be sufficient to induce apoptosis in the expanded population of monocytes and macrophages (Mangan and Wahl, 1991).

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Erythropoietin

Myeloid compartment

Lymphoid compartment

GM-CSF, G-CSF

Fig. 2 Survival factors and population control. The “social control hypothesis” suggests that all cells express a default apoptosis program and will undergo cell death unless directed not to do so by survival signals (Raff, 1992). This mechanism would limit population sizes according to the availability of survival factors. If the expansion of each the various subpopulations of hematopoietic cells is dependent upon a specific subset of viability factors, then the concentrations of these factors will dictate the composition of the mature hematopoietic population. Thus the stem cell population size will be dictated by the availability of survival factors such as IL-3, GM-CSF, G-CSF, and Steel factor. Early committed progenitor cells would also be dependent on these and other cell type-specific factors. Erythroid-, myeloid-, and lymphoid-specific survival factors will dictate the boundaries of their respective populations. Cell production that is in excess of available survival factors will result in apoptosis.

B. Survival Factors and Stem Cell Viability The survival of hematopoietic progenitor cells is dependent upon cell-cell interactions in the microenvironment of the bone marrow, and on the presence of the CSFs IL-3, G-CSF, and GM-CSF (Osmond, 1993; Williams et al., 1990; Rodriguez-Tarduchy et al., 1990). Erythropoietin (Epo) is also an important survival factor for erythroid progenitor cells (Koury and Bondurant, 1990; Spivak et al., 1991). It has been suggested that Epo levels are insufficient for the majority of the Epo-dependent progenitors, and that a minority of the erythroid progenitor cells are responsible for normal erythroid production. Apoptosis is therefore considered to be a major component of normal erythropoiesis (Koury and Bondurant, 1990). A number of cytokines, including IFN-a/P, IFN-y, TNF-a, and trans-

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forming growth factor-pl (TGF-pl), have been shown to negatively regulate progenitor cell viability. CD34+ cells exposed to IFN-y or TNF-a were found to upregulate Fas expression on their cell surfaces, suggesting that the ligation of the Fas receptor is responsible for the induction of apoptosis by these negative regulators (Nagafuji et al., 1995; Maciejewski et al., 1995a). The inhibitory hematopoietic effects of IFN may be at least partially mediated by the transcription factor IRF-1 (Sato et al., 1995), and inducible nitric oxide synthase has been suggested to be an important mediator of cytokine-induced hematopoietic suppression (Maciejewski et al., 1995). Bcl-x, is expressed in immature hematopoietic cells and is prevalent in primitive hematopoietic precursors (Park et al., 1995). In contrast to mice deficient in BCL-2, which can progress through development, BCL-x, Knockout mice die at embryonic day 13, suggesting that BCL-X, may be an important survival gene in the early stages of hematopoiesis (Motoyama et al., 1995).

C. Survival Factors and Myeloid Differentiation While the importance of hematopoietic cytokines in mediating cell viability is now well established, their role in mediating differentiation is becoming increasingly controversial. Three models have been proposed regarding the role of cytokines in hematopoietic differentiation. The instructive model suggests that cytokines induce differentiation and direct lineage commitment, whereas the stochastic model suggests that cytokines permit the viability and proliferation of intrinsically committed cells. The hybrid model, as its name suggests, predicts that a combination of both external cytokines and internal genetic programs direct lineage commitment. In order to establish the authenticity of one of these models, several groups have transfected “antiapoptotic” genes into hematopoietic progenitor cells and then examined their ability to undergo differentiation in the absence of cytokines. Transfection of BCL-2 into a multipotent hematopoietic cell line (FDCPMix) alleviated the cells’ growth factor dependency. Morphological examination found that the transfected cells were capable of undergoing multilineage differentiation in serum-deprived conditions, suggesting that growth factors do not direct differentiation but are important for maintaining cell viability (Fairbairn et al., 1993). Another group generated both BCL-2 and BCL-x transfectants from the multipotent cell line 32Dc13 (Rodel and Link, 1996). 32D-Bcl-2 and 32D-Bcl-x clones acquired some of the features of granulocytic differentiation, namely, nuclear fragmentation, but were unable to produce myeloperoxidase in the absence of G-CSF. Rodel and Link’s data suggest that complete maturation of 32D cells to granulocytes is dependent upon both intrinsic cellular programs and the presence of G-CSF.

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The 32Dc13 cell line has also been transfected with the Bcl-2 homolog A1 (Lin et al., 1996). A1 was originally isolated from a cDNA library prepared from differentiating bone marrow cultures (Orlofsky et al., 1991). The same group also transfected the 32Dc13 cell line with BCL-2 and generated another transfectant that contained both A1 and BCL-2. In the absence of growth factor, the A1 transfectants exhibited granulocytic maturation and produced myeloperoxidase. In contrast, the BCL-2 transfectant and BCL-2-A1 double transfectant showed relatively few morphologically mature cells and a complete absence of myeloperoxidase. Thus the effect of Bcl-2 was dominant to that of A1 and inhibited complete maturation. Previous studies have suggested that Bcl-2 is downregulated in myeloid cells that have been induced to differentiate (Hockenbery et al., 1991; Naumovski and Cleary, 1994), whereas A1 expression in 32D cells may be upregulated by G-CSF (Lin et al., 1993). The expression of both A1 and BCL-2 transcripts was therefore compared in differentiating 32D cells by the Lin et al. group. Bcl-2 expression started to decline after 2 days in culture with G-CSF; in contrast, A1 mRNA levels increased throughout differentiation. Myeloperoxidase activity was also induced from day 2 onward. Why differentiating cells should switch from one survival regulator to another is unclear. It was suggested that it may be due to changes in apoptotic stimuli likely to be encountered by differentiating cells, or it may reflect some incompatibility of Bcl-2 activity with later developmental stages (Lin et al., 1996). The reasons behind this switch may become clearer with a better understanding of the biochemical functions of Bcl-2 family members. What is clear from these studies is that the effects of G-CSF can be effectively mimicked by Bcl-2 family members, thus favoring a stochastic model for the role of cytokines in differentiation. This does not exclude the possibility that cytokines may play an important role in directing early lineage commitment. Several groups have investigated the importance of other regulators of apoptosis in myeloid cell lines and in normal hematopoietic cells. Expression of c-Myc was shown to be high in proliferating myeloblasts and HL60 cells but dropped upon differentiation (Liebermann and Hoffman-Liebermann, 1989; Collins, 1987), suggesting that c-Myc may not be required for apoptosis following terminal differentiation. Mature human neutrophils (but not monocytes or eosinophils) express Fas ligand on their surface and are susceptible to Fas-induced cell death. The coexpression of both Fas and FasL may contribute to their relatively rapid turnover (Lilesand Klebanoff, 1995). The viability of myeloid cells can therefore be influenced by a variety of cytokines and gene products. Further research is necessary to establish the possible interactions between the extracellular survival signals and internal survival genes.

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D. T-cell Differentiation T lymphocytes are responsible for antigen-specific cell-mediated immunity. The T-cell receptor (TCR) recognizes small peptides contained within the cleft of antigen-presenting molecules called the major histocompatibility complex (MHC) on the surfaces of other cells. CD8-expressing T cells (mostly killer T cells) recognize peptides contained within MHC class I molecule clefts, and CD4-expressing T cells (mostly helper cells) recognize peptides contained in clefts of MHC class I1 molecules. The TCR complex is associated with the CD3 complex, which couples the binding of antigen-MHC to intracellular signaling. Activated CD4+ T cells secrete cytokines that mediate the B-cell immune response, T-cell inflammation, and differentiation of killer T cells. Immature T cells originate in the bone marrow and migrate to the thymus, where they undergo gene rearrangements, proliferation, and differentiation. T cells that recognize self-MHC I and I1 molecules are positively selected (Von Boehmer, 1994), and T cells that recognize self-MHC plus other selfantigens are eliminated by negative selection (Nossal, 1994).Extensive apoptosis in the thymus ensures that the only T cells that reach the periphery are those that have produced receptors that recognize self-MHC molecules harboring non-self-antigens. A number of studies have demonstrated that autoreactive immature T cells are induced to undergo apoptosis by the same mechanism that activates mature cells, that is, ligation of the TCR-CD3 complex with specific antibodies (Smith et al., 1989; McDonald and Lees, 1990; Shi et al., 1991). The mechanism by which the same signal produces different effects in immature and mature cells is the subject of intensive investigation. One line of evidence suggests that the coupling of TCR and CD3 is incomplete in immature cells. In both cell types, antibodies to the total TCR-CD3 signaling complex induce a large calcium ion flux, whereas anti-TCR antibodies only induce a calcium flux in mature T cells and not immature thymocytes (Finkel et al., 1989). Other studies employing transgenic or “knockout” mice suggest that auxiliary molecules such as CD4, CD8, CD28, and leukocyte function-associated antigen-1 participate in the clonal deletion of autoreactive thymocytes (Fung-Leung et al., 1991; Rahemtulla et al., 1991; Punt et al., 1994; Carlow et al., 1992). CD45 has also been shown to play an important auxiliary role in the positive selection of thymocytes (Kishihara et al., 1993). Recent studies have suggested that the orphan steroid receptor Nur77 plays a critical role in TCR-mediated cell death. Nur77 is expressed at high levels in T-cell hybridomas and apoptotic thymocytes, but not in growing T cells or stimulated splenocytes. The use of a dominant negative mutant (Woronicz et al., 1994) and antisense Nur77 constructs (Liu et al., 1994)

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demonstrated that Nur77 was essential for activation-induced cell death but not for apoptosis induced by glucocorticoids. Downregulation of Myc with antisense oligonucleotides demonstrated that Myc expression is also required for activation-induced apoptosis in a T-cell hybridoma but does not affect another outcome of activation: the production of lymphokines (Shi et al., 1992). BCL-2 gene family members may also influence thymocyte viability. Bcl-2 is expressed in developmentally early thymocytes but diminishes at the stage of negative selection (Veis et al., 1993). In addition Bcl-xs peaks at the same stage at which Bcl-2 is downregulated (Boise et al., 1993). In transgenic mice overexpressing Bcl-2 in T cells, self-reactive T cells showed prolonged survival but eventually died by negative selection. T cells also showed resistance to apoptosis induced by radiation, glucocorticoids, and anti-CD3 (Strasser et al., 1991a). In BCL-2 knockout mice, both B and T lymphocytes are severely depleted through excessive apoptosis (Nakayama et al., 1993). Fas expression can influence mature T-cell viability. Loss of function mutations in lpr or gld mice result in severe lymphoproliferative disorders. Despite high expression of Fas in the thymus, mutations in these genes do not appear to affect elimination of self-reactive T cells in the thymus. A deficiency in Fas does, however, affect activation-induced cell death of autoreactive T cells in the periphery (discussed later in Section V.B, and reviewed in Rathme11 and Goodnow, 1995).

E. Cytotoxic T Lymphocytes Cytotoxic T lymphocytes may induce apoptosis in target cells by two separate pathways, secretory and ligand induced. In the secretory pathway, cytotoxic granules containing the lytic protein perforin and a series of serine proteases (granzymes) are released from the lymphocyte and enter the target cell through pores produced in the target cell’s membrane by perforin. Apoptosis is then initiated by an as-yet unknown mechanism. Ligand-induced cell death results from cross-linking Fas receptors on target cells with FasL expressed on the surfaces of cytotoxic lymphocytes (reviewed in Berke, 1995).

F. B-Cell Differentiation B lymphocytes are the principal mediators of the specific humoral response to infection. B-lymphocyte maturation progresses through antigen-independent and antigen-dependent stages. Antigen-independent stages of differentiation take place in the bone marrow and are influenced by stromal interactions and hematopoietic growth factors. Immunoglobulin heavy chain and

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light chain genes are rearranged at this stage, and successful rearrangements result in the surface expression of immunoglobulin (Ig) M. B-cell precursors that fail to produce IgM undergo apoptosis in the bone marrow. The IgM+ B cell then migrates from the bone marrow to the periphery (spleen and lymph nodes), where it undergoes antigen-dependent differentiation. Immature B cells expressing receptors that bind to self-antigens become functionally silent (anergy) or undergo apoptosis. Further maturation of the IgM+ B cell results in the coexpression of IgD. Activation of mature B cells by antigen leads to proliferation and differentiation into antibody-secreting plasma cells or memory B cells, depending on the presence of T-cell-derived cytokines. Thus B cells also have a maturation-dependent response to ligation of their receptors. Less is known about how these responses (i.e., death, anergy, or activation) are mediated in B cells, but the presence of auxiliary molecules is also thought to play an important role. Mature B cells may escape apoptosis following stimulation with cytokines (Holder et al., 1992) or by ligation 6f other surface molecules such as CD40 ligand on activated helper T cells (Tsubata et al., 1993; Clark and Ledbetter, 1994), or CD2 (reviewed in Baixeras et al., 1994). Bcl-2 expression has been implicated in mediating B-cell survival. Overproduction of Bcl-2 can prevent apoptosis of B cells in vitro (Nunez et al., 1990). In addition, many of the stimuli that rescue B cells from apoptosis, including CD40 and CD2, also upregulate Bcl-2 expression (Liu et al., 1991; Baixeras et al., 1994).Targeted overexpression of Bcl-2 in mice extends normal B-cell survival (Nunez et al., 1991)and produces B-cell lymphomas (McDonne11 and Korsmeyer, 1991). Bcl-2 overexpression has been reported not to affect deletion of bone marrow B cells, but to inhibit clonal deletion of self-reactive B cells in the periphery (Nisitani et al., 1993). Mice with loss of function mutations in BCL-2 (knockout mice) can progress through development but have severe immune function failure due to loss of mature B and T cells (Kamada et al., 1995; Nakayama et al., 1993). Ligation of CD40 rapidly induced the appearance of the Bcl-x, protein in B-cell lymphoma cells and rendered the cells refractory to anti-Ig-induced cell death (Choi et al., 1995). Peripheral blood B cells were also shown to upregulate Bcl-x, upon surface immunoglobulin (IgM) cross-linking, CD40 signaling, or lipopolysaccharide stimulation (Grillot et al., 1996). Chimeric mice that lacked Bcl-x expression in the lymphoid system indicated that Bclx expression is required for maintaining the life span of immature but not mature lymphocytes (Motoyama et al., 1995).Bax-a mRNA and protein expression were shown to be upregulated upon sIg induction of apoptosis in a human B-cell line (Bargou et al., 1995). BAX knockout mice show normal development but display lymphoid hyperplasia consistent with a role for Bax in the promotion of apoptosis (Knudson et al., 1995). Another study has investigated the role of Bcl-2 homologs in mediating the

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survival of human peripheral blood B lymphocytes. Mcl-1 expression decreased in cells undergoing apoptosis in medium, and even more markedly in cells treated with the apoptotic stimuli TGF-(3 or forskolin (an activator of adenylyl cyclase). In contrast, the survival signals IL-4, anti-IgM antibodies, and 12-0-tetradecanoyl phorboll3-acetate (TPA) prevented the decline in Mcl-1 levels. The levels of Bcl-2, Bcl-x, and Bax did not correlate with cell survival in this system (Lomo et al., 1996). Expression of Fas has been implicated in the elimination of self-reactive B lymphocytes in a state of anergy. When these cells reach the spleen, they are killed by T cells in a manner that is dependent upon Fas expression by B cells and FasL expression by T cells (Rathmell et al., 1995; Rathmell and Goodnow, 1995).

G. Affinity Maturation During T-cell-dependent antibody responses, antigen-specific B cells undergo affinity maturation in the germinal centers (GCs). Somatic hypermutation in rapidly proliferating cells creates a greater heterogeneity in antigen binding specificity. B cells with a high affinity for antigen are positively selected for further maturation into memory cells; nonselected cells die by apoptosis. Interactions with cell adhesion molecules (CD1l d C D 18-ICAM-1, CD49d-VCAM1)on follicular dendritic cells (Koopman et al., 1994) or CD40 on T cells (Foy et al., 1994; Korthauer et al., 1993) have been reported to influence B-cell rescue. An analysis of gene expression in GC centroblasts found high levels of cMyc, p53, Bax, and Fas in the absence of Bcl-2, suggesting that the predominance of positive regulators of apoptosis may contribute to the increase in spontaneous apoptosis in the GC (Martinez-Valdez et al., 1996).

H. Downregulation of an Immune Response After an immune response has served its purpose, the expanded population of lymphocytes returns to basal levels. This population shrinkage may be a result of decreased levels of survival factors (as discussed in Section IV.A) or increased levels of negative regulators such as TNF (Zheng et al., 1995). Alternatively, the upregulation of Fas on activated B and T lymphocytes may make a major contribution to their deletion (Vignaux and Golstein, 1994; Dhein et al., 1995). An examination of the development and maintenance of the effector cells in the hematopoietic system reveals an extraordinary array of physiological mechanisms that are responsible for either inducing or preventing apoptosis. The variety of external signals suggests that there may be multiple path-

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ways to cell death. Of particular interest is the apparent ease with which cells can change the expression of molecular “apoptosis regulators” throughout their differentiation, and thus alter their intrinsic susceptibility to apoptosis. It is likely that malignant hematopoietic cells also avail of such mechanisms in order to escape physiologically induced or drug-induced apoptosis.

V. DISRUPTION OF APOPTOSIS IN HEMATOPOIESIS It is evident that the regulation of apoptosis is central to the development and function of the hematopoietic. system. Deregulation of apoptosis in hematopoiesis has been shown to play a major role in the pathogenesis of a variety of well-known disorders, including inflammatory diseases, autoimmune disorders, acquired immunodeficiency syndrome (AIDS), and hematological malignancies. This section briefly discusses the contribution of deregulated apoptosis to the former disorders, and then examines in some detail the role of antiapoptotic events in the development of hematological malignancies.

A. Inflammation Accumulation of eosinophilic granulocytes has been associated with a variety of chronic inflammatory disease, particularly in allergic manifestations such as bronchial asthma and atopic dermatitis (Holgate, 1993; Kroegel et al., 1994).Inhibition of apoptosis has recently been implicated as a key pathogenic event in eosinophilia (Simon and Blasser, 1995). The hematopoietins IL-3, IL-5, and GM-CSF have been shown to prolong eosinophil survival and abrogate apoptosis. Conversely, the negative regulator TGF-p can abrogate the survival effects of these hematopoietins, and the autocrine production of GM-CSF and IL-5 by eosinophils (Alam et al., 1994). The role of TGF-p in the resolution of inflammation is evident from the fatal inflammatory cell response and tissue necrosis in TGF-p knockout mice (Shull et al., 1992). Other negative regulators of apoptosis (TNF-a, IFN-y) have been shown to induce Fas expression on their target cells (Nagafuji et al., 1995). In addition, cytokine-deprived eosinophils have been shown to upregulate expression of Fas antigen, suggesting that the induction of apoptosis in these cells may be mediated by a Fas pathway (Druilhe et al., 1996). It is possible that a decreased responsiveness to TGF-p or a decrease in susceptibility to Fas-mediated apoptosis may contribute to eosinophilia. Alternatively, eosinophilia may be a consequence of a defect in another cell type. For example, high numbers of CD4-CD8- T cells were found in two patients with associated hypereosinophilia. One patient had idopathic hypereosinophilic

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syndrome and the other was infected with human immunodeficiency virus (HIV)-1. The CD4-CD8- T cells from both patients were highly activated and excessively produced the eosinophil survival factors IL-3, IL-5, and GMCSF. The activated T cells did not, however, express functional Fas. In one case Fas mRNA was absent, whereas in the other soluble Fas molecule antagonized normal signaling of Fas antigen. Thus defective Fas-mediated apoptosis in T cells resulted in the sequential deregulation of apoptosis in eosinophils (Simon et al., 1996).

B. Autoimmunity Two mouse models of systemic autoimmunity, l p and ~ gld mice, were found to have reduced expression of Fas or a mutation in FasL, respectively (Nagata and Suda, 1995). T-cell-specific expression of wild-type Fas protein in lpr mice can abrogate the onset of the autoimmune disorder (Wu and Levine, 1994). The lpr mice produce autoantibodies against DNA and chromatin, similar to the human systemic lupus erythematosus (SLE) disorder, but also show lymphadenopathy and expanded populations of CD4-CD8- T lymphocytes, which is not a feature of SLE. More recently, Fas mutations have been identified in children with systemic autoimmune disease that has symptoms similar to those in lpr mice, including lymph node enlargement with CD4-CD8- T lymphocytes (Rieux-Laucat et al., 1995; Fisher et al., 1995). Evidence suggests that autoreactive antibodies are not all removed in the thymus, and that Fas-induced apoptosis is important for their deletion in the periphery. Normally, when CD4+ T cells are activated by antigen binding in the periphery, they rapidly divide to form an expanded clone. In the absence of costimulatory signals (e.g., IL-2; triggered by the presence of microorganisms), the T cell will undergo apoptosis by an autocrine or paracrine process involving the simultaneous expression of Fas and FasL. A deficiency in Fas would allow autoreactive T lymphocytes to continue proliferating unchecked and thus have the potential to promote autoantibody production by self-reactive, Fas-deficient B cells (reviewed by Rathmell and Goodnow, 1995). While Fas clearly plays a major role in the pathogenesis of autoimmune disease, the expression of dominant negative Fas mutations in disease-free adults suggests that other factors may contribute to the severity of autoimmunity (Rieux-Laucat et al., 1995; Fisher et al., 1995).

C. Acquired Immunodeficiency Syndrome The progressive depletion of CD4+ T cells is a central feature in the pathogenesis of AIDS. It has been proposed that cell depletion in HIV-infected in-

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dividuals is due to the inappropriate induction of apoptosis in response to activation signals that would normally promote survival, differentiation, or proliferation (reviewed by Gougeon,l995; Ameisen et al., 1995a; Zagury et al., 1995). The expression of a viral envelope transmembrane gp120-gp41 complex, and subsequent binding to the CD4 receptor of uninfected T cells, has been shown to directly induce apoptosis (Ameisen, 1992). In addition, HIV particles release gp120, which can bind CD4 and prime uninfected T cells for apoptosis in response to T-cell activation (Gougeon and Montagnier, 1993). The consequence of T-cell apoptosis is an inability to mount an immune response upon antigen challenge.

D. Apoptosis in the Acquisition of Malignancy The development of hematological malignancy has long been regarded as a multistep evolution involving the acquisition of multiple cooperating genetic lesions. New insights into viral transformation, and various studies of in vitro cell transformation employing cooperating proliferative and antiapoptotic oncogenes, have provided a rationale for this requirement. Evidence suggests that the corruption of a cell’s proliferative machinery will automatically direct the cell to undergo apoptosis in the absence of a secondary survival signal. If survival signals are normally provided by external viability factors, then the concentration of these factors will always constrain any dividing cell population. Overriding a cell’s natural propensity to undergo apoptosis must therefore be an important step in the development of any malignancy. The mechanisms by which this is achieved will undoubtedly depend on the components of survival pathways in any given cell type. For example, a cell may undertake the autocrine production of its own survival factor, or alterations in cell surface receptors could result in the constitutive activation of a survival pathway. Alternatively, the cell may block apoptosis further downstream by upregulating negative regulators of apoptosis, such as Mcl1, Bcl-xL, or Bcl-2. What follows is an overview of studies suggesting that genetic changes that deregulate survival factors (apoptosis genes or cytokines) may play a significant role in the pathogenesis of human leukemia. 1. B-CELL LYMPHOMA

The first indication that deregulation of apoptosis may be important in the development of hematological neoplasia came from studies with the BCL-2 proto-oncogene. The BCL-2 gene is overexpressed as a result of the t(14:18) chromosome translocation found in most follicular lymphomas and some diffuse large-cell lymphomas. The contribution of Bcl-2 to the development of neoplasia has been assessed in transgenic mice. Expression of high levels

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of Bcl-2 initially resulted in a polyclonal lymphoid hyperplasia. The development of clonal B lymphomas after a long latent period (- 15 months) was associated with secondary genetic changes. Half of the high-grade lymphomas also had a c-MYC translocation. Direct evidence for the cooperation of MYC and BCL-2 was provided by MYC-BCL-2 double-transgenic mice. These mice rapidly developed malignant lymphomas that appeared to be derived from a lymphomyeloid progenitor cell (reviewed by Cory et al., 1994). Mice transgenic for MYC alone did not show hyperplasia despite an augmentation in proliferation. The increased rate of proliferation was effectively counterbalanced by an increased rate of cell death (Langdon et al., 1986). Thus a genetic lesion that promotes cell viability can interfere with apoptosis induced by a deregulated oncogene, and facilitate the development of a malignant lymphoma. 2. CHRONIC MYELOID LEUKEMIA Chronic myeloid leukemia (CML) is a disease characterized by the progressive accumulation of myeloid cells in the peripheral blood and bone marrow. During the indolent stage of the disease (chronic phase), functionally and morphologically mature myeloid cells comprise the majority of the population. The increase in mature myeloid cells is considered to be a consequence of an expanded population of progenitor cells. In particular the numbers of CFU-GM (colony-forming units committed to the granulocyte and monocyte lineage) circulating in the peripheral blood is increased 100-fold (Lepine and Messner, 1983). Chronic-phase CML is relatively easy to treat, and many patients maintain a near-normal quality of life. After a median interval of approximately 4 years, the disease becomes more aggressive, and the myeloid cells lose their ability for terminal differentiation. The rapid accumulation of blasts, which may be of myeloid, lymphoid, erythroid, or megakaryocytic lineages, eventually leads to fatal bone marrow failure. This stage of the disease is referred to as blast crisis and is morphologically similar to acute leukemia. About one third of patients undergo a lymphoblastic transformation that resembles acute lymphoblastic leukemia; the majority undergo transformation to acute myeloid leukemia. Secondary acute leukemias derived from CML are notoriously difficult to treat, and survival at this stage rarely exceeds 12 months (Spiers, 1992). The hallmark of CML, found in over 95% of patients, is the Philadelphia chromosome, which results from a reciprocal translocation between chromosomes 9 and 22. This translocation produces the BCR-ABL fusion gene and constitutive expression of the Bcr-Abl tyrosine kinase. Various studies with transfected cell lines and transgenic or transplanted animals have examined the role of Bcr-Abl in the etiology of CML. Enforced expression of BCR-ABL cDNA in long-term bone marrow cultures conferred a growth

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advantage and allowed clonal population expansion. The cells notably lacked malignant potential and were unable to form tumors in nude mice (McLaughlin et al., 1987; Young and Witte, 1988). This phenotype could be regarded as a good approximation to chronic-phase CML (i.e., a growth advantage in the progenitor cell pool). A number of groups have demonstrated that overexpression of Bcr-Abl in mice induces a CML-like illness (Gishizky et al., 1993; Daley et al., 1991; van Etten, 1993). In one study, the transplanted mice could be divided into early- and late-onset groups, both of which exhibited granulocytic hyperplasia but appeared to represent distinct disease types, analogous in many respects to the chronic and acute phases of human CML. Only cells from the late-onset CML syndrome could efficiently propagate the disease when transplanted into secondary and tertiary recipient mice. The inability to transplant the disease from mice developing the early-onset CML-like syndrome suggested that this disorder may originate from progenitor cells with limited replication capacity that have undergone clonal expansion but are not immortalized (Gishizky et al., 1993). The gradual expansion of cell populations with limited replication capacity is consistent with studies that suggest that Bcr-Abl can negatively influence the ability of cells to undergo apoptosis, and in this capacity it can cooperate with a variety of other oncogenes in cellular transformation (e.g., myc; see Sections 1II.C and 1II.E). Thus the constitutive antiapoptotic activity of the Bcr-Abl tyrosine kinase may play a major role in expanding the progenitor cell compartment in CML. Blastic transformation, including loss of differentiation and gain of proliferative capacity, is likely to be a consequence of additional genetic lesions. In about 80% of cases, additional nonrandom cytogenetic abnormalities are found at the aggressive stage of the disease that have almost certainly contributed to the evolution of the disease. These include trisomy 8 or 19, isochromosome 17q, and an extra copy of the Philadelphia chromosome. Abnormalities in MYC and RAS are infrequent (McCarthy et al., 1984; Liu et al., 1988; Ahuja et al., 1991), whereas abnormalities in p53 are found in 15-30% of patients in the accelerated phase of blast crisis (Nakai et al., 1992; Neubauer et al., 1993). Deletions and altered expression of interferon genes have also been reported in patients in blast crisis (Neubauer et al., 1990; Wetzler et al., 1991). The cooperation of Bcr-Abl with additional genetic lesions undoubtedly facilitates the evolution of a highly aggressive and drug-resistant disease. This underscores the importance of developing therapy that will eradicate the Philadelphia clone in the chronic phase. 3. CHRONIC LYMPHOCYTIC LEUKEMIA

Chronic lymphocytic leukemia (CLL)is characterized by the accumulation of large numbers of monoclonal lymphocytes in the peripheral blood. Most

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cases involve B lymphocytes, although approximately 5 YO involve T cells. CLL lymphocytes are morphologically mature but functionally inactive and long lived. A candidate normal counterpart for the B-CLL lymphocyte is an anergic self-reactive CD5+ B cell (Caligaris-Cappio, 1996).Variable cytogenetic abnormalities are found in 56-65% of CLL patients. Trisomy 12 and a breakpoint involving the long arm of chromosome 14 (14q+) or 13 (13q+) are frequent abnormalities present in 18, 13, and 10% of patients, respectively (Juliusson et al., 1990). The molecular mechanisms underlying these chromosome abnormalities in CLL are unknown. Involvement of the RAS gene has been postulated because of its location, but no RAS gene mutations were identified in 93 patients (Neri et al., 1988; Browett et al., 1989). The 14q+ abnormality involves a breakpoint at 14q32 at the site of the Ig heavy chain gene. Translocations such as t(11;14)(q13;q32) may juxtapose critical genes to the Ig heavy chain gene. Candidate genes located on 11q13 include BCL-I, CD20, ZNT-2, HST, SEA, and PRAD-1 (reviewed in O’Brien et al., 1995). Translocation of the BCL-2 gene is infrequent in CLL, but expression of the gene at the RNA and protein levels is increased compared to normal CD5+ B cells (Schena et al., 1992) and to normal lymphocytes (Mariano et al., 1993; Hanada etal., 1993).The upregulation of Bcl-2 may therefore contribute to the excessive life span of CLL lymphocytes in vivo. Hematopoietic cytokines have been shown to promote viability in B-CLL lymphocytes. A study of 30 B-CLL patients found that half of the malignant CD5+ populations released G-CSF after in vitro activation. In contrast, CD5+ B cells did not produce G-CSF following activation. G-CSF did not affect the proliferation of seven GM-CSF+ B-cell populations in culture, but rescued three of them from apoptosis (Corcione et al., 1996). IL-4 has been shown to enhance the survival of CLL cells in culture, whereas normal peripheral blood cells were unaffected (Mainou-Fowler et al., 1995). IL-2, IL4, IFN-y, and IFN-a can also prevent spontaneous or hydrocortisone-induced cell death of CLL lymphocytes (Fournier et al., 1995; Jewell et al., 1994; Huang et al., 1993). The protective effects of both IFN-a and IL-4 have been associated with an increase in Bcl-2 expression (Jewell et al., 1994; Dancescu et al., 1992). Another study has suggested that IL-7 gene expression may be required for the maintenance of CLL viability in vivo. IL-7 transcripts were rapidly downregulated preceding apoptosis of CLL lymphocytes in culture. However, IL-7 gene expression could be retained and apoptosis prevented by culturing the CLL cells on a monolayer of EA.hy926 human umbilical cord endothelial hybrid cells. As cell-cell contact was required, integrins expressed on the cell surface were suggested to play a significant role in CLL cell viability (Long et al., 1995). Fas (CD95)is weakly expressed on the surface of most B-CLL cells but can be upregulated in vitro by stimulation with IL-2 or Staphylococcus aureus

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Cowan. Upregulation of CD95 allowed Fas-mediated apoptosis and coincided with a downregulation of Bcl-2 mRNA (Mapara et al., 1993). In contrast, upregulation of Fas by IFN-a or IFN-y resulted in Fas-positive cells that did not undergo apoptosis in the presence of anti-Fas monoclonal antibody. It was suggested that the upregulation of Bcl-2 by these interferons may over ride the cytotoxic signaling from Fas (Panayiotidis et al., 1995). The progression of CLL has also been associated with an increase in cytogenetic abnormalities (O’Brien et al., 1995; Gale et al., 1994). Mutations in p53 have been found in 10-15% of CLL patients and are associated with a more aggressive form of disease and resistance to chemotherapy (El Rouby et al., 1993). Unlike CML, CLL does not evolve into acute leukemia, although in rare cases blastic transformation may occur as a terminal lymphoma (Richter syndrome). The disease does, however, frequently evolve resistance to cytotoxic drugs that may have successfully controlled the white cell count for many years. More aggressive cytotoxic regimens may have some success at this stage, although disease-related death is common. Bone marrow failure and immune deficiency are usually responsible (Sawitsky and Rai, 1992). It is conceivable that the progression of CLL is facilitated by the prolonged life span of the original CLL clone, allowing the accumulation of additional genetic lesions. 4. ACUTE LYMPHOBLASTIC LEUKEMIA

The Philadelphia translocation occurs in about 20% and 5% of adult and paediatric acute lymphocytic leukemia (ALL) patients, respectively. Compared to Ph- ALL patients, who respond very favorably to chemotherapy, Ph+ ALL patients are very difficult to treat and are seldom cured (Clarkson et al., 1995). The antiapoptotic activity of p190 Bcr-Abl may play an important role in both the development of the disease and the drug resistance associated with Ph+ ALL. High levels of Bcl-2 expression in ALL cells were found to correlate with prolonged survival when cultured in the absence of growth factors (Campana et al., 1993). Elevated Bcl-2 expression was also associated with leukemic growth and survival of a lymphoblastoid cell line in mice (Pocock et al., 1995). A recent study has examined the clinical relevance of BCL-2 overexpression in 52 cases of childhood ALL (Coustan-Smith et al., 1996). Bcl-2 expression was found to be higher in B- and T-lineage ALL compared to normal lymphoid progenitors and mature resting B and T lymphocytes. Higher Bcl-2 levels were associated with an improved ability to survive in the absence of serum-derived growth factors, but did not correlate with disease aggressiveness or with in vitro resistance to cytotoxic drugs. Thus the influence of Bcl-2 on susceptibility to apoptosis was dependent upon the nature of the apoptotic stimulus. An intriguing observation in this study was

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the significant association ( p = 0.01) between low levels of Bcl-2 and the presence of the Philadelphia chromosome. These authors suggest that the upregulation of Bcl-2 may be a necessary leukemogenic event to protect lymphoid progenitors with defective rearrangements from undergoing apoptosis. In patients with a Philadelphia chromosome, the antiapoptotic effects of Bcr-Abl may be sufficient to prolong the life span of cells with nonfunctional gene rearrangements. This study therefore suggests that an antiapoptotic event may be an important factor in the etiology of ALL. Fas has been shown to be constitutively expressed in 21 of 30 T-ALL patients; however, the majority of Fas-positive T-ALL patients were resistant to anti-Fas-mediated apoptosis. Resistance was independent of Bcl-2 expression. Inhibition of protein synthesis induced sensitivity to Fas-mediated apoptosis in most T-ALL cases, suggesting that resistance to Fas-induced apoptosis in ALL is maintained by an active cellular program (Debatin and Krammer, 1995).

5. ACUTE MYELOID LEUKEMIA Expression of Bcl-2 in acute myeloid leukemia (AML) blast cells has been correlated with poor clinical outcome (Campos et al., 1993; Maung et al., 1994; Bensi et al., 1995). The latter study found that Bcl-2 was present in 87.3% of AML cases at presentation and in 100% of relapses. Relapses always had higher percentages of positive cells than those at onset. Autonomous in vitro growth of blast cells from AML patients has been associated with a lower remission rate and a higher relapse rate (Hunter et al., 1993). A study by Russell et al. (1995)demonstrated that AML blasts with autonomous growth are relatively resistant to the induction of apoptosis, and that this is related to the autocrine production of GM-CSF. Autocrine GM-CSF was found to upregulate expression of Bcl-2 (Russell et al., 1995). Another preliminary study has also suggested that Bcl-2 can be upregulated by G-CSF in vivo (Andreeff et al., 1995). Expression of Fas was examined in 47 cases of AML. Thirteen of these cases were found to be positive, but no significant difference in Fas expression was found between those patients who entered complete remission and those with refractory or relapsed AML (Munker et al., 1995). 6. PRELEUKEMIA

The term “preleukaemia” (or myelodysplastic syndrome [MDS]) refers to a group of clonal hematopoietic disorders that are characterized by morphological abnormalities and ineffective hematopoiesis. MDS frequently evolves into AML, and rarely into ALL (Geary and Macheta, 1992). Several studies of preleukemia suggest that the peripheral blood cytopenias found

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in many of these patients may be a direct result of elevated apoptosis (Clark and Lampert, 1990; Yoshida, 1993; Raza et al., 1995). Elevated apoptosis can be a feature of cells with deregulated oncogenes (e.g., M Y C or RAS).The potential involvement of RAS is suggested by the high incidence of RAS mutations in MDS (40%) (Jacobs and Culligan, 1992).In addition, the frequent loss of 5q31.1 (and thus IRF-1; see Section 1II.D) in MDS and leukemia may cooperate with deregulated RAS and allow disease progression (Tanaka et al., 1994). Additional evidence suggests that an antiapoptotic event may play a major role in the transformation of MDS into acute leukemia. In one patient diagnosed with MDS, the acquisition of p190 BCR-ABL coincided with transformation to ALL (Kohno et al., 1996). In another MDS patient, p210 BCR-ABL was identified at the stage of transformation to AML (Nakamura et al., 1991). Although Bcr-Abl has been infrequently reported in secondary acute leukemia derived from MDS, these case studies provide a valuable insight into the potential contribution of an antiapoptotic event to disease progression. 7. CHRONIC VERSUS ACUTE LEUKEMIA: SOME HYPOTHETICAL MODELS

Despite the growing consensus that the acquisition of a fully transformed phenotype may require both proliferative and antiapoptotic genetic lesions, the nature of chronic and acute leukemias suggests that the order in which these events occur may be important in defining the phenotype of the leukemia. For example, the accumulation of noncycling cells in chronic leukemias and the indolent nature of these diseases in the early stages suggest that their early lesions are antiapoptotic. Blast crisis in CML is likely to be a consequence of additional mutations (Fig. 3.1). In contrast, the nature of acute leukemias (their proliferative capacity and fast clinical progression) suggests that they may have sustained early genetic lesions associated with proliferation and/or differentiation. For example, an early genetic lesion in Burkitts lymphoma is activation of the proto-oncogene MYC. This lymphoma has a high growth rate and clinical progression, and can be fatal within weeks or months without adequate treatment. M Y C translocations have also been associated with B- and T-ALL (Rabbitts, 1991, 1994). The data on preleukemia (MDS) are also conducive to such a model. In MDS, the transformation from a high level of apoptosis and peripheral blood cytopenia to an accumulation of highly proliferative blasts in the bone marrow is likely to involve the acquisition of an antiapoptotic event (Fig. 3.2). It is possible that, in the evolution of de novo ALL and AML, proliferative-differentiation defective and antiapoptotic events occur simultaneously (Fig. 3.3).

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It has been previously noted that one of the differences between acute and chronic leukemias is the nature of the genes involved in chromosome translocations. That is, translocations in acute leukemias more often involve genes that encode nuclear transcription factors (e.g., MYC, MML, PML, PLZF, ETO, AML-1, CBF, RAR, ENV-1 ), whereas translocations involving cytoplasmic proteins (Bcl-2, Bcr, Abl) are more often a feature of chronic diseases (Rabbitts, 1991). Whether the disease distinctions should be defined as proliferative-differentiation defective early lesions (MDS and de novo acute leukemia), and antiapoptotic early lesions (chronic leukemia) will probably await the development of some complex mouse models.

8. DRUG RESISTANCE IN LEUKEMIA Resistance to cytotoxic therapy is still the major cause of treatment failure in leukemia. Some leukemias are almost immediately assigned to poor prognostic groups (e.g., Ph+ ALL, blast crisis CML), suggesting that drug resistance or “antiapoptosis” is closely linked to the pathogenesis of the disease. Deng and Deisseroth (1995) examined the influence of Bcl-2 family members on chemosensitivity in good (inversion 16, translocation 8;21), and poor (monosomy 5, monosomy 7) prognostic groups in AML. The predominant family members in chemosensitive AML were Bcl-x, and Bcl-x,, which were present in approximately equal amounts. In chemoresistant AML, Bcl2 and Bcl-x were predominant and Bcl-x, was expressed at approximately four times the level of Bcl-x, (Deng and Deisseroth, 1995). Thus negative regulators of apoptosis may have been important in the development of AML in these patients and may also play a crucial role in their chemoresistance. It is also conceivable that drug resistance acquired after several courses of chemotherapy is a consequence of selection for an increasingly antiapoptotic leukemic clone. It is now apparent that many (if not all) of the cytotoxic drugs used in the treatment of leukemia kill cells by inducing apoptosis. These include cytotoxic drugs with a wide range of primary targets-for example, dihydrofolate reductase inhibitors (Lorico et al., 1988); topoisomerase I1 poisons i

Fig. 3 Apoptosis in leukemic evolution: some hypothetical models. The nature of various hematological malignancies suggests that the order in which proliferative and antiapoptotic genetic lesions occur may be important in defining the phenotype of the leukemia. ( I )The primary lesion in CML (Bcr-Abl) appears to be antiapoptotic, resulting in a survival advantage and an expanded population of myeloid cells. Blast crisis in CML is thought to be a consequence of additional mutations, leading to a loss of differentiation and increased proliferative capacity. (2)High levels of apoptosis in MDS suggest early oncogene deregulation. The transformation to a highly proliferative disease such as AML or ALL is likely to involve the acquisition of an antiapoptotic event. (3)In de novo ALL and AML, proliferative-differentiationdefective and antiapoptotic events may occur simultaneously.

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(Walker et al., 1991; Ling et al., 1993); nucleoside analogs; cytosine arabinoside, fludarabine, and 2-chloro-2'-deoxyadenosine(Tosi et al., 1994; Robertson et al., 1993); microtubule poisons; vinblastine and vincristine (Martin and Cotter, 1990); and the DNA-damaging agents cisplatin and alkylating agents (Eastman, 1990; Frankfurt et al., 1993; Begleiter et d., 1994). A study by Friesen et al. (1996) has demonstrated that the mechanism by which the cytotoxic drug doxorubicin induces apoptosis in human T-cell leukemia cell lines involves upregulation of FasL. Doxorubicin-induced apoptosis could be inhibited by blocking anti-CD95 antibody fragments. Methotrexate also stimulated upregulation of FasL (Friesen et al., 1996). Whether or not this mechanism is cell type specific or applies to other cytotoxic drugs remains to be established. Modulation of the expression of genes involved in apoptosis has been shown to affect chemosensitivity. Transfection of the wild-type p53 gene into a small-cell lung cancer cell line increased cellular sensitivity of cisplatin (Fujiwara et al., 1994), and transfection of BCL-x, into MCF-7 (breast cancer) cells increased their sensitivity to etoposide (Sumantran et al., 1995). In contrast, transfection of the apoptosis inhibitor BCL-2 into a murine lymphoid cell line conferred resistance to nitrogen mustard and camptothecin (Walton et al., 1993), and antisense BCL-2 oligonucleotides increased the sensitivity of AML blasts to ARA-C (Keith et al., 1995). Overexpression of BCL-x, in tumor cell lines reduced the cytotoxicity of bleomycin, cisplatin, etoposide, and vincristine (Minn et al., 1995), and downregulation of Bcr-Abl with antisense sequences rendered K562 cells more susceptible to induction of apoptosis by chemotherapeutic agents (McGahon et al., 1994). Clinical studies have also shown an association between the expression of genes involved in apoptosis and response to therapy. p53 mutations have been found in 15-30% of CLL patients (Imamura et al., 1994; Gaidano et al., 1991; Fenaux et al., 1992) and are associated with poor clinical outcome (El Rouby et al., 1993). p53 mutations are found in 5-10% of AML patients (Imamura et al., 1994; Fenaux et al., 1991) and have been shown to be a strong prognostic indicator (Wattel et al., 1994); high expression of BCL-2 in AML has also been associated with a poor clinical response (Campos et al., 1993; Maung et al., 1994). The efficacy of cytotoxic therapy may therefore be heavily dependent upon the intrinsic ability of a cell to engage a program for cell death in response to a cytotoxic insult.

VI. FUTURE PERSPECTIVES The primary objective of cytotoxic drug therapy is the induction of cell death in the tumor population, with minimal damage to normal tissues. Un-

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fortunately, the majority of chemotherapeutic regimens in current use are nonselective in their toxicity, often targeting DNA or other important components of cellular metabolism. Consequently, the dose must be limited, and their effectiveness is often compromised by their harmful side effects, particularly in elderly patients. The development of more specific cytotoxic regimens would therefore be of significant therapeutic value. The observation that specific cell populations may be dependent upon a restricted group of viability factors suggests that there could be considerable potential in a better awareness of tissue-specific (or cell type-specific) viability factors. If the viability of a given malignant cell population is dependent upon a specific cytokine, it may be possible to quench the effects of that factor with anti-cytokine antibodies or the addition of a soluble receptor. Specific receptor antagonists would also be valuable in this respect. This type of therapy has already proven useful in the treatment of some prostate and breast tumors that are dependent upon androgen and estrogen, respectively. Antiandrogens, suramin, and tamoxifen can antagonize the effect of survival factors and induce apoptosis (Kyprianou et al., 1990,1991; Sklar et al., 1993; Warri et al., 1993). Multiple myeloma cells are heavily dependent on the presence of IL6, and the severity of the disease has been shown to correspond to the concentration of IL-6 in the circulation. Anti-IL-6 or anti-IL-6 receptor antibodies were successful in reducing tumor development both in vitro and in vivo (Vink et al., 1990; Bataille et al., 1989). Viability factors may also be useful for protecting specific subpopulations of cells from drug toxicity. GMCSF has proved useful in the treatment of chemotherapy-induced neutropenia, and in promoting the growth and viability of progenitor cells after bone marrow transplantation (Gisselbrecht et al., 1994). Cytokines that induce apoptosis are also potential therapeutic agents, although their use may be limited by the toxicity of effective doses. However, Tos et al. (1996) demonstrated that retroviral-mediated transfer of TNF-a induced apoptotic cell death in a human lymphoma T-cell line. The concentrations of the cytokine released were 100-fold less than normally required to induce apoptosis in parental cells when applied exogenously (Tos et al., 1996). This approach would be restricted by the target specificity of the retrovirus or the accessibility of a localized tumor. In vitro experiments suggest that it may be possible to genetically manipulate cells in vivo and thereby alter their “apoptotic threshold.” Thus transfection with positive regulators of apoptosis, or antisense molecules to negative regulators of apoptosis, may lower the threshold at which cells will commit to apoptosis. The use of specific DNA molecules in vivo has, however, been limited by technical considerations such as gene delivery and target specificity. One particularly encouraging study has shown that a recombinant Bcl-xs adenovirus could selectively kill carcinoma cells without any effect on hematopoietic progenitor cells (Clarke et al., 1995).The uniqueness

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of the chimeric gene BCR-ABL makes antisense therapy an attractive option for CML. Antisense BCR-ABL junction sequences combined with drug therapy achieved efficient elimination of Ph+ leukemic cells in mice (Skorski et al., 1993). A BCR-ABL antisense oligomer has recently been used for autologous marrow purging. Three of four patients were entirely or predominantly Ph- at 3 months (De Fabritiis et al., 1995). A number of tyrosine kinase inhibitors have also been shown to inhibit Bcr-Abl and induce differentiation in cell lines (Honma et al., 1989; Constantinou et al., 1990; Anafi et al., 1993). One report has described the development of an Abl-specific tyrosine kinase inhibitor that induces apoptosis in Bcr-Abl+ cell colonies derived from the marrows of CML patients, but not in cells taken from normal marrows. Preliminary data have shown little, if any, toxicity in animals (Druker et al., 1996). This compound therefore shows considerable potential for use as a therapeutic agent in Bcr-Abl-expressing leukemias. Another study has shown that a specific Jak-2 tyrosine kinase inhibitor, AG-490, inhibited growth of ALL cells in vitro and in vivo by inducing apoptosis, with no deleterious effects on normal hematopoiesis (Meydan et al., 1996).These studies illustrate that a better understanding of the molecular defects in malignant cells can allow the development of more selective cytotoxic drugs. The past several years have seen a huge increase in information regarding the physiological control of cell death. Many new molecular players have been identified, but their biochemical functions have yet to be established. Further effort is required in order to gain a better understanding of the role of these apoptosis regulators in the pathogenesis of neoplastic diseases, and the many other disorders in which apoptotic cell death plays a significant role.

ACKNOWLEDGMENTS We would like to acknowledge the EU Biotech program, Irish Cancer Society, Health Research Board of Ireland, and Children’s Leukaemia Research Project for their generous financial support. We are also grateful to Dr. David Bowen for helpful advice and comments.

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Sumantran, V. N., Ealovega, M. W., Nunez, G., Clarke, M. F., and Wicha, M. S. (1995). Cancer Res. 55,2507-2510. Tanaka, N., Ishihara, M., Kitagawa, M., Harada, H., Kimura, T., Matsuyama, T., Lamphier, M. S., Aizawa, S., Mak, T. W., and Taniguchi, T. (1994). Cell 77, 829-839. Tanuma, S., and Shiokawa, D. (1994).Biochem. Biophys. Res. Commun. 203,789-797. Tanchi, T., Boswell, H. S., Leibowitz, D., and Broxmeyer, H. E. (1994).J. Exp. Med. 179, 167-175. Tos, A. G., Cignetti, A., Rovera, G., and Foa, R. (1996).Blood 87,2486-2495. Tosi, P., Visani, G., Ottaviani, E., Manfroi, S., Luigi Zinzani, P., and Tura, S. (1994).Leukemia 8,2076-2082. Tsubata, T., Wu, J., and Honjo, T. (1993).Nature (London) 364, 645-648. van Etten, R. A. (1993).Leukemia Lymphoma ll(supp1. l ) , 239-242. Vaux, D. L., Haecker, G., and Strasser, A. (1994). Cell 76, 777-781. Veis, D. J., Sentman, C. L., Bach, E. A., and Korsmeyer, S. J. (1993). Cell 151, 2546-2554. Vignaux, F., and Golstein, P. (1994).Eur J. Immunol. 24, 923-927. Vink, A., Coulie, P., Warnier, G., Renauld, J.-C., Stevens, M., Douckers, D., and Van Snick, J. (1990).J. Exp. Med. 172,997-1000. Vogt, M., Lesley, J., Bogenberger, J. M., Haggblom, C., Swift, S., and Haas, M. (1987).Oncogene Res. 2,49-63. Von Boehmer, H. (1994). Cell 76,219-228. Walker, P. R., Smith, C., Youdale, T., Leblanc, J., Whitfield,J. F., and Sikorska, M. (1991).Cancer Res. 51, 1078-1085. Walton, M. I., Whysong, D., O’Connor, P. M., Hockenbery, D., Korsmeyer, S. J., and Kohn, K. W. (1993). Cancer Res. 53, 1853-1861. Wang, H.-G., Millan, J. A., Cox, A. D., Der, C. J., Rapp, U. R., Beck, T., Zha, H., and Reed, J. C. (1995).J.Cell Biol. 129,1103-1114. Wang, Z.-Q., Auer, B., Stingl, H., Berghammer, D., Haidacher, M., Schweiger, M., and Wagner, E. F. (1995). Genes Deu. 9,509-520. Warri, A. M., Houvinen, R. L., Laine, A. M., Martikainen, P. M., and Harkonen, P. L. (1993). J. Natl. Cancer Inst. 85, 1412-1418. Wattel, E., Preudhomme, C., Hecquet, B., Vanrumbeke, M., Quesnel, B., Dervite, I., Morel, P., and Fenaux, P. (1994).Blood 84,3148-3157. Wetzler, M., Kurzrock, R., Lowe, D. G., Kantarjian, H., Gutterman, J. U., and Talpaz, M. (1991).Blood 78,2400-2406. White, E. (1996). Genes Deu. 10, 1-15. White, E., Livanos, E. M., and Tlsty, T. D. (1994). Genes Deu. 8, 666-677. Williams, G. T., Smith, C. A., Spooncer, E., Dexter, T. M., and Taylor, D. R. (1990).Nature (London) 343,76-79. Woronicz, J. D., Calnan, B., Ngo, V., and Winoto, A. (1994). Nature (London) 367, 277-28 1. Wu, X., and Levine, A. J. (1994).Proc. Natl. Acad. Sci. U.S.A. 91,3602-3606. Wyllie, A. H., Arends, M. J., Morris, R. G., Walker, S. W., and Evans, G. (1992).Semin. Immunol. 4,389-397. Yang, E., Zha, J., Jockel, J., Boise, L. H., Thompson, C. B., and Korsmeyer, S. J. (1995). Cell 80,285-291. Yew, P. R., and Beck, A. J. (1992).Nature (London) 357, 82-85. Yin, X. M., Oltvai, Z., and Korsmeyer, S. (1994).Nature (London) 369, 321-323. Yonish-Rouach, E., Resnitzky, D., Lotem, J., Sachs, L., Kimchi, A., and Oren, M. (1991).Nature (London) 352,345-347. Yoshida, Y. (1993).Leukemia 7,144-146. Young, J. C., and Witte, 0. N. (1988).Mol. Cell. Biol. 8,4079-4087.

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CyclincDependent Kinase Regulation during GI Phase and Cell Cycle Regulation by TGF+ Michael 1. Ravitz and Charles E. Wenner Department of Biochemistry, Roswell Park Cancer Institute, New York State Department of Health, Buffalo, New York 14263

I. Introduction 11. Cyclins and Cyclin-Dependent Kinases A. Control of the Mammalian Cell Cycle by cdks

B. Cdk Regulation during G1 and S Phases C. Downstream Effectors of G1 Phase cdks D. TGF-pl and TGF-p Signal Transduction E. Effects of TGF-p1 on G1 Phase Progression F. Effects of TGF-p on G1 Phase cdk Activity G. Cyclins, cdks, and Cancer 111. Conclusions References

The aim of this review is to provide insight into the molecular mechanisms by which transforming growth factor-p (TGF-P) modulates cell cycle progression in different cell types. Particular attention is focused on the differences between these mechanisms in cells of epithelial origin and in mesenchymally derived cells. This is important because many transformed epithelial cells lose responsiveness to the growth-inhibitory effects of TGFp, thus generating a more fibroblast-like phenotype. Loss of negative growth control, including a lack of response to growth-inhibitory factors, is a common feature of many tumor cells. G1 phase cyclin-dependent kinases (cdks) and their inhibitors (ckis) are central to the pathways that regulate commitment to cellular division in response to positive as well as negative growth effectors. Many checkpoints are deregulated in oncogenesis, and this is often due to alterations in cyclin-cdk complexes. The loss of R-point regulation, in particular, can allow cell growth and division to proceed autonomously of external signals. This may occur due to either the aberrant expression of positive regulators, such as the cyclins and cdks, or the loss of negative regulators, such as the ckis. Beginning with a survey of the role of the role of cdks in the mammalian cell cycle, the review examines how cdk activity is modulated by cyclin binding, phosphorylation, and ckis, including the Ink4 proteins and the closely related inhibitors p2lC'p1 and ~ 2 7 ~ ' pParticular l. attention is paid to the role of pnK'p1 and p2lCip1 in the mechanism of TGF-p-induced suppression or stimulation of the cell cycle and how these mechanisms contrast between epithelial cells and cells of mesenchymal origin. Other aspects of TGFp signal transduction are discussed, including its effects on cyclin and cdk expression in various cell types, and the downstream targets of cdks and their modulation by TGF-p

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and other growth factors are also discussed. These include proteins of the retinoblastoma family, and the related modulation of the transcriptional activity of the E2F family members. Finally, the role of cell cycle regulatory proteins in oncogenesis is reviewed in view of the findings described here.

I. INTRODUCTION Members of the cyclin-dependent kinase (cdk) family of serine-threonine kinases are known to be key regulators of eukaryotic cell cycle transit. Different combinations of cdk catalytic subunits and their activating cyclin subunits operate at control checkpoints during the cell cycle to integrate mitogenic and antiproliferative signals that converge at these checkpoints. Checkpoint controls also ensure that the initiation of particular cell cycle events is dependent upon the successful completion of others. Once activated, cdk-catalyzed phosphorylation of downstream effector targets results in the modulation of transcriptional events necessary for transit to the next stage of the cell cycle, in part by inactivating retinoblastoma protein family members that suppress transcription from promoter elements in specific genes. Transforming growth factor-pl (TGF-p1) is a pleiotropic growth factor that can either stimulate or inhibit growth, depending on the cell type and growth conditions. TGF-P can affect the eukaryotic cell cycle throughout most of the G1 phase, largely through its effects on the assembly and activation of cdks that operate at a checkpoint during this period to control Sphase entry. TGF-p exerts these effects through a variety of transcriptional, translational, and posttranslational mechanisms, depending on the cell type, growth conditions, and time of operation in G1 phase. These mechanisms include the ability of TGF-p to positively or negatively affect the regulation of cdk inhibitors (ckis). Because many tumorigenic cell lines have lost responsiveness to the growth-inhibitory effects of TGF-Pl (Masui et a/.,1986; Shipley et al., 1986; Knabble et al., 1987; Haddow et al., 1991; Manning et al., 1991), an understanding of the molecular mechanisms by which TGF-f3 affects cell growth will also provide insight into the molecular events involved in neoplastic transformation. Loss of negative growth control, including a lack of response to growth-inhibitory factors, is a common feature of tumor cells. G1 phase cdks and ckis are central to the pathways that regulate commitment to cellular division in response to positive as well as negative growth effectors. Checkpoint controls become deregulated in oncogenesis, often due to alterations in cyclin-cdk complexes. The loss of restriction point regulation, in particular, can allow cell growth and division to proceed autonomously of external signals. This may occur due to either the aberrant expression of positive regulators, such as the cyclins and cdks, or loss of negative regulators, such as the ckis.

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This review describes the current knowledge on the regulation of cdks operating in G1 phase, and how their activity is modulated during cell cycle regulation by TGF-P. Most of the work performed to date has concentrated on the regulation of cdks by TGF-P in cells of epithelial origin, where TGFP inhibits growth. There are several reasons, however, why it is important to study cells of mesenchymal origin. Growth suppression by TGF-P is lost following the establishment of fibroblastic cultures primary fibroblasts (Sorrentino and Bandyopadhyay, 1989). Since the critical difference between these stages is the acquisition of an immortalized phenotype, understanding the molecular basis for such a differential regulation by TGF-P may provide clues not only to TGF-P action, but also to the changes that occur during the immortalization of cells that accompanies oncogenesis. On the other hand, immortalized, nontumorigenic mouse epidermal cells still respond to TGFP by cessation of growth, whereas in a series of malignant carcinoma lines the response is substantially reduced (Haddow et al., 1991). Haddow et al. also found that, of two nontumorigenic papilloma lines tested, one retained complete sensitivity to TGF-P, whereas the other showed a lack of response similar to that observed in carcinomas. From this they conclude that growth control by TGF-P is lost at a relatively late stage of carcinogenesis in this system. Similarly, the TGF-P-sensitive cell line ANC1 was derived from a relatively large, late-stage adenoma, indicating that loss of response to TGF-P occurs at a relatively late stage in colorectal carcinogenesis (Manning et af., 1991).The latter authors also demonstrated that a loss of sensitivity to TGFP accompanies the conversion of the ANC1 adenoma cell line to a tumorigenic phenotype, suggesting that the loss of response to TGF-P occurs during the benign-malignant transition. In addition to a lack of TGF-P-mediated growth suppression, transformed epithelial cells can acquire other characteristics often associated with fibroblasts. Some nontumorigenic papilloma lines are not only refractory to growth inhibition by TGF-P, they are even slightly stimulated by it (Brown et af., 1993). The acquisition of morphological characteristics associated with fibroblasts can also occur. For example, undifferentiated spindle cells, which possess a fibroblast-like morphology, arise in vivo by progression from squamous carcinoma cells (Buchmann et af., 1991; Brown et al., 1993). These studies suggest that a comparison of the differential regulation of epithelial cells versus fibroblastic cells by TGF-P could provide clues to the progression of cancer. The genetic or epigenetic basis underlying differential sensitivity of epithelial versus fibroblast-like cells to TGF-P has not been established. Although the retinoblastoma susceptibility gene product (pRB) has been postulated to mediate the negative effects of TGF-P, intact pRB is present in both TGF-(3-sensitive and TGF-@-resistantcell lines (Haddow et al., 1991). Thus, it is important to assess whether the pathways that affect pRB function, in particular those involving the G1 phase cdks, are involved in differential cellular responses to TGF-P. Furthermore, in Balb/MK kera-

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tinocytes, TGF-P can still inhibit G1 progression when added at a time in late G1 phase when cell cycle progression is not blocked by 5,6-dichlorobenzimidazole riboside (DRB), an inhibitor of RNA polymerase II-mediated transcription (Alexandrow and Moses, 1995). Their finding that the keratinocytes remain sensitive to TGF-P-mediated inhibition up until the time of DNA replication, even in the presence of DRB, suggests that ckis in particular may have an involvement in the late G1 phase regulation of these pathways by TGF-P. Other work has indicated that receptor mutations can affect TGF-P signaling in cancer cells (see Section 1I.D).

11. CYCLINS AND CYCLIN-DEPENDENT KINASES

A. Control of the Mammalian Cell Cycle by cdks The eukaryotic cell cycle is divided into four distinct phases that together last approximately 24 hours (Fig. 1).Newly divided mammalian cells must either commit to a new round of DNA replication during S phase or else become quiescent during the initial growth phase, called G1. In the absence of mitogenic factors, or in terminally differentiated cells, cells withdraw from the cell cycle and remain arrested at a point very early in this phase, termed GO. The continuous presence of serum growth factors is required for a defined period during G1 phase for quiescent cells to enter the cell cycle, or for the continued progression of cycling cells. On the other hand, intracellular signals, such as the presence of DNA damage, will halt the cell cycle during the G1 phase, allowing DNA repair if possible, or else apoptosis. Passage through the major transitions and checkpoints in the cell cycle is contingent upon the activity of various cdks. Thus the balance between growth-stimulatory and growth-inhibitory signals, whether of intracellular or extracellular origin, modulates the activity of the kinases, thus governing progression through G1 phase via a cascade of phosphorylation events. In the yeast strain Schizosaccharornycespornbe, both the G2-M and G1-S transitions are triggered by the activity of a single protein kinase termed cdc2 (cell division cycle 2; Nurse and Bissett, 1981), which binds to and is activated by different cyclin regulatory subunits during the yeast cell cycle. In higher eukaryotes, cdc2 regulates the G2-M transition, primarily in association with cyclin B, while a homologous but distinct kinase operates at the G1-S transition (Fang and Newport, 1991). This latter kinase, p33cdk2,was the second member of the cdk family to be cloned, shares 65% sequence identity with ~ 3 4 ' ~(Tsai ' ~ et al., 1991), and is known to form complexes with cyclins A and E (Giordano et al., 1989; Dulic et al., 1992; Koff et a/., 1992; Pagano et al., 1992a,b) as well as with cyclin D1 and to a lesser extent

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Fig. 1 The mammalian cell cycle. G1, First growth phase; S, DNA synthesis phase; G2, second growth phase; M, mitosis.

with cyclin D3 (Xiong et al., 1992). Like cdc2, cdk2 is able to readily phosphorylate histone €31 both in vivo and in vitro, thus forming the basis of the histone H1 kinase assay.

B. Cdk Regulation during GI and S Phases 1. C d k ACTIVATION BY CYCLIN BINDING In general, cdk2 is expressed in GO cells, and, like cdc2, is modestly upregulated during growth factor stimulation, while showing little variation in cycling cells (Draetta and Beach, 1988; Pagano et al., 1992a; Firpo et al., 1994; Ohtsubo et al., 1995). This makes the levels of cyclin present at a given time in the cell cycle critical to cdk activation (Desai et al., 1992; Hunt, 1989). Successive waves of cdk2 activity are thereby generated as the cyclin protein levels oscillate, with each successive cyclin generating a new wave of activity capable of targeting either the same or new, distinct substrates. Cyclin A, for example, is first synthesized as cells enter S phase, at which time it activates cdk2 and targets it to complexes containing the transcription factor E2F (Pagano et al., 1992b). Activity of the cycliin A-cdk2 holoenzyme in S phase is necessary for the completion of DNA synthesis, and expression of plasmids encoding antisense cyclin A mRNA or injection of anti-cyclin A antibodies halts the progression of mammalian fibroblasts into S phase (Girard et al., 1991; Pagano et al., 1992a).

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G1 phase-specific cyclins C, D1, D2, D3, and E were first identified by their ability to rescue the growth of yeast deficient in G1 cyclins (Koff et al., 1991; Leopold and O’Farrell, 1991; Lew et al., 1991; Xiong et al., 1991). This brought the attention of investigators to cyclin E, whose message levels are seen to peak by the time of the “START” point in the budding yeast Saccharomyces cerevisiae (Koff et al., 1991; Lew et al., 1991). The analog of START in eukaryotic cells is the so-called restriction point (R-point) in the late G1 phase, at which cells become committed to DNA synthesis and no longer require the presence of serum growth factors (Pardee, 1974). The Rpoint occurs within a period typically from 2 to 4 hours before the onset of S phase (the G1-S transition). Cell cycle progression through the R-point and/or the G1-S transition is thought to be controlled by the activity of G1 phase cyclins and cdks. An accumulation of unstable proteins is required to pass the R-point, and these are now known to be the G1 cyclins, which can regulate one or more cdks to drive cell cycle progression in a rate-limiting manner. In mammalian fibroblasts, microinjection of anti-cyclin E antibodies prevents entry into S phase, while overexpression of cyclin E in the same cells causes early progression into S phase, demonstrating a rate-limiting function for cyclin E in G1 phase progression (Ohtsubo and Roberts, 1993; Resnitsky et al., 1994; Ohtsubo et al., 1995). Subsequent studies have shown that the cyclin E protein and its associated histone H1 kinase activity accumulate periodically during the cell cycle, with peaks near the G1-S transition, preceding those of cyclin A (Dulic et al., 1992; Koff et al., 1992; Tsai et al., 1993). A similar role has been proposed for cyclin D1, as microinjection of antibodies against cyclin D1 during G1 phase prevented S phase entry (Baldin et al., 1993). However, the antibodies are ineffective when injected in late, rather than early, G1 phase, indicating that the primary function of D-type cyclins is to stimulate G1 phase progression, rather than to promote the G1-S transition. Indeed, constitutive overexpression of cyclin D1 in mammalian fibroblasts accelerates the G1 transition and causes premature entrance into S phase, indicating a rate-limiting role for cyclin D1 as well as for cyclin E in G1 phase transit (Quelle et al., 1993; Resnitsky et al., 1994). By contrast, in the HBL human mammary epithelial cell line, stable overexpression of a cyclin D1 cDNA prolongs the S phase and inhibits cell growth (Han et al., 1995). Also, overexpression of cyclin E in the HC11 mouse mammary epithelial cell line is associated with growth inhibition and increased expression of p27 (Sgambat0 et al., 1996). Nevertheless, several lines of evidence suggest that D and E cyclins regulate different aspects of G1 phase. In the first place, D-type cyclins appear earlier than cyclin E, in mid-G1 phase, at which time they activate entirely different cdk family members. These are cdk4, originally identified as PSK-

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53 (Hanks, 1987; Matsushime et al., 1992, 1994), and the functionally related kinase cdk6 (Meyerson and Harlow, 1994). D cyclins localize to the nucleus and reach peak levels by late G1 phase, at which point they must be degraded in order for S phase to continue (Baldin et al., 1993). Cyclin D l-cdk4 complexes accumulate at the G1-S transition in stimulated cells and decline in early S phase, in correlation with cdk4 message levels. This indicates that newly synthesized cdk4 is preferentially bound by cyclin D1 because the overal level of cdk4 remains constant through the cell cycle (Matsushime et ul., 1992). These findings are consistent with the activation of ~ 3 4 by‘ D~ cyclins ~ ~ in mid-G1 phase of the cell cycle (Matsushime et al., 1994).In vitro, cdk4 can efficiently phosphorylate only pRb, other pRb family members, and the transcription factor E2F-1 (Matsushime et al., 1992, 1994; Fagan et al., 1994; Kato et al., 1993; Serrano et al., 1993),as opposed to cdk2, which can phosphorylate histone H1 as well as pRb. In vivo, overexpression of cyclin Dl-cdk4 but not cyclin E-cdk2 in human U2-OS osteosarcoma cells leads to phosphorylation of the Rb family member p107 (Biejersbergen et al., 1995). Biejersbergen et al. also found that only cyclins D1 or D3 overexpressed with cdk4 can fully release Saos-2 osteosarcoma cells from a pl07-induced block to G1 phase progression, although a partial release can also be effected by cyclin E-cdk2. Second, since both cyclins D and E independently control the rate of G1 progression, they presumably regulate different G1 transitions. This idea is given further support by the fact that cyclin E, but not cyclin D1, is essential for entry into S phase in mammalian cells lacking functional pRb (Ohtsubo et al., 1995). Finally, Dtype cyclins have short half-lives (<25 min), so withdrawal of growth factors during G1 phase prevents their steady accumulation and correlates with the failure of growth factor-deprived cells to progress past the R-point (Matsushime et ul., 1991). Therefore, D-type cyclins are regulated by extracelMar signals and display only mild oscillations with the cell cycle (Lukas et al., 1995; Ravitz et al., 1995), as opposed to the periodic expression of cyclins E, A, and B. Cyclins D and E might fulfill different functions required for R-point control, with cyclin A triggering S phase. Alternatively, cyclin D may be the actual R-point “sensor,” resulting in activation of cdk4 and allowing passage through the R-point. This would commit the cells to continued cell cycle progression, resulting in the activation of the cyclin E-cdk2 complex in late G1 phase. Once activated, the length of time until the onset of S phase may be determined, at least in part, by the level of active cyclin E-cdk2 kinase (Ohtsubo et al., 1995). Thus, D-type cyclins might act upstream of cyclin E as targets of growth factor-induced signals, while cyclin E might govern the actual onset of DNA replication, perhaps by controlling the actual “firing” of the replication origins. Following this, cyclin A replaces cyclin E during S phase as the primary activator of cdk2, ensuring the or-

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derly progression of S phase-specific transcriptional events either by maintaining the phosphorylation state of cyclin E-cdk2 substrates or by targeting new substrates.

2. Cdk ACTIVATION BY PHOSPHORYLATION Although cyclin binding alone allows for some enzymatic activity (Connell-Crowley et al., 1993), additional covalent modifications are required for full activation of a cdk. The phosphorylation of a conserved threonine residue is known to be necessary for cdk activation. In human cdc2, this residue is Thr-161, while in cdk2 and cdk4 the homologous residues are Thr160 and Thr-172, respectively (Gu et al., 1992; Kato et al., 1994; Matsuoka et al., 1994). This phosphorylation event is catalyzed by the cdk-activating kinase (CAK), previously named MO1.5 kinase, which also activates M phase-promoting factor (cyclin B-cdc2) in Xenopus (Ducommun et al., 1991; Fesquet et al., 1993; Poon et al., 1993; Solomon et al., 1993). MO15 has been renamed cdk7, as its activity is maintained by association with a novel cyclin, cyclin H (Fisher and Morgan, 1994; Makela et al., 1994). Activation of MO15 by cyclin H requires the presence of a conserved residue, Thr-170, which may itself be a target for phosphorylation (Fisher and Morgan, 1994). Immunoprecipitable CAK activity, however, can be detected in quiescent cells and does not change throughout the cell cycle (Brown et al., 1994; Matsuoka et al., 1994; Poon et al., 1994; Tassan et al., 1994). Thus the phosphorylation of Thr-l60/161/172 during the normal vertebrate cell cycle is more likely to be triggered by other events, such as cyclin binding and/or the loss of cdk inhibitory proteins (discussed later). The conserved threonine residue is contained within the T loop, a region of the cdk protein that blocks its substrate binding site. Although this threonine is inaccessible to solvent in the crystal structure of cdk2, the T loop is quite flexible, and its conformation may change sufficiently upon cyclin binding to allow for phosphorylation of this residue (De Bondt et al., 1993; Morgan and De Bondt, 1994).This may explain the absolute requirement of cyclin binding for CAKcatalyzed phosphorylation of cdc2 (Fisher and Morgan, 1994). CAKcatalyzed phosphorylation of cdk2, on the other hand, can occur in the case of both cyclin-bound and monomeric cdk2, possibly owing to a more flexible T loop (Fisher and Morgan 1994). While cyclin binding could affect CAK access to the conserved threonine, it is also possible that phosphorylation of this residue by CAK affects the stability of cyclin binding. Ducommun et al. (1991) used coimmunoprecipitation methods to show that mutation of Thr-161 in human cdc2 abolished cdc2 binding to cyclin A. This result was also obtained by Desai et al. (1995). However cyclin B-cdc2, cyclin A-cdk2, and cyclin E-cdk2 can form with high affinity in the absence of phosphorylation or other cellular components

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(Desai et al., 1992, 1995; Solomon et al., 1992; Atherton-Fessler et al., 1993). Fisher and Morgan (1994)have proposed that the activation of cdk2 by CAK can occur via two pathways. In the first pathway, available to both cdc2 and cdk2, the heterodimeric cyclin-cdk2 complex is targeted. In the second pathway, available only to cdk2, CAK targets the cdk2 monomer, yielding a phosphorylated intermediate that is inactive until subsequent cyclin binding.

3. Cdk INHIBITION BY PHOSPHORYLATION Human cdc2 and cdk2 can be inhibited by phosphorylation at two sites near the amino terminus, Thr-14 and Tyr-15 (Gu et al., 1992). By contrast, cdk4 has only the tyrosine residue in this region (Matsushime et al., 1992). These residues are located beneath the T loop and are therefore inaccessible to solvent. This may explain the dependence of these phosphorylation events on the prior binding of cyclin (Solomon et al., 1990; Atherton-Fessler et al., 1993). In cdk2, these residues hang from the ceiling of the ATP binding site and are well positioned to affect cdk activity when phosphorylated (De Bondt et al., 1993); however, Tyr-15 phosphorylation does not appear to affect ATP binding (Atherton-Fessler et al., 1993). Phosphorylation of these residues may function to prevent premature activation of CAK-phosphorylated and cyclin-bound kinases, and their dephosphorylation by the dualaction phosphatase cdc25B is tantamount to cdc2 activation at the G2-M transition in eukaryotes. A similar function exists for the cdc25A dual-action phosphatase, which is proposed to activate cdk2 in late G1 phase by dephosphorylating the enzyme on the same two residues (Hoffman et al., 1994; Jinno et al., 1994). 4. Cdk INHIBITION BY ckis

i. Ink Family Inhibitors Ink4a (p16) was originally identified by virtue of its accumulation in cells transformed by DNA tumor viruses (Serrano et al., 1993; Xiong et al., 1993b). Ordinary diploid fibroblasts contain quaternary complexes consisting of cdk4, cyclin D1, proliferating cell nuclear antigen (PCNA), and the general cki p21 (discussed later). In transformed cells, this complex is “replaced” by binary complexes containing only cdk4 and p16, which probably exists as parallel complexes in normal cells. All Ink family members, including p14, p15, p16, and p18, are capable of forming such binary complexes exclusively with cdk4 or the functionally related kinase cdk6, causing the exclusion of D cyclins and thereby inhibiting cdk4kdk6 activity (Guan et al., 1994). Since the function of the retinoblastoma tumor suppressor protein has been compromised in the virally transformed cells, and since other

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pRb-deficient cells also show accumulation of p16, it has been proposed that p16 functions in a negative feedback pathway that shuts off cyclin D-cdk4 following the phosphorylation of pRb, which would otherwise suppress p l 6 transcription. Indeed, p16 is normally expressed only in late G1 and S phases, at a time when pRb is hyperphosphorylated (Tam et al., 1994; see later). Like p16, p l 5 (Ink 4b) exclusively inhibits cdk4 by competing with cyclin D for cdk4 binding. While p16 appears to function in an intrinsic pathway governing cell cycle progression, p l 5 appears to be involved in regulating the response of the G1 phase cell cycle machinery to extrinsic signals, such as TGF-P (Hannon and Beach, 1994).

ii. p2 1, p27, and p57 p21ciP1 forms ternary complexes with cyclin A-cdk2, cyclin E-cdk2, cyclin D-cdk4, and cyclin D-cdk6 by tightly binding to and inhibiting the catalytic subunits of these complexes (Xiong et al., 1992,1993a; Harper et al., 1993). The formation of ternary rather than binary complexes indicates that p21 associates preferentially with cyclin-cdk complexes, rather than with the catalytic subunit alone. p21 is upregulated by the tumor suppressor gene product p53 in a pathway responding to DNA damage (El-Deiryet al., 1993; Dulic et al., 1994)and prevents DNA synthesis via its association both with the cdks required for G1 phase transit and with PCNA, the processivity factor for DNA polymerase 6 (Flores-Rozas et al., 1994; Waga et al., 1994). p21 is also upregulated in irreversibly postmitotic or nondividing cells, such as senescent cells (Noda et al., 1994),and terminally differentiated cells, including muscle cells, where it is upregulated in a p53-independent manner regardless of the presence of the muscle-specific transcription factors MyoD and myogenin (Halevy et al., 1995; Parker et al., 1995). The latter two states of growth arrest differ from quiescence in that p21 levels remain elevated in each case, whereas p21 levels are generally low in quiescent cells and are upregulated following mitogen stimulation, as observed in fibroblasts and T lymphocytes (Firpo et al., 1994; Li et al., 1994a; Noda et al., 1994; Nourse et al., 1994). ~27~'P was l originally identified and purified as a heat-stable protein that appeared in inactive complexes of cyclin E-cdk2 accumulating in MvlLu mink lung epithelial cells arrested by TGF-f3 or by contact inhibition (Polyak et al., 1994a), and in lovastatin-arrested HeLa cells (Hengst et al., 1994). p27KiP21cDNA was cloned independently by two different methods (Polyak et al., 1994b; Toyoshima and Hunter, 1994). The techniques yielded identical murine cDNAs, each possessing an open reading frame that encodes a predicted protein of 197 amino acids and is highly conserved, while the human cDNA predicts 198 amino acids (Polyak et al., 1994b). ~ 2 7 ~ ' p l shares 44% amino acid identity with p21 in a 60-residue aminoterminal segment, which is essential for binding to and inhibiting the activity of cyclin-cdk complexes (Polyak et a/.,199413; Toyoshima and Hunter, 1994).

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~ 2 7 ~ ' Pforms l tightly bound ternary complexes with and inhibits the activity of the same spectrum of cyclin-cdk complexes as p21, also in a stoichiometric manner (Elledge and Harper, 1994). In human mammary epithelial cells, p27 can inhibit fully functional cyclin-cdk complexes in which cdk2 is already phosphorylated on Thr-160. This is caused by p27 binding, which sterically blocks substrate binding or alters the conformation of the catalytic domain, rendering it inactive (Slingerland et al., 1994). The same findings have also been reported for the inhibition of cyclin E-cdk2 by p27 when the immunosuppressent drug rapamycin is used to prevent mitogenic stimulation of T cells by interleukin 2 (IL-2) (Firpo et al., 1994). In MvlLu cells and macrophages, p27 also has an additional mode of inhibition. In macrophages, the association of cyclin D-cdk4 with p27 sterically prevents its activation by CAK (Kato et al., 1994). In MvlLu cells, association of p27 with newly formed cyclin E-cdk2 complexes can also block phosphorylation of cdk2 on Thr-160 by CAK, thus preventing its activation (Koff et al., 1993; Polyak et al., 1994b; Slingerland et al., 1994). The dual effects of p27 on cdk activation and cdk-catalyzed phosphorylation might thus be due to the inhibitor occupying the catalytic cleft, resulting in either or both effects. Both modes of action also appear to be available to p21 (Zhang et al., 1994).The presence of cyclin may stabilize the binding of p27, as p27 binds to cyclin-cdk complexes with greater affinity than to either subunit alone (Polyak et al., 1994a). Conversely, it has been suggested that the binding of either p21 or p27 might stabilize the association between the catalytic and regulatory subunits (Guan et al., 1994). Finally, Toyoshima and Hunter (1994)have presented evidence that the inhibitor is capable of binding to various cyclins alone. This interaction, although of doubtful significance in terms of the previous model, could be effected through posttranslational modification of the cyclin subunit itself, such as the phosphorylation of cyclin E (Dulic et al., 1994) or of cyclin D1 (Matsushime et al., 1991). As is the case for p21, the overexpression of ~ 2 7 ~ ' blocks pl progression of cells through G1 phase (Polyak et al., 1994b; Toyoshima and Hunter, 1994). p27 and p21, however, respond to different signals. p21 appears to function largely in intrinsic pathways that monitor the cells' internal state, such as the detection of DNA damage in the p.53 pathway. p27, on the other hand, appears to be involved in pathways that sense both mitogenic and antiproliferative extrinsic signals. The mechanism by which p27 inhibitory activity is downregulated during mitogenic stimulation in T cells and macrophages or induced in quiescent fibroblasts and epithelial cells is not fully understood. p27 inhibitory activity can be recovered both from quiescent MvlLu cells and from proliferating MvlLu cells following heat treatment of the extracts, indicating that the protein is constitutively present, albeit in a form presumably sequestered by cyclin D-cdk4 (Polyak et al., 1994a). This finding is consistent with the fact that Kip1 message levels are

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similar in exponentially growing and contact-inhibited MvlLu cells, and do not change when cells are released from contact inhibition by replating at low density in the presence of serum, whether in the presence or absence of TGF-f3 (Polyak et al., 1994b). Similarly, in Swiss 3T3 fibroblasts, the p27 protein level is reported not to change during the cell cycle or following serum stimulation of quiescent cells (Toyoshima and Hunter, 1994).Nonetheless, there is mounting evidence that differential regulation of p27 protein levels by various mechanisms can contribute to cell cycle control. While the p27 message is expressed constitutively in both serum-deprived and proliferating fibroblasts and myoblasts, the steady-state p27 protein level increases when cells are confluent and arrested or differentiated in low-serum medium (Halevy et al., 1995; Ravitz et af., 1995,1996). These results suggest that regulation of p27 also occurs at a posttranscriptional or even posttranslational level. Ubiquitin-mediated proteolysis (Hochstrasser, 1995) has been shown to be important for initiation of DNA synthesis in S. cerevisiae, where the cki p4OSic1 must be degraded in order to activate the Clb5-cdc28 kinase that is required for entry into S phase (Schwob et al., 1994). p4OSic1plays a role in the yeast cell cycle analogous to that of mammalian p27, which is also degraded by the ubiquitin-proteosome pathway (Pagano et af., 1995). These authors found that, in both normal human fibroblasts and human osteosarcoma MG-63 cells, the ubiquitin-conjugating enzymes Ubc2 and Ubc3 exhibit less p27 ubiquitinating activity in quiescent than in proliferating cells, accounting for the longer p27 half-life measured in quiescent cells. In S. cerevisiae, it has also been demonstrated that the cki FARl is phosphorylated just before it disappears at START, suggesting that phosphorylation may target it for degradation (McKinney et af., 1993). Differential synthesis also plays a role in the regulation of ~ 2 7 ~ ' abunpl dance. Kato et al. (1994) have reported that, in macrophages, p27 synthesis decreases by about 50% after quiescent cells are stimulated with colonystimulating factor-1 (CSF-1) alone, but increases two- to threefold in cells treated with CSF-1 plus 8Br-cAMP, a nonhydrolyzable derivative of CAMP. Unlike CAMP, rapamycin treatment of these cells does not induce p27, but instead appears to abrogate the CSF-1-induced reduction in p27 synthesis. Hengst and Reed (1996) reported that an increase in the rate of p27 translation as well as increased half-life of the p27 protein was detected in density-arrested human diploid fibroblasts. Together, these findings indicate that the abundance of p27 is regulated at the level of both translation and turnover. There is also some precedent for transcriptional control of ckis in the budding yeast S. cerevisiae, where mating pheromone leads to transcriptional upregulation of the cki FAR1, in addition to its effects on FARl protein stability (McKinney and Cross, 1995). These results indicate that many eukaryotic cells might regulate p27 inhibitory activity by regulating p27 synthesis at some level in addition to regulating its degredation.

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Experimental manipulation of cells often results in cells stalled in the G1 phase with cyclin-bound and appropriately phosphorylated cdks that are, nevertheless, nonfunctional, presumably due to the presence of saturating levels of p27. In peripheral blood T cells, which depend on two mitogenic signals, p27 levels are initially high in quiescent cells. p27 levels remain high after antigenic stimulation in the absence of IL-2, which results in the synthesis of nonfunctional cyclin D-cdk4, cyclin E-cdk2, and cyclin A-cdk2 complexes. Following this, the application of IL-2 causes the rapid elimination of p27, leading to activation of the aforementioned complexes and entry into S phase (Firpo et d., 1994; Nourse et al., 1994). The immunosuppressant rapamycin blocks G1 phase progression by preventing the IL-2-mediated drop in p27 levels (Nourse et al., 1994). Similarly, p27 levels are high in unstimulated macrophage cultures, and decline during G1 progression induced by CSF-1. Inducers of CAMPlead to increased levels of p27, counteracting the effect of CSF-1 and arresting the cells in G1 phase. This results in the accumulation of inactive cyclin Dl-cdk4 complexes that are not phosphorylated on Thr-172 but that contain p27 (Kato et al., 1994). Similar inactive complexes of cyclin D-cdk4 or cyclin E-cdk2 have been reported to accumulate in serum-deprived, quiescent fibroblasts overexpressing cyclin D or cyclin E, respectively (Quelle et al., 1993; Matsushime et al., 1994; Resnitsky et al., 1994; Ohtsubo et al., 1995). In the case of cyclin E overexpression, cdk2 in complexes with overexpressed cyclin E shows the increased mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis characteristic of Thr-160-phosphorylated cdk2 (Ohtsubo et al., 1995), indicating that some other factor is involved in preventing holoenzyme activation. It would appear that p27 is a likely candidate. Similarly, Hengst et al. (1994)have also found that HeLa cells arrested in G1 phase by the antimitotic drug lovastatin contain inactive cyclin A-cdk2 and cyclin E-cdk2 in complexes with a heat-stable cyclin-cdk inhibitory activity, again probably p27. These latter authors also find that the inhibitor accumulates in NHFl human fibroblasts subjected to density-mediated growth arrest. These results suggest that p27 may represent a final level of control in order for cells to cycle through G1. A close relative of p21 and p27, p57, is also able to inhibit the kinase activities of cyclin D-cdk4, cyclin D-cdkb, cyclin E-cdk2, and cyclin A-cdk2, as well as to associate in vivo with cdk2, cdk4, and cyclins A, E, and D1. Transfection of p57 into mink lung cells or into human Saos-2 osteosarcoma cells induces G1 arrest (Lee et al., 1995; Matsuoka et al., 1995). This indicates that p57-mediated inhibition of the cell cycle does not depend on either p53 or pRb, both of which are absent in Saos-2 cells. The ratio.of cyclin-cdk complexes to ckis is a critical parameter for G I progression, which suggests that changes in the abundance of cdks andor cyclins can affect the inhibitory activity of p27 and the other ckis, in addition to changes in the steady-state levels of the inhibitors themselves. As

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noted earlier, the cdk inhibitory protein p21 is induced when quiescent fibroblasts and T lymphocytes are stimulated to proliferate by mitogenic signals. This apparent paradox is potentially resolved by the fact that addition of more p21 to the active extracts obtained from such proliferating cells extinguishes their kinase activity (Zhang et al., 1994; Harper et al., 1995), suggesting a stoichiometric requirement for two p21 units per unit of kinase for inhibition. Like p21, the stoichiometry of p27 in cyclin-cdk complexes may determine whether it acts as an inhibitor or not. While p21 levels are generally low in quiescent cells and rise under the influence of mitogens, p27 tends to accumulate in quiescent cells, and mitogenic stimulation causes the amount of p27 to fall significantly during the GO-S phase transition. Nevertheless, p27 continues to be synthesized in proliferating cells, suggesting the possibility that it may have its expression regulated periodically. In elutriated HL60 leukemia cells, where cell size correlates with cell cycle position, or in HeLa cells synchronized in M phase with nocodozole, p27 levels are maximal in G1-enriched fractions and decline as cyclin E accumulates (Hengst et al., 1994). Similarly, cycling T cells continue to express some p27, albeit at substantially lower levels than in quiescent cells (Nourse et al., 1994). These findings suggest that p27 may play a role in the timing of cyclin-cdk activation in proliferating cells. Together with p21 and possibly the Ink family members, p27 may prevent the premature activation of cyclin-cdk complexes in mid- to late G1 phase. Basal levels of p27 together with other ckis may provide a threshold that must be exceeded before cyclin-cdk complexes can become activated during entry into the cell cycle and/or during G1 progression. Early in G1 phase, p21 and p27 are present in molar excess to any cyclin D-cdk4 that is in the cell, and p21 stoichiometrically inhibits the activity of that complex. As cells enter the cell cycle and proceed toward midG1 phase, p27 is sequestered by accumulating cyclin D-cdk complexes (Fig. 2), with cdk4 activity first becoming evident as a critical threshold set by p21 and/or other ckis is exceeded. The subsequent activation of cyclin E-cdk2 would then result from redistribution of p27 to cyclin D-cdk4 (Firpo et al., 1994; Kato et al., 1994; Slingerland et al., 1994). Similarly, induction of p27 and/or other ckis, or diminution of cyclin-cdk levels, could lead to cell cycle arrest by allowing the amount of cyclin-cdk complexes in the cell to fall below the inhibitory threshold set by these inhibitors. The close adjustment of the levels of p27 and the various cdks in this model suggests that a small increase in available p27 could cause a strong inhibition of cdk2 activity, leading to cell cycle arrest. Indeed, the results of Reynisdottir et al. (1995) indicate that the total cdk pool in exponentially growing MvlLu cells can absorb a twofold increase in p27 levels, whereas a further increase, however modest, has a profound inhibitory effect. According to Reynisdottir et al., this increase in p27 levels becomes absorbed by other cdks besides cdk2, so an even smaller increase should inhibit all cdk2 activity when other cdks do not participate in binding p27, as is the case for cdk4 and cdk6 in TGF-P-treated cells (see

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Cell Cycle Regulation by TGF-P

R Point

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I

i

i

+isve ' QQ 4g4 I

I Inactive

I

I I

I

I

[a.0

01

I

om**

P a l P n pool

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'

I

I Active

'

I I

Fig. 2 An inhibitory threshold set by p21 and p27. In mitogen-stimulated cells entering the cycle, cyclin D-cdk complexes sequester p21 and p27 molecules. As the amount of cyclin D-cdk complexes exceeds a threshold imposed by the fluctuating levels of the two inhibitors, cyclin Ddependent kinase activity becomes detectable in mid-G1 phase and continues to increase as cells progress toward S phase. Titration of p21 and p27 would enable cyclin E-cdk2 complexes to assemble in a functionally active form as cells approach the G1-S boundary, thereby setting a dependence of cyclin E-cdk2 activity on formation of the cyclin D-cdk complexes. Both the stoichiometric and catalytic functions of cyclin D-cdk complexes are proposed to be necessary for G1 progression (Sherr and Roberts, 1995).

later). This hypothesis is consistent with a report that the adenoviral E1A oncoprotein binds to and inactivates p27, at least in vitro (Ma1 et al., 1996).

C. Downstream Effectors of GI Phase cdks The phosphorylation state of the tumor suppressor gene product p l loRb is cell cycle dependent (Buchkovitch et al., 1989; DeCaprio et al., 1989).Hypophosphorylated pRb negatively regulates cell cycle progression from G1 to S phase (DeCaprio et af., 1989).This inhibition of cell growth by pRb depends on the integrity of its pocket region, necessary for pRb to interact with the ElA, T antigen, and E7 oncoproteins from DNA viruses, as well as with the transcription factor E2F (Chellappan et al., 1991; Hiebert et al., 1992; Qian et al., 1992; Qin et al., 1992).Two other relatives of pRb, p107 (Ewen et al., 1991; Zhu et al., 1993) and the closely related p130 (Hannon et al., 1993; Li et al., 1993; Mayol et al., 1993),also associate with adenoviral E1A via pocket domains. Hyperphosphorylation of pllORb during late G1 phase results in the loss of its growth-suppressive properties (Laiho et al., 1990; Hinds et af., 1992; Ewen et al., 1993a). The activation of ~ 3 4 by' D~cyclins ~ ~ in mid-G1 phase

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Michael I. Ravitz and Charles E. Wenner

of the cell cycle is well correlated with the timing of the first phosphorylation of pRb, which, like p107, stably binds to D cyclins directly (Matsushime et al., 1992, 1994; Dowdy et al., 1993; Ewen et al., 1993a; Kato et al., 1993). In insect Sf9 cells coinfected with baculovirus vectors encoding pRb, cdk4, and D cyclins, complexes between cyclins D2 or D3 and pRb are abolished when cdk4 phosphorylates pRb. If a kinase-defective mutant is used in place of wild-type cdk4, stable ternary complexes form instead (Kato et al., 1993). pllORbis also a substrate of cyclin E-cdk2 kinase, at least in vitro (Akiyama et al., 1992), and this may be the cause of the hyperphosphorylation and functional inactivation of pllORb that occurs by the time of appearance of cyclin E and p33cdk2activation in late G1 phase, as observed both in MvlLu mink lung epithelial cells and in C3H 10Ti mouse fibroblasts (Koff et al., 1993; Kim et al., 1994). This is close to the time of the R-point, both in epithelial cells and in fibroblasts (Dulic et al., 1992; Koff et al., 1992; Geng and Weinberg, 1993; Ravitz et al., 1995). Like the D-type cyclins, cyclin E leads to pRb phosphorylation when it is overexpressed in Rb-negative Saos-2 human osteosarcoma cells cotransfected with plasmids encoding pRb (Hinds et al., 1992; Ewen et al., 1993a).Therefore, cyclin E-cdk2 together with cyclin D-dependent kinases may contribute to Rb phosphorylation late in G1 (Hinds et al., 1992; Hatakeyama et al., 1994). It is possible that the first phosphorylations of pRb are caused by cyclin D-cdk4, and then an identical spectrum of phosphorylated residues may be maintained by subsequently activated cyclin E-cdk2 complexes, which most likely phosphorylate other key substrates as well. Alternatively, cyclin D-cdk4 complexes may modify pRb in a way that makes it a target for further, qualitatively distinct phosphorylations by cyclin E-cdk2. In addition to the G1 phosphorylation events, further phosphorylation of pRb is known to occur during S phase and in G2-M (DeCaprio et al., 1992). Dephosphorylation of pRb occurs during anaphase under the action of a type 1 phosphoprotein phosphatase (Durfee et al., 1993; Ludlow et al., 1993). Hypophosphorylated p l loRb,found predominantly in GO and early G1 phase, can bind to and inhibit trans-activation by the transcription factor E2F-1 (Hamel et al., 1992; Hiebert et al., 1992). Phosphorylation of pllORb is known to convert the E2F binding site from a negative to a positive cisacting element (Weintraub et al., 1992). E2F-1 was the first cloned member of the E2F family of transcription factors (Li et al., 1994b), which now includes E2Fs 1-5. Each member of the E2F gene family requires heterodimerization with a DP family member (DP-1) for efficient DNA binding, pRb binding, and trans-activation of target promoters (Biejersbergen et al., 1995; Lam and La Thangue, 1994). Both E2F-1 and DP-1 have been shown to be phosphoproteins. Phosphorylation of E2F-1 at Ser-332 and Ser-337 occurs in late G1 phase and prevents its interaction with pRb (Fagan et al., 1994).This mechanism may supplement or fine tune the termination of their interaction caused by phosphorylation of Rb by G1 phase cdks and leading

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to an increase of E2F-1 transcriptional activity at this time. On the other hand, the phosphorylation of E2F-1 at Ser-375 potentiates the pRb-E2F-l interaction (Peeper et al., 1995). DP-1 is also phosphorylated during S phase by the action of the cyclin A-cdk2 complex, which binds via cyclin A to the amino-terminal domain of E2F-1. This event inhibits DNA binding by E2F1, and may result in a termination of E2F-1-mediated transcription during the S phase (Krek et al., 1994). Each member of the E2F family is tightly regulated by association with a protein of the pRb gene family (Fig. 3). The various E2F-DP complexes have different capacities to bind with different pocket proteins in vivo, and the specificity of binding is mediated by the E2F subunit (Wu et al., 1995). E2F1, E2F-2, and E2F-3 are able to interact with pllORb, but not with the Rb family members p107 and p130, which instead interact with other E2F species (Dyson et al., 1993; Lees et al., 1993). In particular, p107 interacts with E2F-4, and its overexpression inhibits E2F-4-mediated trans-activation of CAT reporter plasmids in C33A cervical carcinoma cells (Biejersbergenet al., 1994).p130 also interacts with E2F-4-DP-1, and is able to suppress E2F+mediated trans-activation in U2-0s osteosarcoma cells (Vairo et al., 1995).p130 is also a potent inhibitor of E2F-5-mediated transcription (Hijmans et al., 1995), and, like p107 and pRb, can arrest the growth of certain cells, including the Rb-negative cell line Saos-2 (Hinds et al., 1992; Ewen et al., 1993a; Zhu et al., 1993; Claudio et al., 1994; Vairo et al., 1995). In addition, Vairo et al. have shown that cotransfection of E2F-4 with p130 in Saos-2 cells overrides pl30-induced cell cycle arrest. E2F-5, which preferen-

E2F Complex

1%

>

h

v

h

01

v

h

>S

Cell Cycle Phase Fig. 3 Summary of the temporal appearance of DRTF1-E2F complexes during cell cycle progression. Free DRTFl-E2F, which begins to appear during G1 (dashed arrow) and peaks during S phase (open arrow), is widely believed to be transcriptionally active (Lam and La Thangue, 1994).

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Michael 1. Ravitz and Charles E. Wenner

tially binds to p130, is more closely related to E2F-4 (78% identity) than to E2F-1 (57% identity) (Hijmans et al., 1995). E2F-1 binds to promoter elements in many genes that become activated at or near the G1-S transition, such as dihydrofolate reductase, thymidine kinase, and cyclin A (Mudryj et d., 1990). E2F sites are also located in other genes of interest, such as c-myc, N-myc, c-myb, B-myb, cyclins D1 and A, and the epidermal growth factor receptor gene (Mudryj et al., 1990; Motokura and Arnold, 1993; Henglein et d., 1994). Work by Duronio and O’Farrell (1995) indicates that cyclin E expression during the G1-S transition of cells in the developing embryo of the fruit fly Drosophila is E2F dependent, suggesting that there is an E2F binding site in the promoter for cyclin E as well. It is possible that the cyclin D-cdk4-catalyzed phosphorylation of pRb and/or p107 in mid-G1 phase (Matsushime et al., 1994; Biejersbergen et al., 1995)might cause the release of transcriptionally active E2F, thereby leading to the transcription of cyclin E in late G1 phase. It has been observed that overexpression of the proto-oncogene myc upregulates E2F activity and cyclin A mRNA (Jansen-Durr et al., 1993). Furthermore, TGFp, which prevents phosphorylation of pRb during inhibition of epithelial cell growth (Laiho et al., 1990), also downregulates cyclin A mRNA (Barlat et al., 1993). These findings suggest that induction of cyclin A by the time of S phase entry might be a response to the further release of E2F that occurs in late G1 phase under the action of cyclin E-cdk2. Thus complexes between hypophosphorylated pRb and E2F-1 accumulate during G1 phase but are unable to form once pRb is phosphorylated by G1 cyclins. Even more transcriptionally active E2F-1 accumulates during the transition from GO-early G1 to S phase by a protein synthesis-dependent mechanism of E2F-1 gene transcription (Slansky et al., 1993). This mechanism may involve autoinduction of the E2F-1 gene, as there are E2F sites in the E2F-1 promoter itself and E2F-1 transcription is stimulated by the deregulated expression of G1 phase cyclins (Johnson et al., 1994). The centrality of E2F-1 in the Rb pathway is highlighted by the fact that microinjection of E2F-1 cDNA into REF-52 rat embryo fibroblast cells that have been made quiescent by serum deprivation induces S phase (Johnson et al., 1993). Similarly, introduction of an E2F-1 expression plasmid into human osteosarcoma cells can override a pRb-imposed block of the growth of these cells (Qin et al., 1994). These results suggest that E2Fs are dominant determinants of G1 progression that represent an endpoint for much of the G1 cdk cascade, and may even be the only rate-limiting effectors of pRb function. The fact that pllORb is still found in complexes with E2F during S phase indicates that not all the Rb is hyperphosphorylated at the time of the G1-S transition, as only hypophosphorylated Rb can bind to the E2F transcription factor (Schwarz et al., 1993). This indicates that the activation of E2Fresponsive genes in late G1 is not controlled exclusively according to the

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original model of p l loRbphosphorylation. Schwarz et al. present evidence that, during the late G1 phase of the cell cycle, E2F is complexed with p107, which, like p l loRb, is able to suppress E2F-mediated transcription from CAT promoter constructs. They also found that, during S phase, p107 is localized to stable E2F-containing complexes that also contain cdk2 and its S phase regulatory subunit, cyclin A, which binds to p107 via the spacer in its pocket region (Ewen et al., 1992). A similar stable complex has been reported to exist in mid- to late G1 phase in HL60 cells, except that cyclin E exists in place of cyclin A (Lees et al., 1992).These latter authors also demonstrate that the complex found by Schwarz et al. is present in S phase, and the appearance of the different cyclin-cdk2 holoenzymes in the E2F-plO7 complex correlates with the kinetics of the synthesis of these cyclins. It is not known, however, whether the appearance of new complexes occurs only after the disappearance of the former complexes, or if the transition is more gradual. The role of cdks in the aforementioned complexes is also not known at present; however, it is possible that they serve to phosphorylate p107 and/or E2F-DP-1. This could modulate either the transcriptional activity of E2F-DP-1 and/or the formation or dissolution of the complexes. The phosphorylation of p107 is cell cycle regulated. A high level of both phosphorylated p107 and E l A-associated kinase activity is detected during S and G2-M in a HeLa cell line derivative expressing adenoviral E1A protein (Herrmann et al., 1991).In NIH 3T3 fibroblasts, the phosphorylation of p107 observed by mid-G1 phase is correlated with an increase in the protein levels of cyclin D1, which, together with cdk4, may be targeting p107 as well as pRb (Biejersbergen et al., 1995). In addition, these authors found that, although the expression of p107 also increases throughout the later portion of G1 phase, this does not result in the increase of E2F-pl07 complexes detected by mobility shift analysis in the same cells. However, they found a reoccurrence of E2F-pl07 complexes as cells enter S phase, suggesting that, while the majority of newly synthesized p107 is phosphorylated, a drop in cyclin Dl-cdk4 activity by S phase may allow some of the newly synthesized p107 to bind to E2F-4 at this time. This is consistent with the additional finding of Biejersbergen et al. that phosphorylation of p107 results in the loss of its ability to associate with E2F-4. Another possible role for E2F-pl07 could be to direct cdks to adjacent targets on the DNA, such as other transcription factors. These G1 phase-specific complexes may be replacing analogs that contain the Rb-related protein p130, particularly if the cells have been well arrested in a GO state prior to cell cycle activation (Cobrinik et al., 1993). The existence of the three pocket proteins in different, independent complexes has been substantiated by the work of various authors (Bandara and La Thangue, 1991; Chellappan et al., 1991; Mudryj et al., 1991; Cao et al., 1992; Devoto et al., 1992; Shirodkar et al., 1992; Cobrinik et al., 1993). Thus, E2F complexes containing p130 and/or p107 probably exist in paral-

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Michael 1. Ravitz a n d Charles E.Wenner

lel with pRb-E2F-l complexes, which are detected during mid- to late G1 phase and into S phase (Fig. 3). As implied earlier, distinct forms of E2F are probably involved in each of these temporally evolving complexes. Indeed, the role in GO of the E2F site in the c-myc promoter may reflect its regulation by the pl30-bound form of E2F that predominates at that time (Cobrinik et al., 1993; Biejersbergen et al., 1994). Finally, the specific E2Fs may have preferential target genes, possibly due to subtle variations in the consensus E2F binding site (Li et al., 1994b).

D. TGF-f3I and TGF-f3 Signal Transduction TGF-P1 is a pleiotropic, 25-kDa, homodimeric growth factor involved in controlling cell cycle progression, cell differentiation, cell adhesion, and extracellular matrix deposition in a variety of cell lineages (Massague, 1990; Roberts and Sporn, 1990). The autocrine-paracrine effectors .of the type P transforming growth factors, TGF-P1, TGF-P2, and TGF-P3, are members of a larger superfamily of peptides that control cell proliferation, differentiation, and developmental processes (Laiho and Keski-Oja, 1992; Massague, 1992; Kingsley, 1994). Signal transduction by TGF-P1 is initiated following ligand binding to heteromeric complexes of high-affinity TGF-P cell surface receptors. Type I and I1 receptors are N-linked glycoproteins (Cheifetz et al., 1986), each with a cytoplasmic domain that shows homology to serinethreonine kinases (Lin et al., 1992; Wrana et al., 1992; Franzen et al., 1993; Carcamo et al., 1994; Kingsley, 1994; Ten Dijke et al., 1994). The type I and type I1 receptors interact with each other directly (Franzen et al., 1993). Whereas the type I1 receptor can bind ligand by itself, the type I receptor requires the type I1 receptor to bind ligand (Wrana et al., 1992) and the type I1 receptor also determines ligand specificity (Ebner et al., 1993). On the other hand, type I1 receptor requires the type I receptor in order to signal and for signal specificity (Carcamo et al., 1994). Phosphorylation of the type I receptor by the type I1 receptor may initiate signaling by the heterodimeric complex (Wrana et al., 1994). The type I11 TGF-P receptor is proposed to be involved in regulating the access of ligand, particularly TGF-P2, to the other TGF-P receptors (Wang et al., 1991; Lopez-Casillas et al., 1993). Attempts have been made to determine the presumed substrates and/or interacting proteins of the TGF-P receptors. A putative substrate that associates with and is phosphorylated by the TGF-P receptor complex has been identified (Chen et al., 1995). The TGF-P type I1 receptor cytoplasmic domain interacts with TGF-P receptor-interaction protein-1 (TRIP-1), containing five WD domains common to adaptor proteins mediating protein-protein interactions (Neer et d., 1994). Possibly, the role of TRIP-1 functions as an adaptor protein in TGF-P signaling analogous to Grb2 in the ras pathway (Chen et al., 1995).

Cell Cycle Regulation by TGF-P

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Genetic screens have also been used to identify other interacting proteins with the cytoplasmic components of the TGF-p type I receptor. The immunophilin FKBP-12 interacted with the TGF-p type I receptor, suggesting that FKBP-12 may play a role in type I receptor-mediated signaling (Wang et al., 1994). The cytoplasmic portion of the type I receptor was also found to interact with p2lRaSfarnesyl transferase a (Serra and Moses, 1996).The authors also presented evidence that Ras may be directly involved in TGF-p signaling. Recently the Smad family of evolutionary conserved proteins has been implicated in TGF-p superfamily signal transduction. One of these, DPC4 (deleted in pancreatic carcinoma) is a candidate tumor suppressor gene that is mutated or deleted in breast, lung, head and neck, and ovarian cancer and fifty per cent of pancreatic malignancies (Hahn et al., 1996; Massague, 1996). Smadl is phosphorylated and moves to the nucleus by TGF-P family members (Chen e t al., 1996). Chen et al. (1996)further demonstrated that Smad2 forms part of a transcriptional activation complex in the nucleus which is responsive to TGF-p and activin signaling. Furthermore, Zhang et al. (1996) have shown that ligand binding by TGF-p is associated with the presence of phosphorylated hMAD-3 (Smad2 homolog). When truncated at their carboxy terminal, hMAD-3 and DPC4 act as dominant negative inhibitors of the normal TGF-p response. In addition, hMAD-3 and DPC4 synergize to induce TGF-P responsive genes (Zhang et al., 1996). Also, wt DPC4 restores TGF-p responsiveness in breast epithelial cells lacking the DPC4 gene (Hata et al., 1997). These results are consistent with the finding that TGF-P binding induces the association of DPC4 with Smad2 suggesting that mad proteins may function in heterodimeric complexes during the activation of TGFp responsive gene elements, possibly in association with other transcription factors (Zhang et al., 1996; Hata et al., 1997). Complete loss of one or more TGF-p receptors occurs in certain neoplastic cells, including neuroblastoma, retinoblastoma, breast carcinoma, and several leukemic cell lines (Keller et al., 1988; Kimchi et al., 1988; Zugmaier et al., 1989). It has been proposed that loss of TGF-P receptors could be a mechanism whereby certain preneoplastic cells escape from growth inhibition by TGF-P during tumor progression. Although the loss of TGF-P signaling during transformation may be attributed in part to either changes in upstream signaling as mentioned earlier or to alterations in cdk activities (see later), there is also evidence for specific frameshift or point mutations in TGF-p type I and I1 receptors. For example, Markowitz et al. (1995) have observed that the type I1 TGF-P receptor is inactivated in some colon cancer cells. The loss of the type I1 receptor is associated with microsatellite instability, namely the RER' (replication error) phenotype but not the RER- phenotype. Although the genetic evidence is compelling, the large number of potential mutations in the RER' phenotype does not necessarily indicate that the type I1 receptor mutation is necessary for tumorigenesis. It is apparent that more extensive studies are required before the loss of type I1 receptor

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can be directly associated with the development of colon cancer. In another study of eight human gastric cell lines, four of the seven that were resistant to TGF-P inhibition were found to have genetic alterations at type I1 receptor gene loci (Park et al., 1994). This is a relatively small sampling and requires further study. It is of interest that overexpression of human cyclin D1 reduces the TGF-fi type I1 receptor and growth inhibition by TGF-P1 in an immortalized human esophageal epithelial cell line (Okamoto et al., 1994). With respect to alterations in the type I receptor, Kim et al. (1996) reported that a prostate cancer cell line that lost sensitivityto TGF-P inhibition exhibits mutations in the type I receptor gene. This finding is in contrast to reports that TGF-P inhibits the proliferation of most prostate cancer cells in vitro.

E. Effects of TGF-f! 1 on GI Phase Progression 1. INHIBITORY VERSUS STIMULATORY EFFECTS OF TGF-f3 ON CELL GROWTH TGF-P1 was originally identified for its ability, in the presence of epidermal growth factor (EGF) or TGF-a to enhance anchorage-independent growth of normal rat kidney (NRK) fibroblasts, and to confer a reversibly transformed phenotype on these cells (Roberts et al., 1981). The effects of TGF-P on cell proliferation are highly dependent on a cell’s type, state of differentiation, and growth conditions, including the presence of other growth factors. While TGF-P1 inhibits the growth of normal cells of epithelial origin (Silberstein and Daniel, 1987; Russell et al., 1988; Moses et al., 1990), its ability to stimulate mesenchymal cell growth has also been well documented, particularly in rodent and human fibroblasts (Shipley et al., 1985; Leof et al., 1986; Soma et al., 1989; Kim et al., 1993,1994). However, the positive action of TGF-P1 seen in these cells appears to depend, at least in part, on the particular conditions of the cell culture. For example, application of TGF-P to C3H 10Ti mouse embryonic fibroblasts can result in either inhibition (Schwarz et al., 1988) or stimulation (Kim et al., 1993, 1994) of DNA synthesis. Cell density and effective concentration may each play a role in the bilateral responses to TGF-P (Linda and Majack, 1989; Battegay et al., 1990). On the other hand, the effect of cell density could also be related to a reduction in available TGF-P receptors at the cell surface, resulting in decreased binding of ligand (Rizzino et al., 1988). Even the source of the fibroblast-like clone can be a factor in the observed response to TGF-(3. TGF-P stimulates growth of fibroblasts from very early human fetuses but inhibits the growth of fibroblasts derived from fetuses of slightly later gestational age (Hill et al., 1986). Furthermore, while TGF-P inhibits the G1-S transition in primary and secondary rodent fibroblasts, this

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effect is lost following immortalization during the establishment of cultures (Sorrentino and Bandyopadhyay, 1989). Other effects of TGF-P in cell growth or differentiation depend on its interactions with other growth factors present. TGF-P stimulates the growth of NRK cells in the presence of EGF in soft agar, but both inhibit the growth and antagonizes the mitogenic action of EGF when these cells are grown in monolayer culture (Roberts et al., 1985). The effects of TGF-P on muscle cell differentiation also depend in part on the presence of other mitogens. In the absence of serum mitogens, TGF-P inhibits myogenic differentiation, whereas in their presence, it induces the differentiation of myoblasts to muscle cells (Zentella and Massague, 1992). 2. EFFECTS OF TGF-P ON CELL CYCLE KINETICS OF C3H 1OTi CELLS

Although C3H 10T; cells are not the reference standard for mesenchymal cells, they do possess several features, including ease of synchronization, that make them particularly useful in the study of the effect of TGF-P on cellular growth. While TGF-P is able to stimulate the growth of C3H 10Ti mouse fibroblasts, it does so with kinetics approximately 4h slower than that of other mitogens such as EGF, platelet-derived growth factor (PDGF), or serum (Kim et al., 1993, 1994). TGF-/3 interferes with the action of many different mitogens, including PDGF and EGF (Roberts et al., 1985; Anzano et al., 1986). In C3H 1OT4 cells, TGF-p also delays by approximately 4 h the stimulation of DNA synthesis induced by these .mitogens (Kim et al., 1993). In this case, however, synergy between the two growth factors is observed, yielding a greater stimulation of both DNA synthesis and pRb phosphorylation than that caused by application of either growth factor alone. The origin of this delay is not completely understood. In AKR-2B fibroblasts, delayed mitogenesis resulting from TGF-P is thought to be mediated indirectly by induction of c-sis mRNA and secretion of PDGF-like proteins (Shipley et al., 1985; Leof et al., 1986). However, polyclonal anti-PDGF-AB antibodies, which are able to inhibit PDGF-BB-induced DNA synthesis in the C3H 10Ti cell line, do not inhibit TGF-P-induced proliferative responses in these cells (Kim et al., 1993).The mechanism responsible for the observed delay appears to act at a point distal to the activation of immediate early genes, as the administration of TGF-P to cells 3-5 h following application of EGF still results in a delay in EGF-induced DNA synthesis, indicating that the delay and subsequent synergy of EGF-induced c-myc expression by TGF-P is not obligatory for this effect (Kim, 1993). Thus the interruption or delay of mitogen-induced DNA synthesis by TGF-P may occur in a substage of G1 near the G1-S boundary. On the other hand, c-myc may play some role in the growth-stimulatory responses to TGF-P, as exogenous expression of c-myc is required to

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allow TGF-P to induce anchorage-independent growth of both AKR-2B and C3H 10Ti fibroblasts (Leof et al., 1987; Alexandrow et al., 1995).

3. INHIBITORY EFFECTS OF TGF-P IN EPITHELIAL CELLS TGF-P inhibits the growth of MvlLu mink lung epithelial cells and keratinocytes by stopping these cells in late G l phase (Laiho et al., 1990; Pietenpol et al., 1990; Howe etal., 1991; Eblen et al., 1994). TGF-P can reversibly inhibit S phase entry when added to cultures both early and late in the G1 phase. While one report demonstrates that a human keratinocyte cell line, HaCaT, loses sensitivity to TGF-P approximately 6 h prior to the G1-S transition (Geng and Weinberg, 1993), in BALB/MK mouse keratinocytes and MvlLu cells, TGF-P can inhibit G1 phase exit when added as late as 1-2 hours prior to S phase, the same point at which the cells become arrested when TGF-P is added at earlier times (Laiho et al., 1990; Howe et al., 1991). Beyond this point, which is temporally equivalent to the R-point, TGF-P is no longer capable of inhibiting growth. In keratinocytes, one of the earliest effects of TGF-P action is to inhibit the transcription of the immediate early gene c-myc (Pietenpol et al., 1990), which contains the consensus E2F binding sequence in its promoter (Mudryj et al., 1990). The importance of c-myc in the growth-inhibitory response to TGF-P is suggested by the finding of Alexandrow et al. (1995) that inhibition of keratinocyte growth by TGF-P is blocked by overexpression of an estrogen-inducible form of c-myc. However, Longstreet et al. (1992) found that transfection of mink lung epithelial cells with a c-myc oncogene maintains sensitivity to TGF-P1 growth arrest. The effect of TGF-P on c-myc expression can be abrogated by DNA viral-transforming proteins, which suggests that growth inhibition by TGFP in BALB/MK keratinocytes depends on the presence of wild-type pRb capable of binding to E2F (Pietenpol et ul., 1990). Other early effects of TGFp include its ability to affect the transcription of delayed early genes such as D cyclins, while, later in the cell cycle, TGF-P appears to function largely through the modification of cdk activity by various mechanisms, including modulation of cki activity (see Section II.F.4).

F. Effects of TGF-P on G I Phase cdk Activity 1 . EFFECTS OF TGF-P ON pRb PHOSPHORYLATION

pRb is a primary downstream target of TGF-P action, substantiated by the fact that overexpression of the transcription factor E2F, known to interact with pRb, overcomes TGF-P-mediated growth arrest in MvlLu cells (Schwarz et al., 1995). Treatment of C3H 10Timouse fibroblasts with TGFP leads to both the increased expression of pRb and pRb phosphorylation

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(Kim et al., 1994). On the other hand, application of TGF-p to cells of epithelial origin, such as MvlLu cells, prevents hyperphosphorylation of pRb, which normally occurs at about the time of the R-point, during late G1 phase (Laiho et al., 1990). Application of TGF-p to cells following this period no longer results in inhibition of the phosphorylation of pRb. Thus, at least some of the signals that TGF-p sends to epithelial cells should be mediating their effects at a time in the cell cycle similar to that at which Rb kinase(s) become active. The first evidence for such regulation was supplied by Howe et al. (1991),who demonstrated that, in MvlLu epithelial cells, TGF-p prevents the phosphorylation and activation of ~ 3 4 ' ~in' association ~ with the reversible inhibition of cell growth by TGF-P at a time close to the G1-S ~ to remain relboundary. The steady-state protein level of ~ 3 4 ' ~is' known atively constant throughout the cell cycle (Draetta and Beach, 1988), and Howe et al. also found that TGF-p has no effect on the total protein level of this enzyme. By contrast, Eblen et al. (1994), using the same MvlLu clone as Howe et al., have demonstrated that TGF-p downregulates p34CdC2synthesis in addition to inhibiting cdc2-associated histone H1 kinase activity. It has also been shown in MvlLu cells that the formation of active cyclin E-cdk2 complexes is inhibited by TGF-p, despite the accumulation of cyclin E to normal levels (Koff et al., 1993). Similarly, TGF-p prevents the accumulation of active cyclin D-cdk4 complexes in MvlLu cells by depressing cdk4 synthesis at the level of translation during mid- to late G1, without affecting D cyclin levels (Ewen et al., 1993b). Ewen et al. also demonstrate, however, that while constitutive expression of cdk4 renders MvlLu cells resistant to the growth-inhibitory effects of TGF-p, allowing for cdk2 activation and hyperphosphorylation of pRb, constitutive expression of cdk2 does not. In addition, cdk4 overexpression reduces the serum requirement of cells released from contact inhibition. These results suggest that cyclin D-cdk4 operates temporally upstream from cyclin E-cdk2, as part of a feedforward mechanism responsible for cyclin E-cdk2 activation. Ewen et al. further suggest that an inhibition of cdk4 synthesis is primary to the action of TGF-p in these cells, and leads to the inhibition of cdk2 activity and pRb phosphorylation. 2. EFFECTS OF TGF-P ON p27 EXPRESSION AND DISTRIBUTION Levels of cdk inhibitory activity, attributable at least in part to p27, are elevated in quiescent MvlLu cells and decrease as cells progress through G1 phase, coincident to the activation of cyclin E-cdk2. The inhibitory activity of p27 increases the longer cells are in the growth-arrested state. Inactivation of p27 during the cell cycle correlates directly with the progressive loss of sensitivity to TGF-p-mediated growth arrest as cells approach the R-point (Polyak et al., 1994a; Slingerland et al., 1994). In principle, cyclin D-cdk4

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complexes could phosphorylate cdk2 itself or an intervening CAK, thereby triggering the activity of the cyclin E-cdk2 kinase. However, nonproliferating MvlLu cells arrested either by TGF-P treatment or by contact inhibition contain an inhibitor of cdk2 activation, a 27-kDa protein that binds to cyclin D-cdk4 as well as to cyclin E-cdk2. Polyak et al. obtained data suggesting that the cyclin D-cdk4 complexes acted stoichiometrically in binding and sequestering the inhibitory components in the extracts. Furthermore, p27 inhibitory activity can be recovered both from quiescent MvlLu cells and from proliferating MvlLu cells following heat treatment of the extracts. This indicated that the protein is constitutively present, consistent with the result that TGF-P treatment has no effect on K t p l message levels in this cell type. Together with the results of Ewen et al., these results suggest the following model whereby TGF-@inhibits the phosphorylation of pRb by G1 phase cdks and induces G1 arrest in cells of epithelial origin, such as MvlLu cells (Fig. 4a). TGF-P represses cdk4 synthesis, preventing the redistribution of p27 to cyclin D-cdk4 complexes so that it continues to saturate and inhibit cyclin E-cdk2 (Polyak et al., 1994a). Conversely, during G1 progression, the sequestration of p27 by cyclin D-cdk4 would thereby promote cyclin E-cdk2 activity, helping to establish a temporal order for the activation of these kinases. This model explains how steady-state p27 levels can remain constant in proliferating MvlLu cells, as well as in Swiss 3T3 fibroblasts, where the upregulation of cyclin D1 upon serum-induced stimualtion is proposed to cause a redistribution of p27 to cyclin Dl-cdk4 complexes, resulting in the activation of cyclin E-cdk2 (Polyak et al., 1994a; Toyoshima and Hunter, 1994). However, a completely different mechanism operates in the case of C3H 1OT4 mouse embryonic fibroblasts, where mitogenic stimulation by TGF-@does not have a major effect on either cdk4 or cyclin D1 levels but does lead to the striking downregulation of the steady-state level of p27 (Ravitz et al., 1995). This downregulation occurs at a postscriptional level, probably involving the ubiquitin-proteosome pathway (Fig. 4B) (Ravitz, 1996; Ravitz et al., 1996; Pagano et al., 1995). However, the possibility remains that modulation of synthesis also plays a role in the regulation of p27 activity in many cell types, as discussed in Section II.B.4.k Application of epidermal growth factor does not lead to the rapid downregulation of p27 observed in C3H 1OT4 fibroblasts following treatment of cells with TGF-P (Ravitz et al., 1996).EGF treatment upregulates the steadystate level of cyclin D1 in C3H 1OT4 cells, with a magnitude much greater than that induced by TGF-@(Ravitz et al., 1996). Furthermore, this ability of EGF to induce cyclin D1 is abrogated by the simultaneous addition of TGF-P, suggesting a possible explanation for the TGF-@-induced delay of EGF-stimulated cell cycling as discussed in Section 11.E.2. Therefore, p27 downregulation is only a minor component of, and not central to, the mechanism of cdk activation by EGF, whereas it is central in the case of TGF-P.

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Cell Cycle Regulation by TGF-P R-pt

1

G1

TGF-~

A

inactive

*S

sGtiYe

UBlQUlnN

G1

f

TGF-p

B

Fig. 4 (A) TGF-P inhibits cell proliferation via the regulation of G1 phase cdks. In epithelial cells, TGF-P can depress the synthesis of cdk4. TGF-P treatment also induces certain cells to increase steady-state levels of pl5. Together, these effects of TGF-P prevent the sequestration of ~ 2 7 ~ ' Pfrom ' cyclin E-cdk2 by the cyclin D-cdk4 holoenzyme, either preventing or rendering ineffective CAK-mediated phosphorylation and activation of these kinases. (B) TGF-P induces cell proliferation via the regulation of G1 phase cyclin-dependent kinases. In C3H 1OT&fibroblasts, which are examples of mesenchymally derived cells, TGF-P leads to a mild induction of D cyclins, while leading to the striking downregulation of p27 and a loss of this protein from complexes with cyclin E-cdk2 kinase. This downregulation of ~ 2 7 ~ ' poccurs ' posttranslationally, probably via the ubiquitin-proteosome pathway (UBIQUITIN).

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Both the “sequestration” pathway and a pathway for protein degradation may operate in the cases of TGF-p and EGF, and it is suggested that crosstalk between the signal transduction pathways for these two growth factors is possible. It is further speculated that both pathways may also exist in epithelial cells as well, with the degradative pathway hitherto undetected. Despite the close structural and functional homology between p27 and p21, the latter authors also found that, in C3H 1OT&cells, TGF-p has no observable effect on p21 steady-state levels, and application of EGF also has no effect on the steady-state levels of this protein.

3. OTHER EFFECTS OF TGF-P ON CYCLIN AND cdk EXPRESSION While TGF-f3 treatment prevents the formation of active cyclin E-cdk2 complexes, it does not prevent the normal accumulation of cyclin E in MvlLu cells released from GO by replating at low density in serum-containing medium (Koff et al., 1993). Nevertheless, TGF-p prevents the appearance of cyclin E in serum-stimulated HaCaT human keratinocytes, at both the mRNA and protein levels (Geng and Weinberg, 1993). This effect is lost after a time in late G1 phase when cyclin E normally reaches maximal levels in these cells, corresponding roughly to the R-point. TGF-p also prevents the induction of cyclin A, which, like cyclin E, has nearly undetectable levels of message in cells arrested in GO. Downregulation of cyclin A by TGF-p has also been observed in Chinese hamster lung fibroblasts by Barlat et al. (1993). Since TGF-p loses its inhibitory effects by late G1 phase, prior to the onset of cyclin A synthesis, the effect on cyclin A expression is probably secondary to a cell cycle block incurred at an earlier stage, namely the loss of cyclin E expression. Geng and Weinberg also found that, while the expression of D cyclin mRNAs was high in GO cells and showed a moderate (two- to threefold) induction with cell cycle progression, TGF-p treatment had very little effect on them. By contrast, TGF-P treatment of rat intestinal epithelial cells inhibits cyclin D1 expression at both the message and protein levels, both in exponentially growing and GO-arrested cells, while having no effect on cyclins D2 or D3 (KO et al., 1995). Interestingly, TGF-P did block a fourfold serum induction of both cdk2 and cdk4 mRNAs observed in HaCaT cells (Geng and Weinberg, 1993). TGF-p treatment, however, has no effect on cdk4 mRNA or protein levels in exponentially growing rat intestinal epithelial cells (KO et al., 1994). Similarly, in proliferating HaCaT cells, as well as in proliferating MvlLu cells, TGF-p does not affect the steady-state protein levels of cdk4 or cdk6, except as a late effect following cell cycle arrest as cells fall into the quiescent state (Reynisdottir et al., 1995). Such a reduction in the levels of cdk4 and cdk6 proteins may be an effect of TGF-p-induced cell cycle arrest, rather than a cause, in contrast with the ability of TGF-p to

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inhibit cdk4 synthesis in MvlLu cells emerging from quiescence during release from contact inhibition (Polyak et al., 1994a; Reynisdottir et al., 1995). The varying effects of TGF-P on cdk4 might be explained by noting that it has its suppressive effect on cdk4 synthesis when cells are emerging from quiescence but it is without such effects when added to actively proliferating cultures. Due to the rate-limiting nature of cyclin expression during G1 phase cdk activation, a key effect of TGF-p may be to prevent cyclin E accumulation and subsequent cdk2 activation; however, changes in the level of either subunit of cyclin D-cdk4 could affect the p27 threshold level and provide a potential mechanism for TGF-P action. 4. EFFECT OF TGF-P ON p15 LEVELS AND cki DISTRIBUTION Although initial studies indicate that p53-independent induction of p21 plays a role in TGF-p-induced cell cycle arrest, at least in keratinocytes (Reynisdottir et al., 1995) and ovarian carcinoma cells (Elbendary et al., 1994), the primary cki targets of TGF-P appear to be p27 and the recently described inhibitor p l 5 (Ink4b).TGF-P treatment of HaCaT human keratinocytes induces Ink4b mRNA transcription by nearly 30-fold, causing increased binding of p15 to cdk4 and cdk6 in vivo and resulting in a loss of their kinase activity (Hannon and Beach, 1994). In this study, levels of cdk4 protein and mRNA did not vary with TGF-P treatment, in contrast to the results obtained in MvlLu cells (Ewen et al., 1993; Polyak et al., 1994a,b) and in the same cell line by different investigators (Geng and Weinberg, 1993). In addition, TGF-P treatment had no effect on the Kip2 message level in this cell type, indicating that any effect of TGF-P on p27 would have to occur at a posttranscriptional level. While p15 may be the dominant effector of TGF-P-mediated cell cycle arrest in human keratinocytes, its action may be coordinated with that of p27. In growing epithelial cells, sequestration of the majority of p27 by cyclin D-cdk4 will render the kinase either active or inactive, depending on the stoichiometry of p27 binding (i.e., on its partitioning between the different cdks). Upon TGF-P treatment, the induced p15 would bind cdk4 and/or cdk6, destabilizing the D cyclins and displacing the bound p27. This would then leave p27 free to bind to and saturate cyclin E-cdk2, rendering it inactive (Fig. 5). Evidence for such a cooperation between p27, p21, and p l 5 ckis has been provided by Reynisdottir et al. (1995), who found that, in both MvlLu cells and HaCaT ketatinocytes, TGF-p causes the induction of p l 5 with a concomitant redistribution of p27 from cdk4 and cdk6 to cdk2. In MvlLu cells, this leads to a four- to sixfold increase in cdk2-bound p27, which they predict should be sufficient to extinguish cdk2 activity completely (see Section II.B.4.ii). In the case of the HaCaT cells, which possess inactive p53 (Lehman et al., 1993), TGF-P also appears to upregulate p21. This suggests that p21 contributes to cdk2

Michael I. Ravitz and Charles E.Wenner

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4iuMs=h

+TGFB 4P15

Phosphorylation supporting G1- S

b1

GI arrest Cdk inhibition

K-15 * KCplS

Quiescence Cdk down-regulation

Fig. 5 The proposed model for TGF-P-induced cell cycle arrest of epithelial cells via cdk inhibitors. Exponentially growing epithelial cells contain p27 distributed in complexes with cyclin D-cdk4, cyclin D-cdk6, and cyclin E-cdk2. TGF-P addition causes a rapid elevation of pl5, which binds to and inhibits cdk4 and cdk6 and displaces bound p27. In MvlLu lung epithelial cells, the displaced p27 binds to cdk2 complexes, leading to their inhibition. In HaCaT human keratinocytes, the increase in plS is accompanied by an increase in p21, which binds to and inhibits cdk2. Thus both cell lines are growth arrested via the combined action of an Ink4 inhibitor (pl5) and a CipKip inhibitor (p27 andlor p21). Following cell cycle arrest, cdk expression declines as cells fall into the quiescent state. For purposes of clatity, cdk6 is not depicted in the diagram (Reynisdottir et af., 1995).

inactivation, together with p27 displaced from cdk4 and cdk6 by pl5. Reynisdottir et al. also found that recombinant p l 5 inhibits p27 binding to cdk4 in vitro, and overexpression of p15 in MvlLu cells induces the transfer of p27 from cdk4 to cdk2, providing further support for this model. However, whether the actual stoichiometry of p27 with respect to the various cyclin-cdk complexes is in agreement with the model outlined in Figure 5 awaits confirmation. 5. DISRUPTION OF THE ~ 2 7 ~ ' GENE p' AND RELATION TO CELL CYCLE ACTIVATION MODULATED BY TGF-P AND EFFECTORS OF G 1 PROGRESSION

Studies by Nakayama et al. (1996), Kiyokawa et al. (1996), and Fero et al. (1996) indicate that mice lacking ~ 2 7 ~ ' have P l enhanced growth. Tissues that express p27 at the lowest levels exhibit increased cell proliferation and increased cell number. As previous reports suggested that inhibition by TGFp is mediated, at least in part, by ~ 2 7 ~ ' pNakayama l, et al. (1996) studied the effect of TGF-P in T-cell proliferation on p27-'- mice. They demonstrated that TGF-@inhibits the stimulatory effects of IL-2 observed in T cells derived from lymph nodes of mice lacking ~ 2 7 ~ ' as p lwell as it does in T cells

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from control mice. The inhibitory effect of rapamycin, which prevents IL-2induced downregulation of p27 (Nourse et al., 1994), was also observed in lymphocytes isolated from p27-I- mice in a dose-dependent manner similar to that of wild-type T cells. This indicates that the inhibitory effect of rapamycin on T-cell growth does not require (Nakayama et al., 1996). In mouse embryonic fibroblasts (MEFs) prepared from p27-’- mice, the capacity for G1 arrest by contact inhibition or serum starvation is not affected (Kiyokawa et al., 1996). There is neither a significant increase in S phase duration nor an acceleration in G1 phase in these cells. Therefore, it would appear that p27 may be dispensable for the mediation of arrest by serum deprivation or contact inhibition as well as for inhibitory responses by TGF-p. Kiyokawa et al. (1996) have also reported that p27-I- MEFs, when deprived of serum or grown to confluence, reenter S phase when released from quiescence and, in addition, activate cdk2 with kinetics similar to those of the wild-type controls (unpublished data from the Koff laboratory). On the other hand, these workers have found an increase in the fraction of S phase cells in p27-I- thymus, suggesting that p27 affects the balance between proliferating and nonproliferating cells, as least in this organ. Although these studies do not rule out the possibility that p27 contributes to G1 phase arrest by contact inhibition, serum deprivation, or TGF-p in the wild-type mice, they provide evidence that other regulators of cell cycle progression may compensate to propagate antimitogenic signals by TGF-p when p27 is not available. For instance, perhaps p l 5 and/or p21 could compensate for p27 by acting redundantly in a similar pathway, or different inhibitory pathways could be involved for these effectors.

G. Cyclins, cdks, and Cancer Cancer, or neoplasia, is a heterogeneous group of pathological states in which cells fail to properly control their proliferation, leading to high numbers of abnormal cells that subsequently invade surrounding tissues. Cancer cells undergo rapid and uncontrolled cellular division, which is often associated with a failure to differentiate properly. They also possess increased survival potential, due at least in part to decreased apoptosis and a failure to appropriately monitor the fidelity of DNA replication. These characteristics arise as a result of accumulating genetic lesions that occur during the oncogenic process. Such lesions include the acquisition of dominantly acting oncogenes, which promote cellular division, and the concurrent loss of growth-inhibitory tumor suppressors or antioncogenes. Oncogenes can be growth factor analogs, growth factor receptors, signal transducers, or nuclear transcriptional activators (Baserga and Rubin, 1993), which operate upstream of the cdks, as well as cyclins and cdks themselves. Tumor sup-

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pressors include downstream targets of cdks, such as p53, pRb, and p130. pRb is defective or deleted in retinoblastomas, small-cell lung carcinomas, and many sarcomas and bladder carcinomas (Horowitz et al., 1990). p130 has been mapped to human chromosome 16q12.2, a region frequently altered in breast, ovarian, hepatic, and prostatic carcinomas, and is also found mutated in a nasopharyngeal carcinoma-derived cell line in which it is expressed only at low levels (Claudio et al., 1994). In general, any change of function in a downstream component of a regulatory chain may abrogate the requirement of the upstream components, resulting in loss of control over cell division. As mentioned in Section I, loss of checkpoint control may result from either the aberrant expression of positive regulators, such as the cyclins and cdks, or the loss of negative regulators, such as the ckis. Cyclin D1 was discovered in parathyroid carcinomas, where it is found overexpressed as the PRADl oncogene at chromosome 11q13 (Motokura et al., 1991; Motokura and Arnold, 1993). Overexpression and/or stabilization of cyclin D1 mRNA transcripts has also been found in gastric, esophageal, breast, and squamous cell carcinomas, as well as in B-cell lymphomas (Lammie and Peters, 1991; Jiang et al., 1992; Gillett et al., 1994), sometimes due to gene amplification at 11q13. Similarly, the cdk4 gene, which maps to chromosome 12, is amplified in many glioblastomas and some gliomas (He et al., 1994; Schmidt et af., 1994). Cyclin A is overexpressed in some hepatocarcinomas because it lacks a cyclin destruction box due to genomic insertion by hepatitis B virus (Wang eta]., 1992). This might in principle be the cause of transformation in these cells, as a nondegradable cyclin A could prematurely activate cdk2 and bypass R-point regulation due to its aberrant presence in G1 phase. The fact that TGF-P decreases the expression of cyclin E in certain cell types (Geng and Weinberg, 1993) suggests that overexpression of cyclin E might also override growth-inhibitory signals that converge near the Rpoint. Cyclin E is overexpressed in many types of human tumors, including breast carcinoma cell lines and mammary tumors, sometimes due to gene amplification (Buckley et af., 1993; Keyomarski and Pardee, 1993; Keyomarsi et al., 1994; Khatib et al., 1993). However, there is no direct evidence that cyclin E acts as a proto-oncogene. The involvement of ckis in the negative regulation of cell proliferation suggests that they may also function as tumor suppressor genes. The inhibitor p21, which maps to chromosome 6 ~ 2 1 . 1 ,has a potential role in tumorigenesis on the basis of its transcriptional activation by p53. However, to date there is no evidence that the gene encoding p21 itself is mutated in human tumors. Ink4a, which encodes p16, on the other hand, maps along with Ink4b to the chromosomal region 9p21, termed multiple tumor suppressor (MTS). This region is frequently found mutated in tumor cell lines derived

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from familial melanomas and gliomas (Kamb et al., 1994; Nobori et al., 1994), and also bladder carcinoma (Orlow et al., 1994). Moreover, homozygous or hemizygous deletions of Ink4a appear in the majority of gliobalstomas examined (Schmidt et al., 1994). There is some discrepancy over whether these defects occur in primary tumors. Deletion of Ink4a appears to be restricted to subsets of tumor cells that retain functional pRb, and vice versa (Otterson et al., 1994), providing evidence for the feedback loop discussed in Section II.B.4.i. The fact that ectopic expression of p16 (as well as the closely related inhibitor p18) suppresses cell growth with a correlated dependence on endogenous wild-type pRb indicates that p16 is a true tumor suppressor. It further suggests that pRb is the sole substrate of cyclin D-cdk4. Many transformed cells fail to arrest in response to TGF-P treatment and are no longer subject to contact inhibition, suggesting that p27 and/or p l 5 function could be targets of oncogenic transformation. Because the genes encoding p l 5 and p16 are so closely linked that deletions in the MTS region most frequently involve both genes, the loss of both p15 and p16 may be important to the development of certain tumors. Indeed, both tumor-derived cell lines and the primary tumors from non-small-cell lung cancer patients show homozygous deletions of not only INK4a but INK4b as well (Washimi et al., 1995).While TGF-P can inhibit the growth of melanocytes, many metastatic melanoma cells are TGF-P resistant (Rodeck et al., 1994), suggesting that p15 as well as p16 has been affected. Dual mutations of these two tumor suppressors would wipe out both intrinsic and extracellular modes of cdk control simultaneously, giving the cancer cell considerable growth advantage. A variety of cancers also show abnormalities at 12~12-12~13.1, the chromosomal location of Kip1 (Ponce-Casteneda et al., 1995). Deletions and rearrangements in this region occur frequently in acute myeloid leukemia (Chan et al., 1992) and in peritoneal mesothelioma (Decker et al., 1990). The nonrandom inversion i(12p) occurs in 85% of male germ cell tumors (Rodriguez et al., 1992) and has also been reported in ovarian teratoma and malignant ovarian neoplasms (Spelman et al., 1990,1992). Furthermore, introduction of a portion of chromosome 12 (12pter-12q13) into a human prostate cell line suppresses tumorigenicity in athymic nude mice (Berube et al., 1994). Nevertheless, several authors have independently noted the absence of structural mutations at the p27 gene locus in a variety of tumors analyzed (Bullrich et al., 1995; Pietenpol et al., 1995). However, p27 acts stoichiometrically to inhibit cyclin-cdks, and even modest changes in its levels might have a major impact on G1 phase progression. Thus, it is possible that posttranscriptional or posttranslational modifications, or even altered patterns of p27 expression, could occur during tumorigenesis or tumor pro-

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gression. It is also possible that other gene products lying upstream or downstream of the p27 pathway may be affected in certain human cancers-for example, transcription factors that regulate p27 transcription. Human p57Kip2is also a candidate tumor suppressor protein, owing to its chromosomal location at 1 1 ~ 1 5a, region known to undergo frequent deletions or rearrangements in many forms of human cancer, including Wilms tumor, and in patients with Beckwith-Wiedemann syndrome, which predisposes them to rhabdomyosarcoma and other tumors (Koi et al., 1993; Koufos et al., 1985).

Ill. CONCLUSIONS The findings presented in this review show that extracellular signals, including TGF-P and EGF, induce or inhibit cell proliferation via the regulation of G1 phase cdks (Fig. 5 ; Table I). The ckis have opened up new concepts as to how cdks are inactivated outside of cyclin degradation or posttranslational modification. Alterations in the balance of active cyclin-cdk complexes through regulation and/or redistribution of ckis may be a common theme in cellular growth control. Such details of G1 phase cdk regula-

Table I Key Cell Cycle Regulatory Proteins Protein

Cell cycle role

Function

Regulation"

P53 PRb p107 p130 cdc2 (cdkl) cdk2 cdk4/6 cdk7 (CAK) E2Fl-S Cyclin A Cyclin B1,2 Cyclin D1,2, 36

G1-S Mid-late G1-S G1-S GO41 M Late G1-S Mid-late G1 All phases G1-S S M G1 G1 G1 GI-S G1

Transactivate p21 transcription Repress transactivation by E2F-1 Repress transactivation by E 2 F 4 Repress transactivation by E2F-4/S PO, of bistone H1, lamins, etc. PO, of pRB, other pocket protiens? PO, of pRB, titration of p21/p27 Activation of cdks 1-6 by PO, Transcriptional activation Activation of cdk-2 (cdc2) Activation of cdc2 Activation of cdk4/6 (cdk-2) Inhibits cdk4/6 Inhibits cdk4/6 Inhibits cdk2/4/6 and PCNA Inhibits cdk4/6 and cdk2

DNA damage-induced Inactivated by PO, PO, by K2-E or -A? PO, by K416 or K2? Cyclins A, B; PO, Cyclins A, E; PO, Cyclin D1-3; PO4 Constitutive E2F-1 autoregulated Tranx; ubiquitin Tranx; ubiquitin Tranx; PEST Upregulated by TGF-P pRb blacks tranx Transcribed by pS3 TGF-0; ubiquintin

151mk4B

p161nk4A P21C'P' ~27~'p'

'PO,, phosphorylation, tranx, transcription; PESI, PEST-targeted proteolysis; ubiquitin, degradation hy thr ubiq uirin-prottoaome pathway; K4/6, cdk4/6; K2 E, eyclin E-cdk2, K2-A, cyclrn A
tion by extracellular signals are central to an understanding of how cells integrate mitogenic and antiproliferative signals with the cell cycle machinery. Addendum: The crystal structure of the human ~ 2 7 ~ ' kinase pl inhibitory domain bound to the phosphorylated cyclin A-cdk2 complex has recently been determined. The amino terminus of p27 binds to cyclin A at conserved cyclin box residues possibly competing with substrate binding, while its carboxyl terminus binds to and rearranges the amino terminal lobe of cdk2, and inserts into the catalytic cleft preventing ATP binding (Russo, A. A., Jeffrey, P. D., Patten, A. K., Massague, J., and Pavlevich, N. P., Nature 382: 325-331, 1996).

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The Natural Somatic Mutation Frequency and Human Carcinogenesis Andrew 1. C. Simpson Laboratory of Cancer Genetics, Ludwig Institute for Cancer Research, 01 509-010, Srio Paulo, SP, Brazil

I. Introduction 11. Somatic Mutation of Microsatellite Sequences A. Microsatellite Instability as a Result of DNA Mismatch Repair Deficiency B. The Somatic Mutation Rate is Controlled at the Level of DNA Repair C. Detection of Microsatellite Alterations by the Polymerase Chain Reaction D. Microsatellite Alterations in Tumors May Not Always Be Due to Mismatch Repair Deficiency E. Estimate of the Frequency of Microsatellite Alterations in Normal Tissue 111. Somatic Mutation of Minisatellite Sequences IV Somatic Mutation o f the HPRT Gene A. HPRT Mutation in Epithelial Cells B. HPRT Mutation in Peripheral Blood Lymphocytes V. The Frequency of Somatic Mutations in Solid Tissues Can Account for Multistep Carcinogenesis VI. Cellular Proliferation as a Risk Factor for Cancer VII. Germline Mutations A. Evolutionary Selection of the Human Germline Mutation Rate B. Estimates of the Human Germline Mutation Rate C. Evidence That Germline Mutations Are Due to Endogenous Mutations D. The Relationship between Germline and Somatic Mutations VIII. The Mutation Rate as the Fundamental Biological Pacemaker IX. The Importance of Measuring Somatic Mutation Rates X. The Mutational Clock and Cancer Prevention References

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Much recent attention has been paid to the important role of the DNA mismatch repair system in controlling the accumulation of somatic mutations in human tissues and the association of mismatch repair deficiency with carcinogenesis. In the absence of an intact mismatch repair system, cells accumulate mutations at a rate some 1000 times faster than normal cells, and this mutator phenotype is easily measured by the detection of the formation of new variant alleles at microsatellite loci. However, the mismatch repair system is not 100% efficient, even when intact, and the pattern of microsatellite alterations in a wide variety of tumors is consistent with these being due to clonal amplification from tissues that are genetically heterogeneous at microsatellite loci rather than

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mismatch repair deficiency in the tumor itself. On this basis, it can be estimated that the mutation frequency of microsatellites in normal human tissues is approximately per locus per cell. Similarly, a frequency of mutation at minisatellite loci in normal tissues of around lo-' per locus per cell can be estimated. Such elevated levels of mutation are consistent with a recent study of the frequency of HPRT mutation in human kidneys that demonstrated these to be frequent (average 2.5 X lop4 in individuals of 70 years or more) and exponentially related to age. Taken as a whole, the data suggest that somatic mutation in human epithelial cells may be some 10-fold higher than in peripheral blood lymphocytes and that the underlying rate of spontaneous mutation is sufficient to account for a large proportion of human carcinogenesis without the need to evoke either stepwise alteration to a mutator phenotype or clonal expansion at all the mutation steps in carcinogenesis. The exponential increase in mutation frequency with age is predictable on the basis that the mutation rate is controlled at the level of repair and that mutation in genes that affect the efficiency of these processes will gradually increase the underlying rate. In addition, the age relatedness of mutation frequency strongly supports the concept that mutation is cell division dependent and that cellular proliferation per se is an important risk factor for cancer. Comparison of somatic mutations with those in the human germline mutation suggests common mechanistic origins and that the high levels of somatic mutation that occur are a direct reflection of the germline mutation rate selected over evolutionary time. Thus, the somatic accumulation of mutations can be seen as a natural process within the human body and cancer a normal part of the human life cycle. This point of view may explain why it has been so difficult to significantly reduce cancer incidence and suggests that, for this to be achieved, the means of altering the natural somatic mutation rate needs to be identified.

I. INTRODUCTION Malignant tumors develop in humans for the most part in individuals of 55 years of age or more (Parker et al., 1996) and consist of enormous clonal expansions of cells that have accumulated mutations in defined subsets of genes (Nowell, 1976; Fearon and Vogelstein, 1990). Mutation accumulation occurs throughout life and is the underlying cause of cancer (Vogelstein and Kinzler, 1993), but as yet the dynamics of this process are poorly understood. It is perhaps surprising that we know almost nothing concerning the frequency and dynamics of human mutations in the normal tissues where cancer commonly occurs, such as the colon, breast, and prostate. Without careful and detailed analysis of these phenomena and the resultant genetic heterogeneity of normal somatic tissues, the etiology of the common human cancers will remain obscure. Perhaps more importantly, our ignorance of somatic mutation frequencies under different circumstances and in different tissues may lead (or have led) to extremely biased conclusions concerning the most likely strategies for reducing the incidence of cancer. Analysis of the literature leads to the observation that an entire field of study, which we might call the population genetics of somatic cells in solid

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tissues, has not been developed and that calculations of cancer probability may have been made with misleading data. The aim of this review is to examine what we know (and what we do not know) and what might be deduced indirectly concerning the frequency of somatic mutations, the area of both greatest importance and our greatest ignorance at this point in time, in terms of understanding human carcinogenesis. The initial portion of the discussion centers on an examination and reinterpretation of recent data concerning the instability of microsatellites in human tumors, which are consistent with normal human tissues being generally highly genetically variable. Subsequently, other avenues of research are reviewed that indicate the presence of frequent mutations in human tissues, the number of which increases exponentially with age. The likelihood of the high level of somatic mutations being due to spontaneous events is discussed, and the underlying rate related to that in the germline. On the basis of these comparisons, a simple model of the human life cycle is elaborated. This model proposes that the overall human DNA mutation rate, manifest both in germline and somatic cells, is the fundamental biological pacemaker that defines not only the rhythm of evolutionary change but also the longevity of individual members of the species, limited at least in part by the development of fatal mutation-dependent pathologies such as cancer.

11. SOMATIC MUTATION OF MICROSATELLITE SEQUENCES

A. Microsatellite Instability as a Result of DNA Mismatch Repair Deficiency A series of studies reported in 1993 showed that the short, repetitive, and highly polymorphic DNA sequences known as microsatellites can exhibit length alterations in colorectal tumors (Aaltonen et al., 1993; Thibodeau et al., 1993; Ionov et al., 1993). The frequent association of such microsatellite instability with the inherited form of the disease was a key step in the identification of the genes mutated in the germline of individuals with hereditary nonpolyposis colorectal cancer (HNPCC) (Fishel et al., 1993; Leach et al., 1993; Papadoupolos et al., 1994; Bronner et al., 1994; Nicolaides et al., 1994). The genes in question, hMSH2, hMLHl, hPMS1, and hPMS2, encode components of the DNA mismatch repair system, and the loss of both alleles of any one of such genes leads to 100 to 1000-fold increases in microsatellite alterations as well as mutations in marker genes such as hypoxanthine phosphoribosyltransferase (HPRT) (Shibata et al., 1994; Bhattacharyya et al., 1994,1995; Eshleman et al., 1995; Heale and Petes, 1995).

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It is suggested that the mismatch repair genes act in a way similar to tumor suppressors in that mutations are recessive and the loss of both alleles of any of these genes allows rapid mutation accumulation and increased risk of cancer. In HNPCC, one of the alleles is mutated in the germline and the other lost through somatic mutation. In somatic tumors with mismatch repair deficiency, both alleles are lost through somatic mutation (Nicolaides et al., 1994; Leach et al., 1993; Papadopoulos et al., 1994; Hemminki et al., 1994). There are a number of reviews of the interrelationship of mismatch repair deficiency, microsatellite instability, and carcinogenesis (Fishel and Kolodner, 1995; Kolodner, 1995; Dunlop, 1996; Simpson, 1996).

B. The Somatic Mutation Rate Is Controlled at the Level of DNA Repair Of general importance, the study of microsatellite instability and replication error consolidates the concept that the somatic mutation rate is controlled at the level of DNA repair. Thus, the DNA mismatch repair system reverses errors that result from misincorporation by DNA polymerases and that are missed by the polymerases’ proofreading function. The mismatches may be at single bases or due to misalignment of a number of nucleotides during replication, which is apparently enhanced at short repetitive sequences such as microsatellites (Kolodner, 1995). Other DNA repair systems also participate in the control of the mutation rate. The base excision repair system repairs small lesions that result from endogenous processes such as the deamination of DNA bases (most frequently at cytosine and 5-methylcytosine), and the nucleotide excision repair system acts on bulky helixdistorting lesions such as the dipyrimidine photoproducts produced by ultraviolet light (Seeburg et al., 1995; Lehmann, 1995). Nucleotide excision repair deficiency is also associated with cancer. In the same way that mutation in the mismatch repair genes predisposes to colon and other epithelial cancers in HNPCC patients, xeroderma pigmentosum (a rare autosomal recessive disease typified by a number of clinical features, including hypermutability and proneness to ultraviolet light-induced skin cancer) is due to loss of nucleotide excision repair capacity (Lehman, 1995). In Bloom syndrome, where cells from affected individuals exhibit elevated levels of chromosomal breaks, gaps, and somatic crossovers and where the clinical features again include elevated risk of developing cancer, the mutated gene reveals motifs homologous to the RecQ/Sgsl helicase subfamily (Ellis et al., 1995). This helicase is also involved in DNA repair (Rothstein and Gangloff, 1995). Likewise, the gene responsible for Werner syndrome, which causes premature aging and a variety of epithelial and nonepithelial cancers, also appears to be a helicase (Yu eta]., 1996). Finally, the increased incidence of

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tumors in individuals suffering from ataxia-telangiectasia is associated with abnormally high sensitivity to ionizing radiation (Taylor et al., 1996).

C. Detection of Microsatellite Alterations by the Polymerase Chain Reaction The relative ease of measuring microsatellite alterations has facilitated the analysis of such events in a wide variety of tumors. HNPCC families exhibit a relatively broad spectrum of tumors: colon, stomach, endometrium, ovary, pancreas, and upper urinary tract (Lynch et al., 1993). Microsatellite instability has been observed not only in these tumor types but in essentially all tumor types examined. In all cases, analysis has been undertaken by comparing the polymerase chain reaction (PCR) products of microsatellite loci amplified from DNA extracted from tumor and normal tissues. When microsatellites detected in the amplification products derived from tumor DNA differ from those derived from the DNA of normal tissues, the tumor is said to exhibit microsatellite instability. The interpretation of such experiments is that (i) microsatellite alterations do not occur in normal tissues, (ii) tumors with altered microsatellite alleles are genetically unstable, and (iii) mutations in components of the mismatch repair system are responsible for the instability and have contributed to tumor formation. These conclusions may not always be correct. The reason for possible errors of interpretation lies in the methodology used for detection of microsatellite alterations. Invariably some 50 ng or more of normal and tumor tissue are amplified by PCR, and the size of the microsatellite alleles determined by gel electrophoresis. PCR is a competitive process (Gilliland et al., 1990; Piatak et al., 1993; Caballero et al., 1995). Coamplified sequences that constitute less than about l/lOth of the starting mixture are difficult to detect among the reaction products. Given that there are two normal alleles at each microsatellite locus, and mutations can be gains or losses at either allele, 40% of loci could be mutated but the mutations not detected by the PCR assay. This effect is exacerbated by the fact that the assay for the most part analyzes dinucleotide repeats, which are highly prone to cause slippage during amplification, so that from each allele a series of products result, which also reduces the sensibility of detection of microsatellite variation. Indeed, Foucault et al. (1996) have estimated that 6% of starting DNA must represent the same variant allele to be detected in a microsatellite amplification assay and that as much as 50% of the DNA could be altered by either loss or gain of alleles and not be detected. This has in fact been directly demonstrated in a study of microsatellite alterations in phenotypically normal cells from HNPCC patients (Parsons et al., 1995). In this study, highly diluted samples of DNA (containing 0.5-3 genome equivalents) extracted from Epstein-Barr

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virus-transformed lymphocytes and colon epithelium and nonepithelium from HNPCC patients were amplified, and up to 53% of samples were found to harbor alterations, although these were not evident in the undiluted DNA samples. The epithelium-derived DNA samples generally exhibited higher frequencies of alterations than nonepithelial samples. Two normal patients were also studied and, interestingly, in one of these 4% of samples for one of the loci studied were found to contain alterations. Overall, this study clearly demonstrates the shortcomings of amplifying high concentrations of DNA in order to detect microsatellite variability.

D. Microsatellite Alterations in Tumors May Not Always Be Due to Mismatch Repair Deficiency In cell lines prone to mutations at microsatellite alleles due to mismatch repair deficiency, the mutation rate can be as high as lop2 mutations per locus per generation at dinucleotide repeats (Bhattacharyya et al., 1994; Shibata et al., 1994). In tumors with demonstrated mutation in mismatch repair genes, the new alleles commonly appear as relatively faint ladders above or below the normal alleles, which is consistent with the accumulation of mutations, at the rates estimated, during clonal expansion of the tumor (Liu et af., 1995). Furthermore, in such cases the majority of loci studied exhibit instability (Liu et al., 1996). In many studies of tumors, particularly those that do not form part of the HNPCC spectrum, the pattern of microsatellite instability observed is quite different. Commonly, a single mutant allele is observed at a given locus, with the concomitant loss of intensity of the normal allele. The mutant allele may be at some distance from the normal allele, and frequently only one of many loci studied exhibits an alteration. Alterations of this kind can be seen in the studies of Wooster et af. (1994), Gao et al. (1994), Dams et al. (1995), and Yee et al. (1994), for example. This pattern of mutation cannot easily be explained on the basis of accumulated mutations during the clonal expansion of a genetically unstable tumor. An alternative explanation is that the tumor is in fact relatively stable and that the microsatellite alterations observed are due to clonal expansion of an already altered cell expanded from heterogeneous normal tissue and are not due to mismatch repair deficiency in the tumor. This idea is supported by the observation that, in primary tumors and metastases where instability is detected in both tumor types, exactly the same pattern of alteration is observed (Ishimaru et af., 1995). In addition, defects in the mismatch repair genes are not found in many sporadic tumors that exhibit microsatellite instability (Katabuchi et al., 1995; Bubb et al., 1996). The mutation in the microsatellite itself provides no selective advantage that would account for clonal ex-

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pansion, thus raising the possibility that tumors can be used to sample such neutral (and thus overall) mutation frequencies in normal tissues.

E. Estimate of the Frequency of Microsatellite Alterations in Normal Tissue When enough microsatellite loci are examined, all tumors appear to have some level of alteration. The most clear-cut example of this lies in a study of esophageal adenocarcinoma (Gleeson et al., 1996). In this study, 139 microsatellite loci were studied in 17 cases of esophageal adenocarcinoma arising from Barrett metaplasia esophagus, a clonal expansion that undergoes successive changes involving metaplasia, dysplasia, and finally malignant carcinoma (Zhuang et al., 1996). In one adenoma, widespread alterations were observed, with 43% of the loci examined exhibiting alterations that consisted of multiple new alleles. The other 16 cases all exhibited alterations in at least one locus, but these were typically limited to single new alleles at the loci affected. A possible interpretation of the data is that the single case with 43% of altered alleles represents a tumor with mismatch repair deficiency, whereas the others reflect the heterogeneity of the tissue from which the tumor arose. Indeed, the authors allude to this by stating that low-level microsatellite alterations may reflect the inherent instability of these polymorphic markers. In those cases of low-level alterations, the frequency of mutations at dinucleotide repeat loci is 1.6% and at tetranucleotide repeat loci is 5.2%. On this basis, the frequency of dinucleotide repeat alterations in the esophagus can be estimated as 1.6 X lop2 and that of tetranucleotide repeats as 5.2 X lop2.These are very high mutation frequencies. Other studies of microsatellite alterations have analyzed far fewer loci, with the result that in the majority of tumors no alterations are observed. Nevertheless, analysis of reports of a variety of tumor types reveals that the same general pattern prevails, with some tumors exhibiting microsatellite alterations at most loci studied and others in which only one or two altered loci are observed. Table I shows calculated levels of mutation frequency at microsatellites for a variety of tissues based on the argument that tumors permit the sampling of neutral mutation frequencies. In the calculations, all tumors that exhibited alterations at more than 40% of loci studied are omitted since they may represent tumors that do have mismatch repair deficiency. The calculation is thus made by dividing the remaining number of altered loci by the total number of loci studied. Many different loci, with possibly different intrinsic mutation rates, are analyzed in the different studies, and the number of loci analyzed varies greatly. Furthermore, the use of 40% as the value for exclusion is obviously arbitrary. The indirect nature of the data and the vari-

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Table I Estimates of Mutation Frequencies in Human Somatic Tissues Sequence

Cell type

MutatiodCells

Reference

Microsatellites Microsatellites Microsatellites Microsatellites Microsatellites Microsatellites (dinucleotide) Microsatellites (tetranucleotide) Microsatellites Microsatellites Microsatellites Microsatellites Microsatellites (dinucleotide) Microsatellites (tetranucleotide) Microsatellites Microsatellites Microsatellites Microsatellites Microsatellites Microsatellites Microsatellites Minisatellites

Brain Brain Brain Bladder Breast Cervix

1.9X 1.25X10-2 1.5~10-~ 3.8x10-2 8.8x10-3 2x10-3

Dams et al. (1995) Zhu et al. (1996) Wooster et al. (1994) Mao et al. (1996) Wooster et al. (1994) Larson et al. (1996)

Cervix

4 ~ 1 0 - ~

Larson et al. (1996)

Colon Colon Esophagus Esophagus Esophagus

2.7X10-2 2.6X10-2 3X1OW2 3X10-2 1.6x

Suzuki et al. (1994) Ishimaru et al. (1995) Meltzer et al. (1994) Nakashima et al. (1995) Gleeson et al. (1996)

Esophagus

5.2x10-2

Gleeson et al. (1996)

Kidney Lung Lung Lung Ovary Pancreas Soft tissues Various

2x10-2 3.5X 5 ~ 1 0 - ~ 1.2x 10-2 4.1 x 1.3X10-2 9.2~ 1x10-1

Uchida et al. (1994) Shridhar et al. (1994) Ryberg et al. (1995) Fong et al. (1995) Wooster et al. (1994) Brentnall et al. (1995) Wooster et al. (1994) Matsumura and Tarin

HPRT HPRT

Kidney Lymphocytes

(1992)

Martin et al. (1996) Reviewed in Albertini et al. (1990) and Cole and Skopek (1994)

ation in methodology preclude meaningful comparisons of mutation frequencies in different tissues, and no attempt to correlate frequency with age has been made. Furthermore, it must be remembered that in all cases cancer has developed in the tissue being studied, possibly elevating the values. It can be noted that Table I does not include endometrial or gastric cancer. Although these tumor types form part of the HNPCC spectrum and frequently exhibit microsatellite instability, this appears always to be present in a high percentage of the loci studied in each tumor where it is detected, thus precluding the calculation of normal tissue heterogeneity on the basis developed (Burks et al., 1994; Rhyu et al., 1994). Nevertheless, comparison of the different values shows a uniformly high mutation frequency in all the tissues studied that can be averaged at about lop2.

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111. SOMATIC MUTATIONS OF MINISATELLITE SEQUENCES Somatic mutations in minisatellite sequences are also detectable by the comparison of tumor and normal DNA from the same individual. In analysis of tumors from 32 patients using the probes 33.15 and 33.6, Matsumura and Tarin (1992) detected alterations in hybridization patterns in 27 patients, with up to four alterations in the same tumor. Approximately 10 bands per analysis are discernible, and the total number of alterations (either losses or gains of bands) in primary tumors is 67, giving an overall mutation frequency of about 10%. The paper interprets the observations made in the light of alterations associated with tumorigenesis on the basis that tumors have a higher rate of mutations than normal tissue, and thus the very high rate of mutations observed might have been expected from this point of view. The alternative interpretation is again that the alterations are in fact representative of the heterogeneity of the cells in the normal tissue from which the tumor arose. Consistent with this hypothesis are the clear-cut differences between the hybridization patterns of normal and tumor DNA, which are thus likely to represent the respective cell populations as a whole. In addition, identical patterns were seen in the primary tumor and in lymph node metastases in 10 of 17 cases where mutations were observed. If the alterations were ongoing events taking place in the unstable tumor cell population, then this observation would not have been expected. Indeed, given that tumor growth involves the production of around lo9 cells from a single original, then the difference in percentage of alterations between primary tumor and metastases is in fact low. Moreover, the majority of cases where the metastasis and primary tumor differ involved the failure to detect mutations in the former, which could be accounted for by signals below the limit of sensitivity due to the lower percentage of tumor cells in the lymph nodes. It is also relevant that five benign fibroadenomas studied all exhibited minisatellite alterations. Thus the very high value of lo-' may be estimated for the variation in minisatellite sequences in normal human tissues.

IV. SOMATIC MUTATION OF THE HPRT GENE A. HPRT Mutation in Epithelial Cells The HPRT gene is a widely used model for the study of somatic mutation in humans. The advantages of this gene are that it is X-linked and thus there is only one functional copy of the gene per cell, and also that cells with in-

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activating mutations are easily selected due their resistance to the toxic effects of purine analogs (Cariello and Skopek, 1993). The introduction of a dinucleotide repeat sequence into a marker gene transfected into a mammalian cell line demonstrated that mutations occur at least 100 times more often in a microsatellite than in the average gene (Farber et al., 1994). Comparison of HPRTand microsatellite mutation rates in the same cell line with mismatch repair deficiency puts the relative rate at about 1000 (Bhattacharyya et al., 1994,1995). If we take the mutation freper locus per cell, quency of a microsatellite in normal tissue as being per cell for a then we could predict a mutation frequency of around marker gene such as HPRT A single study of HPRT mutation frequency in a normal tissue other than blood has been reported in which human kidneys were used (Martin et al., 1996). In this first direct analysis of somatic mutation frequencies in normal epithelial cells, a frequency of mutations rising from about 5 X lops in donors in the first decade of life to about 2.5 X lop4 for donors of 70 or more years was observed. Immunocytochemical analysis of the selected clones confirmed their epithelial character. These data are thus compatible with the frequency of mutation deduced from the clonal expansion of somatic cells with micro- and minisatellite alterations as set out earlier. Most importantly, the HPRT data are directly comparable with similar studies undertaken with human lymphocytes. Such comparison reveals mutations in the kidney epithelium to be some 10-fold higher than those for lymphocytes. The authors argue against methodological explanations, such as differential thresholds of sensitivity or colony-forming efficiency, for this difference. Analysis of the mutational spectrum at the level of cDNA sequencing in 14 mutant cell lines revealed the presence of small mutations in all cases, six of which were splice junction mutations and eight of which were nucleotide substitutions and a 4-bp and a 1-bp deletion. The analysis of micro- and minisatellite alterations exhibited by tumors does not allow deductions concerning the dynamics of mutation to be made. However, the study of HPRT mutations in the kidney directly addresses this question in that kidneys from individuals ranging from less than 10 to more than 70 years of age were used. The results, not unexpectedly, demonstrated an increase in mutational frequency with age. However, statistical analysis of the age-related increase demonstrated that it was exponential rather than linear, with an increase of about 1%per year of age. This finding is of fundamental importance and is directly compatible with the exponential nature of the increase of incidence of human cancer with age (Renan, 1993). In this context, the study also showed that kidneys where renal carcinoma was present exhibited a mutation frequency approximately double that of cancer-free kidneys even though the cells were always taken from a histopathologically normal portion of the diseased organ. This relationship between HPRT mutation frequency and cancer strengthens the argument

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that cancer incidence reflects overall mutational levels, which can be made on the basis of the similar relationship of both variables with age. On the other hand, the increase of mutational frequency with age being due to most older kidneys containing a cancer was ruled out since addition of a variable to the analysis to identify kidneys containing a carcinoma substantially improved the fit of the data to an exponential mode. A final noteworthy observation made in this pioneering study was that, when several independent tissue samples were taken from the same organ, very similar mutation frequencies were measured. This suggests that the mutation frequency is characteristic of the organ as a whole. As discussed earlier, it is now clear that the mutation rate is controlled at the level of DNA repair rather than being defined by the number of potential mutation-causing events that occur. On this basis, we would expect mutation accumulation to be an exponential process since mutations (and thus reduced efficiency) in the genes that control the mutation rate will continually tend to increase the overall rate. The more mutations that have occurred, the higher the percentage of repair genes mutated and thus the higher the mutation rate, leading to an exponential increase in mutational frequency. The repair systems are complex, and genes that do not directly encode components of the systems, such as those encoding transcription factors and other proteins necessary for the synthesis of repair components, can also be presumed to be involved in the maintenance of their efficiency. Furthermore, the loss of one allele of the genes that encode the components of the systems may cause partial increases in the mutation rate (Parsons et al., 1995). Thus a gradual and continual increase in mutation rate with age, rather than stepwise alterations, is expected.

B. HPRT Mutation in Peripheral Blood Lymphocytes With the exception of the studies cited previously, the analysis of human somatic mutations has almost invariably been undertaken with cells from the peripheral blood due to the relative ease of obtaining samples. The HPRT locus has been the most thoroughly studied (see Cole and Skopek, 1994, for an extensive review), and the average frequency of mutations for normal individuals is 5 x lop6per cell (Table I). As noted earlier, this frequency is significantly different from that found in the kidney and also from that which might be predicted from the study of micro- and minisatellites in tumors. The basis of the difference probably lies in the fact that blood is an extremely complex, dynamic tissue where continual clonal selection takes place, and it is entirely reasonable that it might not be possible to extrapolate the data concerning mutation frequencies from lymphocytes to other cell types. Consistent with the lower mutation frequency in blood is the fact that tumors

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that arise from the accumulation of multiple mutations are not associated with hemopoietic cell lineages. Lymphocytic malignancies typically arise from single mutational events, particularly chromosome translocations. Nevertheless, although quantitatively different from those in solid tissues, the data on mutation frequencies in peripheral blood add considerably to an overall view of somatic mutation and support some of the conclusions derived earlier. First, the mutation frequency of HPRT in lymphocytes increases with age, with the average value for cord blood being 0.96 X l O V , that for children being 3.02 X loy6, and that for adults 8.35 X lop6, with an increase of 1-5% per year. Second, there is a very large range of values within age groups, with the frequency varying over two orders of magnitude in different donors. Third, although smoking (as an important source of external mutagens) was found to tend to increase mutation frequency, the effect was relatively small (-1.5-fold increase as compared with nonsmokers) and not seen in all studies. Other external mutagens, such as radio- or chemotherapy or exposure to ionizing radiation, had more pronounced but highly variable effects, with inconsistent data between studies and significant overlaps between control and exposed groups. However, a number of studies show large increases in mutation frequency immediately after exposure in some individuals (Cole and Skopek, 1994). In the study of HPRT mutations in lymphoctyes, a wide variety of mutations have been detected, including large and small deletions, frameshifts, and single base substitutions as well as mutations that cause aberrant mRNA splicing, which have been well documented in an analysis of the HPRT data base (Cariello and Skopek, 1993). Approximately 15% of the mutations in adults are large deletions or other complex alterations detectable by Southern blotting, the remainder being more subtle mutations not detectable by this methodology. Interestingly, in newborn infants the situation is quite different, with about 75% of mutations involving gross structural alterations. A distinct deletion hotspot is evident in that about half the mutants have a deletion of exons 2 and 3. The deletion endpoints have been sequenced and show strong homology with the consensus sequence for V(D)J recombinase. This enzyme is responsible for rearrangements in T-cell receptor genes and is very active during fetal development and in very young children. It appears that this enzyme can act on other substrates, causing the type of mutation seen in HPRT, and indeed may be responsible for the distinct profile of pediatric tumors, which frequently involve large structural changes such as translocations. The fraction of HPRT mutants with gross structural alteration is highest at birth and broadly declines with age, whereas the total frequency of HPRT mutation increases with age. However, a more detailed analysis of the age distribution of exon 2 and 3 deletions showed that in fact there is a significant increase in the number of mutations of this kind up to 5 years of age, with subsequent decline (Finette et al., 1996). The age profile

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22 I

of these mutations matches well that of acute T-cell leukemia, which is also characterized by V(D)J recombinase-mediated recombination, thus strongly supporting the argument that such mutations are responsible for at least some childhood cancers. These data further indicate that mutational frequencies in lymphocytes may be untypical but support the notion that underlying mutations in normal tissues define cancer risk and type and also that the mutations in a marker gene such as HPRT can be used to understand the general pattern of mutations and the associated risk of cancer.

V. THE FREQUENCY OF SOMATIC MUTATION IN SOLID TISSUES CAN ACCOUNT FOR MULTISTEP CARCINOGENESIS The estimated values for the mutation frequencies of microsatellites, minisatellites, and a 1.3-kb open reading frame of lop2, lop1, and 2.5 X lop4, respectively, in the epithelial cells of normal adult tissues mean that we can consider normal tissues to be enormously genetically heterogeneous. The human body contains about 1014 cells. Let us consider a small hypothetical tissue composed of epithelial cells comprising 1/100th of the adult body and thus containing 1 O I 2 cells. In such a tissue, in the absence of any selective X cells will contain mutations in proliferation, 2.5 X lo8 (2.5 X any given gene of the size of HPRT Furthermore, taking the average mutation frequency at 70 years or more as approximately 2.5 X lop4 for a gene of 1.3 kb, we can then take a value of approximately 2 X lo-’ as an estimate of the frequency of mutations per nucleotide. Many tumor suppressor genes are among the largest genes known, and their size alone thus makes them more likely targets for inactivating mutations. The A P C gene, for example, the inactivation of which is critical to the development of colorectal cancer, has an open reading frame of some 8500 nucleotides and an overall size of 9 kb could be taken to include control regions (Groden et al., 1991). The estimated frequency of an inactivating mutation in this gene would be 1.7 X per cell. Let us take a simple model for the formation of a tumor that involves the loss of both alleles of two suppressor genes of the size of APC. The frequency of this combination of mutations is (1.7 X 10-3)4, or Thus there may be no need to evoke elevated rates of mutation 8.3 X to account for multistep human carcinogenesis since carcinomas derive from single cells. The underlying mutation frequency and resultant cellular heterogeneity appear to be more than sufficient to provide the mutations necessary even for the complex and statistically improbable process of multistep carcinogenesis. The calculation assumes only the involvement of tumor suppressor genes

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on the basis that this combination of mutations is sufficient to cause clonal expansion. If the tumor expands to a size of only lo8 cells (a tumor of 1cm3 has about lo9 cells), this would generate sufficient mutations to make the combination of mutations in any pair of genes of approximately 1 kb in the same cell highly probable [(2.5 X 10-4)2, or 1.25 X lo-*] without any increase in the rate of new mutations. These mutations could then turn a benign expansion into a transformed metastatic tumor. In addition, the same probability has been assumed for the inactivation of both alleles of the two hypothetical genes, which may not be the case. The inactivation of tumor suppressor genes commonly occurs through a point mutation in one allele and deletion of the second (Fearon and Vogelstein, 1990). The frequency of the loss of the second allele following point mutation of the first suggests that this is a more common event than a second point mutation, and thus that the probability of occurrence is higher than estimated. It should also be noted that, following the loss of both alleles of APC, clonal expansion does occur prior to the loss of DCC, for example, again making the probability of the loss of the second tumor suppressor gene more likely (Vogelstein and Kinzler, 1993). In summary, although it is widely stated that the frequency of mutations in the human body is too low to account for the incidence of cancer (Loeb, 1991), and transformation to an elevated level of mutations in tumors is required to account for the number of mutations that occur in multistep carcinogenesis, the information discussed here does not support this conclusion and indicates that the aging human body accumulates sufficient mutations to account for the number of mutations required for carcinogenesis. Although many studies have suggested the existence of a mutator phenotype due to mismatch repair deficiency in a variety of tumors, the evidence is in fact indirect and does not necessarily demonstrate the presence of mismtach repair deficiency as an important underlying cause of somatic multistep cancers. Indeed, a very small minority of tumors may have this characteristic, and it is noteworthy that, even in HNPCC patients, not all tumors exhibit the high levels of microsatellite instability due to loss of both alleles of a mismatch repair component. It is also noteworthy that the higher levels of mutation frequency now becoming apparent mean that clonal expansion is not required at all steps in the process of tumorigenesis (Nowell, 1976).

VI. CELLULAR PROLIFERATION AS A RISK FACTOR FOR CANCER If normal tissues are extensively genetically heterogeneous and if mutation frequency increases exponentially with age (and thus the number of cell divisions), then it follows that cellular proliferation per se is a risk factor for

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the development of cancer. This idea has received wide support (PrestonMartin et al., 1990).There are extensive epidemiological data supporting the connection between accelerated cell division due to hormone action, drugs, infectious agents, chemical irritants, and physical injury and increased risk of cancer (Preston-Martin et al., 1990; Ames and Gold, 1990). Indeed, Ames and Gold proposed that many carcinogenic agents are in fact mitogens, and not mutagens, which supports the argument that cancer predominantly results from naturally occurring mutation events rather than exposure to manmade mutagens, and thus that cancer is essentially a naturally occurring event. As pointed out by Farber (1995), the implications of this argument are very far reaching indeed, as a new and radically altered orientation toward cancer prevention would follow. A simple view of the effect of accelerated cell division is that the age of a tissue in biological terms is proportional to the number of cell divisions that it has undergone and that increased cell division thus prematurely ages the tissue affected, with the additional risk of the association of the correct mutations to favor tumorigenesis. Abnormal cell proliferation is also important for the formation of tumors in that it allows competition between cell lineages to occur, and those with the advantage of more rapid proliferation will predominate. These lineages could be those with mutations that form part of the tumorigenic permutations. The idea of competition between lineages can be identified in the work of Martin et al. (1974), who showed extensive heterogeneity in fibroblast cultures and suggested that, as a result, hyperplastic foci may be monoclonal or oligoclonal. More recent data concerning microsatellite alterations in pancreatitis and ulcerative colitis are consistent with the monoclonality of proliferation (Suzuki et al., 1994; Brentnall et al., 1995). In both of these studies, nonmalignant, proliferative conditions were found to exhibit what has been referred to as microsatellite instability in a very high proportion of cases. Following the argument propounded earlier that altered microsatellite profiles in tumors may actually reflect monoclonal expansion from a highly heterogeneous tissue rather than instability in the tumor per se, the detection of altered microsatellites when total DNA is amplified from the proliferating tissue demonstrates that the alteration is indeed present in a large percentage of the proliferating cells. This would occur if the proliferating cells originate from a cell that has microsatellite alterations or if alterations occurred in one of the very first cell divisions that occurred following the onset of proliferation. In either case monoclonality or very limited oligoclonality is required. Thus these proliferations are in fact nonmalignant tumors. It is significant that in both cases the proliferative condition presents an increased risk for the subsequent development of malignant tumors. In this context, the study of pancreatitis and pancreatic cancer also showed an equal percentage of both conditions with K-ras mutations in the pancreatic juice (Brentnall et al., 1995).Earlier studies had indeed shown ras mutations in the pancreatic juice of patients with no evidence of pancreatic

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cancer who subsequently developed malignant tumors in this tissue at 18 and 40 months of follow-up (Berthklkmyet al., 1995). Another example demonstrating the monoclonality of cellular proliferation comes from the study of Barrett esophagus (Zhuang et al., 1996).In this case monoclonality was demonstrated by the presence of identical APC gene alterations in dysplastic and adenocarcinoma foci of all informative cases; these alterations were also identified in adjacent metaplastic tissue in some cases. Monoclonality was verified by the same X chromosome inactivation pattern in carcinoma, dysplasia, and metaplasia in female cases. In addition, in another cancer site where increased cell proliferation has been related to the development of cancer, the endometrium, monoclonality of nonmalignant cellular proliferation has been demonstrated (Jovanovic et al., 1996). Analysis of X chromosome inactivation as well as microsatellite alterations demonstrated the monoclonality of endometrial carcinoma, endometrial polyps, and atypical endometrial hyperplasias. The argument that cellular proliferations tend naturally to be monoclonal unites the competing hypotheses of the field theory of carcinogenesis and of monoclonal expansion. This is evidenced by the data reviewed here in connection with endometrial proliferation, where the general stimulus of the organ to proliferate (field effect) results in many foci of monoclonal expansion. A separate example comes from the analysis of skin lesions at sun-exposed sites (Ren et al., 1996). In this analysis, all portions of the same tumor, irrespective of morphological grade, always carried the same p53 mutation indicating monoclonality. Other p53-reactive areas of the same individual, on the other hand, had different p53 mutations. Thus, different areas of the same affected tissue can have separate monoclonal expansions due to the stimulus to proliferate. In the case of skin lesions, the selective proliferative advantage of cells with p53 mutations is explained by the observation that ultraviolet light induces p53-mediated apoptosis in skin cells, and those cells with mutations in this gene escape such programmed cell death and thus proliferate preferentially in the replacement of the skin layers lost through sunburn (Ziegler et al,, 1994).

VII. GERMLINE MUTATIONS A. Evolutionary Selection of the Human

Germline Mutation Rate Mutations occur in somatic tissues and cause cancer. They also occur in germline cells, where they are responsible for the heterogeneity of individual members of the species and thus for evolution. It has been widely argued that

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the rate of germline mutation is controlled and that the present rate in humans has evolved due to the selective pressure of opposing forces. For example, Sommer (1990)pointed out the importance of the evolution of an optimal mutation rate. Too little mutation would result in the extinction of the species since it would not be able to adapt to environmental alterations, and too much mutation would lead to an increase in morbidity due to genetic defects. Radman et al. (1995)have put forward a similar view with the added observation that Escherichzu coli actually has a system, the sos system induced under conditions of stress, for increasing variability and thus possible accelerated adaptation. It has also been argued that the human germline mutation rate is unusually high compared with those of other species. For example, the average mutation rate per nucleotide in primitive eukaryotes such as yeast and Neurosporu is approximately 1O-lo, close to the value in humans (see Section VI1.B). However, since the size of the human genome and the number of expressed human genes is very much higher, the genome rate in humans is greatly elevated (Crow, 1993). This is not the case among haploid microbes. Drake (1991)noted that among such organisms the mutation rate per nucleotide varies by a factor of over 6000, whereas the genome rate varies by only about 2.5-fold. Perhaps as a consequence of the elevated rate in humans, it has been estimated that about half of human conceptions are not implanted in the uterus but result in unrecognizable spontaneous abortion (BouC et ul., 1985), and also that about 5% of human births have a recognizable malformation (McKeown, 1976).

B. Estimates of the Human Germline Mutation Rate A wide variety of experimental approaches have been adopted in order to investigate the rate of human germline mutation (see Table 11). Analysis of microsatellite variations in tumors led to the proposal that the frequency of somatic mutations in these sequences is very high. In the most careful of a number of analyses of germline mutations at microsatellite loci, Weber and Wong (1993)found mutation rates ranging from 0 to 8 X in 28 repeats of either two or four nucleotides contained in loci on chromosome 19, with per locus per gamete per generation. The rate meaan average of 1.2 X sured for tetranucleotide repeats (2.1 X is significantly greater than In this context, some tetranuthat for dinucleotide repeats (5.6 X cleotide repeats have been found to have even higher mutation rates, with the most unstable identified to date exhibiting between 0.56 and 1.4 x mutations per generation (Talbot etal., 1995). The dinucleotide data of Weber and Wong (1993)agree well with several earlier observations, including those made while generating a second-generation linkage map of polymorphic (CA), repeats in the human genome where, in 1.7 x lo5 parent off-

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Table 11 Estimates of Human Germline Mutations Rates ~

~___

Locus Type Microsatellites (dinucleotide) Microsatellites (overall) Microsatellites (tetranucleotide) Microsatellites (dinucleotide) Minisatellites Polypeptides Sentinel phenotypes

Mutationdindividuav generation 1x10-3 2.1~10-3 1.2~10-3 5.6X 4 ~ 1 0 - ~ 1.8X10-s 2x10-5

Reference Weissenbach et al. (1992) Weber and Wong (1993) Weber and Wong (1993) Weber and Wong (1993) Jeffreys et al. (1988) Neel et al. (1986) Mohrenweiser (1994)

spring allele transfers analyzed, a mutation rate of 1 x was observed (Weissenbachet al., 1992). Similar data are available for minisatellite sequences. These also exhibit a high rate of spontaneous mutation, and rates as high as 5 X per gamete have been detected for the most variable loci, with an average of 4 X l o p 3 (Jeffreyset al., 1988).The compatible value of 8 X mutations per gamete was also subsequently determined by the analysis of minisatellite variation in individual sperm (Jeffreyset al., 1994). Comparison of germline and somatic mutations at loci detected by the same probe (33.15)shows that, whereas Matsumura and Tarin (1992)detected an overall somatic mutation frequency of about 5% at these loci comparing tumor and normal DNA, Dubrova et al. (1996)demonstrated the germline mutation rate of 1 X lop2 for these sequences. At a total genome level, and undertaking analysis over an evolutionary time frame, comparison of synonymous base substitutions (which represent the rate of total point mutations) in humans and chimpanzees gives a rate of per year (Li and Graur, 1991).If we take the mean divergence of 1.3 x generation time as being 15-20 years, then the total mutation rate is approximately 2 X lop9per nucleotide per generation. In an analysis of spontaneous mutations in normal individuals measured by altered electrophoretic mobility of 36 polypeptides in a total of 539,170 locus tests, the rate per and the rate per nucleotide locus per generation was estimated at 1.8 X at 1 x lop8per generation (Neel et al., 1986). Other electromorph data have arrived at exactly the same rate of 1 x lop8for the rate of mutation per nucleotide per generation (reviewed by Mohrenweiser, 1994). Monitoring the frequency of sentinel phenotypes (new cases of genetic traits associated with complete infertility), where all cases of dominantly inherited diseases and one third of sex-linked diseases can be assumed to be due to new mutations, has resulted in estimates for the mutation rate that vary by a factor of more than 100 for the 12-15 loci usually monitored, but the average from a series

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of studies is 2 x per locus per generation (reviewed by Mohrenweiser, 1994).In this context, the incidence of Lesch-Nyhan syndrome of 1/100,000 is of interest due to the wealth of data concerning the frequency of somatic mutation at the HPRT locus. Since the gene is X linked, the estimate for the number of new mutations will be 3.3 x Analysis of other inherited diseases has yielded similar data. For example, in a review of the mutation rate at the factor IX locus, detailed in molecular terms, an aggregate mutation rate for all types of mutation of 3.6 X lou9 per nucleotide per generation was calculated (Sommer, 1995). Earlier analyses of this locus had produced estimates of 2.6 X l o p 6 mutations per locus per generation and an and analysis estimated de novo nucleotide substitution rate of 3.1 X of two severe dominantly inherited hemoglobinopathies resulted in estimates of 3 X lou6 mutations per locus per generation (reviewed by Mohrenweiser, 1994).

C. Evidence That Germline Mutations Are Due to Endogenous Mutations Taken as a whole, these data clearly show that there is a finite and constant rate of mutation in the human germline. The consistency of the data are impressive and support the hypothesis that the frequency of human germline mutation is controlled and is not merely the reflection of external factors. The principal stimulus for the majority of the studies of human germline mutation frequencies cited has been the measurement of increase in mutation rate following environmental exposure to mutagens. However, only the analysis of minisatellites has provided experimental evidence that germline mutation rates in humans can be increased by external factors. Children born in heavily polluted areas of Belarus after the Chernobyl accident were found to exhibit a frequency of new mutations that was twice as high as in a control population (Dubrova et al., 1996). In all other studies no detectable difference was found following exposure to mutagens, and thus the observed mutation rate reflects spontaneous mutations resulting from natural events or endogenous processes. This conclusion is supported by analysis of the types of mutation detected. For example, analysis of mutations in the gene encoding factor IX shows that 25% of all independent mutations causing hemophilia B are transitions at CpG, presumably resulting from spontaneous deamination of endogenously methylated 5-methylcytosine. This pattern of mutation is indistinguishable in different racial and geographical groups, ruling out increased mutation due to mutagen exposure specifically associated with race or lifestyle. Furthermore, the pattern of recent mutation is compatible with the ancient pattern that fashioned the G + C content of 40% and the nonrandom dinucleotide frequencies in DNA

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(Sommer, 1995). Although the exact pattern of mutation varies from locus to locus, presumably in function of gene structure and disease characteristics, a nonrandom frequency of transitions at CpG is uniform in analysis of point mutations in human genes (Sankaranarayanan, 1993).

D. The Relationship between Germline and Somatic Mutations Somatic mutation frequencies in epithelial tissues in older individuals are about 10-fold higher than the corresponding germline mutation rates (Tables I and 11). This observation is consistent with the hypothesis that both somatic and germline mutations result from the same underlying and continuous processes. The frequencies of mutations in somatic tissues measured in individuals in the later decades of life are expected to be significantly higher since mutation accumulation is exponential and the germline rate reflects mutation frequencies at the reproductive age of about 30 years. If the same mechanisms are responsible for both categories of mutation, we would expect the characteristics of somatic and germline mutations to be similar. This turns out to generally be the case, although since the two types of mutation are selected on quite different bases (the appearance of disease or resistance to purine analogs in culture in the case of HPRT), some variation is to be expected. This is particularly the case in terms of splicing mutants where entire exons are lost, which appear to be a more common component of the somatic mutation spectrum than the germline spectrum, and in this context the influence of V(D)J recombinase-induced mutants in lymphocytes is relevant (Cariello and Skopek, 1993). Nevertheless, if we consider only point mutations in HPRT, C:G-to-T:A transitions at CpG represent 33% of such mutations in the germline and 36% of such mutations in lymphocytes (Cariello et al., 1992; Cole and Skopek, 1994). The mutational data base of HPRT mutations in other tissues is as yet extremely small and does not permit meaningful analysis. However, a separate source of information particularly relevant to the present discussion comes from the study of the p53 gene, which is frequently mutated in human tumors and which exhibits a high percentage of informative point mutations (Harris, 1993). In this case we can use the tumor-derived information not only to access the somatic mutational spectrum but also to investigate whether tumors are likely to be caused by spontaneous mutations. The baseline germline data in this case come from the study of individuals with the Li-Fraumeni syndrome, who carry a germline mutation in p53 and who have predisposition to develop a variety of tumors, particularly sarcomas and breast cancer (Birch, 1996). In these patients, 53% of all p53 mutations are G:C-to-A:T transitions at CpG (Greenblatt et al., 1994). This percentage matches well with that in colon

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cancer, where 47% of all pS3 mutations are of this type and can thus be proposed to arise principally from endogenous mutations. Consistent with this proposition is the fact that the overall incidence of colon cancer has been relatively stable over time. In contrast, lung cancer has increased enormously over the last SO years and is clearly associated with cigarette smoking. In this instance, where an external carcinogen is clearly implicated, only 9% of mutations are G:C-to-A:T transitions at CpG (Greenblatt et al., 1994). Overall, at least 20% of pS3 mutations are G1C-to-A:T transitions at CpG in ovarian, pancreatic, gastric, prostate, brain, breast, endometrial, thyroid, hematological, and cervical carcinomas as well as sarcomas, which we might hypothesize arise primarily due to endogenous mutations. Great caution obviously must be applied to this interpretation, and it does not mean that external factors do not contribute to carcinogenesis in these cases. For example, if accelerated cell proliferation is playing a significant role in cancer development, this will not in itself alter the pattern of mutation. In this regard, the incidence of stomach cancer is extremely variable in time and between populations, clearly suggesting an important role for environmental factors, although the percentage of CpG transitions is very high. Other cancers have much lower levels of CpG transitions, consistent with an important role for external mutagens. The list of such cancers includes those of the skin, head and neck, bladder, and liver (Greenblatt et al., 1994). Thus we cannot state as yet what percentage of somatic mutations are due to endogenous processes, but it would seem most probable that the majority fall into this category. Furthermore, at least some common forms of cancer that develop through a multistep process, such as colorectal cancer, appear to be due to endoenous mutations. In the case of microsatellite alterations, we know that these are caused by polymerase slippage and can thus be classified as endogenous. It is also of interest that the small deletions and insertions that account for around 11% of p53 mutations are also due to DNA polymerase slippage and misalignment (Greenblatt et al., 1996), although the authors point out in this case that such slippage may also be due to damage caused by environmental mutagens. Somatic mutation frequency is cell division dependent and thus increases with age. If germline and somatic mutations are mechanistically related, we would expect the same to be true of germline mutations. Moreover, in humans it is estimated that the number of cell divisions between zygote and egg is 24 and largely independent of age, whereas the number of divisions prior to a sperm at puberty is estimated at 36 and thereafter rises by about 23 divisions per year. Thus at age 20 the number of cell divisions responsible for sperm production is about 200 and at 30 is about 430 (Vogel and Rathenberg, 1975). If inherited mutations result from events that occur during the cell divisions involved in gametogenesis, we would thus expect a disproportionate contribution from the male germline and also an increased age of nor-

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ma1 fathers of new mutant phenotypes, an idea suggested as long ago as 1912 (Weinberg, 1912). In his discussion of this topic, Crow (1993)cites a number of examples supporting this idea. One is that the data on paternal age for acrocephalosyndactyly and achondroplasia and on maternal grandparental age for hemophilia give an average increase of about 6 years as compared with controls, and other conditions give a lesser increase (Morch, 1941; Vogel and Rathenberg, 1975). Importantly, Crow (1993) has also pointed out that the increase in disease is not linear with the calculated number of cell divisions leading to sperm production. In achondroplasia, myositis, and Marfan syndrome, the increase from 30 to 45 in the age of the father results in about a fourfold increase in disease, whereas the relative increase in cell divisions over this age is 1.8 (Vogel and Rathenberg, 1975; Model1 and Kuliev, 1990). Thus the average increase in mutations may in fact be a quadratic and nonlinear function of age beyond puberty. Evidence that the male germline mutates at a faster rate than the female germline was first presented by Haldane (1935,1937) in his study of hemophiliacs, an dlater support came from the study of Lesch-Nyhan syndrome (Vogel and Motulsky, 1979). More recently, Dryja et al. (1989)presented evidence that the majority of new mutations responsible for ,bilateralretinoblastoma arose in the male germline, and in keeping with the proposal that these mutations occurred during gametogenesis is the observation that most germline mutations in the retinoblastoma gene are indeed due to replication errors (Hogg et al., 1993). Likewise, errors at microsatellite loci were found to be more frequent in the male as compared to the female germline (Weber and Wong, 1993). For minisatellites, some show a large male preference, whereas others do not (Jeffreys et al., 1988, 1991; Vergnaud et al., 1991; Henke et al., 1993). Analysis of germline mutations in the HPRTgene showed that, despite relatively low numbers of samples per mutation type, the sex ratio varies dramatically with different types of mutation. Transitions at CpG, the result of spontaneous mutation, are predominantly found in the male germline, for example, whereas deletions are distributed more or less equally between male and female germlines (Sommer, 1995). These observations allow the hypothesis that point mutations accumulate during spermatogenesis, whereas deletions may be associated with meiosis. Thus, although it appears that some mutations do not accumulate during gametogenesis, most types of mutation do. This conclusion is also supported by the study of germline mutations in mice, where the results from a number of laboratories give a similar estimate for mutation frequency in the male germline of 0.75 to 1.2 X lop5 (Favor, 1994). but a frequency in female germ cells of 0.2 X Thus the majority of mutations are likely to be the result of events that take place during cell division and DNA replication and thus are common to both germline and somatic cells. The germline theory postulates that ani-

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mals have two distinct categories of cells: those that make up the germline and those that form somatic tissues. The germ cells form the male and female gametes, whereas the somatic cells make up all other tissues, including the gonads. During embryogenesis, a small number of cells, known as the primordial germ cells, are set apart. The number of these primordial germ cells is limited in all animals, and in mammals there are only a few dozen or less. In some species, such as birds and mammals, germ cell differentiation occurs late in embryonic development; in others, such as sponges, differentiation only occurs in adult life (Denis and Lacroix, 1993). Thus, although germ cells and somatic cells have some fundamental differences, such as the former’s apparent immortality and ability to undergo meiosis, they are produced by essentially the same developmental processes, and there is no reason to suppose that the processes and enzymatic systems controlling their replication and thus probably their rate of mutation are different. Direct evidence linking the same DNA repair mechanisms to the two types of mutation comes from knockout mice deficient in p M S 2 that exhibit elevated mutations in both normal tissue and sperm as well as having elevated levels of cancer (Baker et al., 1995).

VIII. THE MUTATION RATE AS THE FUNDAMENTAL BIOLOGICAL PACEMAKER Taken as a whole, the comparison of germline and somatic mutations allows the hypothesis that the overall mutation frequency forms a continuum within the individual, is controlled at the level of DNA repair, is due primarily to endogenous mutational events, and has been selected for on the basis of promoting evolution. A model of the human life cycle based on mutation frequency is shown in Figure 1. The salient features are the lower level of germline mutations than somatic mutations, the exponential nature of mutation accumulation, and the continuity of germline and somatic mutations. There is continuity because germline and somatic mutations are due to the same underlying mechanisms, the lack of complete repair of altered DNA. It is argued here that the level of spontaneous somatic mutations that are present in the epithelial tissues of the aged are sufficient to account for the incidence of cancer that exists. The implication from this model is that cancer is a predictable and inevitable facet of the human life cycle. Cancer is not responsible for all deaths in the aged, and, in the United States, heart and cerbrovascular diseases are also major causes of death (Parker et al., 1996). This may at first sight diminish the relevance of the argument that the underlying spontaneous mutation rate is a major limiting factor in the human life cycle. However, it is becoming apparent that many

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Somatic mutations

0

Germline mutation rate

YEARS

Fig. 1 The relationship between the germline mutation rate and somatic mutations.

other age-related diseases may also be mutation dependent, but that the crucial target for mutations in disease other than cancer is not the nuclear genome but rather the mitochondrial genome (Wallace, 1992). The paradigm propounded by Wallace (1992) is that oxidative phosphorylation, which is the main source of energy (ATP)for a variety of organs, including the brain, striated muscle, kidney, and pancreatic islets, and involves a complex series of both nuclear and mitochondrial genes, declines with age due to the accumulation of mutations. The principal cause of the decline may be the accumulation of oxidative damage in the mitochondrial DNA and its effects, manifested in diseases such as diabetes mellitus, Parkinson disease, and Alzheimer diseases as well as ischemic heart disease. Thus, in postmitotic cells, the accumulation of mutations in the mitochondrial genome rather than the nuclear genome appears to be the phenotypic reflection of the mutation accumulation model illustrated. Although prior to the 1990s it was widely assumed that damaged mtDNA was not repaired, the 1990s have seen a number of studies that demonstrate that this is not the case and that mitochondria have the capacity to repair several types of damage in their genomes (Pettepher et al., 1991; LeDoux et al., 1992, 1993; Driggers et al., 1993). Most recently, evidence has been presented that in fact the same mechanisms of repair of oxygen-induced damage are operative in both the nucleus and mitochondria (Driggers et al., 1996). Such repair is effected by genes encoded for in the nuclear genome, and it is of importance to note that xeroderma pigmentosum patients suffer from not only increased incidence of cancer but gradual and massive loss of neurons with accompanying mental deterioration (Cleaver and Kraemer, 1989). Thus the hypothesis of the central role of endogenous mutations in defining the human life cycle is strengthened and, as stated by Lindahl (1993), the spontaneous decay of

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DNA is likely to be a major factor in mutagenesis, carcinogenesis, and aging. There are two fundamental characteristics of life, reproduction and change. Our genetic material, DNA, is organized in such a way as to facilitate both. The double helix allows information conserving replication (Watson and Crick, 1953), but the energy of interaction between nucleotides is such that alterations (or mutations) are frequent during replication (Loeg et al., 1974) and the structure of nucleotides makes them susceptible to mutations through chemical reactions with ubiquitous components of the environment, such as oxygen and water (Loeb, 1989; Lindahl, 1993). Life has been argued to represent a complex phenomenon to ensure the continued existence of DNA; it is proposed that organisms are simply DNA's way of ensuring its own propagation and that natural selection acts at the level of the gene and not the individual (Dawkins, 1976). Natural selection occurs because genes are variable; variation occurs because of mutation, but mutation is responsible not only for evolution but also for cancer and other ageassociated diseases. DNA is selfish (Dawkins, 1976; Doolittle and Sapienza, 1980; Orgel and Crick, 1980): It favors its own continued existence in the evolutionary time frame by finely controlled frequencies of germline mutation (Sommer, 1990; Radman et al., 1995). Subsequent to reproduction, continued somatic mutation in the individual (a consequence of required germline mutation) is of little direct consequence to the continued existence of the DNA molecule in the species; it is, however, of immense importance to the individual (Szilard, 1959; Curtis, 1966; Orgel, 1973; Morley, 1982; Burnett, 1984). Individuals, however, are dispensable in DNA's self-serving survival strategy. Cancer in the aging individual may thus be a direct consequence of the selfishness of DNA.

IX. THE IMPORTANCE OF MEASURING SOMATIC MUTATION RATES Although it is possible to arrive at the plausible hypothesis that cancer is an inevitable and predictable component of the natural human life cycle, it is clear that this view must be supported by infinitely more experimental evidence than is currently available, and the serious development of the field of population genetics of somatic cells, particularly in those tissues where cancer commonly develops, is overdue. To undertake work of this kind, the basic requirement is that individual cells should be compared in order to measure heterogeneity and genetic variability. The technology for undertaking the genetic analysis of whole genomes from single cells already exists and exploits the power of the PCR to greatly amplify the genetic material from sin-

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gle nuclei prior to analysis (Zhang etal., 1992).Furthermore, it has also been demonstrated to be possible to select individual cells from histological sections and undertake analysis of mutations in the context of B-cell development in human germinal centers (Kiippers et al., 1993). Thus there are no methodological impediments that prevent the instigation of serious analysis of genetic variability in solid tissues. We need to confirm that mutations are indeed generally accumulated in these tissues in the exponential manner described, and we need to measure the relationship between the accumulation of mutations in cancer-associated genes and carcinogenesis. To understand the relationship between these phenomena, we need to know if mutation accumulation is tissue dependent and confirm the relationship between proliferation and mutation. Stimulated by the Human Genome Project, the technologies for DNA analysis are evolving at a tremendous rate, and studies that may be considered unreasonably labor intensive today may be readily undertaken in the near future.

X. THE MUTATIONAL CLOCK AND CANCER PREVENTION Once practical methodologies for measuring accumulated mutation frequencies in solid tissues are available, and if they indeed demonstrate a causal relationship between mutation accumulation and carcinogenesis,they would have important and immediate clinical applications in cancer prevention. In particular, the notion of somatic predisposition would become consolidated and preventive measures in high-risk tissues considered. Efficient methods to measure mutation frequency would also make the increased consideration of chemopreventive therapies more likely, as an objective rationale to undertake treatment would exist as well as the means to measure effect. Furthermore, such methods would be fundamental to the rational development of measures that might reduce the mutation rate. Sporn (1992) penned a compelling personal essay on the need to put increased effort into the prevention of cancer and the treatment of preneoplastic lesions, and has argued that excessive concentration on treatment has held up overall progress toward reducing the cancer burden. His views represent those held by many, but the lack of decline in major forms of cancer may also reflect the intrinsic nature of these pathologies, and even the treatment of early lesions may meet with frustration in terms of effective control of cancer moribidity and mortality. Excess cancers caused by exposure to external carcinogens can be eliminated in time by public health measures, but those that reflect the intrinsic disintegration of our genetic constitution will not be avoidable by any presently known strategy. Although it may be pos-

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sible to delay onset and surgically cure individual lesions that are diagnosed at early stages, the future is likely to see the overall increase of individuals with second and third primary cancers with decreasing disease-free intervals between lesion development. Sugimura (1992) noted that 8% of patients at the National Cancer Center in Tokyo were being treated for such tumors in 1992, as compared with 2%in 1962. The rational goal must be to reduce the spontaneous accumulation of DNA mutations. Some chemopreventive measures, such as treatment with tamoxifen, may do this by reducing cellular proliferation, and the concept of using either retinoids or tamoxifen to suppress carcinogenesis has existed for over 20 years (Sporn, 1996). Are there other drugs with carcinogenesissuppressive effects that could be due to reduction of mutation accumulation? Are there lessons to be learned from human tissues that exhibit high levels of proliferation but a low incidence of cancer, such as the small intestine? Can lessons be learned from long-lived species such as plants and some birds, fish, and reptiles? As a final note, a very simple and immediately applicable measure may be available that would indeed reduce the incidence of cancer and prolong life and that has a rational basis in the reduction of mutation accumulation: dietary restriction. Dietary restriction is the experimental manipulation most clearly associated with an increased life span (McCay, 1952; Weindruch and Walford, 1988), and rodents that are restricted to a dietary intake of 60% of that of controls have a prolonged life span of approximately 40%. Dempsy et al. (1993) showed that this prolongation can be directly accounted for by a markedly decreased age-associated accumulation of mutations. The mechanistic basis of this observation is not clear, and the simple explanation that most mutations result from dietary factors may not be true. The detailed analysis of the alteration in mutations in this situation may, however, point the way forward.

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CD44: Structure, Function, and Association with the Malignant Process David Naor, Ronit Vogt Sionov, and Dvorah Ish-Shalom The Lautenberg Center for General and Tumor Immunology, The Hebrew University-Hadassah Medical School, Jerusalem 91 120, Israel

I. Introduction 11. CD44 Nomenclature

111. CD44 Biochemical Structure A. Genomic Organization B. Standard CD44 C. CD44 Isoforms D. Association of the CD44 Cytoplasmic Tail with the Cytoskeleton IV. CD44 Expression on Normal Cells A. CD44 Isoforms in the Developing Embryo and during Maturation of the Hematopoietic System B. CD44 Isoforms Expressed on Epithelial Cells and on Other Nonhematopoietic Cells of the Adult C. CD44 Isoforms Expressed on Nonactivated and Activated Hematopoietic Cells V. Hyaluronic Acid Is the Principal Ligand of CD44 A. The Structure of Hyaluronic Acid and Its Distribution in Normal and Tumor-Invaded Tissues B. Evidence That CD44 Is an HA Receptor C. Binding of HA to CD44 Variants D. The Topography of the CD44 HA Binding Sites E. Influence of the CD44 Cytoplasmic Tail and Cytoskeleton-Related Proteins on HA Binding to CD44 F. CD44 Activation Allows HA Binding in Some CD44' Nonbinder Cell Types G. The Effect of CD44 Glycosylation on HA Binding VI. Non-HA Ligands of CD44 VII. Soluble CD44 VIII. Genetic Control of CD44 Expression IX. CD44 Functions A. CD44 Is a Coaccessory or Independent Receptor Involved in Transmitting Growth Signals B. CD44 Is a Homing Receptor C. Cell Binding to Endothelium or ECM via the CD44 Receptor D. Cell Surface CD44 Involvement in HA Internalization and Enzymatic Degradation E. CD44-Dependent Cell Traffic F. CD44-Dependent Cell Aggregation

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David Naor et al. G. CD44 Influence on Hematopoiesis and Apoptosis H. Cytokine and Growth Factor Presentation by CD44 Involvement of CD44 in Physiological and Pathological Cell Activities A. The Role of CD44 in Wound Healing B. Endometrial CD44 Expression during the Human Menstrual Cycle C. CD44' Cytolytic T Cells Protect against Malaria Infection D. CD44 in Rheumatoid Arthritis and Inhibition of Experimental Arthritis with Anti-CD44 mAb E. CD44 and the Immunodeficiency Virus CD44 Association with the Malignant Process in Experimental Models A. Experimental Evidence for CD44 Involvement in Malignant Processes B. Prevention of Primary Tumor Growth andor Metastatic Spread in Experimental Models by Reagents Interfering with the CD44-Ligand Interaction CD44 Expression in Human Neoplasms and Its Correlation with the Malignant Status A. Tumors of the Nervous System B. Head and Neck Tumors C. Respiratory Tract Cancer D. Alimentary Tract Cancer E. Other Alimentary Tract Cancers F. Genitourinary Tract Cancer G. Gynecological Cancer H. Breast Cancer I. Melanomas J. Non-Hodgkin's Lymphoma and Chronic Myeloid Leukemia CD44 Association with Malignancy: Some Practical Comments Conclusions References

CD44 is a ubiquitous multistructural and multifunctional cell surface adhesion molecule involved in cell-cell and cell-matrix interactions. Twenty exons are involved in the genomic organization of this molecule. The first five and the last 5 exons are constant, whereas the 10 exons located between these regions are subjected to alternative splicing, resulting in the generation of a variable region. Differential utilization of the 10 variable region exons, as well as variations in N-glycosylation, 0-glycosylation, and glycosaminoglycanation (by heparan sulfate or chondroitin sulfate), generate multiple isoforms (at least 20 are known) of different molecular sizes (85-230 kDa). The smallest CD44 molecule (85-95 kDa), which lacks the entire variable region, is standard CD44 (CD44s).As it is expressed mainly on cells of lymphohematopoietic origin, CD44s is also known as hematopoietic CD44 (CD44H). CD44s is a single-chain molecule composed of a distal extracellular domain (containing the ligand-binding sites), a membraneproximal region, a transmembrane-spanning domain, and a cytoplasmic tail. The molecular sequence (with the exception of the membrane-proximal region) displays high interspecies homology. After immunological activation, T lymphocytes and other leukocytes transiently upregulate CD44 isoforrns expressing variant exons (designated CD44v). A CD44 isoform containing the last 3 exon products of the variable region (CD44V8-10, also known as epithelial CD44 or CD44E), is preferentially expressed on

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epithelial cells. The longest CD44 isoform expressing in tandem eight exons of the variable region (CD44V3-10) was detected in keratinocytes. Hyaluronic acid (HA), an important component of the extracellular matrix (ECM), is the principal, but by no means the only, ligand of CD44. Other CD44 ligands include the ECM components collagen, fibronectin, laminin, and chondroitin sulfate. Mucosal addressin, serglycin, osteopontin, and the class I1 invariant chain (Ii) are additional, ECM-unrelated, ligands of the molecule. In many, but not in all cases, CD44 does not bind HA unless it is stimulated by phorbol esters, activated by agonistic anti-CD44 antibody, or deglycosylated (e.g., by tunicamycin). CD44 is a multifunctional receptor involved in cell-cell and cell-ECM interactions, cell traffic, lymph node homing, presentation of chemokines and growth factors to traveling cells, and transmission of growth signals. CD44 also participates in the uptake and intracellular degradation of HA, as well as in transmission of signals mediating hematopoiesis and apoptosis. Many cancer cell types as well as their metastases express high levels of CD44. Whereas some tumors, such as gliomas, exclusively express standard CD44, other neoplasms, including gastrointestinal cancer, bladder cancer, uterine cervical cancer, breast cancer and non-Hodgkin’s lymphomas, also express CD44 variants. Hence CD44, particularly its variants, may be used as diagnostic or prognostic markers of at least some human malignant diseases. Furthermore, it has been shown in animal models that injection of reagents interfering with CD44-ligand interaction (e.g., CD44s- or CD44v-specific antibodies) inhibit local tumor growth and metastatic spread. These findings suggest that CD44 may confer a growth advantage on some neoplastic cells and, therefore, could be used as a target for cancer therapy. It is hoped that identification of CD44 variants expressed on cancer but not on normal cells will lead to the development of anti-CD44 reaEents restricted to the neoplastic mowth.

I. INTRODUCTION Cancer progression is dependent on a cascade of genetic alterations, each one essential, but not sufficient, to afford the tumor a growth advantage. The selection of a malignant phenotype, a rare event that occurs in only a small number of cells, is determined by the successful and complete accumulation of many metastasis-supporting activities. These include induction of vascularization, detachment from the primary tumor mass, digestion and invasion of neighboring tissues, the crossing of blood and lymph endothelium (intravasation), and hematogenous or lymphogenous migration toward the target organ. Acquisition of the ability to form aggregates and re-traverse the endothelium (extravasation) allows the tumor cells to be arrested in the capillary beds and then to colonize the secondary tissue (Poste and Fidler, 1980; Nicolson, 1987; Fidler, 1995).Upon analyzing the different phases of tumor progression, it is clear that this process is tightly associated with the activity of various adhesion molecules. In fact, malignant cells exploit the same adhesive functions used by normal cells in maintaining their routine physiological activities. Normal adhesive functions are carried out by distinct families of mole-

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cules, each structurally different from the other. The integrins are composed of two noncovalently associated subunits designated (Y and p. The same p subunit can interact with different (Y subunits and, conversely, the same (Y subunit can combine with different p subunits, yielding multiple heterodimer products. For example, the combination of an cxL subunit with a Pf subunit generates the leukocyte function-associated antigen-1 (LFA-1; ctLPz) integrin, and interaction of the a4 subunit with the p1 subunit yields the very late antigen-4 (VLA-4; a4p1)integrin (Albelda and Buck, 1990; Dustin and Springer, 1991). The integrins (e.g., LFA-1) are implicated in interactions between CD4+ T cells and antigen-presenting cells (APC) or between CD8’ cytotoxic T cells and their target cells (Dustin and Springer, 1991). Some integrins, such as LFA-1 and VLA-4, are also involved in the binding of leukocytes to endothelial cells (Springer, 1994) and in the adherence of various cell types (including neoplastic cells) to extracellular matrix (ECM) components (Ruoslahti and Pierschbacher, 1987; Ruoslahti, 1991; Yamada, 1991). The immunoglobulin superfamily is a functionally diverse group of molecules with variable numbers of extracellular immunoglobulin-like domains (Bierer and Burakoff, 1991; Dustin and Springer, 1991; Springer, 1994). Some members of this family, such as intercellular adhesion molecule1 (ICAM-1) and vascular cellular adhesion molecule-1 (VCAM-l), are integrin counterreceptors: the ICAM-1 of an APC can interact with the LFA-1 of T cells at the antigen recognition phase of the immune response, and the VCAM-1 of endothelial cells binds to the VLA-4 of T lymphocytes at the “arrest” stage of the extravasation process (Dustin and Springer, 1991; Shimizu et al., 1992; Springer, 1994). Other members of this group also interact with one another (e.g., the CD2 of the T cell and the leukocyte function-associated antigen-3 [LFA-31 of an APC; Dustin and Springer, 1991). The Ca2+-independentcell adhesion molecules (CAM) (e.g., neural cell adhesion molecule [N-CAM], Ng-CAM), most of which are localized in neural tissues, are a unique immunoglobulin superfamily subgroup. These molecules are involved in homophilic interactions during histogenesis (Albelda and Buck, 1990). The cadherins are calcium-dependent CAM that contain internal repeats (of -100 amino acids) that do not resemble immunoglobulin domains. Like the calcium-independent CAM, they are also involved in homophilic adhesion and in organizational events occurring in the course of embryogenesis. In addition, the structural integrity and polarity of adult tissues may also be dependent on cadherins (Albelda and Buck, 1990).Selectin molecules are composed of a lectin-like domain, an epidermal growth factor-like domain, and a variable number of complement-regulatory repeat sequences. L-selectins, expressed on leukocytes, and P- and E-selectins, expressed on endothelial cells, are implicated in leukocyte attachment to, and rolling and arrest on, endothelium. This sequence of events leads to extravasation of leukocytes into inflamed tissues and to lymphocyte homing through the high endothelial venule into lymph nodes (Shimizu et al., 1992;

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Springer, 1994). Addressins are mucin-like molecules (heavily O-glycosylated proteins rich in serine and threonine) whose sialylated carbohydrates (sialyl Lewis-like) adhere to selectins, thus allowing leukocyte-endothelial cell interactions (Springer, 1994). Cartilage link proteins and proteoglycan core proteins are ECM components that interact with hyaluronan (Doege et al., 1987; Goetinck et al., 1987; Neame et al., 1987). The CD44 molecule, which is the focus of this article, is a member of this group. Adhesion molecules have both a negative and a positive influence on the malignant process. On the one hand, they (e.g., E-cadherin or aJ3, integrin) prevent the detachment of potential metastatic cells from the primary tumor by maintaining cell-cell contact (Behrens et al., 1989; Qian et al., 1994). On the other hand, as mentioned before, they enable the binding to endothelium of intravasated or extravasated metastatic cells (Nicolson, 1982a,b; Fidler, 1995) and the latter’s migration along blood vessels, lymphatic vessels, and matrices (Nicolson, 1982a; Ruoslahti, 1992, Fidler, 1995). In addition, adhesion molecules are involved in homophilic (Updyke and Nicolson, 1986) and heterophilic (Fidler and Bucana, 1977; Fidler, 1995) aggregate formation, thereby arresting the metastatic cells in remote organ capillaries and conferring immunoresistance on them. They also support the docking of metastatic cells in secondary organs (Liotta and Stetler-Stevenson, 1991; Nakai et al., 1992; Ruoslahti, 1992; Taraboletti et al., 1993). Therefore, like all other factors involved in the malignant process, CAM are essential to, but not sufficient for, neoplastic progression. As their functions are rate limiting, blocking of adhesion molecules at any of the metastatic phases (aside from the first “detachment” step) may grind the entire metastatic cascade to a halt. The various tumor cells exploit different families of adhesion molecules, which function at distinct phases of the malignant process. Furthermore, even the same tumor can express various adhesion molecules that may be utilized at the same or different phases of the metastatic cascade (Zahalka et al., 1995). When evaluating the role of adhesion molecules in the metastatic process, the CD44 family of molecules deserves considerable attention in view of its adhesive, locomotive, and growth-transducing functions as well as its prevalence among cancer cells. The comprehensive review by Lesley et al. (1993a) will provide the reader with the relevant CD44 literature prior to 1993.

11. CD44 NOMENCLATURE

CD44 was first described as brain-granulocyte-T lymphocyte antigen (Dalchau et al., 1980).Like many cell surface and bioactive molecules, CD44 was accorded various structural or functional names that merged to an es-

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tablished single name only after molecular sequencing. The synonyms GP9OHermeS, extracellular matrix receptor 111, homing cell adhesion molecule, phagocytic glycoprotein-1, glycoprotein 85, Ly-24, hyaluronate receptor, HUTCH-1, and In (Lu)-relatedp80 glycoprotein are all included in the CD44 cluster designation assigned by the Third International Workshop on Leukocyte Typing (Cobbold et al., 1987).

111. CD44 BIOCHEMICAL STRUCTURE

A. Genomic Organization CD44 is a family of molecules consisting of many isoforms. The molecular diversity of this glycoprotein is generated by both posttranslation modification and differential utilization of alternatively spliced exons. CD44 is encoded by a single gene, located on the short arm of chromosome 11in humans (Goodfellow et al., 1982) and on chromosome 2 in mice (Colombatti et al., 1982), spanning approximately 50 kb of human DNA (Screaton et al., 1992). CD44 genomic organization, described by Screaton et al. (1992,1993), involves 20 exons in both mouse and humans (Fig. 1B). The first 5 exons coding for the extracellular domain (designated the 5’ constant region) are constant in both species (exons 1-5), whereas the next 10 exons are subjected to alternative splicing. This generates a variable region (see Fig. 1A) containing different exon combinations (Brown et al., 1991; Dougherty et al., 1991; Cooper et al., 1992; He et al., 1992; Jackson et al., 1992; Screaton et al., 1992). Variable region exons are designated V1 (exon 6 on the sequential scale) to V10 (exon 15 on the sequential scale; V stands for variant exon). Note, however, that exon V1 is not expressed in humans (Screaton et al., 1993). Exons 16 and 17 are the first two constant exons of the 3‘ constant region, and they, together with part of exon 5, encode the membraneproximal region of the extracellular domain (with optional inclusion of variant exons). The next domain (i.e., the hydrophobic transmembrane region) is encoded by exon 18 of the 3’ constant region. The cytoplasmic domain is also subject to alternative splicing. Differential utilization of exons 19 and 20 generates the short version (3 amino acids) and the long version (70 amino acids) of the cytoplasmic tail, respectively. The first 3 amino acids, common to both tails, are encoded by exon 18. The DNA sequence of exon 19 carries a long A+T tract, possibly causing instability in the mRNA of the short version. The additional amino acids of the long cytoplasmic domain are encoded by exon 20. The long version of the cytoplasmic tail is much more abundant than the short version (Goldstein and Butcher, 1990; Screaton et al., 1992).

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B. Standard CD44 The most abundant version of CD44 is the standard one (CD44s, 85-95 kDa) (Stamenkovic et al., 1991), which lacks the entire variable region, with exon 5 of the 5’ constant region being directly spliced to exon 16 of the 3’ constant region (Idzerda et al., 1989; Nottenburg et al., 1988; Zhou et al., 1989; Aruffo et al., 1990; Wolffe et al., 1990; Bosworth et al., 1991; Harn et al., 1991; He et al., 1992). CD44s is expressed mainly on hematopoietic cells and is therefore also designated hematopoietic CD44 or CD44H. Northern blot analysis of RNA isolated from hematopoietic cell CD44 revealed three major transcripts in humans (-1.6,2.2, and 4.8 kb) (Goldstein et al., 1989; Stamenkovic et al., 1989; Quackenbush et al., 1990) and three (-1.6, 3.5, and 4.5 kb) (Haegel and Ceredig, 1991) or four (1.6, 3.2, 4.0, and 5.2 kb) (Wolffe et al., 1990) in the mouse. Utilization of multiple polyadenylation signals may explain this heterogeneity (Harn et al., 1991). The human (and primate) CD44s mRNA is translated to 361 (in mouse, 363) amino acids. The predicted size of the core protein is 37-38 kDa (Goldstein et al., 1989; Idzerda et al., 1989; Nottenburg et al., 1989; Stamenkovic et al., 1989; Zhou et al., 1989; Screaton et al., 1992). However, the molecular size of CD44 is considerably increased by posttranslational modifications. Extensive glycosylation (see Fig. 1A) may occur in the mouse at 5 potential N-linked carbohydrate sites and at 10 potential O-linked carbohydrate sites (Zhou et al., 1989), and, respectively, at 6 and 7 potential sites in humans (Goldstein et al., 1989).This posttranslational modification doubles the molecular size of CD44s, bringing it from 38 kDa to 85-95 kDa (Zhou et al., 1989; Lokeshwar and Bourguignon, 1991). Indeed, tunicamycin treatment of mouse CD44 and subsequent immunoprecipitation revealed a 42-kDa molecular species (Lokeshwar and Bourguignon, 1991), a size quite close to the predicted one. Four serine-glycine motifs in the human CD44 extracellular domain and three in the mouse (Goldstein et al., 1989; Stamenkovic et al., 1989; Zhou et al., 1989) can be modified by the glycosaminoglycan (GAG) heparan sulfate (HS) (Brown et al., 1991; Tanaka et al., 1993) or by chondroitin sulfate (CS) (Jalkanen et al., 1988; Stamenkovic et al., 1989, 1991), thereby converting the molecule to a proteoglycan with possibly altered ligand specificity (Faassen et al., 1992; Jalkanen and Jalkanen, 1992). Chondroitin sulfate modification increases the size of the CD44 molecule from 85-95 kDa to 180-200 kDa (Jalkanen et al., 1988). Phosphorylation of the CD44 cytoplasmic tail (Isacke et al., 1986; Kalomiris and Bourguignon, 1988; Neame and Isacke, 1992) is another optional posttranslational modification with potential functional consequences (see Section V.E). The CD44 glycoprotein is an acidic (isoelectric point = 4.2) molecule, its charge largely due to sialic acid (Jalkanen et al., 1988). The t+of CD44 turnover was found to be 8 h (Lokeshwar and Bourguignon, 1991). As previously indicated, the CD44s molecule is composed of several do-

A. GLYCOPROTEIN

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Fig. I Schematic representation of CD44 glycoprotein (A), its exon map (B), and examples of six alternatively spliced transcripts (C). (A) Protein structure. Using disulfide bonds, the amino terminus of the molecule forms a globular domain, or three globular subdomains. The circle and the “downstream” ellipse represent areas that influence hyaluronate (HA) binding (Peach et al., 1993; Zheng et al., 1995). The black track inside the circle refers to a region displaying 30% homology with cartilage link protein and proteoglycan core protein, both showing HA binding ability. The black track at the amino terminus (inside and outside the circle), transmembrane-spanning domain (23 amino acids), and cytoplasmic tail (70 amino acids) represents regions with 80-90% interspecies homology. The alternatively spliced short cytoplasmic domain ( 3 amino acids) is nonproportionately represented by a small bar. The lightly shaded track in the center indicates the nonconserved membraneproximal region, which displays 3 5 4 5 % interspecies homology. The optional variable region, containing various combinations of variant exon products (see part C of legend), is inserted between amino acids 201 and 202 (mature protein) and marked by a zigzag track. The full amino acid sequence of human and mouse CD44s is presented in Zhou et al. (1989) and the nucleotide sequence (including the variable region) of human CD44 in Screaton et al. (1992). 0,potential N-linked glycosylation sites (only those of standard CD44 are shown); X, areas rich in serine and threonine, possible sites for 0-linked glycosylation (those of the variable region are arbitrarily assigned); , potential sites for GAG (CS or HS) incorporation, @, potential sites for phosphorylation (only part of the sites are depicted). The symbols on the standard part of the molecule mostly refer to mouse CD44 (Zhou et d., 1989),whereas those of the variable region are based on information taken from both mouse and humans. (B) Exon map. The filled circles represent exons of the constant regions. Empty circles represent exons that can be inserted by alternative splicing in the variable region. Note that exon V1 is not expressed in the human CD44. LP, leader peptide-encoding exon; TM, transmembrane-encoding exon; CT, cytoplasmic tail-encoding exons. (C) Examples of alternatively spliced transcripts. 1 and 2, standard CD44 with short and long cytoplasmic tails, respectively, which lacks the entire variable region. 3, pMeta-1 (CD44V4-7). Exons V4, VS, V6, and V7 are inserted in tandem between exons 5 and 17.4, pMeta-2 (CD44V6,7). Exons V6 and V7 are inserted between exons 5 and 17. pMeta-1 and pMeta-2 are known as “metastatic” CD44, bccause their cDNA confers, upon transfection, metastatic potential on nonmetastatic rat pancreatic tumor cells (Giinthert et al., 1991).Note that exon 16 is not expressed in either pMeta-1 or pMeta-2.5, epithelial CD44 (CD44V8-10), expressed preferentially on epithelial cells. Exons V8, V9, and V10 are inserted between exons 5 and 16.6, keratinocyte CD44 (CD44V3-10), one of the largest CD44 molecules known. Exons V3 through V10 are inserted between exons 5 and 16.

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mains. The human extracellular domain contains 248 amino acids (mouse, 250; the first amino acid of the mature protein is designated residue 1, and the entire precursor protein sequence is termed the primary protein). Included in this domain is the amino terminus region, a stretch of about 90 relatively hydrophobic residues (amino acids 12-101) that displays an approximately 80-90% sequence similarity among species and also a relatively high homology (-30%) with cartilage link protein and proteoglycan core protein. The amino terminus region contains six cysteine residues, which are possibly utilized to form a globular domain or three globular subdomains. The membrane-proximal region of the extracellular domain is less well conserved, with approximately 3 5 4 5 % interspecies sequence similarity. The single transmembrane-spanning domain contains 23 amino acids in both mouse and humans, with 80-90% homology among different species. The CD44 cytoplasmic tail is highly conserved, as evident from the approximately 80-90% sequence similarity among species (Goldstein et al., 1989; Nottenburg et al., 1989; Stamenkovic et al., 1989; Zhou et al., 1989; Screaton et al., 1992). The human CD44 cytoplasmic tail contains 6 potential phosphorylation sites, and the mouse CD44 cytoplasmic tail 10. At least part of these sites are constitutively phosphorylated (e.g., serines 303 and 305 of the mature protein) (Isacke et al., 1986; Carter and Wayner, 1988; Camp et al., 1991; Lesley et al., 1993a; PurC et al., 1995). Although the cytoplasmic domain includes consensus phosphorylation sites for protein kinase C (PKC) and protein kinase A, as well as for CAMP- and cGMP-dependent protein kinases, there is no evidence that these sites are active (Wolffe et al., 1990; Camp et al., 1991).

C. CD44 Isoforms CD44s is a ubiquitous molecule expressed mainly on leukocytes but also on fibroblasts and cells of mesodermal and neuroectodermal origin. However, CD44 molecules of larger size appear on different normal and malignant cells. Alternative splicing, as well as differential posttranslational modifications (glycosylation and glycosaminoglycanation or, in short, glycanation) of distinct CD44 isoforms, enrich the CD44 repertoire, which, in turn, may increase the optional functions of this molecule. Individual cells, whether normal or malignant, can simultaneously express different CD44 isoforms. Insertion of all the variable region between amino acids 202 and 203 of the mature human CD44 sequence (see Fig. 1A)generates an extra stretch of 381 amino acids (mouse, 423), which show 64% interspecies homology (Screaton et al., 1993). The entire CD44 variable region (exon V1 to exon V10) reveals four additional potential N-glycosylation sites and a large number of 0-glycosylation sites (especially at exons V2 and V8-V10: e.g., V9 and V10

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exon products contain 40% and 30% serine and threonine residues, respectively). In addition, one motif (serine-glycine) for potential insertion of GAG has also been detected in the variable region (Screaton et al., 1993; Bennett et al., 1995b). CD44 variants containing the exon V3 product can be decorated by HS (Bennett et al., 1995a), and variants containing the V6 exon product can be modified on the colon carcinoma cell line by an H blood group sugar (LabarriGre et al., 1994).Interestingly, the use of additional variant exons (e.g., CD44V4-7) may also enhance phosphorylation of the CD44 cytoplasmic tail (Ponta et al., 1994-95). To date, at least 20 different CD44 transcripts have been described. However, this is most likely not the final word. Theoretically, 768 membrane-bound CD44 isoforms can be generated by alternative use of the variant exons (van Weering et al., 1993).Another example of such diversity is N-CAM, which, by alternative splicing, can generate 27 isoforms out of 54 theoretical possibilities (Reyes et al., 1991). In addition to CD44s (described in Section IILB), several other CD44 isoforms deserve special attention (Fig. lC), as they are frequently described in this chapter. Epithelial CD44 (CD44E), which is preferentially expressed on epithelial cells (see Section N.B) (Brown et al., 1991; Dougherty et al., 1991; Hofmann et al., 1991; Stamenkovic et al., 1991; He et al., 1992; Jackson et al., 1992), utilizes exons V8, V9, and V10 of the variable region in conjunction with the 5’ and 3‘ constant regions (Fig. 1C) to generate the CD44V8-10 isoform (130 kDa; Stamenkovic et al., 1991). pMeta-1 and pMeta-2 are known as “metastatic” isoforms because, upon transfection, their cDNAs confer metastatic potential on nonmetastatic cells (Giinthert et al., 1991). In pMeta-1, exons V4, V5, V6, and V7 are inserted between the 5’ and 3’ constant regions, generating the CD44V4-7 variant. Similarly, in pMeta-2, exons V6 and V7 are inserted between the same constant regions to produce the CD44V6,7 variant. Note that “constant” exon 16 is not expressed in the 3’ constant region of the two variants (Herrlich et al., 1993) (Fig. 1C).The longest CD44 isoform was detected in keratinocytes. Here exons V3-V10 are inserted in tandem between the two constant regions to generate the CD44V3-10 isoform (230 kDa) (Hofmann et al., 1991) (Fig. 1C). The long additional sequence (338 amino acids) provides additional glycosylation, particularly 0-glycosylation, sites and an attachment sequence for sulfated GAGS (HS or CS) (Brown et al., 1991; Haggerty et al., 1992; Bennett et al., 1995b), thereby further increasing its molecular size. CD44 variants (abbreviated as CD44v) can be identified by monoclonal antibodies (mAbs)directed against specific exon product epitopes, using flow cytometry or immunohistochemical (IHC)analysis. Such assays provide partial information, as they do not allow the identification of the full-length sequence but rather define exclusive epitope(s) on a specific exon product(s). To illustrate this limitation, exon products identified by antibody are marked by a small horizontal bar above the V affiliated to the exon number. For ex-

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ample, the V6 exon product identified by anti-V6 mAb is registered as Cd44V6 to emphasize that the isoform contains the V6 product. It is not known if this product is included in the sequence of other exons or if it is expressed independently. The missing information can be provided by reverse transcriptase-polymerase chain reaction (RT-PCR) (see later). In addition, mAbs that recognize epitopes on the CD44 constant region do not distinguish between CD44s and CD44v, as this region is shared by all CD44 isoforms. Such antibodies are defined as anti-pan-CD44 mAb (or, in most cases, simply anti-CD44 mAb) and the CD44 recognized by them is designated pan-CD44, or simply CD44. Furthermore, identification, following electrophoresis, of CD44 molecular species larger than 85-95 kDa by antibodies recognizing the CD44 constant region does not necessarily imply that spliced variants are present, since posttranslational modification may also account for the higher molecular size. Full-length CD44 isoform sequences are detected by RT-PCR and nucleotide sequencing. Abbreviated registration of all the exons expressed in a particular CD44 molecule is used to identify the variants (e.g., CD44V4-7 denotes pMeta-1). Finally, for reasons of convenience, we will frequently use the term “exon” in conjunction with protein CD44, although the term “exon product” is more accurate.

D. Association of the CD44 Cytoplasmic Tail with the Cytoskeleton According to several reports, the CD44 cytoplasmic tail interacts with cytoskeleton-related components such as actin, ankyrin, or members of the ezrin-radixin-moesin family (Lacy and Underhill, 1987; Kalomiris and Bourguignon, 1988; Bourguignon et al., 1991,1992,1993; Lokeshwar and Bourguignon, 1991, 1992; Lokeshwar et al., 1994; Tsukita et al., 1994). A number of modifications, including PKC-mediated phosphorylation (Kalomiris and Bourguignon, 1989; Bourguignon et al., 1992), palmitoylation (Bourguignon et al., 1991), and GTP binding (Lokeshwar and Bourguignon, 1992; Galluzzo et al., 1995), may regulate the interplay between CD44 cytoplasmic tail and cytoskeleton-related proteins and, perhaps, the subsequent CD44-ligand interaction. The observation that in some cells a fraction of the CD44 molecules is resistant to extraction by detergent has been interpreted as evidence of CD44 interaction with the cytoskeleton (Tarone et al., 1984; Lacy and Underhill, 1987; Carter and Wayner, 1988; Geppert and Lipsky, 1991). However, in fibroblasts, tailless CD44 mutants, which are not able to interact with the cytoskeleton, have also been found in the detergent-insoluble fraction (Neame and Isacke, 1993; Perschl et al., 1995a), suggesting the interplay of CD44 with lipids of the plasma membrane rather than with the cytoskeleton (Neame et al., 1995). In those cases where an as-

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sociation between CD44 and the cytoskeleton has been demonstrated, there are contradictory findings as to the phosphorylation dependence of this event. Bourguignon and colleagues (Kalormiris and Bourguignon, 1989; Bourguignon et al., 1992)suggest that the association is phosphorylation dependent, whereas other investigators (Camp et al., 1991; Neame and Isacke, 1992) do not favor this implication. Yet another study showed that activation of T lymphocytes with phorbol ester reduces the interaction between CD44 and the cytoskeleton (Geppert and Lipsky, 1991). Again, it is uncertain whether the phosphorylation status of the activated CD44 influences this effect. Variations in the internal andlor external cell environment as well as differences in methodology may account for all these discrepancies.

IV. CD44 EXPRESSION ON NORMAL CELLS A. CD44 Isoforms in the Developing Embryo

and during Maturation of the Hematopoietic System As CAMS are indispensable for the generation of cell communities during fetal development (Fleming et al., 1994),the role of CD44 in this process has obviously attracted much attention. CD44 was detected by indirect immunofluorescence in early, preimplanted human embryos containing one to eight cells. The intensity of expression was maximal at the eight-cell phase and downregulated at the morula, blastocyst, and postimplantation stages (Campbell et al., 1995). During the preimplantation stage, CD44 may be involved in cell-cell homophilic interactions of embryonic blastomers or in the heterophilic adhesion between the inner cell mass and trophectodermal cells. Immunohistochemical studies with variant-specific mAbs revealed the expression of CD44v in 10-week-old human embryos. The predominant CD44v9: isoform was found in the epidermis, trachea, lung, thyroid gland, and mesonephric and paramesonephric ducts. CD44V6: was found in the epidermis and trachea (Terpe et al., 1994b). In newborn rats, the CD44V6 isoform has been identified in basal layers of the epidermis, hair follicles, the lower part of the crypts in the colon mucosa, and ductal epithelia of pancreatic glands (Wirth et al., 1993). Interestingly, expression of V6 exon is a hallmark of the proliferating mobile cells (see Section 1V.C) populating these tissues. Transcript analysis with RT-PCR of 7.5-day-postcoitum total mouse embryos revealed that CD44v (CD44V10 and CD44V8-10) are more intensively expressed in the fetus than is the standard isoform. In situ hybridization, using a V6-V10 sequence as a probe, indicates that the strongest expression of the CD44 variants is confined to the cell layer most proximal

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to the amniotic cavity. Later, between days 9.5 and 12.5 postcoitum, CD44s predominates, but larger variants are found in heart, somites, and limb bud mesenchyme (Ruiz et al., 1995). CD44 glycoproteins have also been detected on fetal rabbit fibroblasts (Alaish et al., 1994), embryonal murine neuronal cells located in the optic chiasm (Sretavan et al., 1994), and (mostly CD44v) embryonal Schwann cells, providing the myelin sheath coating the axons (Sherman et al., 1996). The ductal epithelia of the pancreatic gland of newborn rats express the epitope encoded by CD44 exon 6 (Wirth et al., 1993). A full-length splice variant was detected in the rat apical ectoderm ridge (AER) on day 12.5 postcoitum. The AER promotes the proliferation of the underlying mesenchymal cells in the growing limb. The mesenchyme, however, expresses CD44s only. After treatment with mAb directed against an epitope encoded by the CD44 V6 exon, AER failed to support the outgrowth of the limb bud (Wainwright et al., 1996). Since a member of the fibroblast growth factor (FGF) family is able to replace the AER function (Niswander et al., 1993), and as the proteoglycan version of CD44 can present various growth factors (see Section IX.H), it is tempting to speculate that the role of AER CD44 variants is to present FGF-like growth factors to growing limbs (Wainwright et al., 1996). Embryonic hematopoietic cells vary in CD44 expression, according to differentiation stage. Pluripotent bone marrow stem cells express CD44 (reviewed by Lesley et al., 1993a), and CD44 is detected also on bone marrow cells about to populate the thymus (pre-T cells). The earliest mouse thymocyte population CD4- CD8- CD3- interleukin-2 receptor- [IL-2R-I) expresses CD44s. The differentiated progeny of these double-negative (CD4CD8-) cells undergo a transition from CD44' IL-2R- through CD44- IL2R' to CD44- IL-2R-, becoming double-positive (CD4' CD8+)cells that express the T-cell receptor (Husmann et al., 1988; Penit and Vasseur, 1989; Lesley et al., 1990b; Petrie et al., 1990; Scollay, 1991). At a later stage, CD44 reappears on T cells of mouse strains expressing the CD44.1 allele (e.g., BALB/c mice). In contrast, only small amounts of CD44 are expressed in CD44.2 mouse strains (e.g., C57BLl6 and AKWJ) (Lynch and Ceredig, 1988, 1989). The same sequence of CD44 expression has been observed in the fetal human thymus. The earliest cells migrating into the thymus express CD44. During cell differentiation, CD44 is downregulated or lost (excluding a small subpopulation of thymic non-T cells) and later reexpressed (de 10s Toyos et al., 1989; Horst et al., 1990a; MBrquez et al., 1995), mostly as CD44s (Mackay et al., 1994). CD44 isoform expression during fetal human thymus development has been analyzed by irnmunostaining of frozen tissues. CD44 was detected on both thymic epithelial cells and thymocytes beginning at 8.2 weeks of fetal gestation, the time of initial colonization by bone marrow stem cells. CD44

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variants containing V4, V6, and V9 exon products emerge later, at 10 weeks of fetal gestation, and are confined to terminally differentiated thymic epithelial cells within and surrounding Hassall bodies (Mackay et al., 1994; Terpe et al., 1994b; Pate1 et al., 1995). Early human B cells (CD10') express a low level of CD44. The transition to the CD44-high phenotype occurs relatively late in development, when the cells assume the CD20 phenotype (Kansas and Dailey, 1989). Bone marrow precursors (ie., granulocyte-macrophage colony-forming units and erythroid burst-forming units) express high levels of CD44 (Lewinsohn et al., 1990). Whereas adult rat hematopoietic cells express mostly CD44s (see Section IV.C), a large fraction of newborn bone marrow and mesenteric lymph node cells display,.as shown by flow cytometry, CD44 variants containing the V6 exon product (Arch et al., 1992). This indicates that CD44v expression is shared by both the hematopoietic and the nonhematopoietic embryonic cells.

B. CD44 Isoforms Expressed on Epithelial Cells and on Other Nonhematopoietic Cells of the Adult Comprehensive knowledge regarding cells expressing standard CD44 or its variants at distinct states of differentiation or activation might allow better evaluation of CD44 function. Moreover, quantitative and qualitative comparisons of CD44 expressed on tumors and on the corresponding normal cells may reveal variations that could be exploited for diagnostic and prognostic purposes (see Section XII). Using different techniques, such as mRNA analysis (including RT-PCR) (Stamenkovic et al., 1989; Quackenbush et al., 1990), IHC (Alho and Underhill, 1989), and flow cytometry (Mackay et al., 1994), CD44 was detected on multiple cell types. Earlier and even later studies, using antibodies specific for the CD44 constant region, did not distinguish between the different isoforms. Such CD44 molecules (also designated pan-CD44) have been detected on the epithelium of skin, cheek, tongue, esophagus, vagina, cervix, ovary, intestine, stomach, oviduct, bladder, the tubular region of the kidney, liver bile ducts, long bronchi, salivary gland, thyroid gland, mammary gland, endometrium, epididymis, prostate gland, pancreatic ducts, urinary tract, and Hassall corpuscles in the thymus (Alho and Underhill, 1989; Stamenokovic et al., 1989; Heider et al., 1993b; Behzad et al., 1994; Fox et al., 1994; Mackay et al., 1994; Terpe et al., 1994b; Yaegashi et al., 1995). The molecule is predominantly expressed in regions of active cell growth (Alho and Underhill, 1989; Mackay et al., 1994). The use of variant-specific mAbs and the relevant PCR primers has enabled more rigorous definition of the CD44 isoforms present in the previously

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indicated epithelial tissues. These include, in addition to CD44s, CD44 isoforms expressing different combinations of variant exons such as CD44V3-10 or CD44V8-10, the most representative epithelial isoform (Fig. 1A) (Behzad et al., 1994; Iida and Bourguignon, 1995; Stauder et al., 1995). A CD44 variant containing V6 exon products (CD4486)has been detected in the basal layer of epidermis, on hair follicles, and on cryptic gut epithelium of adult and newborn rats (Wirth etal., 1993).In humans, both CD44V6 and CD44V4 were identified in epithelial cells of skin epidermis (CD4486 was evident particularly in the upper layers; Salmi et al., 1993), hair follicles, esophagus, tonsil, and thymic Hassall’s corpuscles. CD4486 (but not CD44V4) was detected in the epithelium of sweat glands, prostate gland, mammary gland, and lung bronchi. CD44v9 has been found in all of the previously mentioned epithelial tissues as well as in intestine, stomach, pancreatic ducts, the tubular region of the kidney, hepatic bile ducts, thyroid gland, salivary gland, endometrium, epididymis, urinary tract, and epithelial cells of mucosa-associated lymphoid organs (Mackay et al., 1994; Terpe et al., 1994b; Stauder et al., 1995). In another study, IHC staining of normal tissues revealed the presence of CD44v3, V4,5, 8 6 , and 88,9 variants in respiratory epithelium, transitional epithelium, and keratinizing and nonkeratinizing squamous epithelium. Expression of CD44V3, v4,5, and V6 was detected in placental cytotrophoblasts, thyroid follicular epithelium, skin adnexae, the myoepithelial layer of breast and prostate, and thymic Hassall’s corpuscles (Fox et al., 1994). In two studies the crypt epithelium of the gastrointestinal tract and the pancreatic ducts proved to be negative or only weakly positive for CD44 containing V4- or V6-encoded epitopes (Fox et al., 1994; Mackay et al., 1994). However, contradictory findings regarding the presence of CD44 variants in other epithelial tissues have been obtained. For example, thyroid and salivary glands were CD44v4- and CD44v6- in one study (Mackay et al., 1994) but positive in another (Fox et al., 1994). This discrepancy can be attributed to technical differences in methodology, varying degrees of staining sensitivity, or the use of antibodies that recognize different epitopes. The presence of CD44 on endothelium is also a matter of dispute, possibly related to similar causes. Some investigators (Alho and Underhill, 1989; Quackenbush et al., 1990) have reported that endothelial cells do not express CD44, whereas others (Pals et al., 1989a; Fox et al., 1994) demonstrated just the opposite. Furthermore, it has been shown by one group (Mackay et al., 1993), but not by another (Bennett et al., 1995a), that in endothelial cells tumor necrosis factor OL (TNFoL) upregulates CD44. CD44 has also been detected on smooth muscle (Pals et al., 1989a; Picker et al., 1989), fibroblasts (Flanagan et al., 1989; Pals et al., 1989a; Picker et al., 1989),melanocytes (Guo et al., 1994c),adrenal gland, and the choroid of the eye (Kennel et al., 1993). In addition, CD44 has been found in the

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white matter, especially on perivascular astrocytes (Picker et al., 1989; Girgrah et d., 1991b; Asher and Bignami, 1992, Vogel et d., 1992; Moretto et al., 1993; Salmi et al., 1993), and on Schwann cells of the peripheral nervous system in rats (Sherman et al., 1995). Upregulation of CD44V6 and downregulation of CD44s and CD44V9 were observed after incubation of human epithelial cell lines with interferon y (IFNy) (Mackay et al., 1994). Phorbol 12-myristate 13-acetate (PMA) or the combination of TNFa and IFNy enhanced CD44 mRNA and protein expression in murine astrocytes. Furthermore, PMA-stimulated astrocytes also express CD44 variants containing V6 and V10 exons (Haegel et al., 1993). Enhanced expression of CD44 mRNA has been detected also in human lung fibroblasts incubated with IL-la or TNF, and it is further augmented by a combination of the two (Sampson et al., 1992). The 29-kDa fragment of fibronectin or IL-la upregulated the expression of CD44s and CD44V10 in chondrocytes (the latter to a lesser degree), while simultaneously inhibiting proteoglycan synthesis (Chow et al., 1995). Analysis by RT-PCR revealed that, in human MCF-7 breast carcinoma cells, CD44V8-10 (CD44E) and CD44V8,9 isoforms were upregulated after treatment with hyaluronidase (Tanabe et al., 1993).

C. CD44 Isoforms Expressed on Nonactivated and Activated Hematopoietic Cells All types of hematopoietic cells, including erythrocytes, T and B lymphocytes, natural killer cells, macrophages, alveolar macrophages, Kupffer cells, and interdigitating and follicular dendritic cells, as well as granulocytes, preferentially express CD44s (Pals et al., 1989a; Quackenbush et al., 1990; Koopman et al., 1993; Fox et al., 1994; Mackay et al., 1994; Arai et al., 1995; Telen, 1995). CD44V4,5 and V6 have been detected on medullary thymocytes (Fox et al., 1994), and CD44V3, CD44V6, and CD44V10 are present on lymphocytes of reactive lymph nodes (Stauder et al., 1995). These findings lead to the conclusion that the CD44 repertoire of hematopoietic cells is far more restricted than that of epithelial cells. Memory and activated T cells have much higher levels of CD44 than do naive T cells; in addition, they express CD44 variants containing the V9 exon (Mackay et al., 1994; reviewed in Lesley et al., 1993a). Memory or activated CD44' T cells also express high levels of LFA-1, CD2, and CD58 (LFA3, the ligand of CD2) and low levels of CD45RO (a marker of naive T cells) (Sanders et al., 1988; Mackay et al., 1990). In addition, they produce high levels of IFNy (Budd et al., 1987). Sheep memory cells, expressing high levels of CD44 but lacking MEL-14, enter the peripheral lymph node exclusively via the afferent lymphatics (Mackay et al., 1990). The CD44' MEL-

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14- mouse T-cell lymphoma utilizes the same route for lymph node invasion (Zahalka et al., 1995). A substantial change in the CD44 repertoire has been noted after cell activation. Three to 1 4 days after in vivo antigenic stimulation with allogeneic cells, rat T cells, B cells, and macrophages express, in addition to CD44s, CD44 variants containing the V6 exon product, as indicated by RT-PCR or relevant anti-CD44 variant-specific mAb (Arch et al., 1992). Similarly, in vitro mitogenic, allogeneic (mixed lymphocyte reaction), or antigenic activation of human T cells transiently upregulates cell surface expression of CD44 variants containing V6 and V9 exon products, as shown by V6- and V9specific mAbs (Koopman et al., 1993; Mackay et al., 1994). A more recent RT-PCR study (Stauder et al., 1995) implies that nonstimulated cloned T cells express CD44V3, CD44V6, and CD44V10 isoforms. This repertoire is changed after phytohemagglutinin stimulation to include larger variants (CD44V3-8, CD44V24, and CD44V8,9). It should be noted, however, that long-term culture may induce the production of CD44 isoforms that are not present in the primary cells (see introduction to Section XII). Injection of mAb directed against the V6-encoded epitope into mice simultaneously immunized with allogeneic cells or haptenxarrier conjugate inhibited their cytotoxic responses to the relevant allogeneic target cells or the humoral responses to both hapten and carrier (Arch et al., 1992). This finding suggests that cell surface expression of V6 in CD44 molecules is essential to the normal function of cells involved in the immune response. To ascertain the biological significance of lymphocytes expressing CD44 containing the V6 exon, Moll and his colleagues (1996) generated transgenic mouse strains whose T cells constitutively express the rat CD44V4-7 gene product. Lymphocytes of transgenic mice immunized with allogeneic cells or trinitrophenylated bovine serum albumin (TNP-BSA) displayed, following in vitro stimulation with the relevant antigens, earlier and more extensive cell proliferation than did lymphocytes from nontransgenic mice. The cytotoxic response of the transgenic lymphocytes against relevant allogeneic blast cells was also markedly accelerated and increased. Similarly, the primary in vitro proliferative responses of transgenic lymphocytes to allogeneic cells and TNP-BSA were higher and more rapid than those of nontransgenic lymphocytes. In the presence of mAb directed against the V6-encoded epitope of rat CD44v, the enhanced antigen-induced proliferative responses of the transgenic lymphocytes reverted to the level of the nontransgenic lymphocytes. These findings suggest that CD44V4-7 drives the lymphocytes to a constitutive preactivation state (analogous to that in memory T cells), resulting in their earlier entry into the S phase (Moll et al., 1996). It is shown in Section X1.A that tumor cells expressing CD44V4-7 have a growth advantage. CD44 transition has also been demonstrated during B-cell activation and differentiation in the germinal centers of human tonsil. High levels of CD44 are expressed on resting immunoglobulin (Ig) D+ IgM' cells. Following anti-

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gen activation, CD44 is downregulated at the early blast stage, when the blast marker CD38 emerges. During the blast (CD38' IgM') and centroblast (CD38' Ig-) stages, CD44 expression remains low or negative, but it is again upregulated upon transition to the centrocyte level. IgG' and IgA' cells at the postgerminal center stage express high levels of CD44 (Kremmidiotis and Zola, 1995). Similarly, mouse splenic B cells express CD44, which undergoes upregulation after stimulation with lipopolysaccharide (LPS), antiIgD-dextran, the supernatant of cloned Th2 cells, or IL-5 (Murakami et al., 1990; Hathcock et al., 1993). Normal human splenic B cells activated with anti-Ig antibody express increased levels of CD44s, CD44V6, and a CD44 variant containing the V10 exon product (Salles et al., 1993).Whereas resting human peripheral blood B cells express CD44s only, various CD44 variants (CD44E, CD44V10, CD44V6, and CD44V6,7) have been detected after stimulation with PMA, anti-IgM mAb, or IL-2. Epstein-Barr virus (EBV)-negativeBurkitt lymphoma (BL) cells do not express CD44. In contrast, CD44s, CD44E, and CD44V10 were detected in EBV' BL cells and EBV- BL cells infected with EBV (Kryworuckho et al., 1995),indicating that fewer variants are induced after viral activation. Bone marrow human plasma cells constitutively express CD44V9. Pulmonary macrophages express both CD44V6 and CD44V9. CD44 variants expressing V6 and V9 exons are upregulated in myelomonocytic cell lines after in vitro incubation with TNFa and IFNy, as indicated by flow cytometry and RT-PCR analysis (Mackay et al., 1994). It is difficult to ignore the finding that epithelial regions rich in proliferating cells, such as the basal cells of stratified squamous epithelium and glandular epithelium, express high levels of CD44v, especially isoforms containing the V6 exon. Similarly, activated leukocytes and epithelial cells upregulate V6- and V9-containing variants. The extensive locomotive and generative activities within the embryo are also accompanied by marked expression of CD44v. Again, the V6 version is particularly conspicuous. Malignant cells, which share many properties with normal adult and fetal cells of generative tissues, bear similar CD44 isoforms (see Section XII).

V. HYALURONIC ACID IS THE PRINCIPAL LIGAND OF CD44

A. The Structure of Hyaluronic Acid and Its Distribution in Normal and Tumor-Invaded Tissues Hyaluronic acid (HA; hyaluronate, hyaluronan) is a ubiquitous polysaccharide (GAG) consisting of a linear polymer of repeating disaccharide units with the structure (D-glucuronic acid [ 1-p-31 N-acetyl-D-glucosamine [ 1-p-

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4]),. Hyaluronate has a high molecular mass (106-107 Da) (Laurent and Fraser, 1992). It is synthesized by fibroblasts (Teder et al., 1995), chondrocytes (Mason et al., 1989), and mesothelial cells (Honda et al., 1991; Heldin et al., 1992). The production of HA by human lung fibroblasts is stimulated by cytokines TNF, IFNy, and IL-1 and further augmented by a combination of TNF and IFNy, or of TNF and IL-1, as was shown in an in vitro assay (Sampson et al., 1992).Hyaluronate is an important component of the ECM, filling the intercellular spaces. Within the ECM, HA noncovalently interacts with proteoglycans, the binding stabilized by a link protein (Hardingham and Fosang, 1992; Laurent and Fraser, 1992). Hyaluronan is particularly abundant in connective tissues such as skin dermis, smooth muscle, lung, the lamina propria of mucous membranes, and the adventitia surrounding blood vessels (Aruffo et al., 1990). It is also present in the lymph (Laurent and Fraser, 1992)and lymph node matrix (Aruffo et al., 1990).Hyaluronic acid provides cellular support and a water-filled compartment. In addition, HA regulates cell-cell adhesion and the cell’s spatial orientation and traffic, as well as its growth and differentiation (Laurent and Fraser, 1992). Consequently, HA is involved in various biological processes such as inflammation (reviewed in Laurent and Fraser, 1992), wound healing, and tissue remodeling (West et al., 1985; Weigel et al., 1989; Laurent and Fraser, 1992; Oksala et al., 1995), as well as morphogenesis (Laurent and Fraser, 1992). Hyaluronan also interacts with the cell surface to form a “coat,” which may act as a protective cellular barrier (McBride and Bard, 1979; Gately et al., 1984). In addition, it supports the migration of invasive tumors (Toole et al., 1979). Indeed, tumor invasion is sometimes observed in regions with high concentrations of HA, such as the medullary and papillary interstitium of renal tissue, the submucosa of the gastrointestinal tract, around the centrilobular veins, and beneath the capsule of the liver (Sy et al., 1991).Some tumors synthesize and release H A into their immediate environment (Turley and Tretiak, 1985), while others stimulate the production of HA by surrounding fibroblasts (Knudson et al., 1984). Zhang and colleagues (1995) reported that intravenously injected mouse melanoma cells (B16-F1) expressing a high level of HA on their surfaces formed a greater number of lung metastases than did melanoma cells bearing low amounts of surface HA. Collectively, these findings emphasize the central role of HA in metastasis.

B. Evidence That CD44 Is an HA Receptor CD44 is the principal HA receptor, although the molecule can bind other ligands, in some cases at a lower affinity (see Section VI). The CD44 receptor coordinates a minimum of six HA sugar residues (three repeating disaccharides units), but has a higher affinity for longer HA molecules (Underhill

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et al., 1983), with a dissociation constant of 0.3 nM (Bourguignon et al., 1993). A hint that CD44 is a lectin-like receptor for HA came from a sequence comparison between known HA binding proteins (cartilage link protein and proteoglycan core protein) and CD44. The amino-terminal domain of CD44 (amino acid positions 12-101 of the mature protein in the human) displays about 30% sequence homology with the HA binding region of the previously mentioned proteins (Fig. 1A) (Goldstein et al., 1989; Stamenkovic et al., 1989), with the homology increasing to about 50% if conservative amino acid substitutions are considered (Goldstein et al., 1989). The evidence for a receptor-ligand relationship between CD44 and HA has been established by several experimental approaches. It has been demonstrated that the binding of plastic-immobilized or soluble fluoresceinated HA (Fl-HA) to CD44expressing cells can be prevented by some (but not all) anti-CD44 mAbs (Lesley et al., 1990a, 1992; Miyake et al., 1990b; Bennett et al., 1995b; PurC et al., 1995; Zahalka et al., 1995),an excess of soluble HA (Lesley et al., 1990a, 1992; PurC et al., 1995), or pretreatment of immobilized HA with hyaluronidase (PurC et al., 1995; Zahalka et al., 1995). After genetic fusion of cDNA encoding the extracellular domain of CD44 with genomic DNA segments encoding the IgG constant region, the construct was transfected into COS cells. The transfectants yielded a secretory, soluble CD44immunoglobulin G (CD44-lg) fusion protein that bound to lymph node high endothelial cells. Binding was blocked by the inclusion of low concentrations of HA, but not of other GAGS, in the assay system or by pretreatment of the endothelial cells with hyaluronidase (Aruffo et al., 1990). This finding implies that the HA expressed on high endothelial cells is recognized by soluble CD44. CD44- cells transfected with CD44 cDNA acquire the ability to interact with anti-CD44 mAb (Aruffo et al., 1990; Stamenkovic et al., 1991; Lesley et al., 1992) or to bind to lymph node high endothelial cells. Binding to endothelial cells can be inhibited by anti-CD44 mAb, soluble HA, or pretreatment with hyaluronidase (Stamenkovic et al., 1991; Lesley et al., 1992). In addition, transfection of CD44 cDNA into CD44- cells conferred on them the ability to bind F1-HA from solution or to adhere to plastic-immobilized HA. Again, binding was CD44 dependent (Lesley et al., 1992). Perhaps the clinching experiment proving that HA is a ligand of CD44 was the one showing that radiolabeled CD44 purified from placenta is able to bind immobilized HA, and that adherence is inhibited by anti-CD44 mAb, soluble HA, or hyaluronidase (St. Jacques et al., 1993). Similarly, soluble CD44-lg fusion protein bound HA in a cell-free system (Peach et al., 1993). Not all the tested anti-CD44 mAbs can block the binding of HA to cells expressing CD44 (Lesley et al., 1990a, Zheng et al., 1995). This indicates that the topography of the CD44 epitopes and their orientation toward the HA binding site dictate the ability of the relevant antibody to interfere with

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HA adherence. Perhaps even more important, not all CD44-expressing cells are able to bind HA, although some of them acquire this property after activation or chemical modification (see Sections V.F and V.G). A similar type of activation-dependent upregulation of ligand binding has been observed in integrins (O’Toole et al., 1990; O’Toole, 1995).

C. Binding of HA to CD44 Variants The ability of human hematopoietic CD44 (CD44H or CD44s) to bind H A either constitutively or after activation is well established. Such consensus cannot, however, be extended to epithelial CD44 (CD44E, CD44V8-10) and other CD44 variants. A CD44- BL cell line (Namalwa) transfected with human CD44E, CD44V6-10, or CD44V7-10 cDNAs did not display considerable HA-dependent adhesion to lymph node high endothelial cells (Stamenkovic et al., 1991; Bartolazzi et al., 1995). Similarly, the Namalwa transfectants, as well as a melanoma cell line transfected with human CD44E cDNA, did not significantly bind to plastic-immobilized or soluble HA (Sy et al., 1991; Thomas et al., 1992; Bennett et al., 1995b; van der Voort et al., 1995). Insertion of CD44V3-10 or CD44V3,8-10 also failed to confer on B-lymphoma cells the ability to efficiently bind HA (Bartolazzi et al., 1995; Jackson, D. G., et al., 1995; van der Voort et al., 1995). In contrast, when transfected with CD44H cDNA, the same cell lines did bind HA. In addition, CD44E-lg, in contrast to the CD44H-lg fusion protein, does not interact with immobilized HA, as indicated by enzyme-linked immunosorbent assay (ELISA) (Peach et al., 1993; Bennett et al., 1995b). Phorbol ester treatment induced cells transfected with CD44H, but not with CD44E, cDNA to bind HA (Liao et al., 1993; van der Voort et al., 1995). Contradictory results were obtained, however, when a CD44- AKRl cell line of mouse T-cell lymphoma was transfected with the mouse analog of human CD44E cDNA. The mouse CD44V8-10 transfectants, as well as mouse transfectants expressing other CD44 variants, exhibited significant binding to an HA-bearing cell line (He et al., 1992). In addition, a rat pancreatic adenocarcinoma cell line transfected with CD44V4-7 (pMeta-1) (see Fig. 1) displayed enhanced binding of soluble HA (Sleeman et al., 1996). Lymphocytes of pMeta-1-transgenic mice expressing the CD44V4-7 transgene were, however, unable to bind HA (Sherman et al., 1994). Collectively, these results prove that the insertion of additional exon products does not interfere with HA binding. Furthermore, it was found that, in activated human T cells, antibody-induced modulation of V6 or V9 CD44-encoded epitopes markedly reduces their ability to adhere to immobilized HA (Galluzzo et al., 1995), suggesting, rather, that the expression of variant exons enhances the CD44-ligand interaction. These seemingly conflicting findings can be rec-

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onciled by assuming that the specific internal cell environment influences the ability of the CD44 variants to interact with HA. It is likely that different cells synthesize analogous CD44 variants (e.g., CD44E) decorated with distinct glycosylation or glycanation patterns, some of which interfere with HA binding. An experiment supporting this assumption is described in Section V.G. Alternatively, a “binder” CD44 variant may differ from its “nonbinder” analog by the replacement of a few amino acids. Indeed, Dougherty and colleagues (1994) reported that a CD44 variant that differs from human CD44E by three amino acid substitutions was able to bind HA.

D. The Topography of the CD44 HA Binding Sites CD44 belongs to a group of proteins (also known as hyaladherins; Knudson and Knudson, 1993) that share the ability to interact with HA. Included in this group are the receptor for hyaluronate-mediated motility (RHAMM; Turley et al., 1991); cartilage link protein; proteoglycan core protein (aggrecan) (Hardingham and Fosang, 1992); fibroblast versican (LeBaron et al., 1992); hyaluronectin (Delpech and Halavent, 1981); the Ivd4 epitope of endothelial cells (Banerjeeand Toole, 1992);TSG-6, a TNFainducible protein (Leeet al., 1992);and ICAM-1 (the latter was purified from an HA affinity column but the extracellular domain was not analyzed for HA binding) (McCourt et al., 1994). The suggestion has been advanced that these proteins include regions containing positively charged amino acids (arginine and lysine), which interact with the negatively charged hexuronate groups of HA (Hardingham and Fosang, 1992). Each HA binding cluster consists of two basic amino acids, either arginine or lysine, separated by seven nonacidic amino acids (B[X7]B motif) (Yang et al., 1994). In addition, part of the HA binding proteins (i.e., the proteoglycan core and link proteins) contain conserved cysteine residues, which possibly form disulfidebridged loops that are involved in HA binding (Goetinck et al., 1987). The interspecies homologous region of the CD44 extracellular domain (Fig. 1A) also contains six conserved cysteines (Goldstein et al., 1989; Nottenburg et al., 1989; Zhou et al., 1989) that may form a single globular domain (Goldstein et al., 1989)or a structure consisting of three loops (Zheng et al., 1995). At least part of the HA binding capacity of CD44 is confined to these loop(s) (Liao et al., 1993; Zheng et al., 1995). In contrast, the HA binding capacity of the second subgroup of proteins (e.g., RHAMM) is reduction resistant (Hoare et al., 1993), suggesting that their ligand binding is not dependent on an S-S-bonded globular domain. The complete sequencing of the CD44 molecule has allowed the identification of two extracellular regions containing a relatively high number of positively charged amino acids (arginine and lysine) known to be important

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for HA binding by other proteins. Using truncation and site-directed mutagenesis of the human CD44 sequence, Peach and colleagues (1993) found that these two regions are involved in HA binding. One of the clusters (amino acids 21-45 of the primary human CD44 sequence) is included within the link protein homologous region (Fig. lA, circle), where arginine at position 41 predominantly affects HA binding. The second cluster (amino acids 144-167) is located outside the link protein homologous region (Fig. lA, ellipse), where mainly the combination of lysine at position 158 and arginine at position 162 influences HA binding. Both clusters are situated within the interspecies homologous region (Fig. lA, black track at the amino terminus), which is located in the amino-terminal two thirds of the CD44 extracellular domain. Truncation mutagenesis of the membrane-proximal region (Fig. lA, the light shaded track), which follows the conserved amino terminus of human (Peach et al., 1993) or mouse (He et al., 1992) CD44, does not influence HA binding. The membrane-proximal region is the least conserved among species. Using mAbs recognizing defined epitopes of murine CD44s in order to block HA binding to CD44s-expressing cells, Zheng and colleagues (1995) showed that antibodies whose specificity was dictated by a stretch of eight amino acids located at positions 46-53 (of the mature protein) interfere with HA binding. The same effect was achieved with antibodies whose specificity is influenced by histidine at position 83 or valine and threonine at positions 90 and 91, respectively. Although these sites are included in the cartilage link protein homology region, none of them is located in the HA binding cluster described by Peach and colleagues (1993). However, antibodies directed against one CD44 epitope may change the conformation of the molecule so that the HA binding site, which is normally exposed at a different location, is no longer accessible. Another interesting point, deriving from the work of Zheng and colleagues (1995),is related to the fact that different cell types display distinct CD44 epitopes, which, after interacting with the relevant antibody, influence HA binding sites. In conclusion, it appears that the HA binding machinery of CD44 is dependent on two basic amino acid motifs, critically spaced disulfide bonds forming the globular domain(s) (Neame et al., 1986), expression of variant exons (see Section V.C), the conformation and integrity of the entire molecule, and the glycosylation and glycanation status of the protein (Section V.G).

E. Influence of the CD44 Cytoplasmic Tail and Cytoskeleton-Related Proteins on HA Binding to CD44 The observation that the CD44 cytoplasmic tail interacts with cytoskeletal proteins (Section 1II.D) raised the question of cytoskeleton involvement in HA binding. In this connection, a stretch of 15 amino acids (residues

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305-320 of the primary mouse protein) was assigned to the interaction between the CD44 cytoplasmic tail and ankyrin, in view of the finding that the corresponding synthetic amino acid sequence exhibited specific binding to this cytoskeleton-related protein (Lokeshwar et al., 1994). Transfection of Jurkat of COS cells with CD44 lacking the cytoplasmic sequence that contains the ankyrin binding segment resulted in 90% reduction of the cell's ability to bind soluble HA or to adhere to plastic-immobilized HA (Liao et al., 1993; Lokeshwar et al., 1994). Additionally, immunofluorescent staining showed that HA induces patching and capping of CD44 molecules on the surface of mouse T lymphoma cells. Double staining revealed ankyrin accumulation underneath the capped structure of the CD44 HA receptor, suggesting that ankyrin is involved in linking CD44 to the cytoskeleton contractile system. The notion that HA binding and HA-induced receptor capping are dictated by signals delivered from the cytoskeleton is further supported by the observation that both events are inhibited by the microfilament blocker cytochalasin or the calmodulin blocker W-7 (Bourguignon et al., 1993; Galluzzo et al., 1995). Cytoskeleton involvement is also important for the function of a number of other adhesion molecules (such as cadherins) (Geiger and Ayalon, 1992). As opposed to the previously mentioned possibility that interaction with ankyrin is essential to the HA binding function of CD44 (Lokeshwar et al., 1994), another study demonstrated that deletion of the ankyrin binding segment did not prevent HA binding, as indicated by cDNA cell transfection and a ligand binding assay (Perschl et al., 1995b). However, it should be noted that the truncated CD44 molecules were inserted into different types of cells (COS versus AKRl ), and, therefore, influenced by distinct intracellular environments. Further support that CD44-related HA binding may be cytoskeleton independent is provided by experiments with transfected AKRl cells in which all the inserted CD44 was detected in the Triton X-100soluble fraction, yet the cells bound HA (Uff et al., 1995). In addition, incubation with cytochalasin B did not affect the adherence of CD44' M o l t 4 cells to immobilized HA (Murakami et al., 1994). As for cytoskeletal involvement in CD44 binding function, there are also contradictory findings regarding the ability of cells transfected with tailless CD44 constructs to adhere to immobilized HA. Three reports imply that cells expressing tailless CD44 do not adhere, or adhere only marginally, to immobilized HA (Lokeshwar et al., 1994; Pur6 et al., 1995; Uff et al., 1995), whereas other studies maintain just the opposite (Lesley et al., 1992; Thomas et al., 1992). Interestingly, these seemingly conflicting observations even extend to transfectants of the same cell type (i.e., AKR-1 cells; compare Lesley et al., 1992, with Pure et al., 1995). On the other hand, it is clear that cells expressing tailless CD44 do not bind soluble HA, whereas under identical conditions those expressing the wild-type CD44 possess this ability (Lesley et al., 1992, 1993b; Liao et al., 1993; Lokeshwar et al., 1994; Uff et al.,

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1995). It has been shown that, after treatment with enhancing (agonistic) anti-CD44 mAb (e.g., IRAWB14 mAb) or its multimeric (but not monomeric) Fab fragments (Lesleyet al., 1993b), even tailless CD44 transfectants that cannot interact with soluble (Lesley et al., 1992)or immobilized (PurCet al., 1995) HA acquired the ability to bind the ligand. In addition, transfectants expressing the disulfide-bonded dimer of CD44 (constructed by genetic replacement of the CD44 transmembrane region with the corresponding region of the CD3-5 chain), efficiently bound soluble HA, even in the absence of the cytoplasmic domain (Perschl et al., 1995b). It bears mention that the transmembrane region of the CD3-5 chain contains a cysteine residue that forms an intermolecular disulfide bond, generating functional 5-5 homodimers or heterodimers (Weissman et al., 1988). Similarly, cytoskeletonmediated dimerization of CD44 molecules, through disulfide bonding, may allow HA binding under normal conditions. Indeed, Jurkat cells transfected with a human CD44 construct, in which cysteine-286 (primary protein) of the transmembrane domain had been replaced with alanine, failed to bind HA after stimulation with anti-CD3 mAb, whereas those transfected with the wild-type construct displayed efficient binding under identical conditions (Liu and Sy, 1996). Collectively, these results imply that the CD44cytoskeleton interaction is not an absolute requirement for HA binding, as this interaction can be bypassed by cross-linking the CD44 receptor (see later). In summary, outside-in signals provided by anti-CD44 antibody may allow clustering of tailless CD44 molecules on the cell membrane, thus conferring on them the ability to bind HA. The same distributional change may be induced in tailless CD44 by HA immobilized onto plastic or by artificial dimerization of the transmembrane domain and, in wild-type CD44, by inside-out signals delivered from the cytoskeleton. In any event, a threshold level of cell surface CD44 expression is required before HA binding is observed. At concentrations above this level, the amount of bound HA rises as the level of CD44 increases. This correlative effect has been observed in some (Perschl et al., 1995b; Uff et al., 1995), but not in all (Hyman et al., 1991; Galandrini et al., 1994b), cell lines. The primary mouse CD44 sequence is constitutively phosphorylated on serines 325 and 327 of the cytoplasmic domain, even though 10 potential phosphorylation sites are available (Pure et al., 1995). PurC and colleagues demonstrated that transfected AKR-1 cells expressing mutated mouse CD44, with serine substitutions at positions 325 or 327 to prevent phosphorylation, were defective in HA binding and ligand-induced receptor modulation. The ability to bind HA was restored by the addition of IRAWB14 anti-CD44 mAb, suggesting the inability of dephosphorylated CD44 to form clusters, which are essential to the adhesion function. Uff and colleagues ( 1995) reported contradictory findings: AKR-1 cell transfectants expressing human CD44 mutated at positions 323 and 325 (corresponding to positions

<-

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325 and 327 of mouse CD44) were able to bind soluble or immobilized HA, despite the lack of phosphorylation at these sites. If this discrepancy is not due to technical reasons, it is possible that mouse CD44 behaves differently than human CD44, as far as phosphorylation-dependent HA binding is concerned. The clue to understanding the apparently conflicting results described in this section is possible related to the cell-dependent differential mechanisms of CD44 cluster formation leading to cell surface HA binding. In some cell types, cluster formation is conditional upon the interaction between CD44 and the cytoskeleton. In other cells, cluster formation is largely dependent on the phosphorylation status of the cytoplasmic tail, which may or may not interact with the cytoskeleton. In addition, the length of the cytoplasmic tail may also dictate the ability to form stable clusters on the cell surface. Indeed, it has been demonstrated that AKR-1 cells transfected with CD44 containing only the first 16 amino acids of the cytoplasmic domain efficiently bind HA, whereas those containing only the first 6 amino acids do not (Perschl et al., 1995b). While all these parameters are not mutually exclusive, the level of CD44 on the cell surface may also have some bearing on HA binding, and its differential expression in various cell types could also reconcile the contradictory findings obtained by various groups. It bears mention that even tailless CD44 can form clusters and bind soluble HA if a sufficiently high concentration of molecules is expressed on the cell surface (Perschl et al., 1995b). It has been found, however, that tailless CD44 tends to accumulate in the cytoplasm and its half-life is markedly reduced, whereas intact CD44 is localized on the basolateral cell surface (Neame and Isacke, 1993). In addition to their possible influence on CD44 cell surface aggregation, some or all of the previously mentioned factors may induce changes in receptor configuration. Which mechanism is the more important for the acquisition of ligand binding capacity is a topic for further research.

F. CD44 Activation Allows HA Binding in Some CD44+ Nonbinder Cell Types Some CD44’ cell types (e.g., the BW5147 T-cell line, myeloma B-cell lines, and alveolar macrophages) have the capacity to bind HA constitutively (Lesley et al., 1990a; Culty et al., 1994). Such cells were designated “constitutively active” or, in short, “active” by Lesley et al. (1995). Many cell types, however, although expressing CD44, do not bind HA (Lesley et al., 1993a; Katoh et al., 1995).Some of these “nonbinder” CD44’ cells acquire the ability to bind the ligand after external stimulation or deglycosylation (“inducible” group) (see Section V.G), whereas other cell types consistently ignore the activation signal (“inactive” group) (Lesley et al., 1990a, 1993a).

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Given the ubiquity of CD44 and HA, this observation is hardly surprising, as only tight binding regulations may prevent unnecessary or even harmful CD44-dependent cell adherence. Reagents able to induce or enhance HA (both soluble and immobilized) binding to nonbinder CD44-expressing cells include PMA, a PKC activator (Lesleyet al., 1990a; Hyman et al., 1991; Liao et al., 1993; Galandrini et al., 1994b; Murakami et al., 1994; Zahalka et al., 1995), a cytokine (IL-5) (Murakami et al., 1990; Hathcock et al., 1993), a combination of anti-CD3 mAb and IL-2 (Galandrini et al., 1994b), and a select number of agonistic anti-CD44 mAbs (e.g., anti-mouse CD44 I R A m 1 4 and anti-human CD44 F10-44-2) (Lesley et al., 1992; Lesley and Hyman, 1992; Liao et al., 1993; Galandrini et al., 1994b). The PMA activation effect on HA binding has been detected in Jurkat T cells transfected with CD44s, but not with CD44E, although CD44 expression was similar in both cell lines (Liao et al., 1993). These results suggest that the additional region (exon products V8, V9, and V10) interferes with ligand adherence (see Section V.C). Phorbol ester-dependent HA binding of cell surface CD44s is conditional upon de 1zouo protein synthesis, as proved by the finding that the protein inhibitors cycloheximide or anisomycin prevented binding of activated human Molt-4 cells or mouse lymphoma cells to immobilized HA, without affecting CD44 cell surface expression (Murakami et al., 1994; Albaz and Naor, unpublished finding). How activation of PKC by PMA induces HA binding to CD44 remains unresolved. Perhaps PKC is involved in cytoskeleton signaling through a phosphorylated CD44-ankyrin interaction that, in turn, activates CD44 receptor aggregation and allows HA binding. However, as not all CD44-dependent HA binding mechanisms are necessarily cytoskeleton related (see Section V.E), other PKC signaling pathways may be involved. Although some cytokines and growth factors (TNFa, TNFP, basic FGF [bFGF]) affect CD44 expression (Osada et al., 1995) or CD44 glycanation (Romaris et al., 1995), it is not clear which physiological factors (with the exception of IL-5; see later) substitute for phorbol esters in the induction of CD44-dependent HA binding. Serum components may be among the potential candidates, as 3- to 4-day cultivation of human peripheral blood monocytes in medium containing 5 % autologous serum converted them from HA nonbinders to CD44-dependent HA binders (Culty et al., 1994). In searching for physiological factors substituting for PMA, priority should be given to cytokines or growth factors that activate PKC. In this connection, it should be noted that the stimulatory effect of IL-5 is possible PKC dependent (Li et al., 1992). The cytokine IL-5 induced in cultivated enriched murine B cells a CD44 bright, Ia dull, B220 dull subpopulation that responded to restimulation with the same cytokine by proliferation and differentiation to lg-producing cells. This CD44 bright subpopulation also acquired the ability to interact with soluble or immobilized HA, as indicated by binding assays ( 3 days are

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required to induce HA binding by IL-5). All these properties were absent from the remaining subpopulation (CD44 dull, B220 bright) (Murakami et al., 1990; Hathcock et al., 1993). Interestingly, a similar, although not identical, subpopulation of CD44 high HA-binding B cells was detected in spleens of mice undergoing the graft-versus-host (GVH) reaction. The HAbinding B cells secreted large amounts of IgG, including autoantibody specific for single-stranded DNA (Murakami et al., 1991). The intriguing possibility that IL-5 induces this cell population in vivo merits further research. CD44 molecules immunoprecipitated from IL-5-stimulated B cells displayed an apparent molecular size that was 3-5 kDa lower than that of CD44 from unstimulated B cells (80-90 kDa). Treatment with N-glycanase eliminated the difference in molecular weight (Hathcock et al., 1993), suggesting that the CD44 of activated B cells is deglycosylated at N-glycosylation sites. It is shown in Section V.G that deglycosylation gives rise to or enhances CD44dependent binding of HA. Some CD44-expressing cells (e.g., unstimulated B cells and T-cell lymphomas) that do not normally bind HA, or only moderately bind it, acquire or augment this ability after stimulation with agonistic antibody (e.g., IRAWB14.4) (Lesley et al., 1992; Hathcock et al., 1993; Liao et al., 1993). Interestingly, 45% of AKR mouse splenic T cells are CD44+, but practically none of them bind HA before antibody activation, whereas almost all of them bind the ligand after stimulation with this antibody, as indicated by fluoresceinated ligand binding and cell adherence to immobilized HA (Lesley et al., 1992; Lesley and Hyman, 1992). In one instance, it was reported that the effect of antibody activation on HA binding is not pronounced unless the CD44’ cells are prestimulated with both anti-CD3 mAb and IL-2 (Liao et al., 1993). In another experiment, lymphocytes ligated by anti-CD44 antibody displayed enhanced adhesion to endothelial cells. This binding was not blocked by mAbs specific for CD44, LFA-1, ICAM-1, VLA-4, VCAM-1, or CD2, nor did pretreatment of the target cells with hyaluronidase prevent adherence. The investigators suggested that the CD44 ligation induces a novel adhesion pathway mediated by still-unknown interacting molecules (Toyama-Sorimachi et al., 1993). Agonistic antibody may enhance CD44 molecule aggregation on the cell surface to promote HA binding. This notion is supported by the observation that HA binding to HUT-78 T-cell surface CD44 is conditional not only upon the latter’s interaction with the activating anti-CD44 mAb, but also on subsequent cross-linking of this mAb with anti-lg antibody (Galandrini et al., 1994b). It has also been reported that the agonistic antibody effect on HA binding is dependent on calcium mobilization, as it is blocked by the calcium chelator EGTA and enhanced by ionomycin (Galluzzo et al., 1995). This finding suggests that calcium mobilization influences receptor aggregation. Identification of the activating antibody’s natural ligand is a matter for fur-

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ther investigation. Perhaps polymeric HA can, under certain physiological conditions, cross-link CD44 cell surface receptors, thereby allowing them to be activated. Indeed, it has been demonstrated that tailless cell surface CD44 interacts with plastic-immobilized CD44, but not with soluble CD44 (Lesley et al., 1992). In contrast to normal spleen cells, spleens of mice stimulated in vivo by allogeneic cells (Lesley et al., 1994) or the GVH reaction (Murakami et d., 1991) contain CD44' lymphocytes that bind HA. All the cytotoxic T lymphocytes (CTL) generated in the allo-stimulated mice were included in the HA binding subpopulation (Lesley et al., 1994). It is not known what factor (cytokine, growth factor, or ECM component) is responsible for this in vivo transient emergence of splenic CD44' cells that bind HA. In addition, it is not clear if the event is related to the activation of cell surface CD44 binding function or to the expansion of a preexisting, but small, cell subpopulation that constitutively binds HA.

G. The Effect of CD44 Glycosylation on HA Binding CD44 is heavily glycosylated by both N- and 0-linked oligosaccharides. Nascent CD44 (p42) does not bind HA, but all its intermediate precursors along the glycosylation route (pS2, p58) possess this ability (Lokeshwar and Bourguignon, 1991). Although a certain level of cell surface glycosylation is probably required for CD44-dependent HA binding, the complete glycosylation pattern may interfere with the CD44-HA interaction. In B-cell populations, as mentioned before, IL-5 or LPS stimulated the appearance of CD44 bright cells that bind HA. The CD44 of the stimulated cells exhibited less N-glycosylation than did the unstimulated B cells (Hathcock et al., 1993). Removal of sialic acid by neuraminidase proved sufficient to enhance HA binding in the latter cells (Katoh et al., 1995). Furthermore, a glycosylation-defective clone of CD44' Chinese hamster ovary (CHO)cells showed consistent binding of HA, but the wild-type CHO cells displayed wide heterogeneity of this characteristic. An electronically sorted (by fluorescenceactivated cell sorter) subclone of CD44' CHO cells that do not bind HA, as well as other CD44' cell types defective in HA binding, acquired the ability to bind the ligand after treatment with tunicamycin (an inhibitor of N-glycosylation), a mixture of endoglycosidase F and N-glycosidase F (which remove N-linked sugars), or neuraminidase (which eliminates sialic acid) (Katoh et al., 1995; Lesley et al., 1995). In another set of experiments, pre-B lymphoma and fibroblast cell lines expressing inactive CD44 (which does not bind HA), inducible CD44 (which binds HA after treatment with anti-CD44 mAb), or active CD44 (that constitutively binds CD44) were obtained (the latter two by electronic sorting) and their CD44 molecular weights were determined following immunopre-

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cipitation. Inactive CD44 had a higher molecular weight than did inducible CD44, whose molecular weight was higher than that of constitutively active CD44. These differences were assigned to discrete N-glycosylation patterns for each cell type. Indeed, inactive and inducible CD44' cells (that were not activated by antibody) acquired the ability to bind HA after treatment with tunicamycin (Lesley et al., 1995). P-D-Xyloside, which prevents the linking of CS to proteins (Schwartz, 1977), also changed the HA binding status of the cells, indicating that GAGS interfere with the CD44-ligand interaction (Lesley et al., 1995). In all the previously mentioned experiments, reduced glycosylation was proved by measuring the molecular size of CD44 immunoprecipitates before and after the deglycosylation procedure (Hathcock et al., 1993; Katoh et al., 1995; Lesley et al., 1995) or by lectin binding assay (Katoh et al., 1995). Since deglycosylation affects not only the CD44 receptors but also other cell surface glycoproteins, including those that may cooperate with CD44 in HA binding, it is impossible, under such circumstances, to specify the target molecule(s) of the deglycosylation procedure. It has been shown, however, that removal of sialic acid by neuraminidase from CD44-lg fusion protein absorbed by protein A-conjugated Sepharose beads enhances HA binding to the coated beads (Katoh et al., 1995), indicating the direct interference of CD44 carbohydrate with HA binding. Geneticallyconstructed CD44-lg variants containing either V9 (CD44V9-Ig) or V8 and V9 (CD44V8,9-Ig) exon products, as well as CD44H (CD44H-Ig, which lacks the entire variable region) and CD44E-Ig (CD44V8-10-Ig), were assessed by ELISA for their ability to bind HA. A hierarchy was observed: the fewer the expressed exons, the greater the HA binding. As an increasing number of exon products provides a greater number of glycosylation sites, it was suggested that overexpression of carbohydrates disturbs the CD44-HA interaction. Indeed, the CD44E-Ig fusion protein generated in COS cells incubated with phenyl-a-GalNAc (which blocks the incorporation of 0-linked carbohydrates to protein; Kuan et al., 1989) bound HA as efficiently as did CD44H-Ig, whereas the original CD44E-Ig did not exhibit such binding. Although it is not known how CD44E 0-linked sugars interfere with HA binding, it should be noted that the exon V8-10 product has an especially high (40% for V9 and 30% for V10) content of serine and threonine; both are linkers of these carbohydrates. Hence, the binding of HA to CD44 is controlled at two levels of regulation; induction of alternative splicing and expression of glycosyltransferase enzymes (Bennett et al., 1995b). We (Rochman, Naor, and Ish-Shalom, unpublished results) have found that Fl-HA binding to CD44 of LB T-cell lymphoma is dependent on both N-deglycosylation (with tunicamycin) and PMA activation. Attempts to use either of these procedures to confer HA binding have failed. It is still not clear if quantitative, qualitative, or both kinds of changes are involved in the ac-

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quisition of HA binding by LB cells, as PMA both increases CD44 intensity and upregulates expression of variant exons. Several non-mutually exclusive mechanisms may account for the influence of cell surface sugars on CD44-dependent HA binding. Steric interference or conformational changes mediated by carbohydrates might affect the HA binding status of CD44. Sugar side chains could restrict the cell surface mobility of CD44 receptors, or cause charge repulsion (especially by negatively charged sialic acid) between neighboring CD44 molecules, thus preventing the formation of functional, multivalent aggregated receptors (see Sections V.E and V.F). In addition, positively charged amino acids of the CD44 molecule are required for interaction with negatively charged HA groups (see Section V.D). Removal of negatively charged groups (e.g., sialic acid) may increase the overall net positive charge of CD44, thus improving HA binding. Note that the glycosylation status also influences the function of other cell surface adhesion molecules such as integrins and the immunoglobulin superfamily (Diamond et al., 1991; Braesch-Andersen and Stamenkovic, 1994; Zheng et al., 1994).

VI. NON-HA LIGANDS OF CD44 Although HA is the principal ligand of CD44, it is by no means the only one. CD44 can adhere to the ECM components collagen (Carter and Wayner, 1988; Lokeshwar and Bourguignon, 1991; Faassen et al., 1992; Jalkanen and Jalkanen, 1992; Bartolazzi et al., 1994; Romaris et al., 1995), fibronectin (Jalkanen and Jalkanen, 1992; Bartolazzi et al., 1994; Romaris et al., 1995), laminin (Jalkanen and Jalkanen, 1992), and CS (Aruffo et al., 1990; Sy et al., 1991; Peach et al., 1993), albeit, as indicated in some studies, at a low affinity (Aruffo et al., 1990; Peach et al., 1993). According to one report, CD44s binds chrondroitin 4-sulfate more effectively than chondroitin 6-sulfate (Peach et al., 1993), whereas other communications maintain that CD44 molecules do not interact with CS (Miyake et al., 1990b; Murakami et al., 1990, Bartolazzi et d., 1994; Toyama-Sorimachi et d., 1995; Zahalka et al., 1995). The CD44 activation, glycosylation, or glycosaminoglycanation status (see later), or the type of CS used as ligand in the binding assay, may account for these apparent discrepancies. It should be further emphasized that, as the commercial GAG preparations are not always completely purified, nonspecific biological effects must be tightly controlled. Some investigators have reported that CD44 binding to collagen or fibronectin is associated with the CS-glycanated form of CD44 (180-200 kDa) (Faassen et al., 1992; Jalkanen and Jalkanen, 1992; Romaris et al., 1995). Support of cell motility on type I collagen may be an important function of chondroitin-sulfated CD44. In this context, it was noted that inva-

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sive melanoma cells express the CS-glycanated version of CD44 (Faassen et

al., 1992). Mucosal vascular addressin (Picker et al., 1989), serglycidgp600 (Toyama-Sorimachi and Miyasaka, 1994; Toyama-Sorimachi et al., 1995), osteopontin (Weber et al., 1996), and the major histocompatibility complex (MHC) class I1 invariant chain (Ii) (Naujokas et al., 1993) are additional, ECM-unrelated ligands of CD44. Serglycidgp600 is a small chondroitinsulfated proteoglycan stored in the intracellular secretory granules of lymphoid, myeloid, and some tumor cells (Stevenset al., 1988). The chondroitin 4-sulfate chains of serglycin/gp600 are essential to its interaction with CD44. The CD44-related binding of peripheral lymphocytes and CTLs to serglycidgp600 is dependent on their activation by agonistic antibody (see Section V.F). Furthermore, the interaction with serglycidgp600 allows CTL activated with anti-CD44 and anti-CD3 mAbs to release granzyme A (Toyama-Sorimachi et al., 1995), suggesting a physiological role for this proteoglycan. The Ii is a nonpolymorphic glycoprotein associated with class I1 MHC. This chain prevents the binding of inappropriate peptides to class I1 molecules during their early transport in APC (Newcomb and Cresswell, 1993). The small amount of chondroitin-sulfated Ii (Ii-CS) that remains associated with class 11 molecules on the APC membrane markedly enhances its ability to stimulate allogeneic and mitogenic responses in T cells. The allogenic responses can be blocked with anti-CD44 mAb and soluble CD44. Antigenpresenting cells expressing nonsulfated Ii-mutated molecules did not enhance the stimulation of T-cell responses, indicating that the linkage of Ii to CS is essential to the accessory effect of this glycoprotein (Naujokas et al., 1993). Collectively, these findings indicate that Ii-CS can function as an APC costimulatory molecule by interacting with the CD44 of a responding T cell. Osteopontin (Eta-1), a chemotactic phosphoprotein secreted by activated T cells and osteoblasts, is another novel ligand of CD44. It has been shown that the binding of soluble osteopontin to fibroblasts transfected with CD44 cDNA is blocked by anti-CD44 antibody. Additionally, the binding of the transfected cells to immobilized osteopontin is specifically inhibited by the soluble ligand. Moreover, it was found that the migration of CD44transfected fibroblasts in Boyden chambers toward osteopontin was inhibited by anti-CD44 or anti-osteopontin antibodies (Weber et al., 1996), suggesting physiological and pathological roles for the CD44-osteopontin interaction. The polymorphic nature of CD44, a result of alternative splicing as well as of differential glycosylation and glycanation, may account for the multifunctional nature of this molecule (see Section IX) and for its ability to bind many ligands. In view of the central role played by CD44 in many physiological and pathological functions (see Sections X and XI), every effort should be made to identify new ligands of this molecule. In fact, the exis-

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tence of unknown CD44 ligands has been suggested in several experimental models (Culty et al., 1990; Toyama-Sorimachiet al., 1993). For example, although Hermes-3 anti-CD44 mAb, which recognizes epitope(s)on the CD44 nonconserved membrane proximal domain (Goldstein et al., 1989),does not interfere with HA binding, it blocks the adhesion of human lymphocytes to mucosal high endothelial venules (HEV) (Culty et al., 1990). This finding suggests that CD44 interacts with a yet-unknown non-HA ligand.

WI. SOLUBLE CD44 Cell surface CD44, as well as some variant sequences introduced by alternative splicing, are vulnerable to proteolytic cleavage. Therefore, it is not surprising that released CD44 is found in cell culture supernatants, arthritic synovial fluid, and plasma. As the data up to 1993 have already been reviewed by Lesley et al. (1993a), we refer to later findings only. It was found that the serum concentration of soluble CD44 is 2 kg/ml in mouse (Katoh et al., 1994) and 5 p,g/ml in humans (Lesleyet al., 1993a). These levels increase to 3-7 pg/ml in autoimmune mouse strains and up to approximately 10 pg/ml in tumor-bearing mice. In contrast, immunodeficient mice have low concentrations of CD44 (0.5 pg/ml), indicating that immunological and malignant activities can induce shedding of cell surface CD44 (Katoh et al., 1994). Injection of anti-CD44 mAb into mice also causes the release of cell surface CD44 into the serum (Camp et al., 1993).In vitro PMA activation enhances the release of CD44 from the cell surface to the culture supernatant. This effect was abolished by protease inhibitors, suggesting that endogenous enzymes are involved in CD44 cleavage. Indeed, the molecular size of the shed CD44 is smaller than that of membrane-extracted CD44, with the difference detectable only after deglycosylation. Furthermore, an antibody specific for an epitope of the CD44 cytoplasmic tail failed to immunoprecipitate serum CD44, suggesting that soluble CD44 is tailless. The possible physiological role of soluble CD44 has been attributed to its ability to partially inhibit the binding of HA to cell surface CD44 (Katoh et al., 1994). CD44 variants (i.e., CD44V6-10, CD44V7-10, CD44V8-10) display a greater proclivity than does CD44s to be released from transfected Namalwa cells into the medium (Bartolazziet al., 1994,1995). Interestingly,coexpression of the V3 exon with the other exons limits shedding of the variant CD44. The released variant CD44 blocked binding of Namalwa cells expressing CD44s to stromal cells (Bartolazziet al., 1995),suggesting a regulatory role for the shedding process (e.g., in the detachment of potential metastatic cells from the primary tumor mass). Regardless of its physiological role, soluble CD44 could be used, like other adhesion molecules (Gearing and Newman, 1993), as a diagnostic or

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prognostic tool for evaluation of pathological disorders such as rheumatoid arthritis (Haynes et al., 1991) and malignant diseases. A case in point is patients with advanced gastric (or colon) cancer, in whom the concentration of serum CD44 is 10 times higher than normal (Guo et al., 1994b).

VIII. GENETIC CONTROL OF CD44 EXPRESSION Activated ras or src genes induce expression of CD44, as indicated by transfection of these oncogenes into fibroblasts or epithelial cells of the intestinal crypt (Hofmann et al., 1993; Jamal et al., 1994; Kogerman et al., 1996).It should be noted that c-src stimulates some signal transduction pathways that are also activated by c-ras (Qureshi et al., 1992). In the transfected fibroblasts, but not in intestinal epithelial cells, increased transcriptional levels of CD44s were accompanied by the appearance of alternatively spliced CD44 mRNAs and the production of CD44 variant proteins, suggesting that ras overexpression in these cells affects both CD44 promoter activity and the splice control machinery. Co-transfection of a c-Ha-ras-expression plasmid and CD44 promoter-chloramphenicol acetyltransferase (CAT) reporter gene construct into fibroblast cell lines resulted in CAT activity, indicating a transcriptional event. Sequence analysis of the CD44 promoter led to the identification of a binding site element for the AP-1 transcription factor. Indeed, mutations in this region prevented induction of CAT activity in the previously mentioned co-transfection experiment (Hofmann et al., 1993).These findings suggest that ras and src induction of CD44 expression is mediated by the AP-1 transcription factor via the mitogenactivated protein kinase cascade (Seger and Krebs, 1995). Activated ras pleiotropically controls several genes whose products are involved in various aspects of the malignant process, such as cell proliferation (Filmus et al., 1992,1993,1994; Zhao and Buick, 1993), and proteases and metalloproteinases (Denhardt et al., 1987; Garbisa et al., 1987). The CD44 gene, whose products are also involved in the malignant process (Section XI), can now be added to this list.

IX. CD44 FUNCTIONS A. CD44 Is a Coaccessory or Independent Receptor

Involved in Transmitting Growth Signals In addition to its adhesive function, the CD44 receptor also serves as an accessory molecule, cooperating with other molecules in the transmission of

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growth signals delivered from the cell membrane to the cell nucleus (reviewed by Lesley et al., 1993a). Several anti-CD44 mAbs (including V6- or V9-specific mAbs), or their Fab fragments, act in conjunction with immobilized anti-CD2 or anti-CD3 mAbs (usually at suboptimal levels) to enhance T-cell proliferation or IL-2 production (Galandrini et ul., 1993; Lesley et al., 1993a; Galluzzo et al., 1995; Sommer et al., 1995). Addition of anti-CD28 mAb to this assay system markedly augments these cellular activities (Huet et al., 1989; Sommer et al., 1995). The CD44-supported proliferative response is cyclosporin-A sensitive (Sommer et al., 1995). Other anti-CD44 mAbs promote T-cell proliferation of IL-2 production, even if they are the sole stimulator in the in vitro assay system (Galandrini et al., 1993; Lesley et al., 1993a). In some cases, the promoting effect is dependent on the presence of macrophages in the in vitro system (Lesley et al., 1993a; Funaro et al., 1994). On the other hand, some anti-CD44 mAbs or their Fab fragments inhibit CD3-dependent cell proliferation or IL-2 production (Guo et al., 1993, 1994a; Lesley et al., 1993a). Although several anti-CD44 mAbs suppress CD3-dependent IL-2 production or cell proliferation of T-cell hybridoma or peripheral blood lymphocytes, they enhance the corresponding CD2-dependent responses (Rothman et al., 1991; Guo et al., 1993,1994a). The same anti-CD44 mAbs that inhibit the proliferation of CD3-activated T cells induced palmitoylation of the ligated CD44, suggesting that lipid modification of this molecule interferes with signaling pathways (Guo et al., 1994a). Collectively, these findings emphasize the significance of “crosstalk” between CD44 and other cell surface molecules, leading to positive or negative growth-transducing signals. It was further demonstrated that engagement of monocyte CD44 receptor with immobilized anti-CD44 mAb induces the release of TNFa and IL-lP (Webb et al., 1990). Triggering of CTL or natural killer (NK) cell cytolytic activities with anti-CD44 mAb has also been observed (Lesleyet al., 1993a). In this context, it was reported (Seth et al., 1991; Galandrini et al., 1993) that the lytic activity of human CTL against the Fcy receptor-positive EL4 or P815 mouse cell lines is activated by anti-CD44 mAb, used to link the CD44 receptor of the effector cell to the Fcy receptor of the target cells (a procedure known as redirected killing). The anti-CD44 mAb triggers the CTL to release the trypsin-like esterase granzyme. Genestein, a protein tyrosine kinase inhibitor, blocked both the CD44-dependent proliferation and the cytotoxic activity of the effector cells (Galandrini et al., 1993).It was also found that CD44 activation of CTL causes tyrosine phosphorylation of several proteins within the 70- to 150-kDa range, but not of the 21-kDa 5 chain of the T-cell receptor. The same anti-CD44 mAbs did not trigger the cytotoxic activity of NK cells (Galandrini et al., 1993).However, it has also been reported that the CD44-dependent redirected killing of target cells with antiCD44 bispecific mAb is conditional upon IL-2 or IL-12 co-stimulation of the NK cells (Sconocchia et al., 1994).

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Whereas anti-CD44 mAb was unable to redirect the lysis of mouse P815 cells by human NK cells (Galandrini et al., 1993), the same investigators showed that the antibodies induced a rapid increase of intracellular free calcium in the effector cells and enhanced the lysis of NK-sensitive target cells (Galandrini et al., 1994a). Ligation of canine NK cell CD44 with anti-CD44 mAb augmented conjugate formation between the effector and target cells and induced the production of TNFa by the effectors. The released TNFa caused apoptosis in the target cell population (Tan et al., 1995). According to a quite surprising finding (Taher et al., 1996),ligation of CD44 with specific mAb couples this molecule to the T-cell receptor growth signal machinery. Engagement of the T cell CD44 molecule with anti-CD44 mAb induced tyrosine phosphorylation of several intracellular proteins, including ZAP-70 (Rudd et al., 1994). Furthermore, immunoprecipitation has shown that CD44 activation leads to its association with p56lCk,and, according to an in vitro kinase assay, to the simultaneous increase in ~ 5 6 tyrosine " ~ kinase activity. The coupling of ligated CD44 to p56lCkand ZAP-70, which are involved in the growth signal transduction pathway of T cells, may explain its ability to activate IL-2 production and to promote cell proliferation. It was previously demonstrated (Noble et al., 1993) that, following HA stimulation, macrophages are able to express IL-1p, TNF-a, and insulin-like growth factor-1 (IGF-1) mRNAs as well as IGF-1 protein. This activity was inhibited by anti-CD44 mAb, suggesting that CD44 engagement by HA initiates cytokine synthesis. As HA is a natural ligand of CD44, it is hardly surprising that this ligand can substitute for antLCD44 mAb in enhancing cell proliferation, IL-2 production, and granzyme release by anti-CD3 mAbactivated CD44' T cells (Guo et al., 1993; Galandrini et al., 1994b). Hyaluronate-induced IL-2 production was blocked by anti-CD44 mAb (Guo et al., 1993), implying that the CD44 receptor transmits the growth signal. It is conceivable that the widely distributed HA supports the growth of CD44' tumor cells as well. As mentioned previously, the class I1 Ii of APC also stimulates the proliferation of T cells by direct interaction with their CD44 receptor (see Section VI), thus pointing to an additional route for the delivery of growth signals to T cells via CD44. To sum up, engagement of CD44 surface molecules stimulates an increase in intracellular calcium (Bourguignon et al., 1993; Galandrini et al., 1994a)and in protein phosphorylation (Galandrini et al., 1993), with subsequent acquisition of cell effector functions such as HA binding, cell proliferation, or cytotoxicity. However, the linkage between the biochemical events and these cell activities has not been elucidated.

B. CD44 Is a Homing Receptor The homing function of CD44 was extensively explored in the late 1980s and early 1990s. The findings have been comprehensively reviewed by Les-

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ley et al. (1993a), and we confine our discussions to a brief summary. Depletion of CD44’ cells from bone marrow by anti-CD44 mAb (using a “panning” procedure) prevented their ability to reconstitute the thymus of irradiated mice (O’Neill, 1989). This shows that CD44 is a homing receptor for migrating thymus progenitor cells. In contrast, the migration of lymphocytes into the lymph nodes and into other organs was normal, despite the removal of their CD44 receptor by anti-CD44 mAb treatment (Camp et al., 1993). Traffic of mouse lymphocytes to lymph nodes and their adhesion to HEV, as indicated by examination of frozen lymph node sections, was blocked, however, by Mel-14- or LFA-l-specific mAbs (Gallatin et al., 1983; Hamann et al., 1988, 1991; Camp et al., 1993). These findings indicate that the HEVrecognizing receptor of the mouse lymphocyte is selectin or integrin, rather than CD44. Unlike in the mouse, anti-CD44 mAb (e.g., Hermes-1) inhibits binding of human lymphocytes to lymph node HEV, suggesting that CD44 is the homing receptor of human cells (Jalkanen et al., 1987; Pals et al., 1989a). Sheep memory T cells, which express high levels of CD44 but not of Mel14, enter the lymph nodes via the afferent lymphatics, using the bloodtissue axis. On the other hand, naive T cells, which express low levels of CD44 but high levels of MEL-14, penetrate the nodes from the blood circulation via the HEV (Mackay et al., 1990). Similarly, subcutaneously inoculated CD44’ Mel-14- T-cell lymphoma invades the lymph node through the afferent lymphatics (Zahalka et al., 1995). The possibility that Mel-14 is the key to entry via the HEV gate, whereas CD44 is the “password” for passage through the afferent lymphatics, should be substantiated by further experiments.

C. Cell Binding to Endothelium or ECM via the CD44 Receptor

Whereas lymph node homing of lymphocytes via CD44-dependent binding to HEV has been observed in humans but not in mice, the ability of lymphocytes or lymphoma cells to adhere to endothelial cells, in a CD44dependent manner, has been demonstrated in both species (OppenheimerMarks et al., 1990; Lesley et al., 1992). According to one report (ToyamaSorimachi et al., 1993), the binding of lymphocytes to endothelial cells is conditional upon activation of the lymphocytes by anti-CD44 mAb. Another communication showed that the CD44-dependent lymphocyte-ndothelial cell interaction occurred only if the lymphocytes were activated by both phorbol dibutyrate and ionomycin, and the endothelial cells were stimulated by IL-1 (Oppenheimer-Marks et al., 1990). This finding suggests that inflamed endothelium can activate the CD44-dependent binding of lympho-

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cytes. The interaction between cell surface CD44 and endothelium may be vital to the hematogeneic spread of tumors. In addition to CD44, cell surface selectins and integrins also are involved in the adherence of leukocytes to endothelial cells, an event that is essential to intra- and extravasation, as well as to cell traffic along the blood vessels (Springer, 1994). It is likely that expression of CD44 by leukocytes is also requisite for events following endothelial adhesion, including penetration into the ECM of lymphoid tissues and subsequent locomotion inside the matrix. It has been suggested that cell movement in the ECM is guided by local chemotactic signals (Dustin and Springer, 1991),while “stop signals” halt the movement of cells, anchoring them to ECM components (Knudson and Knudson, 1993). Cell surface adhesion molecules, especially p l integrins (Hemler, 1990; Yamada, 1991) and HA binding proteins (including CD44) (Knudson and Knudson, 1993), are involved in cell adherence to ECM. However, HA receptors, including CD44, are used not only to anchor cells (such as chondrocytes, fibroblasts, epithelial cells, and hematopoietic cells) to the ECM, but also to facilitate the assembly and organization of the pericellular matrix (Knudson and Knudson, 1993; Knudson et al., 1993). In vitro experiments have revealed that chondrocytes (Knudson, 1993; Knudson and Knudson, 1993) and synovial cells (Mikecz et al., 1995) exploit the hyaluronan receptor to bind HA, which, in turn, interacts with cartilage proteoglycan (aggrecan) to form the pericellular matrix. This HA-mediated cell-proteoglycan interaction might be essential to the organization and retention of aggrecan molecules within the cartilage ECM, and it could also protect tumor cells from immune surveillance.

D. Cell Surface CD44 Involvement in HA Internalization and Enzymatic Degradation The CD44 cell surface receptor participates in the uptake and intracellular degradation of HA. The production of HA in human lung fibroblasts, induced by a combination of TNF and IL-1, is regulated by its cleavage by lysosoma1 hyaluronidase. Cytokine-induced HA degradation has been found to be correlated with enhanced mRNA expression of fibroblast CD44 and increased binding of HA to its cell surface, suggesting, but not proving, that the intracellular degradation of HA is mediated by the adherence of hyaluronate to the CD44 receptor (Sampson et al., 1992). Unfortunately, it ha5 not been reported whether anti-CD44 mAb can block HA binding and degradation by the lung fibroblasts. According to a more recent communication (Hua et al., 1993), bovine chondrocytes internalize and degrade HA following its binding to the CD44 receptor, as indicated by the ability of antiCD44 mAb and HA hexasaccharides to inhibit both cell surface adherence

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and endocytosis of HA. Cell surface CD44 may thus be involved in the catabolism of HA, enabling its removal from ECM. Like bovine chondrocytes, transformed mouse fibroblasts (SV-3T3),as well as human and hamster alveolar macrophages, also degrade labeled HA following incubation with this ligand (Culty et al., 1992, 1994). Degradation of HA is inhibited by antiCD44 mAb and soluble HA. The CD44 receptor itself does not possess any HA-degrading activity. In addition, cleavage of HA by the macrophages can be blocked by chloroquine and NH,CI, which inhibit lysosomal acid hydrolases, thus pointing to intracellular degradation. Subsequent experiments have revealed that fluorescein-taggedHA is internalized by macrophages and that anti-CD44 mAb blocks this process (Culty et al., 1992), implying the direct involvement of the CD44 receptor in the internalization of HA. Digestion of HA, following its binding to the CD44 receptor, may facilitate locomotion of mobile cells, such as macrophages and invasive tumor cells.

E. CD44eDependent Cell Traffic Cell migration is prominent in areas rich in HA, such as the embryonal limb bud (Toole, 1981, Knudson and Toole, 1988), the adventitia surrounding the blood vessels (Underhill, 1989; Aruffo et d., 1990), wounds undergoing repair (Weigel et al., 1989; Oksala et al., 1995), sites of inflammation (Weigel et al., 1989; Hallgren et al., 1990), and tissues invaded by tumors (Toole et al., 1979; Knudson et al., 1989; Sy et al., 1991).As HA creates a microenvironment with a low resistance to cell traffic (see Section V.A), CD44, the HA receptor, may play an important role in cell motility, a crucial factor in formation of metastases. Several models have been used to prove the role of CD44 in cell migration. It has been shown that 4 weeks are required for complete T-cell recovery of mouse blood, lymph nodes, and spleen subjected to severe T-cell depletion by intravenous injection of anti-Thy-1 antibody. T-cell reconstitution of these organs was markedly delayed when soluble forms of CD44 or Lselectin were injected into the anti-Thy-1 antibody-treated mice (Guo et al., 1994d). In contrast, isotope-labeled lymphocytes, stripped of their CD44 molecules by anti-CD44 mAb treatment, infiltrated normally into mouse lymphoid and nonlymphoid organs following intravenous injection, as indicated by the accumulated organ radioactivity at 1 and 18 h (Camp et al., 1993). Collectively, these results show that the utilization of CAM in longterm lymphoid organ repopulation is substantially different from that in short-term infiltration to the same organs. Whereas long-term cell migration is CD44 and L-selectin dependent, short-term cell migration exploits the LFA-1 integrin but not CD44 (Camp et al., 1993). In another model, injection of anti-CD44 mAb into 2,4-dinitro-l-

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fluorobenzene (DNFB)-sensitizedmice caused the release of CD44 from the cell surfaces of lymphocytes, yet they followed a normal pattern of infiltration into the regional lymph nodes after DNFB challenge. However, the delayedtype hypersensitivity (DTH) of the anti-CD44 mAb-injected mice was retarded by 24 h beyond that in a corresponding group of mice injected with rat IgG (Camp et al., 1993). The injected antibodies may interfere with leukocyte extravasation, cell migration inside the local tissue, cytokine release from infiltrating and local cells, or combinations of these events. Injection of mice with anti-CD44 mAb before the hapten sensitization phase, rather than before challenge, also reduced the 24-h postchallenge cutaneous inflammatory DTH response (Verdrengh et al., 1995). These findings indicate that circulating cell migration in normal mice and mice undergoing a DTH response is not influenced by the loss of CD44, although local DTH activity is substantially delayed. The dependence of cell motility on cell surface CD44 was directly proved by an in vitro “wound” assay. Wounds of approximately 5 mm were made with a plastic scraper in subconfluent monolayers of CD44-“high” and CD44-“low” human melanoma cell clones. Of the two clone types, only the CD44-“high” melanoma cells substantially migrated to the “wounded” space, and cell motility was inhibited by anti-CD44 mAb (Birch et al., 1991). Two modalities used to facilitate cell movement along substrates have been described in in vitro studies. The first, occurring under shear stress flow, is tethering and rolling, which is operative on vascular components. Such cell motility exploits selectins and addressins (Springer, 1994; Lawrence et a/., 1995; Luscinskas et al., 1995), as well as (perhaps to a lesser extent) integrins and integrin ligands (Berlin et al., 1995; Luscinskas et al., 1995), as receptors and substrates for rolling, mediated by an adhesion-de-adhesion process. Integrin ligands are used to halt cell motility and to allow transendothelial migration (Springer, 1994; Lawrence et al., 1995; Luscinskas et al., 1995). The second modality, which takes place in the absence of shear stress flow, is cell “crawling” on ECM components such as collagen (Parkhurst and Saltzman, 1992). The CD44-HA interaction also supports cell traffic under shear stress flow or on ECM components in the absence of flow. Cell movement was analyzed by time-lapse videomicroscopy. Using a parallel plate flow chamber designed to approximate postcapillary physiological flow, DeGrendele and colleagues (1996) described CD44-dependent lymphoid cell rolling on cultured endothelial cells or, directly, on immobilized HA. By adding anti-CD44 mAb, soluble HA, or hyaluronidase to block cell rolling, it was possible to show that lymphoid cell traffic on endothelial cells or HA is dependent on the CD44-HA interaction. Some B- and T-cell lines, as well as normal lymphocytes, acquired the CD44-dependent rolling capacity only after activation with phorbol ester and ionomycin.

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Cell motility in the absence of shear stress flow on immobilized HA or HA incorporated within collagen matrix has also been described. The movement is randomly directed and the speed is much slower (0.5-10 p d m i n ) (Thomas et al., 1992; Fried1 et al., 1995) than that described for locomotion under shear flow (-1200 p d m i n ) (DeGrendele et al., 1996). Human melanoma cells stably transfected with CD44H, but not with CD44E (Thomas et al., 1992), displayed enhanced motility on a HA-coated substrate, as did human melanoma cell lines (Thomas et al., 1993). Cell migration was blocked by CD44-lg fusion protein, anti-CD44 mAb, or soluble HA (but not CS). Melanoma transfectants expressing tailless CD44 did not exhibit enhanced cell motility on HA, yet appreciable binding to this ligand was observed (Thomas et al., 1992). This finding suggests that the association of CD44 with the cytoskeleton is essential to cell movement but not to cell attachment to the matrix component. The intriguing possibility that the cell's transition from a mobile to a sessile state is dependent on the dissociation of CD44 from the cytoskeleton (Thomas et al., 1992)merits further research. A mouse melanoma cell line expressing CD44-related chondroitinsulfated proteoglycan displayed cell motility on type I collagen, as indicated by a Boyden chamber assay. Coupling of CS to core protein, and cell locomotion, were inhibited by P-D-xyloside. The same effect was obtained after treatment with chondroitinase (Faassen et al., 1992). Transforming growth factor-pl enhanced both CS expression and cell migration (Faassen et al., 1993). These findings suggest that the CS-modified version of CD44 plays a role in cell locomotion on type I collagen and that the CS is a critical component in mediating this effect. CD44-dependent tumor cell motility is likely an important factor in at least some metastatic processes.

F. CD44cDependent Cell Aggregation The phenomenon of CD44-dependent cell aggregation has been described in several communications. Spontaneous homophilic aggregation, prevented by the addition of anti-CD44 mAb, was observed in cell suspensions of human melanoma cells that intensively express CD44 (Birch et al., 1991). Whereas the previously mentioned aggregates were formed in the absence of HA, the homophilic aggregation of several CD44' mouse cell lines (e.g., BW5147) is dependent upon the presence of this ligand, which presumably bridges the CD44-expression cells. Again, anti-CD44 mAb blocked aggregate formation (Lesleyet al., 1990a). Addition of anti-CD44 mAb to cell suspensions of a human monocytic cell line (Guo and Hildreth, 1993), Molt4 cells (Murakami et al., 1994), or other hematopoietic cell lines (Cao et al., 1995) caused homophilic aggregate formation, possibly by linking the CD44 receptors of neighboring cells or by activating other adhesion molecules (see

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later). Cytochalasin B blocked this effect (Murakami et af., 1994), suggesting cytoskeletal involvement in aggregate formation (Guo and Hildreth, 1993). In other cases, anti-CD44 mAb activated homophilic T-cell aggregation, which is LFA-MCAM dependent, as antibodies against the integrin or its ligand disturb aggregate formation (Koopman et af., 1990; Rodrigues et al., 1992; Funaro et af., 1994; Vermot-Desroches et af., 1995). The finding that CD44-induced LFA-l-dependent cell aggregation was prevented by inhibitors of PKC (AMG, H7) or microfilament formation (cytochalasin B) (Koopman et af., 1990) suggests that the effect is dependent on protein kinase activity and cytoskeleton integrity. It was further shown that human immunodeficiency virus (HIV)infection of a human monocyte cell line induced downregulation of cell surface CD44- and CD18- (p chain of p2 integrins) dependent aggregate formation (Guo and Hildreth, 1993). The suggestion that CD44 controls the integrin function in this model awaits experimental evidence. We have described spontaneous cell aggregation between T cells and tumor cells in spleen cell suspensions derived from BALBk mice subcutaneously inoculated with LB T-cell lymphoma (Zahalka et af., 1993; Zahalka and Naor, 1994). Intravenous injection of anti-CD18 mAb into LB cell-inoculated mice inhibited both the proliferation of spleen-infiltrating LB cells and tumor cell-splenic T cell aggregate formation, suggesting that the interaction between the two cell types supports local tumor growth. Both events were markedly restored when anti-CD44 mAb was coinjected with anti-CD18 mAb, raising the possibility that CD44 engagement upregulates integrin activity, rendering it less sensitive to the blocking effect of anti-CD18 mAb. Homophilic or heterophilic CD44-dependent aggregation of metastatic cells may protect them from immune surveillance and enhance their entrapment in target organs (see Section I).

G. CD44 Influence on Hematopoiesis and Apoptosis The genesis and death of at least some hematopoietic cells may be influenced by signals transmitted through their CD44 receptor. Administration of anti-CD44 mAb to a mouse Dexter-type long-term bone marrow culture (LTBMC) prevented myelopoiesis of neutrophil granulocytes and macrophages. The production of B-lineage lymphocytes in Whitlock and White LTBMC was also blocked by anti-CD44 mAb. Both cultures were productive after the addition of mAbs against MEL-14 or LFA-1 (Miyake et d., 1990a).Although this study does not provide any direct evidence, it is tempting to speculate that bone marrow stromal cells participate in the induction of the CD44-dependent hematopoiesis process. This supposition is based on

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the finding that stromal cells support in vitro lymphocyte proliferation (Pietrangeli et al., 1988), proving their ability to influence the growth of other cells. In addition, their interaction with. B-cell hybridoma is inhibited by the introduction of anti-CD44 mAb or soluble HA (Miyake et al., 1990a,b), indicating their potential capacity to deliver CD44-mediated signals to other cells. Another experiment (Hamann et al., 1995) showed that immobilized HA augments the proliferation and differentiation of CD34' umbilical cord blood cell progenitors, resulting in fully matured eosinophils. The fact that anti-CD44 mAbs reduced the ligand-induced eosinopoiesis to control levels shows that signals delivered by HA and transduced by the CD44 receptor of the precursor cell are involved in the augmented proliferation and differentiation of the cells. These in vitro models of hematopoiesis may not, however, fully reflect the in vivo process of cell differentiation, as additional hematopoietic signals can be delivered by cells or matrix components not represented in the in vitro assays. Apoptosis (programmed cell death) induced in a mouse T-cell hybridoma cell line by engagement of the T-cell receptor with anti-CD3 mAb, or by dexamethasone treatment, was abolished in the presence of anti-CD44 mAb or immobilized HA (Ayroldi et al., 1995). In contrast, p53-dependent programmed cell death, induced by ultraviolet irradiation, was resistant to the antiapoptotic influence of antibody-activated CD44. Unlike IL-3, activated CD44 did not affect the mRNA expression of bcl-2 (Ayroldi et al., 1995), whose oncoprotein product inhibits apoptosis (Nuiiez and Clarke, 1994). The possibility that the antiapoptotic effect of activated CD44 supports the malignant growth of some transformed cells should be investigated.

H. Cytokine and Growth Factor Presentation by CD44 Cytokines, chemokines, and growth factors may be best presented to traveling leukocytes if anchored to the substratum, thus preventing them from being swept away, as by the bloodstream (Tanaka et al., 1993). Proteoglycans, including CD44, decorated with HS perhaps best satisfy this presenting function, as HS is often used to bind growth factors (reviewed by Bennett et al., 1995a).In accordance with this concept, it has been demonstrated (Tanaka et al., 1993)that the presentation of macrophage inflammatory protein-1 p (MIP-1p) by CD44 immobilized onto plastic markedly enhances the VLA-4-dependent binding of CD8 T cells to immobilized VCAM-1, a ligand of VLA-4 (MIP-1P is both a chemoattractant and cell adhesion inducer). The increased binding was blocked by anti-VLA-4 and VCAM-1 mAbs, as well as by HS proteoglycan and heparin, indicating that sulfated heparin links the CD44 to the chemokine, which, in turn, activates T cells, causing them to adhere to VCAM-1 via their VLA-4 receptor. As MIP-1p is present on lymph node endothelium (Tanaka et al., 1993), the investigators suggested that the

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same mechanism activates passing leukocytes, causing them to bind to the endothelium of inflamed blood vessels. It has also been reported (Bennett et al., 1995a; Jackson, D. G., et al., 1995)that CD44 isoforms containing the V3 exon product are decorated by HS. Radioblotting and ELISA revealed that these CD44 variants bind via their HS bFGF and heparin-binding epidermal growth factor. The researchers advanced the suggestion that one of the functions of V3-expressing CD44 variants is to present HS-binding growth factors to neighboring cells. In line with this concept, they found that the HS-decorated CD44 of keratinocytes (which contains the V3 product), can bind bFGF (Bennett et al., 1995a),which, in turn, may deliver activation signals to cells in inflamed skin. A similar CD44-dependent growth factor-presenting mechanism could account for growth stimulation of tumor cells.

X. INVOLVEMENT OF CD44 IN PHYSIOLOGICAL AND PATHOLOGICAL CELL ACTIVITIES A. T h e Role of CD44 in Wound Healing Wound healing involves several biological functions, such as cell migration, cell proliferation, basement membrane regeneration, and granulation tissue formation. Histochemical analysis of human mucosal would healing has revealed that CD44 and HA are localized during all tissue repair stages in the same region of epithelium, especially around keratinocytes. Whereas in 1-day-old wounds the first migrating epithelial cells are weakly CD44 positive, in 3-day-old wounds they are clearly CD44 positive. The newly formed granulation tissue strongly expresses CD44 and HA. It was inferred from these observations that the keratinocyte CD44-matrix HA interaction plays an important role in wound reepithelization (Oksala et al., 1995). It is probably not just a coincidence that the processes of wound healing, tissue remodeling, embryogenesis, and carcinogenesis share the combination of intensive cell migration and proliferation with high CD44 and HA localization, as the CD44-HA interaction influences cell motility and growth.

B. Endometrial CD44 Expression during

the Human Menstrual Cycle Immunohistochemical analysis of endometrium samples during the various phases of the menstrual cycle revealed that CD44s and, to a certain extent, its V6-containing variant are expressed in the mid and late secretory phases, but not in the proliferative phase, early secretory phase, or atrophic

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phase. It has been speculated that sex steroids influence endometrial CD44 expression, supporting adhesion between the endometrium and ovum during the mid and late secretory phases (Albers et al., 1995; Yaegashi et al., 1995).

C. CD44+ Cytolytic T Cells Protect against Malaria Infection The involvement of CD44' CTL in protection against malaria has been described by Rodrigues and colleagues (1992). Protective and nonprotective CTL clones were isolated from mice immunized with malaria parasite protein. Only the protective clones efficiently inhibited, upon adoptive transfer, the liver stage parasitemia of infected mice, as indicated by a plasmodia1 rRNA detection assay. An in vitro comparison between protective and nonprotective cell clones revealed that the distinct antiplasmodial activity of the former was not associated with a greater cytolytic response or more efficient cytokine release. However, T-cell protective clones expressed CD44 more intensively than did T-cell nonprotective clones, and they also more efficiently formed conjugates with parasitized hepatocytes following their adoptive transfer to infected mice. Following cell transfer, electronically sorted CTL expressing low levels of CD44 did not prevent liver parasitemia, whereas, under similar circumstances, the sorted high-CD44 expressors exhibited efficient antiplasmodial activity. This led to the assumption that the ability of protective CTL clones to inhibit intrahepatocytic stages of malaria is associated with their efficient CD44-dependent migration along the liver parenchyma.

D. CD44 in Rheumatoid Arthritis and Inhibition of Experimental Arthritis with AnticCD44 mAb There have been several reports of enhanced CD44 expression on rheumatoid synovium cells (Haynes et al., 1991; Takahashi et al., 1992; Johnson et al., 1993; Kelleher et al., 1995). In one of them it was suggested that cell surface CD44 of activated or memory lymphocytes is used for homing to the synovium (Kelleher et al., 1995). However, the alternative possibility that CD44 is upregulated only after the lymphocytes lodge in the synovium cannot be ruled out. It has also been claimed (Henderson et al., 1994)that CD44 expression in arthritic synovium is lower than in normal synovium. Technical reasons (e.g., the use of different anti-CD44 mAbs or variations in methodological approaches) could account for this discrepancy. Rheumatic joints also contain high levels of extractable HA (Pitsillideset al., 1994).The augmented expression of CD44 in joints of patients with rheumatoid arthri-

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tis suggests the active involvement of this cell surface molecule in the disease process. This concept has been challenged in animal models of arthritis. Joint swelling, edema, and leukocyte infiltration, which are the hallmarks of proteoglycan or collagen-induced mouse arthritis, were markedly reduced after injection with anti-CD44 mAb and, to a lesser extent, anti-CD18 mAb (Mikecz et al., 1995; Walmsley, Feldmann, and Naor, unpublished observations). The CD44-specific antibodies induced the shedding of CD44 from both leukocytes and synovial cells, resulting in the accumulation of this molecule in the serum. The investigators suggested that the double-edged antiinflammatory effect of anti-CD44 mAb treatment lies in the latter’s action on both CD44-expressing connective tissue cells and leukocytes (Mikecz et al., 1995). In another study of collagen-induced arthritis, the antirheumatic effect of anti-CD44 mAb was attributed to the high level of IFNy detected in the treated mice (Verdrengh et al., 1995). This finding should encourage an evaluation of the anti-CD44 mAb effect in other T-cell-mediated autoimmune experimental diseases. This suggestion seems feasible, in view of the strong CD44 expression observed following immunophosphatase staining on glial cells surrounding inflammatory lesions in the spinal cord white matter of experimental allergic encephalomyelitis (Haegel et al., 1993), a frequently used animal model of multiple sclerosis.

E. CD44 and the Immunodeficiency Virus CD44 involvement in immunodeficiency virus infections merits serious consideration, due to several observations: the selective infection and depletion of CD44’ memory cells in monkeys by simian immunodeficiency virus (Gallatin et al., 1989; Willerford et al., 1990), suggesting that they serve as a reservoir for the virus; the ability of anti-CD44 mAb to inhibit HIV infection of monocytes and peritoneal macrophages, which may indicate that CD44 serves as a receptor for the virus (Rivadeneira et al., 1995; Levesque and Haynes, 1996); and, finally, the ability of HIV to “steal” functional CD44 from its host cell and present it independently to other cells (Guo and Hildreth, 1995), possibly affording an additional mechanism of infection.

XI. CD44 ASSOCIATION WITH THE MALIGNANT PROCESS IN EXPERIMENTAL MODELS The malignant process resembles, in certain aspects, normal physiological events such as embryo development, wound healing, and routine tissue replacement, all highly dependent on cell growth and cell migration. Another

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hallmark of these physiological functions is the accumulation of HA and CD44 at sites of cell-cell or cell-matrix interactions. Therefore, it is hardly surprising that cell surface CD44 plays an important role in the carcinogenic process, including metastasis formation. We present evidence for CD44 involvement in the malignant cascade in animal models and describe clinical data supporting or refuting this concept. The therapeutic and diagnostic implications of the findings are also discussed.

A. Experimental Evidence for CD44 Involvement

in Malignant Processes Electronic sorting of human and mouse melanoma cells into CD44-high or CD44-low (Birch et al., 1991) and HA-high or HA-low (Zhang et al., 1995) phenotypes was performed in order to verify CD44 and HA involvement in tumor dissemination. Whereas local growth of the human tumor in BALBk nude mice and the mouse tumor in C57BIJ6 mice was not influenced by the phenotype of the sorted cells, CD44-high or HA-high melanoma cells formed considerably more metastatic lung nodules following subcutaneous or intravenous injection than did the corresponding low phenotypes. Furthermore, a human melanoma cell line that was established from an invaded lymph node and expressed high levels of CD44s, which binds HA, was able to form local tumors and metastases in lung and other organs following subcutaneous injection into immunodeficient mice. In contrast, a melanoma cell line established from the primary tumor of the same patient did not express CD44 and failed to form metastases, although it generated . findings suggest, but do not prove, a local tumor (Guo et al., 1 9 9 4 ~ )These that the formation of melanoma metastases is influenced by cell surface expression of CD44 or HA. The conferment of metastatic behavior on a nonmetastatic tumor by transfection of CD44 cDNA isolated from the metastatic phenotype is perhaps the most convincing proof of the malignant potential of CD44. Giinthert, Zoller, Herrlich, and colleagues (Giinthert et al., 1991; Herrlich et aL, 1993; Rudy et al., 1993) generated mAb against a metastatic rat pancreatic adenocarcinoma cell subline that interacted with the metastatic phenotype but not with its nonmetastatic counterpart. The antibody was used to screen a bacterial cDNA expression library of the metastatic tumor and a cDNA clone, ph4eta-1 (CD44V4-7), was isolated. Another cDNA clone, pMeta-2 (CD44V6,7), was reconstructed from PCR products. Upon transfection, pMeta-1 or pMeta-2 (see Fig. lC), linked to simian virus 40 or to the cytomegalovirus promoter, conferred metastatic potential on the nonmetastatic rat pancreatic tumor and on other, but not all (Sleemanet al., 1996), nonmetastatic tumor cell lines, as indicated by the transfectants’ ability to invade

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rat lymph nodes and lung following intrafootpad injection. Corresponding tumor cells transfected with CD44s did not display such an aggressive phenotype (Rudy et al., 1993). The investigators (Gunthert et al., 1991; Herrlich et al., 1993) suggested that the nonmetastatic phenotype retains all the metastatic properties but lacks CD44, and that completion of the missing genetic information by cDNA transfection fully restores the metastatic potential of the tumor. An alternative explanation is that variant CD44 gene is a pleiotropic entity that affects several metastatic properties. Because the acquisition of CD44 isoforms containing the V6 exon product has been associated with both the metastatic status of pancreatic tumor cells and the lymphocyte activation state (Arch et al., 1992; Moll et al., 1996), it was speculated that the neoplastic cells exploit a normal physiological function (e.g., lymph node homing) that is highly dependent on CD44v expression. Whereas the metastatic rat pancreatic tumor predominantly expresses CD44 variants, the corresponding nonmetastatic cells express CD44s only (Rudy et al., 1993), suggesting that the standard isoform is not involved in the dissemination process. Seemingly conflicting observations were reported for human melanoma (Bartolazzi et al., 1994) and BL (Namalwa) (Sy et al., 1991; Bartolazzi et al., 1995) cells transfected with CD44s or CD44E cDNAs. Melanoma cells expressing CD44s impressively displayed accelerated local tumor growth in immunodeficient mice when compared with cells expressing CD44E (Bartolazzi et al., 1994). Similarly, Namalwa cells expressing CD44s showed more efficient local tumor growth and metastatic spread in immunodeficient mice than did parental cells that did not express CD44. Insertion of CD44E (CD44V8-10) or other CD44 variants (e.g., CD44V6-10) into Namalwa cells further reduced their ability to develop in the animals (Sy et al., 1991; Bartolazzi et al., 199.5).Interestingly, Namalwa cells transfected with CD44 cDNA containing the V3 exon in combination with other variant exons infiltrated the bone marrow following intravenous injection more rapidly than did Namalwa cells expressing CD44 isoforms lacking the V3 exon product. The investigators raised the possibility that CD44 variants expressing the V3 exon acquire the ability to recognize novel ligand(s), thereby facilitating homing to the bone marrow (Bartolazzi et al., 199.5).Another BL cell line (P7), which expressed EBV-induced nuclear antigen-1, but neither latent membrane protein-1 (LMP-1) nor CD44, was explored for LMP-1 involvement in CD44 induction and tumor spread. The original tumor developed locally, without further dissemination, after subcutaneous injection into immunodeficient mice, in analogy to the local development of BL in patients. After transfection of LMP-1 cDNA, P7 cells expressed CD44s and acquired the ability to migrate into the thymus and axillary lymph nodes of immunodeficient mice. P7 cells transfected with CD44 cDNA, rather than with LMP-1, cDNA, also exhibited the disseminating phenotype (Walter et al., 199.5),suggesting that the pleiotropic LMP-

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1 gene induced the expression of CD44s, which, in turn, facilitated lymphoma infiltration into lymphoid organs. Melanoma or lymphoma cells expressing CD44 variants, or CD44 mutants that do not bind HA, fail to grow efficiently in immunodeficient mice, whereas cells expressing standard CD44 (wild-type or with truncated tail), which binds HA, are able to develop in the animals (Sy et al., 1991; Bartolazzi et al., 1994, 1995; Walter et al., 1995). This correlation suggests that the CD44s-HA interaction facilitates the local growth of the tumor and its subsequent migration to distal organs. However, this supposition cannot be extended to another neoplastic cell line, a pancreatic CD44v-expressing tumor that, upon transfection with hyaluronidase cDNA, lost its ability to bind HA but not to maintain local tumor growth and lung metastasis. The investigators (Sleemanet al., 1996)surmised that the tumor’s progression was dependent on the interaction between cell surface CD44v and an unknown non-HA ligand.

B. Prevention of Primary Tumor Growth and/or Metastatic Spread in Experimental Models by Reagents Interfering with the CD44-Ligand Interaction Gene transfer experiments have revealed that the local growth and/or metastatic spread of some tumors is dependent to a large extent on their cell surface CD44, possibly through its interaction with HA or other CD44 ligands expressed on endothelium or matrix. If so, it should be possible to prevent tumor growth and/or dissemination by reagents interrupting the CD44-ligand interaction. In line with this concept, it was found that mAb recognizing the V6-encoded epitope of CD44V4-7 (pMeta-1; see Fig. 1C) retarded the killing of rats by metastatic pancreatic carcinoma, and prevented the death of most animals inoculated with pMeta-1 tumor transfectants (see Section X1.A). The anti-variant antibody inhibited metastatic growth in peripheral lymph nodes and lung, but not the metastatic cells’ ability to migrate to the draining lymph nodes (Herrlich et al., 1993; Seiter et al., 1993). It has been shown that local growth and metastatic spread (especially into lung) of a human melanoma cell line subcutaneously inoculated into immunodeficient mice can be inhibited by simultaneous intravenous injection of mAb directed against the CD44 constant region. Even when injection of antibody was delayed by 7 days, metastases did not develop, de. injection of anti-CD44 spite local tumor growth (Guo et al., 1 9 9 4 ~ )Local mAb and its Fab fragments inhibited lymph node, but not spleen, invasion of LB T-cell lymphoma subcutaneously inoculated into BALB/c mice (Zahalka and Naor, 1994; Zahalka et al., 1995). Intravenous injection of mAb directed against the integrin pz chain (CD18) blocked spleen, rather than

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lymph node, infiltration by LB cells (Zahalka et al., 1993, 1995), indicating that spleen and lymph node metastases are guided by different CAMS (CD18 and CD44, respectively). Soluble CD44 (CD44-lg fusion protein) may compete with cell surface CD44 for ligand binding and thereby interfere with tumor growth. In line with this notion, it has been found that continuous infusion of standard CD44-Ig through an osmotic pump prevents tumor development following subcutaneous injection of B16 melanoma cells into mice. A soluble CD44 mutant that failed to bind HA was ineffective in this respect (Bartolazzi et al., 1994). Similarly, when preincubated with soluble CD44s (CD44-Ig), human Namalwa cells transfected with CD44s are unable to grow in immunodeficient mice (Sy et al., 1992). In summing up these findings, we see that reagents antagonizing CD44v interfere with pancreatic tumor growth, whereas those antagonizing CD44s interfere with lymphoma and melanoma development. Hence, different tumors use distinct CD44 isoforms, and perhaps discrete ligands, for primary growth and dissemination.

XII. CD44 EXPRESSION IN HUMAN NEOPLASMS AND ITS CORRELATION WITH THE MALIGNANT STATUS Gene transfer and tumor prevention experiments in animal models have shown the involvement of CD44 isoforms in the development of at least some types of neoplasms. Obviously, the question of CD44 involvement in human malignancy has been addressed in conjunction with its ability to serve as a potential target for therapy as well as a predictive or diagnostic marker. The finding that enhanced CD44 expression is confined to malignant tissues, but not to corresponding normal tissues, as well as the correlation between CD44 expression and the degree of malignancy, would suggest the involvement of this molecule in human neoplastic diseases. The major findings regarding this issue are discussed in reference to specific diseases. For the most part, tumor cell lines have been excluded from the discussion because such cells tend to change their CD44 repertoire and function during culture (see, e.g., Kuppner et al., 1992; Culty et al., 1994; Haramaki et al., 1995; Levesque and Haynes, 1996). Furthermore, variations in culture conditions may produce contradictory results.

A. Tumors of the Nervous System Immunohistochemical analysis of normal brain sections has shown that CD44 is localized mainly in the white matter, whereas less staining is detected

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in the deep gray structure. In the white matter, CD44 is most prominent among perivascular astrocytes, whereas normal cortical glia are negative (Flanagan et al., 1989; Picker et al., 1989). The CD44 positivity of normal astrocytes and its ability to bind HA was confirmed by later studies (Asher and Bignami, 1992; Moretto et al., 1993) in which RT-PCR was used to prove its hematopoietic (standard)type (Nagasaka et al., 1995). Aside from some reservations (see later), it appears that brain tumors support the concept that CD44 is involved in human neoplastic processes. Gliomas, the most frequent human primary brain tumors, express CD44 displaying an HA binding capacity (Girgrah et al., 1991a; Asher and Bignami, 1992; Kuppner eta]., 1992; Radotra et al., 1994)and the ability to transmit migration signals following in vitro interaction with this ligand (Koochekpour et al., 1995). It appears that aggressive tumor types (glioblastomas, grade I11 astrocytomas) express CD44 more intensively than do less aggressive tumors (grade I1 astrocytoma-oligodendrogliomas) and that gliomas, in general, exhibit greater CD44 expression as compared with normal brain or other brain tumors of the central nervous system, such as meningiomas and medulloblastomas (Kuppner et al., 1992). Anti-CD44 mAbs have already been used for radioimaging of human malignant gliomas (Okada et al., 1994; Romeijn et al., 1994). Northern blot analysis and RT-PCR have shown that gliomas express the standard form of CD44 (Kuppner et al., 1992; Li et al., 1993; Nagasaka et al., 1995). In contrast to gliomas, human schwannomas (derived from Schwann cells, the major supportive cell population in the peripheral nervous system) also express CD44 variants, especially those containing epitopes encoded by exon V5, the border between V7 and V8 (V7N8), and V9,lO (Sherman et al., 1995). Notably, in neuroblastomas CD44 expression correlates with favorable prognosis or progression-free survival, whereas the presence of the MYCN proto-oncogene bodes an unfavorable outcome (Christiansen et al., 1995; Combaret et al., 1995, 1996; Gross et al., 1995; Terpe et al., 1995). These findings suggest that the absence, rather than the presence, of CD44 is associated with the aggressiveness of this tumor. Whereas local brain tumors exclusively express standard CD44, most of the analyzed brain metastases of tumors originating in other organs display CD44 variants as well (Li et al., 1995).A correlation has been found between CD44v expression and the metastatic ability of such tumors (Li et al., 1993). Exclusive CD44v expression by brain metastatic cells was detected in two independent laboratories by different assays (IHC, Northern blot, and RT-PCR). CD44 variants have been detected in brain metastases of squamous cell carcinoma of the lung, cervix, and tonsil, as well as in adenocarcinoma of the colorectum, lung, breast, and testis. Metastases of lung largecell, but not small-cell, carcinoma were also positive for CD44v (Li et al., 1993, 1995; Nagasaka et al., 1995). According to one study (Nagasaka et

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al., 1995) in which four specimens were analyzed, brain metastases of melanoma express only the CD44s isoform. In another investigation (one specimen), the melanoma metastases expressed CD44v as well (Li et al., 1995). It should be noted that melanoma cells are of neuroectodermal origin. The results further show that, like keratinocytes, all brain metastases of squamous carcinoma origin express the long CD44v (CD44V3-10). Most of the other types of metastases (derived mostly from adenocarcinomas) express shorter CD44v transcripts expressing various com binations of exons V3, V4,5, V6,7, V8, and V9,lO (Li et al., 1995). Reverse transcriptase-PCR revealed that brain metastases of colon carcinoma express CD44s, CD44V8-10 (CD44E), and CD44V8,9 (Nagasaka et al., 1995). Gliomas, which express CD44s but not CD44v, display different capacities for local invasion, whereas they barely metastasize to other organs. Furthermore, glioblastomas, which express CD44s more intensively than do low-grade astrocytomas, more aggressively invade the brain ECM (Kuppner et al., 1992).These observations can be explained by the finding that CD44s, but not CD44v, is the principal HA receptor (see Section V) and that HA is an important component of the brain’s ECM (Asher et al., 1991; Bignami et al., 1992), a combination that allows effective tumor-matrix interaction. On the other hand, if CD44v is important for the metastatic process of at least some neoplasms (see Section XI.A), this would explain why gliomas are confined to local growth, whereas remote tumors, which do express CD44v, metastasize to the brain. Alternatively, tumor cells metastasizing to the brain may simply preserve their original CD44v markers, which do not have any role in brain infiltration. The finding that brain metastases of melanoma and lung small-cell carcinoma do not express CD44v is in line with the latter suggestion, yet they may use other adhesion molecules for migration.

B. Head and Neck Tumors 1. SQUAMOUS CELL CARCINOMAS

OF THE HEAD AND NECK Whereas CD44v6 epitopes of benign epidermal neoplasm stained with specific anti-V6 mAb, like their normal neighboring counterparts, isoforms containing this exon product were downregulated in all specimens from patients with head and neck squamous cell carcinoma. Notably, differentiated carcinomas expressed more V6 epitopes than did undifferentiated tumors, and all distant metastases were practically negative (Salmi et al., 1993). This finding contradicts other reports (Section XI1.A) describing the expression of CD44 variants in brain metastases of squamous cell carcinomas (Li et al., 1995).

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2. THYROID CARCINOMAS

The vast majority (97%, using IHC) of tumor specimens obtained from patients with papillary thyroid carcinoma stain with anti-pan-CD44 mAb (which is directed against the constant region), and all samples are positive for the V6-containing variant, as indicated by IHC (Figge et al., 1994) and RT-PCR (Ermak et al., 1995). However, it should be noted that some adenomas and goiters have high V6 mRNA levels as well (Ermak et al., 1995). Nonneoplastic follicular epithelium displays less intensive staining with antiCD44 mAb. Specimens of other thyroid cancers (Hiirthle cell neoplasm, medullary carcinoma, and follicular carcinoma) show a CD44 frequency of 50% or less.

3. NASOPHARYNGEAL CARCINOMA Immunohistochemical studies revealed pan-CD44 expression in nonneoplastic lymphoid and stromal cells, as well as in basal and parabasal squamous epithelial cells. The epithelial cells also included exon 9-containing variants, presumably CD44E. All nasopharyngeal carcinomas were stained by anti-CD44V9 mAb, indicating the presence of CD44E andlor other V9containing variants. Eight of 12 tumors were stained, at different levels of intensity, by anti-CD44V6 mAb. Although RT-PCR analysis cannot distinguish between tumor cells and normal cells in the examined specimen, it can define the CD44 repertoire of the tumor tissue. To overcome the problem engendered by the mixed population, tumor cell lines were also included in the survey and the following transcripts were detected: CD44s, CD44V3, CD44V6-10, and CD44V8-10 (CD44E). There was no correlation between CD44 and LMP-1 expression, or between expression of CD44 and disease stage (Brooks et al., 1995).

C. Respiratory Tract Cancer 1. LUNG CARCINOMAS

In normal lung tissue, pan-CD44 has been detected in alveolar macrophages, interstitial cells, and epithelial cells. CD44 variants expressing V6or V9-encoded epitopes were found on epithelial cells only (Kasper et al., 1995). Immunohistochemical analysis revealed the absence of pan-CD44, CD44V3, and CD44V6 in biopsy samples of highly metastatic small-cell lung cancer, whereas most specimens of non-small-cell lung cancer were positive for at least one of these three epitopes (Ariza et al., 1995). Similar findings were observed in lung carcinoma cell lines (Jackson et al., 1994), indicating that CD44 expression does not necessarily correlate with the tumor’s metastatic capacity.

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2. MESOTHELIOMAS According to one report, all the mesothelioma cell lines studied expressed various degrees of functional CD44, whereas normal human mesothelial cell lines did not display this receptor (Asplund and Heldin, 1994). According to another communication, CD44 variants are present in both normal and malignant mesothelial cell lines (Jackson et al., 1994). It should be emphasized, however, that cell lines do not faithfully represent the primary tumors, as culture conditions may induce changes in CD44 repertoire and function (for references, see introduction to Section XII).

D. Alimentary Tract Cancer 1. COLORECTAL CANCER

In normal colonic mucosa, CD44 (standard or variant) is mostly confined to the proliferative zone of the crypts, as indicated by IHC. In other sites, CD44s expression is practically absent and CD44 variants are not detected (Abbasi et al., 1993; Heider et al., 1993b; Wielenga et al., 1993).Using IHC, RT-PCR, and in situ hybridization, some, but not all (see later), investigators have demonstrated that CD44s and CD44v are expressed in the primary malignant colorectal tissue of at least 50% of patients and in lung, liver, and lymph node metastases of all patients (Matsumura and Tarin, 1992; Heider et al., 1993b; Wielenga et al., 1993; Orzechowski et al., 1995; Suh et al., 1995). A small number of patients with early adenoma showed restricted CD44V6 expression in their polyps, with the ratio increasing to 50% in patients with late adenoma. When progression of the disease from nonmetastatic to metastatic carcinoma was followed, the proportion of patients with CD44V6' tumors increased from 50 to SO%, and expression of the variant was more abundant in each sample, as shown by immunostaining (Wielenga et al., 1993) and in situ hybridization (Orzechowski et al., 1995). According to another communication, tissue sections of most patients with metastatic colorectal cancer do not display CD44V6, although sections from earlier stages (of both adenoma and carcinoma) were found to be positive for this variant (Finke et al., 1995). A lack of correlation between CD44 expression and disease stage was reported by two other research groups. The first (Imazeki et al., 1996) described the strong expression of CD44v (including the epithelial isoform) in adematous colorectal polyps, and the second (Jackson, P. A., et al., 1995) demonstrated minimal CD44 staining in metastasizing carcinomas. To add to this confusion, it was further reported that CD44 expression is confined to areas of proliferation, irrespective of tumor stage or differentiation (Abbasi et al., 1993), and that proximal carcinomas express significantly more CD44V5 and CD44V6 than do distal tumors (Mulder et al., 1995). Two groups of investigators reported that the

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survival of patients with CD44E' (Yamaguchi et al., 1996) or CD44V6' (Mulder et al., 1994) primary tumors is shorter than that of patients with CD44- tumors ( p < 0.01 and p < 0.002, respectively). However, the difference in survival between patients bearing CD44V6' tumors and those bearing CD44V6- tumors was not confirmed by another research group (Koretz et al., 1995).These discrepancies may be related to technical reasons or to differences in the criteria (age, disease stage, etc.) used for including patients in the study. Finally, cDNA transfer experiments (see Sections V.C and V.D) have revealed that CD44s expression in human colon carcinoma cell lines is associated with a greater capacity to bind HA and a reduced ability to grow in cultures or in nude mice. These findings suggest that downregulation of CD44s afford the transformed colon cells a growth advantage (Takahashi et al., 1995). In lymphomas and melanomas, CD44s expression instead correlates with the tumor's ability to grow in nude mice (see Section XLA), pointing to the influence of the internal cell environment on the CD44 function. 2. STOMACH CANCER CD44V5 is confined in normal stomach mucosa to the foveolar proliferation zone and to the mucoid surface epithelium, as indicated by IHC staining with CD44V5-specific mAb (Heider et al., 1993a). In contrast, areas of precancerous intestinal metaplasia stain positively with mAbs specific for both V5 and V6 exon products (Heider et al., 1993a; Harn et al., 1995). Gastric adenocarcinoma of the intestinal type is strongly positive for epitopes encoded by exons V5 and V6; whereas diffuse-type tumors predominantly express exon 5 (Heider et al., 1993a; Dammrich et al., 1995; Hong et al., 1995a). This differential reactivity with mAbs specific for V5 and V6 exon products may help to establish a diagnostic tool able to distinguish between the two types of gastric tumors. It is interesting that infiltrative lymph node metastases of both intestinal and diffuse adenocarcinomas express V6 epitopes, suggesting that V6 is important for lymphogenic dissemination (Dammrich et al., 1995). According to an additional study, 50 and 75% of primary and metastatic tumor specimens (from regional lymph nodes, liver, and omentum), respectively, of patients with gastric carcinoma displayed CD44. Expression of exon V9 was in good correlation with that of panCD44. Patients with CD44' tumors exhibited shorter survival than did those with CD44- tumors ( p = 0.003) (Mayer et al., 1993). Using different antiCD44 mAbs for screening, another group reported that patients with panCD44 or CD44V6' gastric tumors survived as long as patients with CD44tumors (Hong et al., 1995a). Reverse transcriptase-PCR analysis has revealed in gastrointestinal tumors and their metastases a more complex pattern and a greater number of CD44 variant transcripts (including CD44E) than in the corresponding nor-

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ma1 tissues (Matsumura and Tarin, 1992; Heider et al., 1993a; Tanabe et al., 1993; Wielenga et al., 1993; Dammrich et al., 1995; Rodriguez et al., 1995). Furthermore, tumor tissues from 16 of 20 patients with colon cancer showed abnormal retention of intron 9 (located between exons V4 and V5) in CD44 transcripts. Such aberrant expression of intron 9 was also identified in the mRNA of esophageal, colon, and breast carcinoma cell lines (Yoshida et al., 1995). The extensive assembly of CD44 variant and aberrant gene products in tumor cells may reflect the release from alternative splicing control, a phenomenon that is still ill-defined but that could eventually be used as a diagnostic tool. The contradictory findings do not, at this stage, allow the establishment of a conclusive concept regarding the role of CD44s or its variants in the development of gastrointestinal cancer or the prognostic value of these molecules. On the other hand, the results are sufficientlyinteresting to encourage intensive research, especially in view of the detection of CD44 mRNA in stool samples from 6 of 11 patients with colorectal carcinoma (Tarin et al., 1995) and of CD44 isoforms in most serum samples of patients with gastric or colon carcinoma (Guo et al., 1994b). As the normal controls were very low or negative in these two studies, it is conceivable that these noninvasive procedures could be applied to diagnosis.

E. Other Alimentary Tract Cancers 1. PANCREATIC CANCERS In a comparison of normal human pancreatic tissue with malignant pancreatic tissue, IHC revealed strong pan-CD44 positivity in the ductal epithelial cells and acini of the healthy pancreas. In contrast, the normal islets were negative. All the pancreatic gastrinomas (gastrin-producing endocrine tumors) tested expressed CD44, whereas almost all other types of endocrine pancreatic neoplasms were CD44-, as indicated by IHC. CD44' tumors tended to metastasize to the lymph nodes, whereas CD44- tumors were locally invasive or metastasized to the liver. Transcript analysis by RT-PCR amplification has revealed that normal pancreatic tissues, as well as all endocrine pancreatic tumors, express CD44s and CD44E mRNAs, with the exception of gastrinomas, which express additional CD44 variants of larger molecular size (Chaudhry et al., 1994). In another study, specimens of primary and metastatic (liver, terminal ileum, omentum, and mesentery) pancreatic adenocarcinoma, as well as noncancerous control specimens, expressed CD44s, CD44V6, and CD44E, as indicated by RT-PCR (Rall and Rustgi, 1995). In addition, samples of such tumors, but not of control pancreas, displayed CD44V9,lO (or CD44V7-9) and CD44V6-10 transcripts according to one report (Rall and Rustgi, 1995)

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and CD44V2-10, CD44V3-10, CD44V4-10, and CD44V5-10 according to another (Gansauge et al., 1995). Although different CD44 variants were assigned to the tumor samples, both reports suggest that exon V6, linked to other exons, is functionally associated with the malignant phenotype, as proved for rat pancreatic adenocarcinoma (Gunthert et al., 1991). Only cDNA transfer experiments in immunodeficient mice can substantiate this supposition. 2. LIVER CANCER In a study of hepatocellular carcinoma (Haramaki et al., 1995),the CD44' noncancerous liver components included lymphocytes, fibroblasts, and Kupffer cells, as shown by IHC staining. Tumor specimens taken from all 11 patients were CD44-, but two tumor samples of peritoneal effusion were CD44'. Reverse transcriptase-PCR analysis did not reveal any apparent differences in CD44 expression between hepatocellular carcinoma and noncancerous tissues. However, contamination cannot be excluded as the tumors may contain normal CD44-expressing cells (e.g., lymphocytes).

F. Genitourinary Tract Cancer 1. RENAL CELL CARCINOMAS

CD44v have not been detected in nonmalignant kidneys or in benign renal cell tumors (oncocytomas). CD44s and CD44v were found, however, in invasive chromophobe cell, clear cell, and chromophilic cell carcinomas, as indicated by IHC and RT-PCR. In the latter two diseases, the expression of pan-CD44 and CD44v (CD44E, CD44V6, and CD44V9) is correlated with tumor grading (Terpe et al., 1996). Another report (Kan et al., 1995) describes the exclusive presence of the exon V10 product in renal carcinoma CD44.

2. BLADDER CANCER Staining with anti-pan-CD44 mAb and mAbs directed against CD44 variants (e.g., those containing V6-encoded epitopes) has revealed that CD44 isoforms are confined to the basal layers of normal bladder epithelium (urothelium), with their expression tapering toward the superficial cell layer. Stromal cells are CD44' (presumably CD44s'). Most neoplastic bladder specimens exhibit strong immunostaining for pan-CD44 and CD44v (including CD4406) in both basal and more superficial cells (Southgate et al., 1995; Sugiyama et al., 1995). Exfoliated tumor cells isolated from the urine

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of bladder cancer patients display CD44 variants that were not detected in exfoliated cells of control urine samples, as indicated at the transcriptional level by RT-PCR (Matsumura et al., 1994) and at the protein level by Western blot analysis (Sugiyama et al., 1995). A comparison of urine specimens of bladder cancer patients with urine specimens from nonneoplastic individuals revealed that CD44 variant transcripts were overexpressed in 40 of 44 (910/,) cancer patient specimens, as opposed to 8 of 46 (17%) control samples. This means that RT-PCR analysis has a sensitivity of 91% and a specificity of 83% (Matsumura et al., 1994). The corresponding Western blot assessment showed 75% sensitivity and 100% specificity (Sugiyama et al., 1995). Both methods were able to detect tumors expressing CD44 variants at early stages of the disease, indicating that the change in the CD44 repertoire begins in the early stages of the neoplastic process. Transcript analysis by PCR amplification of cDNA derived from the mRNA of a bladder cancer cell line revealed the “illegitimate” transcription of an intron located between exons V4 and V5 of CD44. This intron (intron 9) was also detected by RT-PCR in exfoliated cells isolated from the urine of 18 of 30 (60%) bladder cancer patients. Only 1 of 41 urine samples from tumor-free individuals was positive for the intron (Matsumura et al., 1995). This noninvasive technology could be useful for early diagnosis of bladder cancer, but further improvements are required to increase its sensitivity. However, this optimistic report should be weighed against two other communications (Hong et al., 1995b; Southgate et al., 1995) describing reduced CD44v expression in tumors of patients with advanced bladder carcinoma.

3. PROSTATE CANCER In contrast to normal prostatic epithelial cells, cells from a high-grade prostate cancer expressed high levels of CD44, as indicated by IHC (Lokeshwar et al., 1995).

G. Gynecological Cancer 1. UTERINE CERVICAL CARCINOMA

Pan-CD44 has been found throughout all the stromal cells and epithelial layers of normal uterine cervical specimens. CD44 variant epitopes (e.g., v3,4, v6,7, and CD44E) are confined to proliferating basal cells and spinal cells of normal epithelium (Dall et al., 1994). Specimens from at least some patients with uterine cervical cancer also display various CD44 isoforms (e.g., variants containing V5 and V6). Expression of exon 6 is significantly correlated with the presence of metastases in the pelvic lymph nodes ( p =

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0.04), a greater probability of vascular space invasion ( p = 0.04), and shorter survival ( p = 0.03), especially in patients with noninvolved pelvic lymph nodes ( p = 0.01) (Kainz et al., 1995a). The epitope recognized by mAb directed against both V7 and V8 exon products (anti-V7/8 mAb) was present in 94 or 26% of the cancer specimens studied, according to two different research groups (Dall et al., 1994; Kainz et al., 1995a,b). This epitope was, however, absent from almost all samples of normal cervical squamous epithelia (Dall et al., 1994). Immunostaining (Dall et al., 1996) has revealed that the progression of uterine cervical carcinoma from the low-grade squamous intraepithelial stage through the high-grade squamous intraepithelial stage to the invasive stage is followed by a gradual increase in CD44V7/8+ specimens (from 20 through 50 to loo%, respectively).Expression of this epitope also correlates with short survival ( p = 0.02; Kainz et al., 1995a). A gradual rise in the presence of human papillomavirus 16 has also been detected during tumor progression, and it apparently precedes CD44P7/8 expression (Dall et d., 1996). Amplification of CD44 transcripts by RT-PCR showed qualitative and quantitative differences between malignant and control specimens. The cervical cancer samples exhibited a more complex alternative splicing pattern than did the control samples, and about one half of the neoplastic specimens (including all those from patients with lymph node metastases) displayed a CD44 isoform expressing exons V3-10 (Dall et al., 1994). While it is not known whether the change in the CD44 repertoire confers a selective advantage on some cells in the course of carcinogenesis, this alteration might be useful for diagnostic and prognostic purposes. 2. OVARIAN CANCER Immunostaining with polyclonal antibody specific for epitopes encoded by CD44 variant exons V3-10 revealed a detectable reaction with tumor cells in 75% of 44 ovarian cancer specimens. However, only those specimens exhibiting a moderate or strong reaction (25%) were considered CD44v'. The disease-free survival (i.e., survival without evidence of tumor relapse) of the patient population bearing the CD44v' tumors was shorter than that of patients bearing the CD44v- tumors ( p = 0.03). However, upon examining the overall survival instead of the disease-free survival, the findings were not statistically significant. In contrast, when analysis was confined to populations with advanced disease (stages I11 and IV),both the disease-free and the overall survival of patients with CD44v' tumors were shorter than those of patients with CD44v- tumors ( p = 0.02 and 0.03, respectively). CD44v positivity was correlated with median age and inversely correlated with the

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preoperative platelet count, but not with other established parameters of the disease (Uhl-Steidlet al., 1995).The preoperative serum levels of CD44s and CD44v (V5 and V6) were not higher than those of the controls (Sliutz et al., 1995). Although the size of the studied population was too small to allow conclusions to be drawn regarding the significance of CD44v as a diagnostic or prognostic tool in ovarian carcinoma, the results are sufficiently interesting to encourage further study.

H. Breast Cancer Immunohistochemical analysis by several research groups has revealed that CD44 is not expressed by the epithelium of normal breast tissue (Dall et al., 1995; Iida and Bourguignon, 1995; Sinn et al., 1995), with the exception of myoepithelium, which carries variant epitopes encoded by exons V3, V5, and V6 (Fox et al., 1993; Sinn et al., 1995). Another IHC examination demonstrated the presence of CD44s and CD44 variants containing V6 and V9, but not V4, exon products in normal breast epithelium. Hence, V6-encoded epitopes are present in ductal and ductular epithelial cells as well as in myoepithelium (Friedrichs et al., 1995a). CD44s has also been detected in stromal elements such as fibroblasts, endothelial cells, and lymphocytes (Friedrichs et al., 1995a). Iida and Bourguignon (1995) reported that, in contrast to normal breast tissue, high levels of CD44, detected by IHC, are found in metastatic breast carcinoma. Other investigators indicated that about one third to one half of the primary tumor specimens taken from patients with invasive ductal breast cancer were positive for epitopes encoded by exons V3, V7l8, and V10, whereas 70-80% of all primary tumor specimens were positive for epitopes encoded by exons V5 and V6 (Kaufmann et al., 1995a). A similar distribution of CD44 variants was found in patients with invasive lobular carcinomas (Dall et al., 1995) as well as in in situ carcinomas and local recurrent tumors (Sinn et al., 1995). Furthermore, lymph node metastases in all the patients were positive for epitopes encoded by exons V3, V5, V6, and V10, as indicated by IHC (Dall et al., 1995; Kaufmann et al., 1995a; Sinn et al., 1995). An IHC study performed by a different group (Friedrichs et al., 1995a) revealed the presence of CD44v6 and CD44V9 in 45 and 70% of breast tumor specimens, respectively. In their analysis, CD44s and CD44V9 correlated with established risk factors for survival, such as tumor size and histological grading. Although the two research groups (Kaufmann et al., 1995a, vs. Friedrichs etal., 1995a,b) used the same anti-CD44 mAb (but perhaps not the same concentration) and the same cutoff definition of a positive CD44 sample (according to which the sample is considered positive if 2

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5% of the cells are stained positively), they arrived at opposite conclusions regarding the association between CD44V6 expression by tumor cells and survival rate. One group (Kaufmann et al., 1995a) claimed that the survival of breast cancer patients bearing CD44V6' tumors is shorter than that of cancer patients bearing CD44V6- tumors ( p = 0.02), whereas the other group (Friedrichs et al., 1995a,b) did not find such a difference. Heterogeneity in tumor CD44 expression (defined by the Hermes-3 mAb) in different patients, and the association between CD44' tumor expression (50% cutoff point) and greater mortality ( p = 0.05) among patients with ductal breast cancer, was reported in another communication. CD44 positivity has also been associated with aggressive factors such as poor histopathological grading, high mitotic count, and estrogen receptor negativity (Joensuu et al., 1993). It is hoped that the conflicting findings relating to survival will be resolved by expanding the size of the screened population, by using separate screenings of disease subtypes, and by preventing bias by screening representative populations (to this end, see letters exchanged in Friedrichs et al., 1995b, and Kaufmann et al., 1995b). Transcript analysis by RT-PCR has revealed that, relative to normal breast tissues, breast cancer tissues express high levels of CD44s and CD44E (Iida and Bourguignon, 1995).Furthermore, breast cancer tissues, but not the corresponding normal tissues, contain additional multiple species of CD44 variants (Matsumura and Tarin, 1992; Dall et al., 1995; Iida and Bourguignon, 1995; Rodriguez et al., 1995),some of them allegedly new isoforms (Iida and Bourguignon, 1995). The additional variants could be useful in monitoring breast cancer progression or recurrence and, perhaps, will also be helpful in making decisions as to the modality of treatment. However, this consideration should be weighed against the findings of another study, according to which multiple CD44 variant transcripts are present in both normal and malignant breast tissue (Friedrichs et al., 1995a).

I. Melanomas Whereas melanocytes express high levels of CD44s but not of CD44v, melanoma lesions express, in addition to CD44s, CD44 variants containing V5- and V10-encoded epitopes according to one study (Manten-Horst et al., 1995) and V718-encoded epitopes according to another (Korabiowska et al., 1995). Tumor progression from common nevi through early and advanced primary melanoma to metastatic melanoma was followed by a gradual increase in the proportion of patients with VS+lesions (from 16 to 60%) (Manten-Horst et al., 1995). Interestingly, it appears that brain metastases of melanoma express CD44s only (see Section XI1.A).

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J. Non-Hodgkin's Lymphoma and Chronic

Myeloid Leukemia Immunohistochemical findings showed that, in nonmalignant lymph nodes, CD44s is strongly expressed in the follicular mantle, the interfollicular zones, and the sinusoidal compartment, with weak expression in the germinal center. Variant CD44 isoforms were not, however, detected in any compartment (Terpe et al., 1994a). Using mAbs directed against the CD44 constant region, earlier studies described a correlation between disseminated and aggressive phenotypes of the lymphoma and elevated expression of cell surface CD44 (Pals et al., 1989b; Horst et al., 1990b; Jalkanen et al., 1990). Later, it was demonstrated that the majority, or at least a significant part, of the tumor specimens derived from patients with intermediate/high-grade non-Hodgkin's lymphoma react positively with antibodies directed against CD44v epitopes. Most of them are expressed in V3 and/or V6 exon products and a few in the V9 exon product (Koopman et al., 1993; Terpe et al., 1994a; Stauder et al., 1995). Immunohistochemical analysis did not (Koopman et al., 1993), or only rarely (Terpe et al., 1994a; Stauder et al., 1995), could detect CD44v epitopes on tumors of patients with the low-grade phenotype. This finding was not, however, confirmed by another research group that found a higher frequency of CD44V6' tumors in patients with low-grade lymphomas than in patients with the high-grade disease (Ristamaki et al., 1995). It should be emphasized that these investigators used a different screening method and a different anti-V6 mAb, which may have distinct epitope affinity or discrete epitope specificity. Using RT-PCR, a more sensitive method than IHC, CD44v transcripts were detected in both low- and high-grade lymphomas. Low-grade lymphomas express CD44V3, CD44V6, CD44V8, CD44V9, CD44V10, and CD44V8,9 transcripts. An additional set of transcripts (CD44V4-7, CD44V9,lO) has been detected in high-grade lymphomas. An even more complex CD44 pattern, involving additional variant exons, was identified in the more aggressive tumor at the time of relapse (Stauder et al., 1995). Another report (Salles et al., 1993) indicated that, in comparison with primary nodal large-cell lymphomas, extranodal and disseminated large-cell lymphomas display higher levels of CD44s, CD44V6, and V6-containing variants. Irrespective of grade type, the survival of non-Hodgkin's lymphoma patients with CD4486' tumors is shorter than that of those with CD44V6tumors, as indicated in two (Ristamaki et al., 1995; Stauder et al., 1995) independent studies ( p = 0.06 and p < 0.0001, respectively). Moreover, multivariate analysis has revealed that CD4486 is an independent prognostic parameter (Stauder et al., 1995). Collectively, these findings suggest that, in

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non-Hodgkin's lymphoma, the V6 exon product is not only a disease marker but also a functional entity involved in tumor dissemination and, as such, a potential target for therapy with specific antagonistic reagents (for examples in animal models, see Section X1.B). In comparison with their normal counterparts, neoplastic cells of chronic myeloid leukemia patients display a greater proportion and higher levels of CD44 and an increased number of light-density V10' cells (Ghaffari et al., 1995).

XIII. CD44 ASSOCIATION WITH MALIGNANCY: SOME PRACTICAL COMMENTS Despite intensive CD44 analysis in human malignant diseases and the encouraging results obtained in animal models, it is still difficult to state unequivocally that human tumor cells bearing CD44 or its variants have a selective advantage. However, at least part of the findings are sufficiently interesting to encourage further research, including CD44 cDNA transfer to indolent human cancer cells and subsequent evaluation of the transfectant's ability to grow progressively in immunodeficient mice. It appears, however, that CD44 and/or its variants could serve as diagnostic or prognostic markers in at least some human neoplasms, particularly brain metastases and nonHodgkin's lymphomas, as demonstrated by IHC analysis. If diagnosis, and even more so supportive diagnosis, are based on the detection of multiple and complex CD44 variant transcripts by RT-PCR, this list can be further extended to include gastrointestinal cancer, gastrinomas, pancreatic adenocarcinoma, bladder cancer, uterine cervical cancer, and breast cancer. Whereas in all these diseases CD44 (or its variants) may be the marker of unfavorable outcome, in neuroblastoma, expression of CD44 could signify a better prognosis. When compared with IHC, the use of RT-PCR has both advantages and disadvantages. Reverse transcriptase-PCR is a more sensitive method than IHC and, therefore, can be used to identify CD44 transcripts of exfoliated cells in stool and urine samples (this technique detects CD44 mRNA in as few as 10 tumor cells among lo7 leukocytes; Matsumura and Tarin, 1992). In addition, RT-PCR can determine the exact composition of CD44 variants and, therefore, is used to distinguish between CD44s and other CD44 isoforms. However, it should also be borne in mind that detection of CD44 mRNA does not imply that this transcript is expressed as a protein. Reverse transcriptase-PCR is vulnerable to ribonucleases. Therefore, not only must specimens be immediately frozen after removal, but they should not be stored, even at - 70°C, for longer than 1month. Ideally, RT-PCR, or at least

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the RNA extraction and cDNA reverse transcription steps involved, should be performed immediately after sample removal in order to avoid ribonuclease release during freezing (Matsumura et af., 1994; Sugiyama et af., 1995). Unfortunately this procedure is impractical in many cases. Furthermore, if strict precautions are not taken, RT-PCR can amplify trace contaminants to detectable levels. The ability of IHC to discriminate between normal and neoplastic cells is an obvious advantage over RT-PCR, as the tumor samples contain a mixture of both types of cells. However, the fact that antibodies are restricted to the recognition of accessible CD44 epitopes encoded by individual or limited numbers of exons is a disadvantage, as the complete composition of the isoform remains unknown. Furthermore, antibody directed against the constant region of CD44 does not discriminate between standard and variant isoforms. Since various laboratories use different anti-CD44 mAbs at different concentrations, and the percent cutoff points of sample positivity are determined arbitrarily by each research group, IHC is much more dependent on universal standardization than is RT-PCR. The in sittr hybridization technique, based on the “staining” of histological samples with radiolabeled CD44 RNA probes (Orzechowski et al., 1995), may combine a few advantages of IHC and RT-PCR while retaining some of the disadvantages inherent in each method. The ability of cDNA containing the V6 exon to confer, upon transfection, a metastatic potential on nonmetastatic pancreatic rat tumor (Giinthert et af., 1991; Herrlich et af., 1993; Rudy et af., 1993) has attracted much attention and prompted investigators to focus their studies on a search for a correlation between V6 expression on human neoplastic cells and the malignant activity of the disease. This molecule gained further interest when it was discovered that T-cell activation in both mouse and humans is associated with the transitional expression of V6-containing variants and that specific mAbs inhibit various immunological functions of the activated cells (Arch et af., 1992; Moll et al., 1996). Yet the interest engendered by the V6 exon should not cause other CD44 variants to be overlooked, as the functional superiority of distinct CD44 isoforms is likely to differ from one tumor to another.

XIV. CONCLUSIONS CD44 is a powerful multifunctional molecule that can satisfy at least part of the growth requirements of metastatic tumors. The list of CD44 functions offering a selectivegrowth advantage to neoplastic cells includes cell-cell and cell-matrix interactions, homophilic and heterophilic cell aggregation, anti-

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apoptotic effect, cell motility, HEV-associated homing to lymph nodes, mediation of HA internalization and its subsequent intracellular degradation, presentation of growth factors or chemoattractants, and transmission of growth signals either independently or in collaboration with other surface molecules. Therefore, the ubiquity of CD44 in malignant growths and its involvement in neoplastic processes, as proved in experimental models, are hardly surprising. However, the properties conferring a growth advantage on tumors are by no means confined to CD44; other adhesion molecules, especially integrins and selectins, can also be utilized by the tumor to support the malignant process. Indeed, Driessens and colleagues (1995) demonstrated that, although knocking out of the CD44 gene by homologous recombination hampers the HA binding capacity of mouse lymphosarcoma cells, it does not interfere with their local growth and metastatic spread, which are possibly mediated by integrins, also expressed on the same tumor cells. It appears that both intracellular genetic factors and the selective pressure exerted by the extracellular milieu dictate an adhesion molecule setting that supports tumor growth. If CD44 is involved, such factors may determine which isoform assumes functional superiority. This heterogeneity does not leave room for predictions, but rather emphasizes that the adhesion molecule repertoire of each malignant disease should be determined by laborious analysis of large numbers of specimens. In animal models findings are obviously more conclusive (see Section XI), as inbreeding generates genetically uniform individuals that are maintained under standard laboratory conditions. Yet even the animal models underline the diversity of CD44 isoform usage by different tumors. For example, the metastatic spread of pancreatic carcinoma in rats is CD44v dependent (Giinthert et al., 1991), whereas the growth of mouse melanoma is associated with CD44s (Bartolazzi et al., 1994). The interaction between cell surface CD44 and the matrix or cellular components is essential to normal physiological functions. Therefore, for optimal therapeutic purposes, it is mandatory to identify the functional CD44 variants expressed exclusively on tumor cells. Targeting of such as-yet hypothetical CD44 variants with specific antagonizing reagents (e.g., antibodies) could interfere with tumor spread without causing damage to normal biological activities, which are also CD44 dependent. To date, there are at least 20 known CD44 isoforms, but this number is possibly just the tip of the iceberg. Theoretically, alternative splicing in the variable region of the CD44 molecule could generate hundreds of isoforms, especially in proliferating tumor cells, which are susceptible to genetic alterations. If any of these genetic changes indeed afford the tumor a biological edge, a process of natural selection and stabilization should be expected in tumors expressing the genetically modified CD44. Paradoxically, however, these altered CD44 molecules would also provide an ideal target for focused attack by specific anti-CD44 reagents.

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No less important is the identification of CD44 non-HA ligands, several of which have already been defined, including the ECM components, collagen, fibronectin, laminin, and CS (see Section VI). However, the polymorphic nature of CD44 suggests that this list is far from complete. Such hypothetical, novel CD44 ligands could be involved in tumor cell-tumor cell, tumor cell-normal cell, or tumor cell-matrix interactions, and their identification may allow the design of competitive or antagonistic reagents with potential antimetastatic effects. Autoimmune inflammatory reactions may be associated with CD44dependent migration andor tissue localization of autoreactive lymphocytes (see Section X.D). Similarly, allergic reactions of the skin, lung, or alimentary tract might be associated with CD44-dependent accumulation of leukocytes. The allergy research has barely begun to attract attention, and the possible involvement of CD44 isoforms in the pathology of autoimmune diseases is largely theoretical. The activity of many other diseases may also be associated with CD44 (standard or variant) down- or upregulation, as demonstrated in alopecia areata (Sawaya et al., 1994) and ulcerative colitis (Rosenberg et al., 1995, and editorial commentary by Geboes and Rutgeerts, 1995).In addition, CD44 or its variants may be essential to the mobilization and lodgment of cells involved in transplant rejection or the GVH reaction. Considering these unresolved questions, it is clear that, despite the intensive work behind us, we are just starting to understand the mechanism of action and the significance of the CD44 molecule in both physiological and pathological settings, including its association with the malignant process.

ACKNOWLEDGMENTS K7ethank Drs. Bernhard Holzmann and Elimelech Okon for helpful comments, Mrs. Yaakovah Muller for preparation of the manuscript, and Dr. Alexandra Mahler for her editorial assistance. Work in the authors’ laboratory is supported by The Society of Research Associates of the Lautenberg Center, a grant from Deutsche Krebshilfe in the name of Dr. Mildred Scheel, and an award from Focused Giving Program, Johnson &Johnson.

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Human Papillomaviruses and Cervical Cancer Luisa Lina Villa Ludwig Institute for Cancer Research 01509-010, Siio Paulo, SP, Brazil

I. Introduction 11. Biology of Papillomaviruses 111. Taxonomy and Genomic Variability of Papillomaviruses IV. EpidemiologicalAspects V. HPV Interaction with Cofactors VI. Viral Persistence and Disease Progression VII. Viral Burden and Cervical Disease VIII. HPV in Cervical Screening Programs References

Molecular and epidemiological studies conducted over the last 20 years led to the recognition of certain types of human papillomavirus (HPV) as the etiological agents of cervical cancer, a very common neoplasia, particularly in developing countries. More than 70 HPVs have been described, including both cutaneous and mucosal types. About half of the known HPVs, and an even higher number of variants, have been isolated from genital mucosas. The association of certain types primarily with normal tissues and benign lesions, as opposed to cancer-associated types, has led to the concept of low and high oncogenic risk HPVs, respectively. The latter express oncogenic proteins that interfere with cell growth control functions. As a consequence of the continuous expression of these viral genomes, chromosome instability may occur, leading to fully transformed cells. Studies indicate that persistence of high-risk HPVs may determine progression to more severe stages of cervical disease, while the majority of HPV infections are transient and do not seem to be important in cervical carcinogenesis. The risk for disease progression seems also to be associated with viral burden. Prospective epidemiological studies will contribute to the knowledge of the natural history of HPV infections and provide information on the determinants of viral persistence. Data derived from these studies may define the clinical utility of HPV testing and its use in cervical cancer prevention programs.

I. INTRODUCTION A consensus panel convened by the World Health Organization’s International Agency for Research on Cancer (IARC Working Group, 1995) has concluded that there is now compelling evidence, from both the biological Advances in CANCER RESEARCH 0065-23OW97 $25.00

Copyright D 1997 by Academic Press. All rights of reproduction in any form reserved.

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and the epidemiological standpoints, to consider certain papillomaviruses as carcinogenic in humans. A large series of molecular and epidemiological studies conducted in the last twenty years have confirmed that cervical infection by certain human papillomavirus (HPV) types is a precursor event in the genesis of cervical neoplasia (zur Hausen, 1991, 1996; Muiioz et al., 1992; Schiffman et al., 1993; Bosch et al., 1995; Franco, 1995; Schiffman, 1995). As pointed out by Franco (1995), the magnitude of the association between HPV and cervical cancer is higher than that for the association between smoking and lung cancer and is second only to that of the association between the chronic carrier state of hepatitis B infection and liver cancer. In this review, the molecular and epidemiological aspects that support the etiological role of HPV in cervical carcinogenesis are presented. Data on the clinical utility of HPV testing in cervical cancer prevention programs are discussed.

11. BIOLOGY OF

PAPILLOMAVIRUSES

Papillomaviruses are small, nonenveloped viruses classified in the Papovaviridae family (zur Hausen and de Villiers, 1994; Howley, 1996). These viruses can be found mostly in the epithelia of many different animals, including birds, reptiles, and mammals, being highly specific for their respective hosts. In humans, over 70 papillomavirus types have been defined on the basis of DNA homology, including both cutaneous and mucosal types (Delius and Hofmann, 1994; devilliers, 1994). Sequence data available from most papillomaviruses described have allowed their grouping into taxonomic orders, as discussed later. The HPV genome consists of a circular double-stranded DNA of about 8000 nucleotides, divided into three segments; an early (E) region cornprising six genes, a late region (L) that encodes two structural proteins, and a noncoding region known as the long control region (LCR) (Fig. 1). These genomes are maintained as episomes in the nucleus of normal infected cells. However, in cervical intraepithelial neoplasias (CINs) and even more frequently in cancers, HPV genomes are found integrated into the host chromosomes (Lehn et al., 1988; Cullen et al., 1991). While this event appears to occur at random sites in the cellular genome, the viral DNA integration involves the E l and E2 genes, with important consequences for the regulation of the viral gene expression (see later). Several reviews on the functions of the papillomavirus genes and the LCR are available (Galloway and McDougall, 1989; Schlegel, 1990; Miinger and Phelps, 1993; IARC Working Group, 1995; Howley, 1996). A summary of their main functions and the relevance to cell immortalization and transformation is presented here.

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E4

P97

Fig. 1 Schematic representation of the circular genomic organization of HPV-16. The early (E) and late (L) genes and the long control region (LCR) are indicated.

The late genes, L1 and L2, which sequences are highly conserved among all papillomaviruses (Bernard et al., 1994a),encode the capsid proteins. Following their synthesis, these proteins are directed to the cell nucleus, where viral particle assembly takes place (Zhou et al., 1991). These viral proteins are expressed exclusively in productive infections occurring in differentiated keratinocytes. The product of the E4 gene, which is expressed as a late gene despite its localization in the early region, seems to be involved in the maturation and release of papillomavirus particles. In cultured keratinocytes, the E4 protein is found in association with the keratin cytoskeleton (Doorbar et al., 1991; Roberts et al., 1993), suggesting that it may be involved in virus release in the cornified layers of the epithelium. This process does not seem to be cytolytic. The E6 and E7 genes are located in the 5’ end of the early region. These genes code for multifunctional proteins that interfere with cell growth and are transcribed from the same promoter, giving rise to a polycistronic mRNA. The proteins encoded by the E6 and E7 genes of the high-risk HPV types, and not the low-risk types, are directly involved in cellular transformation. Several in vitro assays have demonstrated that HPV-16 and -38 can efficiently transform established rodent cells (Yasumoto et al., 1986; Bedell et al., 1987; Vousden et al., 1988). However, the E6 and E7 genes of these viruses can only transform primary cells in the presence of an active oncogene (Matlashewski et al., 1987; Phelps et al., 1988; Storey and Banks, 1993).Another important feature of the high-risk E6 and E7 proteins is their ability to immortalize primary keratinocytes from human foreskins or cervical epithelium (Durst et al., 1987; Pirisi et al., 1987; Schlegel et al., 1988;

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Woodworth et al., 1988; Miinger et al., 1989). E6 and E7 proteins can influence transcription from different viral and cellular promoters (Phelps et al., 1988; Lamberti et al., 1990; Desaintes et al., 1992; Lechner et al., 1992; Oshima et al., 1993). As a consequence of the activity of these oncoproteins, genomic instability results, leading to the fully malignant phenotype (Tlsty et al., 1994; White et al., 1994). E6 proteins of the high-risk HPV types bind the tumor suppressor protein p53 (Wernesset al., 1990). This association induces the ubiquitin-dependent degradation of p53 (Scheffneret al., 1990; Crook et al., 1991; Huibregtse et al., 1994), removing the p53-dependent control of the cell cycle. The nuclear phosphoprotein E7 associates with the product of the retinoblastoma gene (pRB) (reviewed in Miinger and Phelps, 1993), a tumor suppressor gene important in the negative control of cell growth. The binding of high-risk HPV E7 releases the E2F transcription factors bound to pRB, which is now free to activate the transcription of several genes involved in the progression of cells through G1 into S phase of the cell cycle. E7 was shown to interact with cyclin A and cyclin-dependent kinase 2, also disturbing cell cycle progression (Tommasino et al., 1993; Vousden, 1994). Another early gene involved in growth stimulation and cell transformation is E5, the product of which is a small hydrophobic protein bound to the cellular membrane (Halbert and Galloway, 1988). HPV E5 has been shown to synergize with epidermal growth factor (EGF) in the stimulation of epithelial cell proliferation. (Leechanachai et al., 1992; Bouvard et al., 1994). In E5-expressing cells, the turnover and phosphorylation of EGF receptors are increased (Straight et al., 1993), possibly leading to an enhanced response to positive extracellular growth signals (Banks and Matlashewski, 1993). The products of the E2 gene are involved in transcriptional regulation of the HPV genome (Thierry and Yaniv, 1987; reviewed in McBridge et al., 1991). When this gene is disrupted, as a result of the integration of the viral genome seen in cervical intraepithelial lesions and cancers, higher levels of E6 and E7 are observed (Sang and Barbosa, 1992). This increase in E6 and E7 correlates with an increased immortalization activity in cell cultures (Romanczuk and Howley, 1992). The replication of the papillomavirus genome is controlled by the proteins encoded by the E l gene (reviewed in Lambert, 1991). Recently, El-E2 heterodimers have been shown to stimulate replication initiation (Sverdrup and Kahn, 1994). Upstream of the early open reading frames resides a DNA segment of about 900 bp that contains the viral origin of replication and the transcriptional responsive elements that regulate HPV gene expression. This is designated as the LCR, where several &-responsive elements both for viral transcription factors (E2) and cellular transcription factors have been mapped. The latter factors include activator protein-1 ,glucocorticoid responsive ele-

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ment, nuclear factor-I, octamer binding factor-1, and transcription repressor YY1 (Chan et al., 1989,1990; Glass et al., 1989; Chong et al., 1990,1991; Sibbet and Campo, 1990; Apt et al., 1993; Cuthill et al., 1993). A very complex interplay of factors is therefore responsible for the regulation of the HPV life cycle. Knowledge of the mechanisms underlying these regulatory processes may contribute to unravel the events involved during malignant progression of HPV lesions.

Ill. TAXONOMY AND GENOMIC VARIABILITY OF PAPILLOMAVIRUSES The phylogenetic trees derived from extensive nucleotide comparisons reflect the relationships between papillomaviruses and their hosts (van Ranst et al., 1992; Bernard et al., 1994a). Chan et al. (1995), based on the greatest diversity between types, distribute all described papillomaviruses into five major branches, of which two contain animal papillomaviruses and three contain HPVs. The largest HPV supergroup includes all genital HPV types, as well as some nongenital HPVs. Most HPV types associated with epidermodysplasia verruciformis, such as HPV-5 and -8, are grouped together in another supergroup. It can be concluded that taxonomic classification based on the nucleotide sequences of several papillomavirus genes clearly correlates with their tissue tropism and oncogenic potential. Based on extensive DNA sequencing analyses performed on hundreds of HPV isolates from clinical specimens and cervical cancer-derived cell lines, a number of variants or lineages have been described in which the nucleotide sequence does not vary more than 2% in coding and 5% in noncoding regions of the viral genome (Bernard et al., 1994b; Galloway, 1994). The most detailed studies of genomic variability have been done with HPV-16 (Icenogleet al., 1991; Chan et al., 1992; Eschle et al., 1992; Ho et al., 1993a; Xi et al., 1993; Yamada et al., 1995), followed by HPV-18 and -45 (Ong et al., 1993). This type of analysis has also been conducted with HPV-6 and -11 (Kitasato et al., 1994; Heinzel et al., 1995), HPV-5 (Deau et al., 1993), and HPV-8 (Deau et al., 1991). Through nucleotide sequence comparisons, the origin and spread of these viruses has been reconstructed. Data obtained from 25 different geographic regions in the world indicate that HPV-16 has evolved along five major branches, two being present mainly in Africa, two in Asia, and one mainly in Europe and India (Ho et al., 1993a). This study revealed that colonization of the Americas by Europeans and Africans is reflected in the composition of their HPV-16 variants. The ancient spread of papillomaviruses and their low rate of evolution suggest that HPVs have coevolved with their natural hosts over a period of several million years. The

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diversity observed within HPV-16, -18, and probably other types was estimated to represent more than 200,000 years of evolution from a precursor genome that possibly originated in Africa (Ho et al., 1993a; Ong et al., 1993). Nucleotide sequence variation has being used as an important tool for epidemiological studies of viral transmission and persistence. Ho et al. (1993b) have indicated the utility of this approach in a study of sexual transmission of HPV-16 among couples in Singapore. The same HPV-16 variant was identified in both partners of 50% of the couples, but the other half carried mismatched virus. In a cohort study of young women, Xi et al. (1995) have found 16 different HPV-16 variants, among which one variant was persisted over time while the other variants were transiently detected. We are using molecular variant analysis in a prospective study of HPV DNA persistence in the uterine cervix (Franc0 et al., 1994; Villa et al., 1997) (see Section Vl). From the studies on the genomic variability of HPVs, novel types have been described that can be placed in phylogenetic trees, indicating that they may represent authentic HPV genomes (Manos et al., 1989, 1994; Ong et al., 1994; Peyton and Wheeler, 1994; Peyton et a/., 1994). These data provide important information for the accurate HPV typing required both for the clinical diagnosis of HPV infections and for large-scale epidemiological studies. However, the risk of cervical disease that can be attributed to these types awaits further epidemiological investigations. Few nucleotide differences found in HPV variants correspond to changes in amino acids, and therefore it is of much interest to define alterations that may interfere with the functional or antigenic properties of specific viral proteins. This is of fundamental importance in the analysis of the oncogenic potential associated with certain variants, still a controversial issue. Genomic variation in the E6 and E7 genes of HPV-18 has not been associated, to date, with pathological grade (McLachlin et al., 1994). On the other hand, analysis of HPV-18 E2 sequence variation in ClN and cervical cancers revealed a subtype with decreased oncogenic potential (Hecht et al., 1995). Nevertheless, further studies are required to determine whether particular variants of high-risk HPVs can predict the risk of cervical neoplasia. This question also has implications in the design of vaccines directed against HPVs. In this context, mutations in L1 or L2 have been shown to interfere with the antigenic properties of these proteins (Kirnbauer et al., 1993). On the other hand, a report by Cheng et al. (1995) indicates that two HPV-16 variants that differ in their L1 protein by seven amino acids are serologically cross-reactive. This finding may indicate that a vaccine developed with a single HPV-16 variant would protect against all HPV-16 infections. Further studies are required before a general conclusion can be drawn concerning the large number of HPV variants described so far, the distribution of which, in diverse populations,

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can be very different. It has been reported that a specific variation in the HPV16 E6 protein, isolated from cervical cancer of HLA-B7 individuals, interferes with the T-cell cytotoxic immune response (Ellis et al., 1995).This finding may have important implications both for the knowledge of the cellular immune response to high-risk HPVs and in the future development of therapeutic vaccines against these viruses. Several research groups are involved in the development of HPV vaccines, and clinical trials are already in progress (Muiioz et al., 1995). Therapeutic vaccines against HPVs are based on the E6 and E7 proteins of the high-risk types and on their cytotoxic T-cell epitopes (Kast et al., 1993). A report indicates the progress in testing a recombinant vaccinia virus expressing these early proteins (Borysiewicz et al., 1996). Prophylactic vaccines directed against the late proteins L1 and L2 were shown in animals to completely protect from the viral infection (Chandrachud et d., 1995; Suzich et d., 1995). It is expected that such vaccines directed against the HPV late proteins will be able to prevent infections in humans.

IV. EPIDEMIOLOGICAL ASPECTS Epidemiological studies have clearly indicated that both HPV infection and cervical cancer are strongly influenced by measures of sexual activity, in the woman and her partner (Franco, 1991; Brinton, 1992; Muiioz and Bosch, 1992; Schiffman, 1994; IARC Working Group, 1995). As represented in Figure 2, persistent infections by high-risk HPV types are important determinants of squamous intraepithelial lesions (SILs). However, additional factors contribute to the multistage process leading to cervical cancer, as discussed later. Conditions leading to immunosuppression increase the risk of acquisition and progression of HPV-associated lesions (IARC Working Group, 1995).An increasing number of studies have shown that renal transplant patients or women infected with the human immunodeficiency virus are at a higher risk of HPV-associated CIN. Molecular epidemiology surveys using the polymerase chain reaction (PCR) to detect the virus have unveiled the sexually transmitted profile of cervical HPV infection (Ley et al., 1991; Schiffman et al., 1993; IARC Working Group, 1995). However, not all PCR-based studies conducted in different populations have uniformly reproduced these results. Data derived from these studies indicate that the association between sexual activity and overall HPV prevalence can be either strong (Ley et al., 1991; Bauer et al., 1993; Wheeler et al., 1993), moderate (Rohan et al., 1991; Hildesheim et al., 1993), or nonexistent (Kjzr et al., 1993). Since misclassification of HPV infection is less of a concern in these studies using PCR compared to previous

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-_---____--____

Persistent

lnvasive

HPV infection with

1

Cofactors: smoking, OC use, parity, other STDs

cancer

1

Fig. 2 Interplay of risk factors in the natural history of HPV infections and cervical neoplasia. (Modified from an original scheme by Dr. Eduardo L. Franco, McGill University, Montreal, Canada.)

studies of employing different methodologies, it is conceivable that the variability among results might be caused by differences across populations in the relative prevalence of HPV types with greater or lesser transmissibility by the sexual route. A recent cross-sectional study that we conducted in Northeastern Brazil provided evidence in this regard (Franco et al., 1995). Infection with low oncogenic risk types was only weakly associated with sexual behavior among women younger than 40, whereas sexual activity variables were strong predictors of infection with HPV types classified as being of high oncogenic risk, regardless of age. A very large survey has shown that the prevalence of specific HPV types in cervical carcinomas may vary according to the geographic origin of the specimen (Bosch et al., 1995). Risk of HPV infection seems also to be independently influenced by other variables, such as parity, oral contraceptive use, and current smoking (Bauer et al., 1993; Schiffman et al., 1995).However, by far the most important determinant of risk of HPV infection is age, with most studies indicating a sharp decrease in prevalence after age 30. The decrease in HPV infection risk with increasing age seems to be independent of sexual activity (Bauer et al., 1993; Wheeler et a/., 1993) and, at least in certain populations, it may be restricted to low oncogenic risk types (Franco et al., 1995). Most of our knowledge concerning HPV-associated pathogenesis is derived from molecular examination of viral genomes and proteins, since these

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Table 1 Classification of Human Papillomavirusesby Oncogenic Risk Oncogenic risk category

HPV type"

Low riskb High-riskc

6, 11, 26,42,44, 54, 70, 73 16, 18, 31, 33, 35, 39,45, 51, 55, 56, 58, 59,66,68

'Types as in de Villiers (1994)and IARC Working Group (1995).The more prevalent types are highlighted, hut frequencies may vary according to geographic region (Bosch et al., 1995). 'Types isolated from benign lesions, mostly genital warts. Types known to he associated with different grades of SILs from the cervix, vulva, vagina, penis, and cervical carcinomas.

viruses are very difficult to cultivate and so far there are no animal models that support HPV infection and replication (Howley, 1996). About half of the known HPVs, and an even higher number of variants, have been isolated from genital mucosas (deVilliers, 1994; IARC Working Group, 1995).Genital HPV types are typically divided into groups based on frequency of association with malignant tumors and, thus, presumed oncogenic potential (Table I). For instance, exophytic genital warts contain HPV-6 and -11, types rarely found in cancers and therefore designated as low-risk types. The high-risk group includes HPV types 16,18, and 45, among others, which are strongly associated with carcinomas and their precursor lesions. Other types, less represented in cancers but very frequent in SILs, are included in the high-risk group, although in some reports they are referred as an intermediate-risk group. Currently, a number of cohort studies are in progress where sensitive and specific viral detection assays are being applied on multiple cervical specimens collected over ti me (IARC Working Group, 1995). Some of these studies will follow up large numbers of women, in different risk areas of the world, aiming to determine the predictive value of persistence of HPV infection with respect to the subsequent occurrence of cervical neoplasia, as discussed later. Several studies rely on different hybridization techniques or type-specific DNA amplification to define the HPV types present in the samples. This can provide an easy means of detecting transient infections once different HPV types are found in consecutive specimens. The difficulty, however, resides in defining persistence, since concordance of types during follow-up does not necessarily represent viral persistence, particularly if a common HPV type is detected, such as HPV-16. We have proposed a solution to this problem (Franc0 et al., 1994) by demonstrating the applicability of DNA sequencing techniques to detect HPV variants in epidemiological studies of the natural history of cervical neoplasia (see later). These variants are identified on the basis of mutational patterns in the DNA (Bernard etal., 1994b), thus allowing interpretation of persistence on firmer grounds.

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V. HPV INTERACTION WITH COFACTORS Mutagens and immunosuppressants, such as those present in smoke constituents, may cooperate with papillomavirus in the induction of malignancies in different ways (Jackson et al., 1993; IARC Working Group, 1995). Detection of higher levels of these compounds in cervical secretions of smokers compared with nonsmokers (Schiffman et al., 1987), and the demonstration of a mutagenic activity in cervical cells similar to that observed in lung tissue (Phillips and NiShC, 1993), point to an important role for these compounds in genital carcinogenesis. In fact, HPV-18-immortalized human foreskin keratinocytes exposed to N-methyl-N-nitrosourea and 12-0tetradecanoylphorbol-13 acetate induced tumors in nude mice (Garrett et al., 1993). Recently, cigarette smoke concentrates were shown to transform HPV-16-immortalized endocervical cells (Yang et al., 1996). Local immunological depletion caused by smoking or mutagenic action of smoke contents could favor virus persistence, contributing to malignant conversion. Hormones interact with papillomavirus genomes, modifying their expression. The upstream regulatory region of papillomaviruses was shown to contain sequences similar to the glucocorticoid responsive element that are indeed inducible by steroid hormones, as shown by the enhancement of HPV-16 transcription upon dexamethasone treatment (Chan et al., 1988). Transformation of mouse primary cells by HPV-16 in combination with an activated oncogene has been shown to be dependent on glucocorticoids: cells became transformed and tumorigenic in the presence of dexamethasone (Crook et al., 1988; Pater et al., 1988). Furthermore, these experiments could be reproduced in the presence of progesterone and progestins, the pharmacologically active component of oral contraceptives (Pater et al., 1990). This effect could be antagonized by RU486, a synthetic steroid that binds to the receptor of glucocorticoids and progesterone (Pater and Pater, 1991). Dexamethasone has also been shown to differentially enhance or inhibit the transcription of HPV E6 and E7 genes in selected HPV-18-positive cervical carcinoma cell lines (von Knebel-Doeberitz et al., 1991). These results may be explained by cis effects exerted by host cell DNA sequences differing among the individual cell lines tested. The levels of the HPV-18 E7 protein in HeLa and C4-1 cervical carcinoma cell lines have been shown to be increased by hydrocortisone, while progesterone, estrogen, or testosterone had no effect (Selvey et al., 1994). No progesterone or estrogen receptors were detected in these cell lines. On the other hand, in human ectocervical cells, a marked increase in HPV-16 mRNA was observed in the presence of glucocorticoid or progesterone (Mittal et al., 1993a). This effect was inhibited by the antiprogestin RU486 and was shown to be dependent on three

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hormone-responsive elements present in the viral regulatory region (Mittal et al., 1993b). Cervical tissues contain estrogen and progesterone receptors, with highgrade cervical lesions exhibiting the highest levels (Monsonego et al., 1991). In this study, cervical carcinomas expressed low levels of progesterone receptors and showed an absence of estrogen receptors. Elevated progesterone receptor levels were more significantly correlated with HPV-16- and -18-positive cervical lesions than to HPV-negative samples. However, additional studies are required to establish the relevance of the association between sex hormones and their receptors in HPV-driven malignant transformation in vivo. The concept that herpes simplex virus (HSV) and HPV may act as syncarcinogens (zur Hausen, 1982) has not been supported by epidemiological studies (Muniioz et al., 1994; Becker et al., 1994). On the other hand, experimental evidence is available showing that segments of HSV-2 (DiPaolo et al., 1990; Dhanwada et al., 1993) or human herpesvirus 6 (Chen et al., 1994) can transform HPV-immortalized cells. The relevance of these interactions in vivo awaits further investigation. The association between HPV and other sexually transmitted agents, such as Trichomonas, Chlamydia, cytomegalovirus, Treponema, and Gardenerella, in the development of cervical neoplasia has been addressed in some epidemiological studies, but so far no clear picture has emerged. The effects of diet and alcohol consumption in the risk of HPV-associated malignant tumors have not been well established so far, and require further investigations.

VI. VIRAL PERSISTENCE AND DISEASE PROGRESSION It has been known for many years that HPV genomes persist in malignant tumors as well as in their derived cell lines (Pfister, 1987; zur Hausen, 1989). However, little is known about risk determinants of persistent HPV infection, since cross-sectional surveys can only probe for overall HPV infection, which may be both transient and persistent. Viral DNA can be detected in 10%-50% of asymptomatic women of reproductive age (Franco, 1991; Schiffman et al., 1993). Interestingly, however, when additional cervical specimens are taken from these women in follow-up surveys, the majority of the infections are found to be transient, and only a small proportion of the women tend to harbor the same HPV type in subsequent specimens (Moscicki et al., 1992,1993; Hildesheim et al., 1994; Franco et al., 1994; Evander et al., 1995; Villa et al., 1997). Hildesheim and colleagues (1994) have

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shown that persistence is higher among older women and tends to decrease with time between samplings. Moreover, persistence is higher among women infected with high-risk HPV types. These findings may suggest that only persistent infections of the cervical epithelium trigger tumor development. Epidemiological studies have indeed indicated that, when a woman is persistently infected with oncogenic types of HPV (about 15 among more than 30 types found in genital mucosas), she is more likely to develop cervical neoplastic lesions than when she is only transiently infected with HPV (zur Hausen, 1991; Koutsky et al., 1992; Schiffman, 1995).Ho et al. (1995) have shown that HPV infection detected in previous visits and that persisted during follow-up was highly predictive of a woman’s short-term risk for developing SILs. Furthermore, persistent high levels of HPV DNA were more predictive of persistent cervical lesion (CIN). Similar results were obtained by Remmink and colleagues (1995) in a study that aimed to evaluate the predictive value of HPV genotyping for progressive CIN disease. They concluded that persistence of high-risk HPV types leads to progression of cervical lesions, which is not observed in the absence of HPV DNA or in the presence of low-risk types. In this study, the effect of HPV persistence in disease progression was influenced by age, number of sexual partners, and smoking. More recently, Brisson et al. (1996) have shown that persistent detection of HPV DNA was higher in the cervices of patients using oral contraceptives for more than 2 years. We are conducting a large cohort study of the natural history of HPV infection and cervical neoplasia in a population of low-income women in Sao Paulo, Brazil, one of the highest risk areas worldwide for cervical cancer (Franco et al., 1997). Two thousand subjects are being followed up over a 5-year period, with prescheduled returns every 4 months in the first year and once yearly thereafter. In each of these visits, cervical smears are taken for Papanicolaou (Pap)cytology and HPV testing by consensus PCR, with subsequent probing for 27 individual types. A blood sample is also obtained for HPV serology testing. We are documenting persistence of infections on the basis of molecular variants of HPV (Franco et al., 1995), which provides a much finer level of detail than simple HPV typing, by sequencing a PCR-amplified fragment of the LCR of the viral genome. Moreover, this may allow us to describe variants of high-risk HPVs with differential oncogenic potential. The LCR sequence analyses completed so far indicate that the same molecular variants were present in all isolates, strengthening the interpretation of persistence. So far, we have found five distinct variants of HPV-16 associated with persistent HPV infections, but only one such HPV-18 variant (Villa et al., 1997). In the study reported by Xi et al. (1995), HPV-16 variants were analyzed by single-stand conformation polymorphism analysis of PCR-amplified LCR fragments, followed by sequencing. In this study, it was observed that one

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particular variant tended to persist over time, which could indicate some biological advantage that would be important in disease progression. Additional studies are required to test this interesting possibility.

VII. VIRAL BURDEN AND CERVICAL DISEASE The number of HPV genomes present in cells has been shown to correlate with the severity of cervical disease, since higher levels of HPV-16, as determined by a semiquantitative PCR technique, were found in high-grade CIN as compared to low-grade lesions (Cuzick et al., 1992, 1994; Bavin et al., 1993; Terry et al., 1993). In cervical cancers, HPV amplification has been shown to directly correlate with retention of El-E2 viral genes (Berumen et al., 1994). Evidence suggests that a high viral burden may predict the persistence of HPV infection in subsequent samplings (Ho et al., 1995; Brisson etal., 1996; Franco et al., 1997). There are, however, important differences in the methods used to quantify viral genomes. We have proposed an HPV DNA quantitation method based on low-stringency PCR that can address with precision the issue of viral burden (Caballero et al., 1995). This quantitative, competitive PCR is internally controlled and therefore is not subject to variations due to the amount of total DNA present in the reaction or the amplification of internal standards. We anticipate that the information on viral burden stemming from ongoing cohort studies will contribute to the development of new strategies for cervical cancer prevention.

VIII. HPV IN CERVICAL SCREENING PROGRAMS The incidence and morbidity due to cervical cancer have declined in the last decades in most countries that perform screening programs that rely on Pap smears for the detection of abnormal cervical cells. However, this neoplasia continues to pose a problem in regularly screened populations, being

a major public health problem in developing countries (Franco, 1993; Birley, 1995).This is due in part to the high frequencies of false-negative results (up to 40%) obtained with the Pap test, because of errors in both the sampling and the interpretation of the smears. As the role of certain HPVs in cervical carcinogenesis becomes indisputable, the detection of HPV in cervical samples is being considered in conjunction with cytology (Reid and Lorincz, 1991; Namkoong, 1995). In a retrospective study, it was clearly shown that detection of high-risk HPVs by PCR can reduce the number of false-negative cytology results (Walboomers et al., 1995).

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Positive predictive values of HPV testing are, however, very low in most asymptomatic women because of the high rates of HPV DNA detection in normal cervical smears (between 15 and 40%).Therefore, the indiscriminate use of an HPV test in conjunction with cytology would be impractical and not cost effective (Nuovo and NUOVO,1991). Moreover, adoption of the Bethesda system for cervical cytology reports (National Cancer Institute Workshop, 1989) resulted in an increase in the overall diagnosis of lowgrade SILs and created a new category for borderline lesions, the “atypical squamous cells of undetermined significance” (ASCUS). While there is consensus that high-grade SILs or more advanced lesions need immediate treatment, uncertainty exists about management options for low-grade SILs and ASCUS (Solomon, 1993). In a study of atypical smears reclassified by the Bethesda system, high-risk HPV types were detected by PCR in 60% of the smears reclassified as SIL, compared with 30% of ASCUS smears and only 10% of negative smears (Sherman et al., 1994). In another study employing hybrid capture to detect HPV DNA, the high predictive value of HPV positivity in patients with ASCUS or borderline Pap smear results were confirmed (Cox et al., 1995). It is therefore suggested that HPV testing should be used to guide the management of these borderline lesions (Ferenczy, 1995; Namkoong, 1995). Another point of concern is that little is known about the natural history of HPV infections. Most HPV-infected individuals do not develop clinical symptoms, and only a small percentage will develop HPV-associated cancer. Except for a few prospective studies, most of the data have come from retrospective studies, which do not provide information on the dynamics of cervical HPV infection in the same individual (Morrison, 1994). A number of research groups are currently engaged in large prospective studies of the predictive value of HPV infection with respect to cervical cancer development (IARC Working Group, 1995). As previously mentioned, a substantial proportion of prevalent cases of HPV infection are transient and are probably of little clinical significance. However, women who harbor persistent infections with high-risk HPV types experience much greater risks of developing cervical lesions. It is therefore expected that research on the determinants of viral persistence will contribute to the formulation of new algorithms for cervical cancer prevention (Schiffman, 1995; Franco et al., 1997).Viral load determination in Pap smears might additionally increase the predictive value of HPV testing in the screening of women at risk to developed high-grade cervical lesions. The prognostic significance of HPV in cervical tumors needs consideration, but so far the evidence is conflicting. Some studies have shown that the absence of HPV in the tumor confers a worse prognosis than if any HPV types are present (Riou et al., 1990; Higgins et al., 1991; DeBritton et al., 1993; Franco et al., 1996a). In contrast, other studies shown an association

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between the presence of high-risk types and poor clinical outcome (Barnes et al., 1988; Walker et al., 1989; Girardi et al., 1992; Rose et al., 1995; Unger et al., 1995).It has been suggested that the detection of HPV DNA in tumorfree regional lymph nodes is a potential prognostic marker for cervical carcinoma relapse (Ikenberg et d.,1996).However, further studies are required to evaluate the significance of these findings for the prognosis of HPV-associated neoplasias. Cervical cancer screening is a major public health and economic problem, mostly in developing countries where the attendance of women to the program is generally poor (Birley, 1995; Pontkn et d., 1995). Any method that could improve the detection of precursor lesions or cancer in its earlier stages must be considered for the reduction of the morbidity and mortality rates associated with this neoplasm. However, before large-scale HPV testing can be recommended, further research is required, including the evaluation of the available methods for HPV detection and the development of new diagnostic tests, such as those based on serology. Several studies used serological assays to detect antibodies against HPV proteins (IARC Working Group, 1995). Currently, the use of an enzyme-linked immunosorbent assay using HPV-16 viral-like particles as antigen (Kirnbauer et al., 1993, 1994) is providing relevant information about HPV infection and associated neoplasia (Muller et al., 1992; Sun et al., 1994; Nonenmacher et al., 1995). Multidisciplinary studies of the natural history of HPV infections are still required to provide information about the viral life cycle, the factors that may influence its maintenance in the individual, and its influence in disease progression. Considering the increasing amount of information generated by several studies around the world, we may expect in the near future to apply new screening strategies for the prevention of cervical cancer. The future development of both prophylactic and therapeutic vaccines may contribute to the control of these common infections and their associated diseases.

ACKNOWLEDGMENTS I am indebted to Drs. Eduardo L. Franco and Andrew J. G. Simpson for critical reading of this review. Maria Stella Leme is acknowledged for the excellent preparation of the manuscript.

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HERe2heu Protein: A Target for Antigenespecific Immunotherapy

of Human Cancer Mary L. Disis and Martin A. Cheever Division of Oncology, University of Washington, Seattle, Washington 981 95

I. Introduction 11. HER-2/neu Vaccines for Cancer Therapy A. Patients with Cancer Have Preexistent HER-Z/neu-Specific Immunity B. Vaccine Strategies to Generate or Augment HER-2/neu Immunity 111. Potential Pitfalls Associated with HER-2/neu-Specific Immunotherapy A. Autoimmune Toxicity B. Immunological Escape IV. HER-2/neu-SpecificAntibodies for Cancer Therapy A. HER-2/neu-SpecificAntibody Therapy Can Interfere with Tumor Growth B. Strategies to “Enhance” HER-2/neu-Directed Antibody Therapy V. Conclusion References

The HER-2/neu oncogenic protein is a tumor antigen. Some patients with cancer have a preexistent immune response directed against the HER-2heu protein. Effective cancer vaccines targeting HER-2/neu will be able to boost this immunity to potentially therapeutic levels. In addition, HER-a/neu-directed monoclonal antibody therapy has been effective in eradicating malignancy in animal models and has shown benefit in the treatment of human HER-2/neu-overexpressingcancers. This review outlines studies that define HER-2/neu-specific immunity in patients with cancer and overviews the current vaccine strategies for generating or augmenting neu-specific immunity. The potential problems associated with eliciting HER-2/neu-specific immunity are addressed, including the question of precipitating autoimmune toxicity against this “self”-protein and the mechanisms of immunological escape that may play a role in preventing effective function of the HER-2heu-specific immune response. Finally, antibody-based HER-2/neudirected therapies are overviewed. HER-2heu is a prototype antigen for groups investigating innovative modifications of monoclonal antibody technology, and cutting edge therapies targeting this antigen are being contemplated for clinical use in the treatment of human malignancy. Immune-based treatments designed to target the HER-2/neu oncogenic protein will soon give the clinical oncologist new therapeutic weapons, directed against a biologically relevant tumor-related protein, with which to fight cancer.

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I. INTRODUCTION The HER-2heu oncogenic protein is receiving increasing attention as a target for antigen-specific immune therapy. The excitement surrounding HER-2/neu-directed immune therapy has been fueled by several recent studies in animal models and in humans establishing that HER-2/neu-specific cancer treatment can be effective. In one study, a monoclonal antibody specific for the extracellular domain (ECD) of rat neu, a homolog to human HER-2/neu, was infused into mice transgenic for rat neu. These animals are destined to develop and die of breast cancer. This antibody to neu dramatically prevented the development of breast cancer (Katsumata et af., 1995). Importantly, in initial human clinical trials, infusion of another HER-2heuspecific monoclonal antibody induced tumor regression in some patients with advanced cancer (Beselga et al., 1996). Clinical responses in patients with advanced cancer using unconjugated antibodies predict that even more effective antibody regimens can be designed. Many approaches to antibody therapy are being contemplated and evaluated. In separate studies, immune responses directed against the HER-2heu protein have been discovered to exist in some patients with HER-2/neu-positive cancer (Disis et al., 1994a; Fisk et af., 1995; Peoples et a!., 1995a). The existent immune responses occur as a consequence of overexpression of HER-2heu by autologous cancer cells. It has not yet been proven that existent immunity to HER-2heu predicts an improved survival, but existent immunity does predict that vaccines will be able to induce and boost immunity to HER-2heu. Existent antibody, helper T-cell, and cytotoxic T-cell immunity to HER-2heu has been detected in humans. Animal models have confirmed that vaccine regimens can induce and boost both antibody and Tcell immunity (Disis et af., 1996b). In humans, vaccine studies are being contemplated and planned by multiple research groups. Given the surety that immunity can develop in humans, the current therapeutic questions are how best to immunize, whether vaccine-induced immunity can mediate a therapeutic effect, and what types of immune responses will be most effective. Part of the enthusiasm for targeting immunotherapy against HER-2heu centers around the biological characteristics of the molecule. HER-2/neu is a member of the epidermal growth factor receptor family and is presumed to function as a growth factor receptor (Bargmann et al., 1986; Coussens et al., 1985). This transmembrane protein consists of a cysteine-rich extracelMar domain that functions in ligand binding and a cytoplasmic domain with kinase activity. Monoclonal antibodies have been generated to the ECD of HER-2/neu protein that are capable of inducing either stimulatory or inhibitory signals to the receptor (Bacus et al., 1992; Lupu et al., 1992). In humans, the HER-2heu protein is expressed during fetal development (Press et al., 1990). In adults, the protein is weakly detectable in the epithelial cells

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of many normal tissues by immunohistochemical staining. The HER-2/neu gene is present, in normal cells, as a single copy (Press et ul., 1990). Amplification of the gene and/or overexpression of the associated protein has been identified in many human cancers. Most effective immune responses involve both antibody and T-cell immunity. The transmembrane nature of HER2heu allows for targeting of antibody therapy as well as T-cell therapy against the protein. The overexpression allows for a relatively selective targeting of malignant cells. Cancers in which HER-2/neu gene amplification and resultant protein overexpression occur include carcinoma of the breast, ovary, uterus, and stomach and adenocarcinoma of the lung (Berchuck et al., 1990,1991; Kern et al., 1990; Slamon et al., 1987; Yonemura et ul., 1991). The clinical consequences of the oncogenic protein overexpression have been best studied in breast and ovarian cancer. HER-2/neu protein overexpression occurs in 2 0 4 0 % of intraductal carcinomas of the breast and 30% of ovarian cancers. It is associated with a poor prognosis in both diseases (Slamon et al., 1989).In breast cancer, HER-2/neu protein overexpression is linked to more aggressive disease, a younger patient, and resistance to standard doses of chemotherapy. This is the type of patient population that would benefit substantially from new treatment strategies. Investigations to be reviewed later justify the view that immunotherapies directed against HER-2Ineu will be useful in at least a subset of patients. First discussed is the concept that vaccines will be able to boost the preexistent HER-2/neu-specific immune responses already identified in some patients and will be able to induce immunity in other patients without preexistent immunity. Several strategies have been proposed to generate or augment HER2/neu-specific immunity. It is yet unknown which strategies will be the most effective and which patient populations will be the most appropriate to target. Second, the potential problems associated with HER-2/neu-specific immune responses are addressed, including the question of precipitating autoimmune toxicity against this self-protein and mechanisms of immunological escape that may be in play for preventing effective function of HER-2heuspecific immunity. Finally, antibody-based HER-2/neu-directed therapies are overviewed. Studies demonstrating that HER-2/neu-targeted antibody therapy can interfere with tumor growth in vitro and in vivo are presented. Many strategies have been proposed to increase the efficacy of monoclonal antibody therapy. HER-2heu is being viewed as a prototype tumor antigen by groups investigating innovative modifications of monoclonal antibody technology. Thus, HER-2heu protein will, in the near future, receive even greater attention as an immunotherapeutic target for the treatment of human malignancy. Future studies of the immunogenicity of this oncogenic protein will help, in general, to define the role of vaccine and antibody therapy against human cancer and specifically against HER-2/neu-positive cancers.

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11. HER-2heu VACCINES FOR CANCER THERAPY Existent antibody and T-cell immune responses directed against the HER2/neu protein are present in some patients with HER-2/neu-overexpressing tumors (Disis et al., 1994a; Fisk et al., 1995; Peoples et al., 1995a). Most patients have very low-level responses, but high-level responses do occur (Disis et af., 1 9 9 6 ~ )Identification . of existent immune responses was surprising as HER-2heu is a self-protein. Immune tolerance should prevent or limit the induction of immunity to self-proteins, and initial attempts to induce immunity in rats to rat neu failed (Bernards et af., 1987; Disis et af., 1996b). The demonstration that some patients have immunity directed against HER2/neu, however, shows that appropriately constructed HER-2/neu vaccines should be able to induce and/or substantially boost immunity to HER-2/neu. Strategies to vaccinate patients with HER-2/neu-positive cancers in remission offer the theoretical advantages of eliciting both antibody and T-cell responses and resulting in long-term immunological memory that may confer lifelong protection against the recurrence of HER-2/neu-mediated malignancy.

A. Patients with Cancer Have Preexistent

HER-2heu-Speciflc Immunity HER-2/neu protein overexpression, in human cancers, has been linked with the possibility of an endogenous immune response to tumor for several years. Investigators have correlated lymphocytic infiltration in breast cancer with HER-2/neu protein overexpression in the primary tumor. One study of over 100 breast tumors demonstrated a strong association between HER2/neu oncogene amplification and dense lymphocytic infiltration ( p = 0.05) (Tang et af.,1990).Other studies have related this observation to patient outcome. HER-2/neu overexpression is often considered to be associated with a poor outcome. However, that simple view was dispelled by an analysis of more than 1000 patients with breast cancer, evaluating the relative significance of HER-2/neu protein overexpression to a variety of prognostic factors (Rilke et af., 1991). That analysis revealed that HER-2heu overexpression is actually associated with a favorable prognosis in stage I patients who have lymphoplasmacytic infiltration in their primary tumor. The observation that HER-2heu overexpression and lymphoplasmacytic infiltration correlate with a good prognosis stimulated the speculation that immunity to HER2/neu might exist and be responsible for the improved outcome. Further analysis of the cellular infiltrate in these tumors revealed a preponderance of macrophages in tumors that overexpress HER-2heu and a preponderance of T cells in tumors that do not overexpress HER-2/neu (Pupa et al., 1996).

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Concurrent evaluation of HER-2/neu protein expression and infiltration and HER-2/neu-specific systemic immunity is needed to allow any conclusions as to whether the infiltration and/or the character of the infiltration is, in any way, connected with the occurrence of HER-2/neu-specific immunity. Existent immunity to HER-2/neu in humans was first reported in patients with breast cancer (Disis et al., 1994a). Both serum antibody and cellular immune responses were observed in a patient with HER-2/neu-overexpressing breast cancer (Disis et ul., 1994a). While the neu-specific antibody response was directed primarily to the intracellular domain (ICD), T-cell responses to epitopes in both the ECD and ICD were identified in this patient. Another study analyzed supernatants of Epstein-Barr virus (EBV)-transformed B cells from breast cancer patients for the presence of antibodies to HER-2heu (Pupa et al., 1993).Antibodies were found in six of seven patients whose tumors had HER-Uneu overexpression plus lymphoplasmacytic infiltration. No antibodies were found in supernatants of EBV-transformed B cells from patients with HER-2heu-negative tumors or in normal donors. In a larger study of well-characterized breast cancer patients, the presence of HER-2heu-specific antibody responses correlated with the presence of . at titers of greater than 1 : l O O breast cancer (Disis et al., 1 9 9 6 ~ )Antibodies were detected in 14 of 127 (11%)breast cancer patients versus 0 of 200 (0%) normal controls. Antibody responses could be substantial, with titers of greater than 15000 in 5 patients. These patients had stage I or I1 disease. The correlation between antibody and breast cancer shows that antibody responses have the potential to serve as a tumor marker for detecting breast cancer. HER-2/neu-specific antibody, however, is not absolutely specific for the presence of breast cancer as lower antibody titers were detected in some normals. HER-2/neu ECD is shed from tumor, and its presence in the sera has been proposed as a tumor marker for breast cancer. The possibility exists that serum antibody to HER-2/neu can clear the serum of ECD in some patients. Thus, detection of both serum ECD and antibody to HER-2heu may provide a more sensitive marker for breast cancer. Antibody responses have not been followed longitudinally in any patients. For patients in remission, the possibility exists that recurrence of a HER-2/neu-positive cancer will be heralded by a change in antibody titer and that changes in antibody titer will provide a more sensitive measure of recurrence in some patients than circulating ECD or other tumor markers. The presence of higher titer antibody correlated with the presence of HER2/neu expression by the primary cancer, but only for early-stage disease. There was no correlation with the presence of HER-2heu expression for advanced-stage disease. Low levels of antibody were detectable in some patients with advanced-stage breast cancer. Advanced-stage patients, however, had levels of antibody immunity specific for Cundida antigen, similar to that seen in a population not affected by cancer. Thus, the blunted antibody im-

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mune response to the HER-2/neu protein in these patients could not be explained entirely by the immunosuppression associated with advanced malignancy. The presence of HER-2heu-specific antibody responses in breast cancer patients and the correlation of antibody with HER-2heu-positive cancer in early-stage patients strongly imply that immunity develops as a result of exposure of patients to HER-2/neu protein expressed by autologous cancer. The most optimistic interpretation of the finding that immunity correlates with HER-2/neu expression in early-stage disease, but not advanced-stage disease, is that the development of an immune response to HER-2heu expressed by growing tumor limits further growth and metastasis. That interpretation may be difficult to establish, but may further stimulate the development and testing of vaccine strategies. The HER-2heu-specific antibody responses demonstrated in the patients described previously were immunoglobulin (Ig) G and IgA. Antibody class switch from IgM to IgG requires CD4' T-cell help. Helper T-cell immunity is often directed against the same protein as the antibody immunity. Accordingly, helper T-cell immunity to HER-2/neu has been detected in some patients with breast cancer (Cheever et al., 1995; Disis et al., 1994a). Initial studies demonstrated a substantial proliferative T-cell response to HER2heu protein in a patient with an antibody response (Disis et al., 1994a). The HER-2/neu was purified from a human breast cancer line. The individual also responded to synthetic peptides representing epitopes derived from the natural sequence of the ECD and the ICD. An analysis of T-cell proliferation to the protein and peptides in a greater number of patients (Cheever et al., 1995) demonstrates that the magnitude of the T-cell response varied greatly. Most patients tested did not respond. A few patients had significant responses, with stimulation indices of greater than 2. Many other patients tested had low-level responses that could be defined only by modified limiting dilution assay techniques (Reece etal., 1993). It is presumed that immunity in patients with low-level immune responses can be boosted using an effective HER-2/neu vaccine. The HER-2/neu protein is large and should have T-cell epitopes available for interaction with all human class I1 major histocompatibility complex (MHC) molecules. Peptide epitopes for the previous studies were chosen by using a computer protein sequence analysis package that used two searching algorithms, based on identifying motifs according to charge and polarity patterns or structure (Feller and Cruz, 1991). Although peptides that bind to class 11 molecules are now known not to necessarily form a-helical orientations, and each MHC molecules has its own unique binding motif, the searching algorithms had empirically been successful in identifying a substantial proportion (50-70%) of helper T-cell epitopes in foreign proteins (Bisset and Fierz, 1993; Roscoe et al., 1994). Further refinement in defining

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individual MHC molecule binding motifs might aid in identifying potential peptide epitopes. Cytotoxic T cells, as well as helper T cells, specific for HER-Yneu have been found to be present in patients with HER-2/neu-overexpressingcancer. Initial studies identified HER-2heu-specific cytotoxic T lymphocytes (CTL) in the malignant ascites of human leukocyte antigen (HLA)-A2 patients who had HER-2/neu-overexpressing ovarian cancers (Ioannides et al., 1993). CTL reactive with autologous ovarian cancer appeared to recognize the peptide p971-980. This peptide was derived from the ICD of HER-2/neu and was shown by computer modeling to contain a Rothbard and Taylor motif (Ioannides et al., 1992), as well as residues that would suggest preferential HLA-A2 binding. Further studies with this peptide revealed it could be used to elicit peptide-specific CTL responses by in vitro stimulations with peripheral blood mononuclear cells (PBMC)obtained from ovarian cancer patients who were HLA-A2 and had HER-2/neu-positive tumors (Fisk et al., 1994). The culture conditions utilized were appropriate for detecting already primed CTL, but were not appropriate for priming in vitro. Thus elicitation of CTL from patients with ovarian cancer under the conditions used provides presumptive evidence that the patients had existent CTL immunity to HER-2heu. HER-2/neu-specific CTL can be identified in patients with a variety of HER-2/neu-overexpressing cancers, including breast, ovarian, and lung (Peoples et al., 1995a; Yoshino et al., 1994).Generation of CTL requires processing and presentation of peptide fragments of HER-2heu by cancer cell class 1 MHC molecules. The HER-2/neu molecule contains many potential peptide fragments capable of binding to particular individual MHC molecules. Development of vaccine and T-cell therapy requires precise identification of CTL epitopes. Recently, two potential “naturally” processed HLAA2-restricted T-cell epitopes have been described for the HER-2/neu protein. One is derived from the ECD of the protein p369-377 (Fish et al., 1995), and the other is located in the transmembrane portion of the protein p654-662 (Peoples et al., 1995a). The peptide p369-377 was first identified based on consensus motifs for binding to HLA-A2. Experiments evaluated 19 peptides from the HER-2/neu protein sequence with HLA-A2 binding motifs for ability to be recognized by CTL specific for HLA-A2-positive, HER-2/neu-positive ovarian cancer cells (Fisk et al., 1995). Binding of p369-377 to HLA-A2 was confirmed in in vitro assays. CTL lines capable of specifically lysing HLA-A2-positive, HER-2heu-positive ovarian cancer cells were shown to lyse an indicator cell line “loaded” with the p369-377 peptide, but not control peptides. As confirmation that the ovarian cancer-specific CTL recognized the peptide, p369-377-“loaded7’ targets were shown to inhibit the tumor-specific killing. Another research group has shown that tumor-infiltrating lymphocyte

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populations derived from HLA-A2-positive patients with HER-2heu-positive breast or ovarian cancers recognize the p654-662 peptide (Peopleset al., 1995a). As confirmation that the p654-662 peptide is a naturally processed epitope, experiments demonstrated that the peptide could be eluted from class I MHC of both breast and ovarian cancer cells (Peopleset al., 1995a,b). Thus, the critical CTL question, whether peptides derived from the HER2/neu protein sequence are processed and presented by class I MHC molecules and can function as targets for specific lysis, is answered. It is not yet known whether the same peptides are presented by tumors sharing the same MHC molecules, or whether there is substantial heterogeneity in the naturally processed and presented peptide epitopes. Relying on the use of CTL from cancer patients to identify CTL peptide epitopes requires that the cancer patients have been immunized by virtue of the presence of overexpressed HER-2heu protein on their own malignant cells. The search for potential CTL epitopes, important for vaccine development, would be simplified if CTL could be generated by immunization in vitro. Studies have shown that in vitro priming to HER-2/neu peptides is possible (Cheever et al., 1995; Disis et al., 1994b), but the priming systems used are still fastidious and priming is very often not detected. Initial priming studies demonstrated that CTL specific for HER-2heu peptides could be elicited by priming in vitro (Disis et al., 1994b). Peptide-specific CTL could be generated after multiple in vitro restimulations to two of four peptides shown to bind to HLA-A2. CTL specific for one of the two peptides could mediate lysis of an HLA-A2 HER-2/neu-positivecancer cell line (Cheever et al., 1995). These results show that many, if not the majority, of the peptides capable of binding to class I MHC molecules do not represent naturally processed CTL epitopes. However, the results do confirm that priming in vitro represents a viable alternative method to identify HER-2heu CTL epitopes. More effective methods of priming in vitro would aid in identifying the best peptide epitopes for HER-2/neu vaccines. The use of dendritic cells (DC) to prime in vitro is the subject of intensive investigation by many groups. Several investigators are attempting to use DC to identify HER-Yneu epitopes. DC are present in peripheral blood in only exceedingly small amounts. However, they can be generated in a variety of ways using cytokines to affect growth and differentiation. The demonstration that CD34' stem cells from cord blood can be incubated with granulocyte-macrophage colonystimulating factor (GM-CSF)and tumor necrosis factor (TNF)to induce differentiation of a stem cell into the DC lineage pathway (Caux et al., 1992) greatly stimulated the development of procedures to expand peripheral blood- (Romani et al., 1994) and bone marrow-derived (Bernhard et al., 1995) DC in vitro. Initial studies to use DC to prime to HER-2heu peptides in vitro used CD34' stem cells derived from bone marrow and from peripheral blood stem cell collections, expanded in the presence of GM-CSF and TNF. Using helper

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T-cell epitopes, the in vitro-derived DC were shown to stimulate HER-2heu peptide-specific helper T-cell proliferative responses (Bernhard et al., 1995). Using HLA-A2-defined CTL epitopes, the in vitro-derived DC were shown to stimulate HER-2heu peptide-specific CTL capable of lysing HLA-A2positive, HER-Uneu-positive cancer cells (Cheever et al., 1995). In more recent studies, the use of GM-CSF in combination with interleukin (1L)-6 allowed a lower background proliferation and thus a higher stimulation index as compared to GM-CSF with IL-4 or TNF (Bernhard et al., 1996). DC can also be generated from PBMC. A technique using elutriated human monocytes obtained from apharesis and treated with IL-4 and GM-CSF results in the generation of large numbers of cells that are consistent in phenotype and function with “activated” CD83’ DC (P. A. Cohen, personal communication). These cells can be used effectively to generate HER-2heu peptide-specific CD8’ T cells in vitro, using PBMC from HLA-A2-normal donors (B. J. Czerniecki, personal communication). In this system, in all donors tested, T cells could be sensitized to p369-377, a known HLA-A2 CTL epitope (Fisk et al., 1995). Peptide-specific CD8’ T-cell responses could be detected in vitro within 8 days of culture. At the moment, HER-2heuspecific CTL have been generated with DC only by using peptides defined to bind to particular MHC molecules. It is as yet unknown whether immunological tolerance will inhibit or prevent priming to whole protein. One of the most remarkable techniques for identification of potential naturally processed class I MHC-presented epitopes is the use of double-transgenic mice (HLA-A2.1 and CD8’) (Lustgarten et al., 1996). These transgenic mice use a mouse T-cell receptor to recognize peptides restricted by the human class I MHC molecule HLA-A2.1. Expression of human CD8’ on the murine cells enhances recognition of HLA molecules on human cells and allows murine CTL to lyse human targets. This technique has been used to identify HER-2heu epitopes. Nineteen potential HLA-A2.1 binding peptides derived from the HER-2heu protein sequence were selected as potential candidate T-cell epitopes and synthesized. The double-transgenic mice were immunized with the peptides. Two of the peptides were found to be immunogenic in the mice, and both peptide-specific CTL lines were able to specifically lyse A2.1 HER-2heu protein-overexpressing tumor cell lines. Not every known HER-2heu CTL epitope was found by this method, but studies with HER-2heu show that the method can readily identify a portion of relevant epitopes.

B. Vaccine Strategies to Generate or Augment HER=2/neu Immunity Studies of HER-2heu vaccines were initially stymied by the presumption that immunological tolerance to this self-protein would prevent immuniza-

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tion. That presumption was based on studies in which rats were injected with a recombinant vaccinia virus vector expressing rat neu ECD (Bernards et al., 1987). The construct was immunogenic in mice, a foreign species, but was nonimmunogenic as a self-protein in rats. Immunization was assessed by antibody and delayed-type hypersensitivity (DTH) assays. Studies confirmed that immunological tolerance to rat neu exists. In those studies, attempts to immunize rats to whole rat neu protein failed; no immunity could be elicited (Disis et al., 1996b). Studies of rat neu are believed to be extrapolatable to humans, since the protein is 89% homologous to HER-2heu at the amino acid level and has a similar level of expression in the same normal tissues as is found in humans. The failure of rats to become immunized following vaccination with rat neu in a vaccinia vector or as a purified whole protein was notably inconsistent with the demonstration that some patients have an existent immune response to HER-2heu. The demonstration of this existent immune response to HER-2heu in some patients encouraged attempts to develop HER-2heu vaccines. One theory of tolerance is that tolerance is directed only to portions of selfmolecules. Immunization to foreign proteins normally elicits immunity to only a subset of potential epitopes, operationally defined as dominant epitopes, whereas other potentially immunogenic epitopes, operationally defined as subdominant epitopes, are ignored. Immunization to self-proteins usually fails to elicit immunity. It has been proposed that autologus T cells recognize and become tolerant to the dominant epitopes of self-proteins but “ignore” the subdominant epitopes (Cibotti et al., 1992; Sercarz et al., 1993). Tolerance to the dominant epitopes is elicited by a variety of mechanisms, including elimination in the thymus or periphery or induction of anergy. Immunity to subdominant epitopes can be elicited by immunization to proteins truncated to not contain the dominant epitopes or to peptides representing the subdominant epitopes alone. Consistent with this theory, tolerance to rat neu in rats could be circumvented by immunization with a peptide-based vaccine (Disis et al., 1996b). Rats were immunized with rat neu peptides designed for eliciting CD4’ Tcell responses. Antibody and T-cell responses specific for both the immunizing peptides and protein were generated in the peptide-immunized animals. No antibody or T-cell responses were observed in animals immunized with intact rat neu protein. These studies do not explain the precise mechanism by which patients become immune by virtue of overexpression of HER-2heu by primary tumor, but do substantiate that immunization is possible and that effective immunization regimens can be devised. The peptides shown to induce immunity in rats were from segments of rat neu that are identical in rats and humans. Moreover, some of the peptides used have been shown to be targets of existent immune responses in particular cancer patients. The neu-specific antibody induced in rats by immu-

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nization to the peptides could immunoprecipitate both rat neu and human HER-2/neu (Disis et al., 1996b). Similarly, the neu-specific T cells elicited in rats by immunization to the peptides could respond to both rat neu and human HER-2heu. Thus, the peptides chosen can be targets for vaccines in both humans and rats. Our group is initiating clinical trials with similar peptides in patients with HER-2heu-positive breast cancer or ovarian cancer to determine whether the peptides can prime the human immune system and whether the peptide-primed T cells can respond to HER-2/neu. Peptide-based vaccines offer an ability to manipulate the immune response through the use of defined immunogenic epitopes. Peptides can direct the response to target the most appropriate portions of the molecule and can guide the immune system to elicit either helper T cells or CTL. Other advantages include relative ease of construction and production, chemical stability, and a lack of infectious or oncogenic potential (Arnon and Honvitz, 1992).Standard methods for immunization of animal or humans to peptides are not yet evident. Few vaccine studies using peptide immunogens have been performed in humans. Two approaches to increase the immunogenicity of HER-2/neu peptides in development are the use of cytokine adjuvants and modification of peptide structure to improve MHC binding. Adjuvants may function in many different ways; they may affect the character and number of antigen-presenting cells (APC) in the inoculation environment, act as a depot to prolong antigen/APC exposure, or affect the pathway by which proteins are processed (Allison, 1994). Studies in rats show that soluble GM-CSF is a potent adjuvant for the generation of immune responses to peptides derived from a rat neu (Disis et al., 1996a). Rat neu peptides inoculated with GM-CSF could elicit a strong DTH response, whereas peptides alone were nonimmunogenic. GM-CSF was tested as an adjuvant because it has been shown in vitro to stimulate the growth of potent dendritic and macrophage APC. GM-CSF has also been shown to increase the immunogenicity of tumors in animal models (Dranoff et al., 1993). Transfecting the cytokine gene into tumor cells, and injecting the transfected cells subcutaneously, results in the production of GM-CSF by the tumor cell itself and enhances immunogenicity. The increased immune response is presumed to be mediated by the role GM-CSF plays in the maturation and function of APC such as DC and macrophages (Reid et al., 1992). In initial studies evaluating the mechanism by which soluble GM-CSF may function as an adjuvant (Disis et al., 1996a), rats were immunized with soluble GM-CSF intradermally or subcutaneously. Following immunization, an increase was observed in class 11-expressing cells in the lymph nodes draining the injection site, a surrogate marker for APC trafficking. GM-CSF given as a “classical” adjuvant (mixed with antigen and injected intradermally), resulted in the generation of both an antibody and a T-cell response to a protein antigen, tetanus toxoid. While the antibody responses to tetanus tox-

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oid induced by alum or complete Freund’s adjuvant (CFA) were of greater magnitude than that induced by GM-CSF, the T-cell responses were comparable. Extrapolating the model to a rat neu peptide vaccine in the rat, soluble GM-CSF was as effective as CFA in inducing a cellular immune response to rat neu (Disis et al., 1996a). The use of cytokines as vaccine adjuvants is appealing because their effect may allow for a choice of which arm of the immune response is enhanced, they may act as chemoattractants for immune cells, and they may further augment the vaccine’s protective effects. The use of other cytokines, in addition to GM-CSF, may allow development of an even more powerful adjuvant effect. GM-CSF may work as an adjuvant because of its in vivo effect on DC. Thus an alternative strategy for vaccination may be to purify large numbers of DC ex vivo and use them as a vaccine vehicle. The use of DC to present HER-2heu proteins or peptides may be a way to use “nature’s adjuvant” to enhance the immunogenicity of a tumor antigen (Steinman, 1991). DC have been shown to be very effective at priming to both peptide (Celluzzi et al., 1996) and protein (Paglia et al., 1996) tumor antigens in animal models in vivo. Several research groups are proposing to test whether immunization with DC plus HER-2heu peptides is an effective method of inducing a potent antitumor response. The use of GM-CSF versus DC as adjuvants for inducing neu-specific responses has not yet been examined and compared in the rat model. The methods must be compared for ability to prime, but also for ability of repeated immunizations to boost neu-specific immunity, in order to determine whether the simpler method (i.e., GM-CSF) will suffice. Another potential method for improving the immunogenicity of a peptide vaccine is to substitute the amino acids in a peptide, prospectively, in order to improve MHC binding without interfering in immune recognition. Immunogenic peptides derived from tumor antigens may bind to MHC molecules with low affinity. HER-2/neu is being used as a model system to determine whether low-affinity binding peptides derived from the HER-2/neu protein sequence can be made more immunogenic by amino acid substitutions (Fisk et al., 1996). Initial reported experiments evaluated 26 HER2heu-derived peptides for ability to bind to MHC class I molecules in vitro. Peptides were identified as high- and low-binding peptides. Subsequent studies identified residues in a defined HER-2/neu CTL-inducing peptide that were of critical importance in immune recognition. Amino acid substitutions at p l and p9 could markedly improve the HLA binding without interfering in immune recognition by neu-specific CTL. It has been proposed that such peptides will be more effective in vaccine formulations than the natural sequence peptides. In rat models, the most effective method of immunization to HER-2/neu is with peptides. However, very few alternative methods have been tested. Vaccinating humans with peptides may prove to be an effective method for

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generating HER-2/neu-specific immunity, but may be limited by having to match peptides used to each individual’s MHC molecules. Vaccine techniques that employ the use of intact protein and the patient’s own processing mechanisms would be preferable. Immunizing to protein would allow a vaccine to be universal for any combination of immune backgrounds. It is possible that immunization of patients with existent HER-2/neu-specific immunity with HER-2heu protein will boost the existent responses. Studies in rats described earlier, however, indicate that immunizing patients who demonstrate tolerance to the protein (i.e., patients with HER-2/neu-positive cancers and no existent immunity) will not be effective. Although immunization with peptides is more likely to be effective in humans, immunization of humans to whole HER-2heu protein must be attempted before it can be dismissed. Immunization with vectors capable of expressing HER-2/neu in vivo have been proposed by several research groups and most likely will be tested in the near future, including immunization with plasmid DNA, RNA, vaccinia virus, and Listeria monocytogenes. These proposed approaches are discussed next. Whole rat neu protein is not immunogenic in normal rats, yet some humans are immunized to HER-2heu by virtue of the protein being overexpressed by autologous tumor. It has been proposed that DNA immunization might best mimic the circumstances of overexpression by tumor. Vaccination by DNA immunization allows for persistent high-level protein expression in vivo by a few cells. The results of HER-yneu DNA immunization, published in abstract form, were very encouraging (Hu et al., 1995). Neu-transgenic mice were immunized with DNA constructs derived from the polynucleotide sequence of neu. The transgenic mice express rat neu on a mouse mammary tumor virus (MMTV) promoter and develop neu-overexpressing mammary cancers approximately 200 days after their birth (Guy et al., 1992). Immunization of transgenic mice with neu plasmid DNA resulted in the generation of neu-specific antibodies and some retardation of the development and/or growth of breast cancer. These results attest that DNA immunization holds great promise. Further refinements in expression promoters, methods of in vivo transfection, and the concurrent expression of cytokines and/or co-stimulatory molecules such as IL-2 and B7 might yield far superior results. Immunizing with transforming DNA capable of integrating into the host genome, such as HER-2/neuYraises significant safety concerns, Investigators are evaluating specific strategies to overcome this potential problem, such as the use of DNA fragments (Conry et al., 1996). One novel proposed strategy is mRNA immunization, which would bypass the possibility of integration of genetic material into the host genome. Preliminary studies using carcinoembryonic antigen (CEA) as a model antigen have shown that mRNA immunization can generate CEA-specific antibody responses (Conry et al., 1996) and might be more appropriate for immunization to HER-2heu.

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A vaccinia-ECD vector was not effective in immunizing rats to rat neu, but vaccinia continues to be an attractive vector for expressing HER-2lneu DNA in vivo. Vaccinia can infect a wide range of human cell types, has a high degree of infectivity, and allows expression of several genes either in one vaccinia vector or in several recombinant vaccinia viruses used together for coexpression at the same site and possibly the same cell. Coexpression of cytokines and/or co-stimulatory molecules has been shown to increase the effectiveness of recombinant vaccinia vectors (Hodge et al., 1995; Rao et al., 1996). Neither has been attempted yet with HER-2lneu. Both might allow circumvention of tolerance. Finally, engineering recombinant vaccinia viruses for exceedingly high levels of expression of the HER-2lneu self-protein might increase the likelihood of immunization to subdominant epitopes of self-proteins such as HER-2heu. A promising and unique biological vector is L. monocytogenes, a grampositive intracellular bacteria. Listeria is strongly immunogenic; the bacteria can escape the phagosome into the cytoplasm and present antigens into both the class I and class II antigen-processing pathways. Studies in in vivo animal models, using colon and renal tumors that express the influenza virus nucleoprotein (Np), demonstrate that immunization with a recombinant L. monocytogenes vector expressing NP can protect mice against tumor challenge (Pan et al., 1995) and can cause regression of established macroscopic tumors. Subsequent investigations in this system have compared the efficacy of NP-L. monocytogenes to NP-vaccinia constructs (Paterson et al., 1996). The NP-L. monocytogenes construct was much more effective than NP-vaccinia in mediating tumor regression of NP-encoding poorly immunogenic tumors. This observation was thought to be due to the role of innate immunity and the stimulation of macrophage to secrete IL-12 by L. monocytogenes. Antibody-blocking studies revealed that the antitumor immunity was mediated by interferon (IFN) y and IL-12. Investigators are currently constructing HER-2lneu-L. monocytogenes vectors for proposed testing in a neu-transgenic mouse model.

111. POTENTIAL PITFALLS ASSOCIATED WITH HER-2/neucSPECIFIC IMMUNOTHERAPY

A. Autoimmune Toxicity Studies demonstrating that no toxicity is observed in patients with existent immunity to HER-2lneu give credence to the concept that HER-2lneu-specific immunity can be used in therapy without destroying normal tissue. The concept was supported by vaccine studies in rats in which the generation of

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neu-specific immunity revealed no evidence of autoimmunity. In the adult, HER-2heu is expressed at low basal levels in a limited number of normal tissues, including skin, digestive tract epithelium, breast, ovary, hepatocytes, and alveoli (Press et al., 1990).When rats were immunized with rat neu peptide vaccines, resulting in the generation of rat neu immunity, these tissues showed no histological evidence of lymphocyte infiltration or tissue destruction (Disis et al., 1996b). The lack of observable toxicity associated with HER-2heu immunity in adult rats and humans is an important demonstration that vaccines in humans may be safe. However, one report described possible fetal toxicity. HER-2/neu protein is associated with the growth and differentiation of fetal tissues and is expressed in increased amounts in the later stages of embryonic development (Press et al., 1990). Theoretically, the maternal HER2/neu-specific antibodies could cross the placenta and react against fetal tissue. This premise was tested by immunizing mice with rat neu cDNA, a foreign protein in the mouse but presumably highly homologous to murine neu (Venanzi et al., 1995). The immunized mice developed HER-2heu-specific antibody responses but did not develop any histopathological evidence of autoimmunity in maternal tissues expressing basal levels of neu. However, 7 of 10 pregnant mice miscarried or had a statistically significant decreased number of pups in a litter when compared to sham vector-immunized animals. Although the study described was small and largely observational, the potential of a decrease in fertility related to the induction of HER-2/neu-specific immunity is a real concern. Breast and ovarian cancers are the two most common cancers in women associated with HER-2heu protein overexpression. Current standard therapies for both malignancies are associated with infertility: oopherectomy and hysterectomy in ovarian cancer and menopause-inducing chemotherapy and hormonal therapy in breast cancer. While the induction of vigorous HER2/neu-specific immunity to treat minimal existing disease or to induce protection from relapse would be acceptable in this population, the effects on fetal tissue may limit widespread HER-2/neu vaccination in a younger “highrisk” female population without existing malignancy. Vaccination of rats induced no evident toxicity. However, the regimens have not yet been tested for efficacy in cancer therapy. Increasing levels of immunity to that necessary to destroy breast cancer tissue in vivo might then also induce toxicity to normal tissue. In animal models, treatment with adoptively transferred T cells immune to tumor antigens is exceedingly more effective than vaccine therapy. The level of T-cell immunity in vivo is in large part a reflection of the number of immune T cells participating in the response. vaccination increases the number of immune T cells, but the ability of repeated vaccination to increase the number of immune T cells is limited by a variety of specific and nonspecific immunoregulatory mechanisms. By

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contrast, immune T cells can be grown in vitro to almost unlimited numbers. Therapy with large numbers of HER-2heu-specific T cells expanded in vivo will provide a greater level of immunity than vaccination alone (Cheever et al., 1986; Chen et al., 1990). The greater level of immunity might occur at the expense of a greater likelihood of autoimmune toxicity. If toxicity does occur, it might not preclude the use of HER-2/neu-specific therapies, as all cancer therapy is predicated on achieving a positive therapeutic ratio and does not require absolute nontoxicity.

B. Immunological Escape The worry for any antigen-specific therapy is the development of antigennegative variants. A unique interplay between IFN and HER-2heu might also limit the effectiveness of vaccine and T-cell therapy. It is known that IFN can reduce expression of HER-2heu protein on human ovarian cancer cells in vitro (Marth et al., 1990, 1992). This phenomenon was specific for the ovarian cancer lines tested and did not occur in the breast cancer lines tested. The reduction of HER-2heu protein overexpression was due to inhibition of HER-2heu gene expression and was dependent on the concentration of IFN. The downregulation of HER-2heu protein expression by IFN may, in itself, be therapeutic for some cancers. In circumstances in which HER2heu overexpression is associated with increased aggressiveness and resistance to chemotherapy, downregulation of HER-2/neu by systemic treatment with IFN might conceivably alter the malignant phenotype and render the malignant cells less aggressive and more chemotherapy sensitive. If the immune system, however, plays a major role in the eradication or containment of HER-2/neu-overexpressing tumors, then the effect of IFN on decreasing HER-2heu protein expression might markedly decrease the ability of immunity to target cancer cells and allow the cancer to grow unchecked. Immune helper T cells and CTL function in immunity, in part, by synthesizing IFN. The degree to which IFN secretion would disallow therapeutic efficacy of HER-2/neu-specific immunotherapy is totally unknown but would deserve further investigation in patients failing immunotherapy. One advantage of therapy against known and defined antigens is that, if therapy fails, the mechanisms of failure can be determined. A second method of immunological escape unique to HER-2/neu overexpression that is receiving experimental attention is the observed resistance of HER-2heu-positive cancer cells to natural killer (NK) cell- and TNF-mediated cytotoxicity (Lichtenstein et al., 1990). The mechanisms of resistance are poorly understood. One initial hypothesis tested was that increased class I MHC surface expression, which has been reported in HER-2/neu proteinoverexpressing malignancies (Lichtenstein et al., 1992),confers resistance to

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NK cell killing. Experiments evaluated the downregulation of class I MHC with pz microglobulin antisense oligonucleotides (Lichtenstein et al,, 1992). Despite marked decrease in class I MHC on the cell surface, the HER-2/neuoverexpressing cell line still remained resistant to NK cell killing as compared to HER-2heu-negative tumors. Subsequent studies have suggested that a decrease in intracellular adhesion molecule-1 expression on the surfaces of HER-2heu-positive ovarian cancer cells might account, in part, for the NK cell resistance (Fady et al., 1993). Other studies have suggested that a relative decrease in NK activity occurs in patients who have tumors that overexpress HER-2heu as compared to patients who have tumors that are HER2/neu negative (Wiltschke et al., 1994).The question of whether NK activity in HER-2/neu-positive cancer patients correlates with existent immunity to HER-2/neu or correlates with ability to immunize patients with HER-2/neu vaccines can now be answered. Persistent low-level immunity in the face of replicating cancer cells is likely to predispose to the development of antigen-negative variants. HER2/neu-specific antibody responses have been found in patients with HER2/neu-negative breast cancer (Disis et al., 1 9 9 6 ~ )It. is possible that immunity resulted in antigen-negative cancers in those patients. Two circumstances in which antigen-negative variants may have been inducedkelected by the presence of immunity are (1)those patients in whom immunity coexists with antigen-negative cancers, and (2) patients with HER-2/neu-positive ductal carcinoma in situ (DCIS) that has progressed as HER-2/neu-negative interductal carcinoma of the breast. The incidence of HER-2/neu protein overexpression is much higher in DCIS than in invasive breast carcinoma (Allred et al., 1992).Immunity to the HER-2/neu protein has not been formally studied in patients with DCIS; however, those studies are underway. Investigations suggest that the level of HER-2/neu-specific immunity detected, as indicated by antibody titer, appears to be greatest in patients with early-stage tumors at the time of diagnosis, albeit small numbers of patients were eval. it is possible that immunity does deuated (Disis et al., 1 9 9 6 ~ )Therefore, velop in some patients at the DCIS stage. An immune response that results in escape of HER-2lneu-negative cancer theoretically might still be beneficial to the patient. HER-2heu-negative cancers are less aggressive than those that overexpress the neu protein, and are associated with a more favorable prognosis, particularly in lymph node-positive disease. Whether the generation of HER-2/neu antigen-negative variants can alter the natural history of the disease in a particular patient is, at present, only speculation. Much has been written over the years on the role of the immunity to cancer. Most current theories of host-tumor interactions, however, were formulated without firm evidence as to the molecular identity of the antigens that are recognized or have the potential to be recognized. Now that some patients with HER-2/neu cancer have been shown to have an immune

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response to HER-2/neu, and now that vaccine studies are coming on line, it is finally possible to explore the role that immunity plays in disease progression in a well-defined antigen system.

IV. HER-2/neu-SPECIFIC ANTIBODIES FOR CANCER THERAPY In animal models, immunotherapy is most effective when used to treat small tumors. HER-2/neu vaccines, therefore, if effective, would be expected to be most effective in preventing recurrence in patients rendered in remission by standard cancer treatments such as surgery, radiation therapy, and/or chemotherapy. Vaccines may be very efficient in preventing the progression of minimal residual disease. Vaccines offer the promise of lifelong immunity and protection mediated by both antibody and T-cell immunity. Although vaccine therapy potentially holds a great theoretical advantage, antibody therapy has already been demonstrated to be effective in humans, albeit in only a small proportion of patients treated. There are a number of innovative strategies under development aimed at increasing the therapeutic efficacy of monoclonal antibodies. Encouraged by positive results in animal models and early human trials, many research groups are using HER-2/neu as a prototype target for developing their own unique approaches to antibody therapy of cancer.

A. HER-Z/neu-Specific Antibody Therapy

Can Interfere with Tumor Growth Antibodies directed against the HER-2heu ECD have the potential to mediate “classical” mechanisms of immune destruction: antibody-dependent cell-mediated (ADCC) immunity and complement fixation. More importantly, antibodies can directly inhibit cancer cell growth by affecting signaling via the HER-21neu receptor. Antibodies to HER-21neu have been shown to have a variety of potential functional effects, including HER-2/neu receptor binding, receptor internalization, an increase or decrease in receptor phosphorylation, and an increase or decrease in cancer cell growth and differentiation (Bacus et al., 1992; Brown et al., 1994; Hurwitz et al., 1995; Tagliabue et al., 1991; Xu et al., 1993). Too few antibodies have been compared to know which of the functional effects are linked and which are independent. Functional antibodies have been shown to be effective in vivo. However, it is not yet clear which functional effects are necessary or sufficient for efficacy in vivo (Shawver et al., 1994). Interpretation of in vivo re-

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sults is further confounded by the possibility that antibody might block the function of the normal HER-2heu ligand(s). The in vivo effects of normal HER-2heu ligands on cancer cells is unknown. The therapeutic efficacy of neu-specific antibodies in vivo was initially established in animal models. Nude mice, implanted with rat neu-transfected NIH 3T3 cells, were treated with neu-specific IgG monoclonal antibodies. None of the antibodies administered alone resulted in the inhibition of tumor growth. If mixtures of the neu-specific antibodies were used, the antibodies exerted a synergistic effect and a substantial fraction of the mice were “cured” (Drebin et al., 1988). The ability of antibody to exert function in vivo has also been established in animal models. Mice transgenic for rat neu on an MMTV promoter are destined to die of neu-overexpressing breast cancer. Investigators demonstrated that 50% of neu-transgenic mice did not develop breast cancer when treated with 7.16.4, a neu antibody directed against the ECD (Katsumata et al., 1995). Of note, infusion of antibody resulted in receptor downregulation in vivo. Furthermore, murine breast cancer isolated from treated mice showed evidence of markedly reduced phosphorylation. Presumably the functional effects were responsible for the dramatic effect on survival. One of the best characterized murine monoclonal antibodies capable of interfering with human HER-2heu function is 4D5 (Hudziak et al., 1989).The 4D5 antibody is an extremely potent inhibitor of the growth of HER-2heuoverexpressing tumor cells in uitro (Sarup et al., 1991). In uitro treatment with 4D5 results in downregulation of surface HER-2heu protein, receptor internalization, and the stimulation of phosphorylation in intact cells. The growth-inhibitory effects of 4D5 were observed for SKBR3, a breast cancer tumor cell line that highly overexpressed the HER-2heu protein, but did not occur for MCF7, a breast cancer cell line that expressed only “basal” levels of the HER-2heu protein (Sarup et al., 1991). The in vivo therapy effects of 4D5 were established in experiments demonstrating that 4D5 induced inhibition of growth of HER-2/neu-overexpressinghuman gastric cancer in a severe combined immunodeficiency (SCID) mouse model (Ohnishi et al., 1995).Antibody treatment had no effect on the growth of HER-2heu-negative tumors. Another functional effect described for HER-2heu that may prove to be critically important for therapy is the ability to render breast cancer cells more sensitive to the effects of chemotherapy. Clinically, HER-Uneu-overexpressing tumors have been associated with resistance to standard doses of cytotoxic agents. In vitro, high concentrations of cisplatin are required to kill breast cancer cells that overexpress HER-2heu. There is a synergistic growth-inhibitory effect of functional antibodies and cisplatin (Hancock et al., 1991; Pietras et al., 1994). In vitro, cotreatment of tumor cells with cisplatin and neu antibody resulted in a 3540% reduction in the repair of cis-

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platin-DNA adducts after drug exposure. Clinical trials in humans with combined antibody and cisplatin are ongoing, but results to date have not been reported. Resistance of HER-2/neu-overexpressing cancer cells to the cytotoxic effect of TNF has been described as a possible mechanism of escape of HER-2/neu-positive cancers from the immune system. Monoclonal antibodies to HER-2heu have been shown to reduce resistance to the cytotoxic effects of TNF (Hudziak et al., 1989). One problem limiting the use of murine monoclonal antibodies in humans is the development of human anti-mouse antibodies (HAMA). Several “humanized” versions of the 4D5 murine ECD-specific monoclonal antibodies were constructed (Carter et al., 1992). These constructs contained the antigen binding loops of the murine antibody with human variable region framework residues plus IgG, constant domains. Several of the constructs did not demonstrate growth-inhibitory effects on HER-2/neu-overexpressing cells in vitro. One construct, denoted rhuMAb HER2, was comparable to the murine monoclonal antibody in the ability to inhibit tumor cell growth in vitro, and was even more efficient in mediating ADCC (Allison et al., 1996; Carter et al., 1992). rhuMAb HER2 has been evaluated in a Phase I1 clinical trial in patients with metastatic breast cancer that overexpresses HER2/neu (Baselga et al., 1996). Forty-six advanced-stage patients, with histories of extensive prior therapy, were treated with weekly infusions of the antibody over an 11-week period. The treatment was well tolerated, with minimal toxicity. Objective tumor responses were seen in 5 of 43 assessable patients, including one patient who had a complete response.

B. Strategies to “Enhance” HER-2heu-Directed Antibody Therapy Antibodies capable of directly perturbing HER-2heu function may prove to play a special and unique role in cancer therapy. Alternatively, many groups are taking the more conventional approach of using antibodies to target immunotoxins, chemotherapeutic agents, and immune effector cells to tumors overexpressing HER-2heu. HER-2heu is an attractive target for antibody immunotherapy in part because it is internalized following antibody binding. Studies of the 4D5 antibody showed that, after binding to HER2/neu-positive NIH 3T3 cells, the antibody was endocytosed via coated pits, routed to lysosomes, and degraded (De-Santes et al., 1992). Internalization is important for therapy with toxins as well as radionuclides. Ricin immunotoxins were shown to kill HER-2heu-positive cells in vitro (Rodriguez et al., 1993). Experiments with multiple tumor cell lines with varied levels of neu protein overexpression revealed anti-HER-2heu ricin immunotoxins significantly inhibited growth only in cell lines with the highest levels of

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HER-2/neu protein overexpression. The inactivity against cell lines with low HER-2heu expression is encouraging for predicting a possible lack of toxicity against normal tissues expressing basal levels of HER-2heu protein. In vivo biodistribution of radiolabeled 4D5 antibody, evaluated in beigehude mice bearing subcutaneous NIH 3T3 HER-2/neu tumor grafts, confirmed a high specificity of localization to tumor (De-Santes et al., 1992). The antibodies used were specific for human HER-2heu. Human biodistribution studies are needed to assure that antibodies do not target to normal tissues with basal levels of expression. Treatment of NIH 3T3 HER-2/neu tumor grafts with radiolabeled 4D5 antibody substantially prolonged survival. However, no mice were cured, possibly as the result of rapid intratumor catabolism of antibody. HER-2/neu antibodies have also been used to successfully target enzyme prodrugs (Eccles et al., 1994) and doxorubicin chemotherapy (Park et al., 1995) to human breast cancer xenografts in mice. Studies targeting doxorubicin used immunoliposomes. This is a promising use of HER-2/neu-specific antibodies for drug delivery. Liposomes conjugated to Fab’ fragments of HER-2/neu-specific antibodies were shown to bind to HER-2heu-overexpressing breast cancer cells with an affinity comparable to that of intact antibody (Park et al., 1995). The immunoliposomes were efficiently internalized into breast cancer cells by receptor-mediated endocytosis via the coated pit pathway and also possibly by membrane fusion. Doxorubicin-loaded anti-HER-2heu immunoliposomes were markedly and specifically cytotoxic against HER-2/neu-overexpressing tumor cells in vitro and were shown to be capable of delivering doxorubicin to human breast cancer xenografts in SCID mice. Methods to target delivery of doxorubicin are of special interest. Although high HER-2/neu expression is associated, in general, with chemotherapy resistance, including to doxorubicin (Tsai et al., 1996), studies associate high-dose doxorubicin with increased survival in patients with HER-2/neu-positive breast cancer (Paik, 1992). One potential problem in targeting tumors with antibodies is the large size of the IgG molecule. IgG penetrates tumors poorly and is slowly cleared from the circulation, which allows accumulation of antibody conjugates in normal organs. Single-chain antibodies are composed of both heavy and light chain variable specificities in a single molecule and thus are smaller and have better pharmacokinetics. Studies with single-chain HER-2/neu-specific antibodies have confirmed rapid serum clearance following intravenous administration in mice, specific binding to human xenografts, excellent tumor-tonormal tissue ratios, and lower nonspecific background as compared to the parental IgG antibody (Adams et al., 1993; Tai et al., 1995). The development of single-chain HER-2/neu-specific antibodies has led to two new and very innovative approaches, “intracellular immunization” and “T-bodies” (i.e., T cells grafted with antibody specificity). Single-chain anti-

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bodies were constructed with an N-terminal leader sequence to target synthesis to the lumen of the endoplasmic reticulum and a C-terminal retention signal to prevent secretion. Intracellular expression of this antibody results in the “trapping” of the ECD in the cytoplasm of the cell, a lack of receptor expression on the surface of the cell, reversion of the transformed phenotype, and marked inhibition of tumor cell proliferation (Beerli et al., 1994; Deshane et al., 1994; Graus-Porta et al., 1995). Recombinant adenovirus expressing single-chain antibody has been tested in vivo and has been shown to induce profound downregulation of cell surface HER-2/neu in an ascites human tumor xenograft model (Deshane et al., 1995). Histological assessment of regressing tumor indicated that intracellular expression of the single-chain antibody mediated apoptotic cell death. Thus, the use of such intracellular expression of antibody represents a powerful approach to study the in vivo function of receptors as well as a potential tool to treat HER2/neu-positive cancers. HER-Yneu single-chain antibodies have also been used to target T lymphocytes. The presumption is that T cells would have better access to tumor than antibody and remain in vivo long after transfer and, therefore, function over a longer time span. Chimeric single-chain receptor genes have been constructed incorporating both the antigen-binding domain of HER-2heu-specific antibodies and the 5 chain signal transduction subunit of the T-cell receptor-CD3 complex. Expression of the chimeric single-chain receptor by T cells has been shown to confer HER-2/neu protein recognition to T cells without the need for antigen processing of HER-2heu and without the requirement of MHC restriction (Moritz et al., 1994; Stancovski et al., 1993). The growth of HER-2/neu-transformed cells in nude mice was markedly inhibited by the adoptive transfer of CTL expressing the HER-2/neu antibody-< chain construct (Moritz et al., 1994). In other studies to promote cell-mediated lysis of cancer cells, bispecific antibodies have been constructed that use HER-2heu ECD antibody on one arm and receptors that interact with the cell-mediated immune system on the other arm. Two such bispecific antibody constructs utilize HER-2/neu ECDspecific antibody with FcyRIII or FcyRI. 2B1 is the bispecific murine monoclonal antibody possessing dual specificity for HER-2/neu and FcyIII receptor (Hsieh-Ma et al., 1992). The purpose of the bispecific antibody is to activate FcyIII receptor-bearing cells and to oppose the activated cells to the HER-2/neu-positive cancer cells. The FcyIII receptor is expressed by macrophages and monocytes, NK cells, and polymorphonuclear leukocytes. In vitro, binding of 2B1 to SKBR3, a breast cancer cell line, was very effective in mediating cytolysis when peripheral blood lymphocytes of either normal individuals or ovarian cancer patients were used (Hsieh-Ma et al., 1992). Systematic administration of 2B1 into SCID mice bearing HER-2heu-overexpressing ovarian cancer xenographs significantly improved survival and

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was associated with no observed toxicity (Weiner et al., 1993). Encouraged by the results in the animal model, a Phase I human clinical trial based on infusion of 2B1 was initiated and completed (Weiner et al., 1995). Significant dose-limiting thrombocytopenia was seen in 2 of 15 patients, both of whom had been heavily pretreated. Non-dose-limiting toxicities included fever, nausea, and leukopenia. Several minor clinical responses were observed. Future studies using antibody in combination with IL-2 treatment are contemplated. One enticing finding is that some of the patients treated with 2B1 bispecific antibody developed antibody immunity to HER-2heu (Gralow et al., 1996). In several patients, immunoglobulin class switch from IgM to IgG was documented, providing strong evidence that they were immunized to the HER-2heu tumor antigen by virtue of the administration of the 2B1 antibody. Presumably, bispecific antibody served to concentrate HER-2/neu protein into APC. Increasing the concentration of antigen can increase T-cell recognition and subsequent priming. Although most patients developed significant HAMA immune responses, a minority of patients developed HER2/neu-specific responses during treatment. The inherent immunogenicity of this murine bispecific antibody would most certainly limit the number of immunizations that could be given to patients to further boost the HER-2heuspecific immune response. However, the generation of new HER-2heu-specific immunity in these patients further validates the concept that vaccines will be able to induce HER-2/neu-specific immune responses in cancer patients. Another bispecific construct, MDX-2 10, using FcyRI as the effector arm, has also completed Phase I trials in humans (Valone et al., 1995). In vitro, MDX-210 effectively directs FcyRI-positive effector cells such as monocytes and macrophages to kill tumor cells that overexpress HER-2/neu. The toxicity of treatment was minimal. Transient monocytopenia and lymphopenia developed without other hematological changes. Localization of MDX-210 could be demonstrated in the tumor tissue of 2 patients. One partial and one mixed tumor response were observed among 10 evaluable patients. Bispecific antibodies may also be used to recruit T cells to the tumor bed. Several constructs utilizing HER-2/neu ECD-directed antibody and antiCD3 have been proposed. One strategy exploited the ex vivo expansion of the T-cell population with OKT-3 prior to in vitro assay with HER-2heuoverexpressing tumor cell lines and the HER-2heu-anti-CD3' bispecific antibody. The targeting of a markedly expanded population of T cells resulted in the delivery of a large number of cytotoxic effectors to the tumor (Nakamura et al., 1992). Subsequent studies from this group of investigators evaluated the use of staphylococcal enterotoxin (SEA) as a superantigen cytotoxic stimulator. SEA treatment of a lymphocyte population resulted in the generation of large numbers of reactive CD4' T cells with significant cyto-

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toxic activity (Kuge et al., 1995). SEA treatment skewed the T-helper population to a T h l phenotype. SEA-conjugated anti-HER-2heu antibody specifically targeted these cytotoxic cells to HER-2/neu-overexpressingtumor. A humanized anti-HER-2heu-anti-CD3’ bispecific antibody has been constructed (Shalaby et al., 1992). Subsequent variations on this humanized construct have resulted in antibodies with markedly improved affinity for binding T cells and improved selectivity for binding cells with high levels of HER-2/neu protein overexpression (Zhu et al., 1995). Such optimization of antibodies may be essential for effective therapy.

V. CONCLUSION The HER-2/neu oncogenic protein is receiving great attention as a tumor antigen. Monoclonal antibody therapy is already in clinical trials. Vaccines will shortly be in clinical trials. Monoclonal antibody therapy has shown efficacy in some patients. There is little question as to whether vaccines will be able to generate immunity, given that some patients with HER-2/neu-overexpressing malignancies already have an immune response directed against the protein. The HER-2heu oncogenic protein makes an excellent tumor antigen for several reasons. It is overexpressed on the cell surface and thus available for antibody attack. It is a growth factor receptor and thus available for therapy with functional antibodies to downregulate cancer cell growth. It is available in both the class I and class I1 MHC antigen-processing pathways, as evidenced by detection of existent HER-2/neu-specific helper T-cell responses and CTL responses in cancer patients. Effective immune responses often involve combined antibody, helper T cell, and CTL responses. All arms of the immune system can be directed against HER-2heu. Tumor-specific antigens have long been sought as immunotherapy targets. Tumor-specific antigens do exist (Disis et al., 1996c), but there is a recent “paradigm shift” (Nanda and Sercarz, 1995)toward the realization that selfantigens can also serve as immunotherapy targets. Generating an immune response to self-antigens is an increasingly important therapeutic concept. Studies generating immune responses to HER-2heu with vaccines or mediating the immune response with antibody or T-cell therapy will help lay the foundation for developing effective immunotherapy targeting other self-proteins as tumor antigens. The safety of generating an immune response against a self-protein is a concern. The overexpression of HER-2heu should allow some selectivity of the immune response without autoimmune toxicity. It is encouraging that large numbers of animal antibody and vaccine studies, and now human antibody trials, show no evidence of toxicity to tissues expressing normal basal

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levels of HER-2heu. This observation, in concert with the demonstration that immunity exists in some cancer patients, lends some surety to the concept that immunity can be generated or boosted without significant effects to normal tissues. This review points out many innovative approaches to target immunotherapy against HER-2heu that are in various stages of development. There is enough ferment in the field and enough approaches are being tested to allow substantial optimism that some of the approaches will prove to be effective in the treatment of human cancer. The expectation is not that HER-2/neu-specific immunotherapy will be effective in all patients, but rather that it will provide incremental benefit as an adjunct to current standard cancer therapy approaches. Tumor immunologists are developing many methods to target cancer antigens. HER-2heu is receiving increasing attention as an immunotherapy target in part because of its biological characteristics. Just as important, there is currently a dearth of other good targets against which cancer immunotherapists can practice their art. Many genomics groups are working with methods to identify all of the genes expressed by individual cancers as well as the tissue of origin. Out of this work will come the identity of many additional ''self''-cancer antigens. The development of immunotherapy against the HER-2heu protein will provide important therapeutic tools for the clinical oncologist. As important, the principles being established for the development of HER-2heu-specific immune-based treatments will allow easier application of immunotherapy to newly discovered cancer antigens.

ACKNOWLEDGMENTS Mary L. Disk is supported by National Institutes of Health Grants KO8 CA61834 and R29 CA68255. Martin A. Cheever is supported by National Institutes of Health Grants R37 CA30558 and R01 CA61912.

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Index

A A1 protein, apoptosis and, 126 Abl tyrosine kinases, apoptosis and, 129 Achondroplasia, age and risk of, 230 Acoustic neuroma, chromosome 3 losses in, 51 Acquired immunodeficiency syndrome, see AIDS Acrocephalosyndactyly,age and risk of, 230 Acute lymphocytic leukemia (ALL), 147-148,150 Acute myeloid leukemia (AML), 148, 197 ACYl gene, 67-68 ACYl protein, 67 Addressins, 245 Adenocarcinoma ,chromosome 3 losses in, 47-48 lung, 55 microsatellite instability and, 215 Adenoma, associated with HNPCC, 96 Adenomatous polyp, mutations and, 111 Adenovirus, apoptosis and, 131, 132 Adhesion molecules, 243-245 Affinity maturation, apoptosis and, 140 AG-490,154 Age, cancer and, 230 AIDS, apoptosis and, 122,142-143 ALL, see Acute lymphocytic leukemia Allelic losses, in tumors, 32-33 AML, see Acute myeloid leukemia Anal canal cancer tumors, chromosome 3 losses in, 48 Aneuploidy, p53 mutations and, 7-8 Anti-CD44 monoclonal antibody, 252, 261, 274,276,277,280 APC gene, mutations, 111,114 APEH gene, 67 APO-1, apoptosis and, 129 Apoptosis, 123-124 ATM gene product, 12 bcl-2 gene, 9, 128, 139-140

CD44 and, 283-284 cytokines and, 133-134, 135, 153 death domains, 129 defined, 121,123 fus gene, 129-130,135,141-142 hematopoietic system, 122-123, 131, 133 affinity maturation, 140 AIDS, 142-143 autoimmunity and, 142 B-cell differentiation, 138-140 B-cell lymphoma, 143-144 cytotoxic T-lymphocytes, 138 immune response downregulation, 140-141 inflammation and, 122,141-142 leukemia, 122,144-152 malignancy, 143 survival factors, 133-135 T-cell differentiation, 137-138 molecular mechanisms, 125 Abl tyrosine kinases, 129 bcl-2 gene family, 125-126 Gas antigen, 129-130 ICE-related cysteine proteases, 130 myc gene, 126-127 p S 3 gene, 126 RAS gene, 127 viruses and, 130-131 morphology, 123 pS3 gene, 8-9, 12, 127, 128 regulation of, 121-122 survival signals, 128, 131, 133-135 of tumor cells, 18 Apoptotic threshold, 153 APR gene, 68 Arthritis, CD44 and, 286-287 Astrocytoma CD44 and, 292 chromosome 3 losses in, 51 Ataxia-telangiectasia (AT), 9-10,213 ATM gene, 12

37 3

374 ATM gene product, 1,2,10 apoptosis, 12 cell cycle control and, 10-16 p53-dependent signal transduction pathway, 9-12 structural and phenotypic homologs, 12-16 Autoimmunity, apoptosis and, 122, 142

Index Brain tumors, CD44 and, 291-293 Breast cancer CD44 and, 301-302 chromosome 3 losses in, 4 8 4 9 HER-2heu gene amplification and, 346-348,357

C

B Baculoviruses, 131 Bad protein, apoptosis and, 126 Bak protein, apoptosis and, 126 Barren esophagus, 215,224 Base excision repair system, 212 bax gene apoptosis and, 9 p53 and, 7 Bax protein, apoptosis and, 126 B-cell lymphoma, apoptosis and, 143-144 bcl-2 gene acute lymphocytic leukemia, 147-148 apoptosis and, 9, 128,139-140 B-cell survival, 139-140 chronic lymphocytic leukemia, 146 drug resistance in chemotherapy and, 152 hematopoiesis and, 135, 136 lymphoma and, 143-144 p53 gene and, 127 BCL-2 gene family, 125-126 bcl-2 protein, apoptosis and, 125, 126 BCL-x gene apoptosis and, 126 hematopoiesis and, 135 BCL-x, gene, 126,135,139 BCL-x, gene, 126,152 Bcr-Abl tyrosine kinase apoptosis and, 129 chronic myeloid leukemia and, 145 myelodysplastic syndrome, 149 Beckwith-Wiedemann syndrome, 198 P2-microglobulin gene, 112 BHRFl protein, cellular proliferation, 131 Bispecific antibodies, 364-365 Bladder cancer CD44 and, 298-299 chromosome 3 losses in, 52-53, 75 B lymphocytes CD44 transition during activation, 258-259 differentiation, 138-140

C3H lOTfcells, TGF-P cell cycle kinetics and, 187,190,191 Cadherins, 244 CAK, see cdk-activatingkinase Cancer, see also Carcinogenesis;individual cancers abnormalities at 12~12-13.1,197 age and risk of, 230 ATM gene product, 17 CD44 and, 243,287-288,291-304 cell cycle control and, 16-17 cellular proliferation as risk factor for, 222-224 cyclin overexpression in, 196 defined, 195 HER-2heu gene amplification, 345 human papillomavirus and, 327-332 loss of TGF-P receptors, 185 malignant phenotype, 243 malignant process, 143-152,243,245, 287-291,304-305 metastasis, 245 mutation rate and, 234-235 mutations and, 243 p53 gene and, 7-8, 16-17 somatic mutations and, 210 TGF-p, 168 Carcinogenesis,see also Cancer cellular proliferation and, 224 heterozygosity and, 29 multistep carcinogenesis and somatic mutations, 221-222 somatic mutations, 210 theories of, 224 Carcinoma, see individual carcinomas Cartilage link proteins, 245 p-Catenin gene, 69 CD36 macrophage surface receptor, apoptosis, 124 CD44,137,242,243,305-307 apoptosis and, 283-284 cancer, 243,287-288,291 bladder cancer, 298-299

Index breast cancer, 301-302 cervical carcinoma, 299-300 chronic myeloid leukemia, 303-304 colorectal cancer, 295-296 head and neck tumors, 293-294 liver cancer, 298 lung carcinoma, 294 malignant process, 245,287-291, 304-305 melanoma, 302 mesothelioma, 295 nasopharyngeal carcinoma, 294 nervous system tumors, 291-293 non-Hodgkin lymphoma, 303-304 ovarian cancer, 300-301 pancreatic cancer, 297-298 prostate cancer, 299 stomach cancer, 296-297 thyroid carcinoma, 294 cytoplasmic tail, 250,252-253,264-267 expression in embryo, 253-255 in epithelial cells, 255-257 genetic control of, 275 isoforms, 257-259 functions, 275,306 cell aggregation, 282-283 cell binding to endothelium, 278-279 cell traffic, 280-282 cytokine and growth factor presentation, 284-285 growth signal transmission, 275-277 HIV infection and, 287 homing receptor, 277-278 hyaluronic internalization and degradation, 279-280 lymphohematopoiesis and apoptosis, 283-284 malaria resistance, 286 menstrual cycle, 285-286 rheumatoid arthritis and, 286-287 wound healing, 285 genomic organization, 247,249,250 glycosylation, 2 70-272 hyaluronic acid, 243,259-272,281-282 isoforms, 242-243,246,250-252 expression, 257-259 ligands hyaluronic acid, 243,259-272, 281-282 non-HA, 272-274,307 nomenclature, 245-246

375 soluble CD44,274-275 standard CD44,247,250 CD44E, 242-243,251 CD44H, 242,262 CD44s, 242,247,250,255,262,264,285 CD44v, 251,253,255 CD44V3-1O9243,251,256 CD44V4-7,251,252,262,290 CD44V5,296 CD44V6-7,251 CD44V8-10,251,256,262 CD95, apoptosis and, 129 cdc2 inhibited by phosphorylation, 173 mammalian cell cycle and, 168 phosphorylation of, 172 cdk2,169 inactivation, 193-194 inhibited by phosphorylation, 173 phosphorylation, 172 cdk4,170-171 inhibition by INK family ckis, 173 TGF-P and, 192-193 cdk6,171 inhibition by INK family ckis, 173 TGF-P and, 192 cdk7,172 cdk42 gene, overexpression in cancer, 196 cdk-activating kinase (CAK), 5, 172 cdki, see Cyclin-dependent kinase inhibitors cdks, see Cyclin-dependent kinases CED-3, apoptosis and, 130 CED-4, apoptosis and, 130 Cell aggregation, CD44 and, 282-283 Cell cycle, GI-S and G2-M phase transition, 2 Cell cycle control, 2 ATM, 10-16 cancer and, 16-17,17-19,195-198 cyclin-dependent kinases, 165-168 downstream effectors, 179-184 G1 and S phases, 169-179 in mammals, 168-169 G1 arrest, 2-7 p53 gene, 2-9 TGF-P activity and, 167, 188-195 transition points, 170 Cell death, see also Apoptosis necrosis, 123 p53 gene and, 1-19 of tumor cells, 17-18 Cell migration, CD44 and, 280-282

376 Cell motility, CD44 and, 282 Cellular proliferation, 131 as cancer risk factor, 222-224 T lymphocytes, 194-195 Cervical carcinoma CD44 and, 299-300 chromosome 3 losses in, 49-50 FRA3B fragile region, 71 human papillomavirus and, 71,321-335 CFS, see Colony-stimulatingfactors Chemokines, CD44 and, 284-285 Chromosome 3 deletion analysis bladder cancer, 52-53 breast cancer, 48-49 cervical carcinoma, 49-50 endometrial cancer, 50 gastrointestinal tumors, 4 7 4 8 head and neck squamous cell carcinoma, 45-47 lung cancer, 35-42 malignant melanoma, 52 malignant mesothelioma, 51 multiple endocrine neoplasia type 2, 52 ovarian carcinoma, SO renal cell carcinoma, 4 2 4 5 salivary gland adenoma, 52 skin cancers, 51 testicular germ cell tumors, 51 thyroid carcinoma, 52 uveal melanoma, 52 in vitro studies, 53-54 esophageal, 4748,115 evolutionary aspects, 73-74 microcell hybrids, 35 p14.2 band, 70-73,76 p21.3 band, 56-58,67-70,75 transfer into human tumor cell lines lung adenocarcinoma, 55 oral squamous cell carcinoma, 55 ovarian cancer, 55 renal cell carcinoma, 54-55 transfer into rodent lines, 56-58 tumor suppression, functional assay, 54-58 Chromosome transfer chromosome 3,54-55 mismatch repair, 55-56 Chronic lymphocytic leukemia (CLL), 145-147,149,150 Chronic myeloid leukemia (CML), 144-145 CD44 and, 303-304

Index Cisplatin, cotreatment of tumor cells with neu antibody and cisplatin, 361-362 Collagen, CD44 binding to, 272-273 Colony-stimulatingfactors (CSFs),apoptosis and, 133 Colorectal cancer (CRC) CD44 and, 295-296 chromosome 3 losses in, 47 etiology, 97 hereditary nonpolyposis colorectal cancer associated with, 95 HNPCC and, 96 microsatellite instability, 100 mutations and, 113 Cowpox virus, 131 CPP32,130 CRC, see Colorectal cancer CrmA protein, cellular proliferation, 131 CTNNBZ gene, 69 Cyclin A, 169 overexpression in cancer, 196 TGF-P and, 192 Cyclin A-cdk2 complex formation, 172-1 73 inactive, 177 Cyclin B-cdc2 complex, formation, 172173 Cyclin k k c 2 complex, 172 Cyclin binding, cdk activation by, 169-172 Cyclin C, 170 Cyclin-cdk2 complex, 168-169 E2F-pl07 complex and, 183 Cyclin-cdk complexes balance of, 198 cell cycle control, 166, 171 p 2 1 gene and, 5-6, 174 p27 and, 175,178 ratio to ckis, 178 Cyclin D, 170, 171 Cyclin D1 overexpression in cancer, 196 TGF-P and, 192 Cyclin D-cdk2 complexes, p21 and, 5 Cyclin D-cdk4 complex inactive, 177 p27 and, 193 phosphorylation by, 189 TGF-P and, 189 Cyclin-dependent kinase inhibitors (ckis), 165 INK family, 173-174 p21,5,174-175

Index p27,175-179,189 ~57,177,178 TGF-p and, 193-194 tumor suppressor genes, 195 Cyclin-dependent kmases (cdks) activation by cyclin binding, 169-172 by phosphorylation, 5, 172-173 cancer and, 195-198 cell cycle control and, 165-168 downstream effectors, 179-1 84 G1 and S phases, 169-179 in mammals, 168-169 inhibition by phosphorylation, 173 INK family, 173-1 74 ~21,174-175 ~27,175-179,189 ~57,175-178 TGF-p activity and, 167, 188-195 Cyclin E, 170, 171, 192 expression during G1-S transition, 182 overexpression in cancer, 196 TGF-P and, 192 Cyclin E-cdk2 complex formation, 172-173 inactive, 174, 175, 177 p21 and, 5 Rb phosphorylation, 180 TGF-p and, 192 Cyclin E-cdk2 kinase, 180, 190 Cyclin G, p53 and, 6 , 7 Cyclin H, 172 Cyclins defined, 5 overexpression in cancer, 196 Cysteine proteases, 130 Cytokines apoptosis and, 133-134,135, 153 CD44 and, 284-285 chronic lymphocytic leukemia, 146 myeloid differentiation and, 135-136 p-53 mediated apoptosis, 9 stem cell viability and, 134-135 Cytotoxic T lymphocytes, 138,349

377 cervical carcinoma, 49-50 endometrial carcinoma, 50 gastrointestinal tumors, 47-48 head and neck squamous cell carcinoma, 45-47 lung cancer and, 35-42 malignant melanoma, 52 malignant mesothelioma, 51 multiple endocrine neoplasia type 2,52 ovarian carcinoma, 50 renal cell carcinoma, 4 2 4 5 salivary gland adenoma, 52 skin cancers, 51 testicular germ cell tumors, 51 thyroid carcinoma, 52 uveal melanoma, 52 in vitro studies, 53-54 comparative genome and RNA analysis, 33-34 localizing tumor suppressor genes, 32 Dendritic cells, HER-2heu epitopes, 350-351 Dexamethasone, HPV and, 330 DNA base excision repair system, 212 damage, 2-7 mutations and, 233 nucleotide excision repair system, 212 radioresistant synthesis, 11 repair systems, 212 DNA mismatch repair, 209,212; see also Mismatch repair genes (MMR genes) biochemical analysis, 98-99 in E. coli, 98 in HNPCC, 93,95 in humans, 98-99 microsatellite instability and, 99-101, 211-213 DNA-PK protein, function of, 13, 14-15 Doxorubicin anti-HER-2heu immunoliposomes and, 363 apoptosis and, 142 DP-1, phosphorylation, 181 Drug resistance, leukemia, 151-152

D

E

Deletion analysis chromosome 3 bladder cancer, 52-53 breast cancer, 48-49

E1A protein, apoptosis and, 130,131, 132 E2F-1, phosphorylation, 181, 183 E2F-2, 182 E2F-3,182

378 E2F-4,182 E2F-5, 182 E2F-DP complex, 181-1 82,183 E2F-pl07 complex, 183,184 E2F protein, 188 apoptosis and, 130, 132 p53 and, 130 EGF, activation, 190 EiB 55K protein, apoptosis and, 131 Elongin complexes, 61, 62 Elongin SIII, 61 Endometrial carcinoma chromosome 3 losses in, 50 mutations and, 113 Eosinophilia, apoptosis and, 141 Epithelial basal cell carcinoma, chromosome 3 losses in, 51 Epithelial CD44, see CD44E Epithelial cells CD44 isoforms expressed on, 255-257 HPRT mutation in, 217-219 inhibitory effects of TGF-P in, 188 somatic mutations and, 210 TGF-P and, 167, 188 Epstein-Barr virus, 131 Erythropoietin, 134 E-selectin, 244 Esophageal adenocarcinoma chromosome 3 losses in, 4 7 4 8 microsatellite instability and, 215 Eta-1, CD44 binding to, 273 Evolution, chromosome 3,73-74 Extracellular matrix receptor 111,246 Extracolonic cancer, HNPCC and, 95-96

F FADD protein, apoptosis and, 129 FAF-1 protein, apoptosis and, 129 Familial adenomatous polyposis (FAP), 94 FAR1 protein, 176 Fas gene apoptosis and, 129-130,135,141-142 B lymphocyte differentiation, 140 chronic lymphocytic leukemia, 146-147 eosinophilia, 142-143 T-cell viability, 138 Fas antigen, p53 gene and, 127 FHIT gene, 71-72 FKBP-12, TGF-P receptors, 185 Follicular thyroid carcinoma, chromosome 3 losses in, 52

Index Founding mutations, 110-1 11 4D5 antibody, HER-2/neu immunotherapy, 362 FRA3B region, 70-71 Fragile histidine triad gene, 71 Fragile sites, 70-71

G G1 arrest, p.53 gene, 2-7, 10 G2-M defect, 11 gadd45 gene, 3 Gastrointestinal tumors, chromosome 3 losses in, 4 7 4 8 G-CSF, chronic lymphocytic leukemia, 146 Genetic instability, see also Microsatellite instability; Minisatellites p53 and, 7-8 Germline mutation rate, 224-227 Germline mutations, 210,211 due to endogenous mutations, 227-228 rate, 224-227 somatic mutations and, 228-231 Glioblastoma CD44 and, 292 mutations and, 114 Glioma, CD44 and, 292,293 Glycoprotein 85,246 GM-CSF acute myeloid leukemia, 148 as immune response adjuvant, 353-354 GNAU gene, 69 GNAT1 gene, 69 GP9OHermeS, 246 Growth factors apoptosis and, 8-9, 15 CD44 and, 284-285 Growth signals, CD44 and signal transmission, 275-277 Growth suppression, by TGF-P, 167 GTBP gene, 100 GTBP protein, 98

H HA, see Hyaluronic acid HAMA, see Human anti-mouse antibodies Head and neck squamous cell carcinoma (HNSCC) CD44 and, 293-294 chromosome 3 losses in, 4547,75, 76 genes for, 93

Index Helicase, 212 Hemangioblastoma, VHL mutations and, 60 Hematopoietic CD44, see CD44H Hematopoietic system apoptosis, 122-123, 131, 133 affinity maturation, 140 AIDS, 142-143 autoimmunity and, 142 B-cell differentiation, 138-1 40 B-cell lymphoma, 143-144 cytotoxic T-lymphocytes, 138 immune response downregulation, 140-141 inflammation and, 141-142 leukemia, 144-152 malignancy, 143 survival factors and myeloid differentiation, 134-135 survival factors and population control, 133-134 T-cell differentiation, 137-138 CD isoforms in, 253-255,257-259 Hemophilia, age and risk of, 230 Hepatocellular carcinoma, chromosome 3 losses in, 51 HER-2heu gene, 345 HER-2heu ECD-specific antibody, 364 HER-2heu protein, 343,344 immunotherapy, 343-345,360 autoimmune toxicity, 356-358 enhancement methods, 362-367 immunological escape, 358-360 single-chain antibodies, 363-364 tumor growth and, 360-362 monoclonal antibody therapy, 343-345, 356-367 preexistent immunity to, 346-351 vaccines for cancer therapy, 346-356 Hereditary nonpolyposis colorectal cancer (HNPCC) 3p21-pl23 gene and, 62 colorectal adenomas and, 96 colorectal cancer associated with, 95 DNA mismatch repair system, 211,212 extracolonic cancer and, 95-96 Lynch MI syndromes, 95-96 MMR genes and, 101-109,111,112-115 tumors in, 213 Herpes simplex virus (HSV), syncarcinogen with HPV, 331

379 Heterozygosity,see also Loss of heterozygosity carcinogenesis and, 29 Histidine triad motif proteins (HIT proteins), 71 HIV, CD44 and, 287 hMLHl gene, 62-64,211 hMSH2 gene, microsatellite instability and, 211 HNPCC, see Hereditary nonpolyposis colorectal cancer HNSCC, see Head and neck squamous cell carcinoma Homing cell adhesion molecule, 246 Homing receptor, CD44 as, 277-278 Homozygous deletions, lung cancer, 4 1 4 2 Hormones, papillomavirus genomes, 330 hPMSl gene, microsatellite instability and, 211 bPMSZ gene, microsatellite instability and, 21 1 HPRT gene, 217-218 somatic mutation, 217-221,228,230 HPV, see Human papillomavirus HPV-16,323, 325,326,330,332, 333 HPV-18, 323,326,330 HPV-E4 gene, 323 HPV E4 protein, 323 HPV-E.5 gene, 324 HPV-E6 gene, 323 HPV E6 protein, 18, 323, 324 HPV-E7 gene, 323 HPV E7 protein, 131, 323, 324 H-Ras protein, apoptosis, 128 Human anti-mouse antibodies (HAMA), HER-2heu immunotherapy, 362 Human papillomavirus (HPV) apoptosis and, 131 cervical cancer and, 71, 321-335 in cervical screening programs, 333-334 E4 gene, 323 epidemiological aspects, 327-329 genome, 322-325 variability, 326-327 infections, 334 long control region, 322-325 taxonomy, 325 tumor development and, 49,53-54 vaccines, 327 HUTCH-1,246 Hyaladherins, 263 Hyaluronate receptor, 246

380 Hyaluronic acid (HA) CD44, internalization and enzymatic degradation, 279-280 CD44 ligand and, 243,259-272,281282

I I1B 19K protein, apoptosis and, 131 IAP protein, cellular proliferation, 131 CAM-I, 244,263 ICE-like protease apoptosis and, 130 Fas and, 129 IFN-u, chronic lymphocytic leukemia, 146 IFN-g, chronic lymphocytic leukemia, 146 IGF-BP3, p53 and, 6, 7 IL-1 converting enzyme, see ICE-like protease IL-3, apoptosis and, 9 IL-4, chronic lymphocytic leukemia, 146 Immune response, downregulation of, 140-141 Immunoglobulins, 244 Immunoliposomes, doxorubicin loaded antiHER-2heu immunoliposomes, 363 Immunotherapy, HER-2/neu protein, 343-345,356-367,360 autoimmune toxicity, 356-360 enhancement methods, 362-367 immunological escape, 358-360 tumor growth and, 360-362 Inflammation, apoptosis and, 122, 141-142 Ink4a cancer and, 196,197 cdk4 inhibition and, 173 Ink4b cdk4 inhibition and, 174 TGF-P and, 193 Ink family, cdk inhibition by, 173-174 In(Lu)-relatedp80 glycoprotein, 246 Integrin a subunit gene, 69 Integrins, 244 Interferon, chronic lymphocytic leukemia, 146 Interferon regulatory factor-1, see IRF-1 Interleukin-3 (IL-3),apoptosis and, 9 Interleukin-4 (IL-4), chronic lymphocytic leukemia, 146 Intracellular immunization, 363 IRF-1, apoptosis and, 128 IRF-I gene, apoptosis and, 128

Index

J

Jak-2 tyrosine kinase inhibitor, 154

K Karyotyping, of tumor cells, 31-32 Kip1 protein, 198

L Laryngeal carcinoma, chromosome 3 losses in, 45-47 Late genes, papillomaviruses, 323 Leukemia acute lymphoblastic, 147-148, 150 acute myeloid, 148, 197 apoptosis and, 122, 144-152 chronic lymphocytic, 145-147,149,150 chronic myeloid, 144-145,303-304 chronic versus acute, 149-151 drug resistance in, 151-152 preleukemia, 148-149,150 LFA-1,244 Li-Fraumeni syndrome, 228 Liposomes, doxorubicin loaded anti-HER2heu immunoliposomes, 363 Ltsteria monocytogenes, as HER-Uneu vector, 356 Liver cancer, CD44 and, 298 Loss of heterozygosity,29, 30, 32 lung cancer, 37-41, 75 renal cell carcinoma, 44-45, 76 Lovastatin, mechanism of action, 177 L-selectin, 244 Lung cancer CD44 and, 294 chromosome 3 losses in, 3542,75, 76 RARB expression, 66 transfer of chromosome 3 into, 55 Ly-24,246 Lymphocytes binding to endothelial cells, 278 B lymphocytes, 138-140,258-259 HPRT mutation in, 219-221 T lymphocytes, 137-139,194-195 Lymphohematopoiesis,CD44 and, 283-284 Lymphoma apoptosis and, 143-144 non-Hodgkin lymphoma, CD44 and, 303-304 Lynch I syndrome, 95 Lynch 11 syndrome, 95,96,113

Index

M Malaria, CD44 and, 286 Malignant melanoma, chromosome 3 losses in, 52 Malignant mesothelioma, chromosome 3 losses in, 51 Malignant phenotype, 243 Malignant process adhesion molecules, 243,245 apoptosis and, 143 B-cell lymphoma, 143-144 leukemia, 144-152 CD44 and, 245,287-291,304-305 Mammalian cell cycle, cdks control, 168-169 Marfan syndrome, age and risk of, 230 Markel cell carcinoma, chromosome 3 losses in, 51 Max protein, apoptosis and, 127-128 Mcl-1 protein, apoptosis and, 126 mdm2 gene, p53-medial growth arrest, 6 MDM2 protein, p53 function and, 18 MDS, see Myelodysplasticsyndrome MDX-210,365 mecl mutants, 13-14 Mecl protein, function of, 13, 14 mei-41 mutants, 13-14 Mei-41 protein, 13 Melanoma CD44 and, 302 TGF-f3 and, 197 Meningioma, chromosome 3 losses in, 51 Menstrual cycle, CD44 expression during, 2 85-2 86 Mesothelioma, CD44 and, 295 Metastasis, adhesion molecules and, 245 MHC Class I1 invariant chain (Ii),CD44 binding to, 273 Microcell hybrids, 35 Microsatellite instability detection by polymerase chain reaction, 213-2 14 DNA mismatch repair deficiency, 99-101, 211-213 mutation rate, 210 in normal tissue, 215-216 not due to mismatch repair deficiency, 214-215 sporadic tumors and, 63-64 TGF-P receptor loss, 185 Microsatellite repeats, 99-100 Minisatellite sequences

38I mutation frequency, 210 somatic mutations, 217 Mismatch repair genes (MMR genes), 95,212 biochemical and functional properties, 98-99 chromosome 3 and, 55-56 HNPCC-related, 101-109, 111, 112-115 identification, 97-98 microsatellite instability, 99-101 mutations, 55-56, 62-64, 105-109 founding mutations, 110-1 1 1 mutator phenotype and tumorigenesis, 311-112 Turcot syndrome, 96 Missense mutations, 105, 107 Mitotic checkpoint, p53 and, 7, 8 M L H l gene, 62-64,98,99 chromosome 3 and, 55-56 founding mutations, 110-11 1 HNPCC, 93-94,95 kindred studies, 103, 104 missense mutations, 105, 107 mutations, 105-109, 114 MLHl protein, 98, 101 MMR genes, see Mismatch repair genes M 0 1 5 kinase, 172 Monoclonal antibody therapy, HER-2heu protein, 343-345,356-367 MORT1 protein, apoptosis and, 129 MSHZ gene, 99 chromosome 3 and, 56 founding mutations, 110-1 11 HNPCC, 93-94,95 HNPCC kindred studies, 103, 104 mutations, 105-109, 113, 114 MSHZ protein, 101 MSH3 gene, 98,100 MSM2 protein, 98 MSTl gene, 67 MTS, see Multiple tumor suppressor Mucosal vascular addressin, CD44 binding to, 273 Muir-Torre syndrome, 96, 113 Multiple endocrine neoplasia type 2, chromosome 3 losses in, 52 Multiple tumor suppressor (MTS), 196-197 Mutation rate, 210-211, 214 age and, 210,230 as biological pacemaker, 231-233 cancer prevention and, 234-235 importance of measuring, 233-234 in normal tissue, 216

382 Mutations, 210; see also Germline mutations; Somatic mutations APC gene, 111 as biological pacemaker, 231-233 cancer and, 243 endometrial carcinoma, 113 founding mutations, 110-111 glioblastomas, 114 hereditary nonpolyposis colorectal cancer and, 93-1 16 missense mutations, 105,107 Muir-Torre syndrome, 113 of p53,7-8 rate, see Mutation rate retinoblastoma, 29 in tumors, 214-215 Turcot syndrome, 114 VHL mutations, 60 mutL gene, 98 myc proto-oncogene apoptosis and, 127-128 E2F upregulation by, 182, 184 Myelodysplastic syndrome (MDS), 148-149, 150 Myeloid cells, differentiation, 135-136 MyoD, 174 Myogenin, 174 Myositis, age and risk of, 230

N Nasopharyngeal carcinoma CD44 and, 294 chromosome 3 losses in, 45-47 VHL mutations and, 60 N-CAM, 244,251 Necrosis, different from apoptosis, 123 Nervous system tumors, CD44 and, 291-293 Non-Hodgkin lymphoma, CD44 and, 303-304 Nucleotide excision repair system, 212 Nur77 protein, 137-138

0 Oncogenes, defined, 195 Oral cavity carcinoma, chromosome 3 losses in, 4 5 4 7 Oral squamous cell carcinoma, transfer of chromosome 3 into, 55

Index Osteopontin, CD44 binding to, 273 Ovarian cancer antigen-negativevariants, 358 chromosome 3 losses in, 50 CD44 and, 300-301 HER-2Ineu protein overexpression, 357 transfer of chromosome 3 into, 55

P p14.2 band, chromosome 3 and, 70-73 p l 5 protein cancer and, 197 cdk4 inhibition, 174 TGF-P and, 193 p16 protein cancer and, 197 cdk inhibition by, 173, 174 p2lC'P1, 174 p21 gene, 4 , 5 cyclin-cdk complexes, 5-6 PCNA and DNA replication, 6 p21 protein, 196 inhibition, 1 7 4 1 7 5 TGF-P and, 193 p21.3 band, chromosome 3,56-58,67-70 p27K'P1 gene, 174175 disruption of, 1 9 4 1 9 5 T-cell proliferation, 194 p27 protein cancer and, 197 cdk inhibition, 175-179, 179 cyclin-cdk complexes and, 175, 178 TGF-P effects, 189-192, 193 p33cdkz,168-169 p4OSLc1,176 p53, E2f and, 130 p53 gene, 1 , 2 adenovirus and, 131 apoptosis and, 8-9, 12, 127, 128 chemotherapy, sensitivity of, 152 chronic myeloid leukemia and, 145 DNA damage, G1 arrest, 2-7 functions of, 2 genetic instability and, 7-8 p57 protein, cdk inhibition, 177-178 ~107,182 pllORbprotein, 179-180 ~130,182,196 p369-377 protein, 349 Pan-CD44,299

383

Index Pancreatic cancer, CD44 and, 297-298 Papillomaviruses, 322; see also Human papillomavirus genome, 322-325 genomic variability, 326-327 late genes, 323 taxonomy, 325 PARP, see Poly-(ADP)ribose polymerase PCNA, see Proliferating cell nuclear antigen PCR, see Polymerase chain reaction Phagocytic glycoprotein-1,246 Pheochromocytoma, 52,58 Philadelphia chromosome, 144, 145, 147 Phosphorylation cdk activation by, 5, 172 cdk inhibition by, 173 p27 and, 175 TGF-P and, 188-189 PI-3 kinases, 12-13, 15 pMeta-l,251,252, 262, 290 pMeta-2,251 PMSZ gene, 98,103,104 PMSl proteins, 98 PMSZ gene, HNPCC kindred studies, 103, 104 PMSR1-7 genes, 98 Poly-(ADP)ribosepolymerase (PARP), 130 Polymerase chain reaction (PCR) CD44 variants, 252 HPV virus, 327 microsatellite instability, detection by, 2 13-2 14 Polyps, mutations and, 111 Population control, social control hypothesis, 133-134 PRA DZ oncogene, overexpression in cancer, 196 Preleukemia, 148-149, 150 Preneoplastic lesions esophagus, 49 lung cancer, 39-41 Programmed cell death, see Apoptosis Proliferating cell nuclear antigen (PCNA) Gadd4S and, 4-5 p21 and, 6 Prostate cancer CD44 and, 299 chromosome 3 losses in, 51 Proteoglycan core proteins, 245 P-selectin, 244 PTPRG gene, 72-73

R rad3 mutants, 13-14 Rad3 protein, 13 RadS3 protein, 14 Radioresistant DNA synthesis, 11 Rapamycin mechanism of action, 195 target of, 13 RARB gene, 66 ras gene CD44 expression and, 275 chronic lymphocytic leukemia, 146 preleukemia, 149 RBZ gene, 28,30 RCC, see Renal cell carcinoma RecQISgsl helicase, 212 Renal cell carcinoma (RCC) CD44 and, 298 chromosome 3 losses in, 42-45, 75 transfer into cell lines, 54-55 3p13-p4 gene, 70 3p21.3 genes, 67-68 VHL disease and, 59-61 Replication error positive (RER+)genes, 62 Replication error positive (RER+) phenotype, 99 colorectal cancer, 100 HNPCC, 100 TGP receptor loss, 185 Representational difference analysis, 33-34 Restriction point (R point), 170 Retinoblastoma age and risk of, 230 mutation and, 29 Retinoblastoma gene (pRb), 324 Retinoblastoma protein, 188 p53-mediated growth arrest, 5-6 phosphorylation, 180 RHAMM, 263 Rheumatoid arthritis, CD44 and, 286-287 Richter syndrome, 147 Ricin, HER-2heu immunotherapy, 362-363 RIP protein, apoptosis and, 129 a-RLC gene, 69 R-Ras protein, apoptosis, 128

S SCLC cell lines, 35-42, 67 SEA, see Staphylococcal enterotoxin

384 Selectins, 244 SEMA-A gene, 69-70 SEMA-IIUF gene, 69-70 Semaphorin genes, 69-70 Seminomas, chromosome 3 losses in, 51 Serglycidgp600, CD44 binding to, 273 Signal transduction ATM gene product, 10 by TGF-Pl, 184-186 Single-chain antibodies, HER-2heu immunotherapy, 363-364 Small-cell lung cancer-derived tumor cells, see SCLC cell lines Social control hypothesis, population control, 133-134 Somatic cell hybrids, tumor growth studies, 34 Somatic mutation rate, 229 DNA repair level and, 212-213 importance of measuring, 233-234 Somatic mutations, 209-21 1 germline mutations and, 228-231 HPRT gene, 217-221 microsatellite sequences, 21 1-216 minisatellite sequences, 217 multistep carcinogenesis and, 221-222 rate, see Somatic mutation rate Squamous cell carcinoma head and neck, see Head and neck squamous cell carcinoma vulval, 50 src gene, CD44 expression and, 275 Standard CD44, see CD44s Staphylococcal enterotoxin (SEA), 365-366 Stem cells, viability, 134-135 Stomach cancer CD44 and, 296-297 chromosome 3 losses in, 4 7 4 8 Survival factors, 128, 131 myeloid differentiation and, 135-136 population control and, 133-134 stem cell viability and, 134-135

T T-bodies, 363 TCR-CD3 signaling complex, 137 Tell protein, function of, 13, 1 4 Testicular germ cell tumors, chromosome 3 losses in, 51 TGF-P,64-65

Index

C3H lOTtcell cell cycle kinetics, 187, 190,191 cancer and, 168 cdk G1 phase activity, effect on, 188-194 cell cycle control, 165, 166-168 cyclin and cdk expression and, 192-193 G1 phase progression and, 186-188 inflammation and, 141 melanoma and, 197 p l 5 levels and cki distribution, 193-194 p27 expression and distribution, 189-192 pRb phosphorylation and, 188-189 T-cell proliferation and, 194-195 TGF-PI, 166 G1 phase progression and, 186-188 signal transduction by, 184-186 TGF-P receptor I1 gene, 64-66, 111 mutations, 113 TGF-P receptors, 184-185 loss of, 185 TGFBR2 gene, 65-66 THRB gene, 66 3p13-pl4 band, tumor suppressor genes from, 70-73,76 3p21 genes, 67 3 ~ 2 1 . 3band, tumor suppressor genes from, 56,67-70,75 Thyroid carcinoma CD44 and, 294 chromosome 3 losses in, 52 T lymphocytes CD44 and, 257-258 cytotoxic, see Cytotoxic T lymphocytes differentiation, 137-138 TGF-p and proliferation of, 194-195 TorlKor2 protein, function of, 13 Transforming growth factor-@,see TGF-P Tumor cells apoptosis, 18 karyotyping, 31-32 Tumors allelic losses in, 32-33 apoptosis and, 18 mutations in, 214 Tumor suppression, 54-56, 195-196 Tumor suppressor genes, 28-30 chromosome 3 and, 75-76 from 3p13-p14,70-73,76 from 3p21.3,56, 67-70, 75 h M L H 2 gene, 62-64 mismatch repair, 55-56

385

Index TGF-P receptor type I1 gene, 64-66 THRB and RARB, 66-67 von Hippel-Lindau disease gene, 58-62 cyclin-dependent kinase inhibitors (ckis), 195 fragile sites and, 70-71 inactivation of, 222 localizing, 30-3 1 allelic loss analysis, 32-33, 75 cell fusion and transfection, 34-35, 5 6-5 7 genome analysis, 33-34 karyotyping, 31-32 pS3 gene, 2-9 RB1,28,30 in rodent lines, 56, 58 Turcot syndrome, 96, 114 Tyrosine kinase inhibitors, 154

U U B E l L gene, 68 UNPH gene, 68-69 Uveal melanoma, chromosome 3 losses in, 52

Vaccines HER-2heu protein, 346-356 human papillomavirus, 327 Vaccinia, as HER-2Ineu vector, 356 VCAM-1,244 VHL disease, see von Hippel-Lindau disease gene VHL protein, 61 Viruses, apoptosis mechanism in host, 130-1 3 1 Vitronectin receptor, apoptosis and, 124 VLA-4,244 von Hippel-Lindau disease gene, 58-62 Vulva1 squamous cell carcinoma, chromosome 3 losses in, SO

w

Werner syndrome, 212 Wound healing, CD44 and, 285

X Xeroderma pigmentosum, DNA repair deficiency and, 212

V

Z

u-Abl gene, apoptosis, 128

Zinc finger genes, 6 7

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