ADVANCES IN CANCER RESEARCH VOLUME 64
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ADVANCES IN CANCERRESEARCH Edited by
GEORGE F. VANDE WOUDE ABL-Basic Research Program NCI-Frederick Cancer Research and Development Center Frederick, Maryland
GEORGE KLElN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden
Volume 64
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Copyright 8 1994 by ACADEMIC PRESS, INC. 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.
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International Standard Serial Number: 0065-230X International Standard Book Number: 0-12-006664-5 PRINTED IN THE UNITED STATES OF AMERICA 94 95 9 6 9 1 98 9 9 Q W 9 8 7 6
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CONTENTS
ix
CONTRIBUTORS TO VOLUME 64 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Interactions between Papillomavirus Proteins and Tumor Suppressor Gene Products KAREN H. VOUSDEN I. 11. 111. IV. V.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human Papillomaviruses ............. Regulation of Cell Growth . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . ViraUHost Protein Interactions HPV Oncoproteins-Tools and ............................. References ...................... . . . . . . . . . . . . . . . . . . . . . .
1 2 5 7 18 19
The Retinoblastoma Tumor Suppressor Protein JEANY. J. WANG,ERIKS. KNUDSEN, AND PETER J. WELCH I. 11. 111. IV. V. VI. VII.
Overview ........................... . . . . . . . . . . . . . . . . . . . . . . Mutation o .... .................. Growth-Inhibitory Activity of RB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Cycle-Regulated Phosphorylation of RB Protein-Binding Function of RB ................................... Regulation of RII Function by Phosphorylation ..................... Future Prospects ................................................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V
25 26 30 32 48 70 75 78
CONTENTS
SH2 and SH3 Domains in Signal Transduction
TONY PAWSON Protein 'I'\rosine Kinases and Their Targets . . . . . . . . . . . . . . . . . . . . . . . . . SHY Doniains . . . . ..... S H 3 and P€1 Donia ............................ Coupling Tyrosine Kinases to Ras . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. SHY-(:ontaining Phosphotyrosine Pliosphatases and the Genetics of Signal Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
87
11. 111. IV.
YO
98 100
104 105
Activation of the Src Family of Tyrosine Kinases in Mammary Tumorigenesis SENTHIL
K.
hfLT'HL!Sb'AMY A N D
WILLIAM J. MULLER
Iiitroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Src Family of Protein T!rosine Kiiiases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Elevation of c-Src Kinase Activity in Primary ,2laniniary Tumors and Tumor-Derived Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1V. Tr;insgenic Mouse Models for Testing the Role of Src Family in 3fanimar) 'lumorigenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . \'. Future Prospects . . . . . . ........................... ....... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.
11. 111.
1I I 112
I17 1 17 120 120
Oncogenic Properties of the Middle T Antigens of Polyomaviruses E'KIEDEMANN KIEFEK, SAKA
I. 11. 111.
I\'. V.
A.
COUKTNEIDGE, AND ERWIN
F.
WAGNER
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (;onsrqucnces of PvntT Expression i r r i'rrw ............ ..... Expression of the Hamster Polyomavirus Middle T Arlcigen in Vivo . . . Analysis of PyniT-Transformed Endothelial Cells . . . . . . . . ..... Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... ..... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
125 133 148 149 153 154
Selective Involvement of Protein Kinase C lsozymes in Differentiation and Neoplastic Transformation
JOANNE GOODNIGHT, HARALD MISCHAK, I. 11.
A N D J.
FKEDERIC MUSHINSKI
Iiitroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P K C Isofornis Involved in Differentiation ..........................
160
176
vii
CONTENTS
111.
Involvement of PKC Isoforms in Tumorigenesis ..................... I v. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
187 196 198
Fcy Receptors in Malignancies: Friends or Enemies? JhNos
GERGELY AND GABRIELLA SARMAY
.................... . . . . . . . . . . . . . . . . . . . . . . I . Introduction Rs . . . . . . . . . . . . . ...... ... I1. Structural Fea 111. Ligand Binding and FcyR Binding Sites ............................ I v. Functions Mediated by Mrmbrane-Bound FcyRs .................... v. Signal Transduction Mediated by FcyRs . . . . . . . . VI . Expression of FcyRs .............................................. ...... .......... VII . Soluble Fcy Receptors . . ......................... VIII . Mechanisms of sFcyR (Ig IX . Regulatory Role of Membrane-Bound and Soluble FcyRs . . . . . . . . . . . . . ... X . Expression of FcRs on Tumor Cells ...................... ............................... XI . ... XI1 . Biological Role of FcR-Mediated Functions in Malignancies . XI11.
References . . . . . . . . . . . . .
............................... ........... ..........
211 212 215 217 219 221 225 227 230 233 235 236 237 238
Dissecting Molecular Carcinogenesis: Development of Transgenic Mouse Models by Epidermal Gene Targeting DAViD
A . GREENHALGH AND DENNIS R . ROOP
I. I1 . 111 . I V.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Development of Single Transgenic Genotypes ....................... Development of Multiple Transgenic Genotypes ..................... Development of a Rapid Screening System for Tumor Promoters and Chemical Carcinogenesis .......................................... V. Summary and Future Prospects . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
247 250 274
INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
297
286
288 290
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CONTRIBUTORS TO VOLUME 64
Numbers in parentheses indicate the pages on which the authors' contributions begin
SARAA. COURTNEIDGE, European Molecular Biology Laboratory, D-691 I 7 Heidelberg, Germany (125) JANOSGERGELY, Department of Immunology, Eotvos Lordnd University, God 2131, Hungary (211) JOANNEGOODNIGHT, Molecular Genetics Section, Laboratory of Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 (159) Department of Cell Biology and Dermatology, Baylor DAVIDA. CREENHALGH, College of Medicine, Houston, Texas 77030 (247) FRIEDEMANN KIEFER,Institute of Molecular Pathology, A-I 030 Vienna, Austrial (125) ERIKS. KNUDSEN,Department of Biology, University of California, San Diego, La Jolla, California 92093 (25) HARALD MISCHAK,Institute for Clinical Molecular Biology and Tumor Genetics, GSF, 0-8000 Munich 70, Germany (159) WILLIAM J. MULLER,Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario, Canada L8S 4KI ( I I I ) J. FREDERIC MUSHINSKI, Molecular Genetics Section, Laboratory of Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 ( I 5 9 ) K. MUTHUSWAMY, Institute f o r Molecular Biology and Biotechnology, SENTHIL McMaster University, Hamilton, Ontario, Canada L8S 4K1 ( I I I ) TONY PAWSON, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1 x 5 , and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5G 1 x 5 (87) DENNISR. ROOP,Department of Cell Biology and Dermatology, Baylor College of Medicine, Houston, Texas 77030 (247) 1
Present address: Ontario Cancer Institute, Toronto, Ontario, Canada M4X 1K9.
ix
X
CONTRIBLTORS
GABRIELLA SARMAY,Vieniia Internatzorial Cooperation Center at Sandoz I;orschuii~,-liutitut,1230 Vienna, Austria (211) KARENH . VOUSDEN, Ludwag Instztutv fot Cancel Research. St. Mury's Hospztul Medzcul School, London W2 1P G , Englmd (I) ERWINF. W A G N ~ R Instilute , of Moleculur Pathology, A-1 030 Vtenria, Auytrza (125) ~ E A NY J. WAXL.Departmeiit of Biology, c'riivel Tits of Cnlafornin, Sun Diego, IA Jollu, Culifiwnta 92093 (25) R.1tu J. W E L C H , Depm trneiit of Bzolo
INTERACTIONS BETWEEN PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSOR GENE PRODUCTS Karen H. Vousden Ludwig Institute for Cancer Research, St. Mary's Hospital Medical School, London W2 1PG, England
I. Introduction 11. Human Papillomaviruses A. Human Tumor Virus
B. Normal Viral Life Cycle C. High-Risk HPV-Encoded Oncoproteins 111. Regulation of Cell Growth A. Control of the Cell Cycle B. Tumor Suppressor Genes IV. ViraVHost Protein Interactions A. Activities of E7 B. Activities of E6 C. Function of HPV-Encoded Oncoproteins in the Normal Viral Life Cycle D. E6 and E7-Targeting a Common Pathway? V. HPV Oncoproteins-Tools and Targets References
I. Introduction
Human papillomaviruses (HPVs) are small DNA viruses that have provided unique insight into the mechanisms that regulate growth and oncogenic progression of human cells. Most members of this large group of common viruses pursue a life cycle that has little serious impact on the host, infecting epithelial tissue and producing self-limiting hyperproliferations, more commonly known as warts (von Knebel Doeberitz, 1992). T h e intense interest in these viruses has resulted from the observation that some of the genital HPV types cause lesions that are not strictly benign; the malignant progression of HPV-infected cells appears to give rise to almost all cervical cancers (zur Hausen, 1991b; Munoz and Bosch, 1992). The importance of this relationship is obvious, since cervical cancer is the second most common cause of cancer death among women worldwide and the most common cancer (without sex adjustment) in parts of the developing world (Parkin et al., 1988). Other viruses 1 ADVANCES IN CANCER RESEARCH, VOL. 64
Copyright 0 1994 hy Academic Press, Inc. All rights of reproduction in any form reserved.
2
KAREN H. VOC‘SDEN
such as hepatitis B virus, Epstein-Barr virus, and human T cell leukemia virus type 1 (HTL\’-l) have also been strongly implicated in the development of several human cancers (zur Hausen, 1991a); the longterm goal of preventing these cancers by prophylactic vaccination to eliminate viral infection is further advanced for some of these viruses than for the HPVs. Unlike the situation for these other viruses, however, scientists have an increasingly clear understanding of how the HPVs contribute to oncogenesis at the molecular level and how several virally encoded proteins exhibit activities consistent with a role in malignant progression. These viral proteins therefore provide targets for the development of therapeutic drugs and the potential to treat, as well as prevent, the majority of cervical cancers. II. Human Papillomaviruses A . H U M A N~ r U M O KVIRUS
Epidemiological studies predicted the involvement of a transmissible agent in the etiology of cervical cancer many years before the role of certain genital HPV types was clearly identified (zur Hausen, 1976). Several different HPV types infect the genital tract and only some of these, the so-called high-risk HPV types, are associated with malignancies (De Villiers, 1989). High-risk HPVs, most frequently HPV16 or 18, are evident in over 90% of cervical cancers; these viral types have been intensively studied. T h e second group of genital HPV types, the low-risk viruses, is found almost exclusively in benign lesions. T h e most common of these are HPVG and I 1 and although there is rarely evidence of malignant progression, the appearance of often large genital warts illustrates the ability of these viruses to induce abnormal growth and hyperproliferation. ‘The natural history of infection with these HYV types is slowly being elucidated, although rhc absence of serological tests to determine incidence of infection has somewhat hampered these studies. As might be expected from a sexually transmitted agent, the incidence of genital HPV infection is rather low until early adulthood, when the commencement of sexual activity is paralleled by a rapid rise in infection which apparently declines in subsequent years (Schiffman, 1992). Less than 5 4 of healthy women over 40 years old show any evidence of HPVIG or 18 infection (de Sanjose et al., 1992),in stark contrast to those with cervical cancers, 90% of which are positive for these HPV types (van den Brule et al., 1991). T h e detection of high-risk HPV in many more young women than will be expected to develop cervical cancer indicates that infection with
PAPILLOMAVIRUS PROTEINS AND T U M O R SUPPRESSORS
3
these HPV types is not sufficient for full malignant progression. Evidence now suggests that these viruses give rise to cervical intraepithelial neoplasia, previously recognized as premalignant lesions that sometimes, although not always, progress to invasive carcinoma (Koutsky et al., 1992). T h e epidemiology is consistent with the notion that high-risk HPV infection only becomes a problem when viral infection becomes persistent and additional oncogenic events accumulate (zur Hausen, 1991b). HPV infection therefore only contributes to part of the multistep oncogenic process (Vogelstein and Kinder, 1993). The nature of such additional events is not clear, although evidence implicates environmental carcinogens through mechanisms such as mutation of cell genes and immune suppression (Jackson and Campo, 1993). Although a role for HPV infection is clear in most cervical cancers, a small but consistent proportion of these malignancies arises without strong evidence of HPV involvement. It is possible that as yet unidentified HPV types are involved in these tumors or that viral sequences have been lost during progression. T h e epidemiology of these cancers is somewhat different from that of the HPV-positive cancers, however, and it seems more likely that in rare cases these malignancies can arise through HPV-independent mechanisms (Riou et al., 1990; Higgins et al., 1991). B. NORMAL VIRALLIFECYCLE
Despite the frequency with which HPV infection occurs in vivo, these viruses are extremely difficult to culture in experimental systems. Consequently very little is known about their natural replication. Two important points are clear, however. The first is that malignant progression is not part of the normal life cycle of any of the genital HPVs. The loss of normal differentiation associated with malignant progression precludes virion production, which requires terminally differentiated keratinocytes (Dollard et al., 1992; Meyers et al., 1992). In many cases cancer cells contain only incomplete viral genome9 that have become integrated into host chromosomes (Cullen et al., 1991; Das et al., 1992). Since all virally encoded proteins must principally play a role in viral replication, any oncogenic activity exhibited by them should be viewed as an unfortunate manifestation of their normal function. That any viral activity exists only to participate in malignant progression seems extremely unlikely. Related to this first idea is the second point of importance when considering the normal viral life cycle, which is that both high- and lowrisk viruses share the ability to disrupt the normal regulation of cell growth and to give rise to hyperproliferation lesions. These viruses in-
4
KAREN
H. VOUSDEN
fect cells that embark on a program of differentiation to form an epithelium consisting of predominantly nondividing cells. T h e small HPV genome cannot encode all the proteins necessary for its own replication; the viral activities that have been identified as potentially oncogenic probably play a principal role in the induction of unscheduled host cell division, thereby allowing the virus to utilize the host replicative machinery. It is therefore of interest to note that both high- and low-risk viruses display some ability to deregulate normal growth control in cultured cells (Storey et al., 1990; Halbert et a[.,1992). All the HPV types are likely t o use similar mechanisms to perturb normal epithelial cell proliferation. The oncogenic potential of the high-risk viruses is paralleled, however, by substantially higher in vitl-o transforming and immortalizing activities. T h e overall difference between the high- and low-risk viruses is likely to reflect the sum of many parameters including viral protein function, control o f - viral gene expression, target cell type, and immune response to infection. T h e consequences of these differences to the normal viral life cycle remain unclear. (;.
HIGH-RISKHPV-ENCODED ONCOPROTEINS
The ability of DNA from the high-risk HPV types to transform and immortalize cells in culture allowed the identification of E6 and E7 as the principal viral oncogenes (Vousden, 199I), although other viral proteins such as E5 ma) also contribute to oncogenesis (Banks and Matlashewski, 1993).Each of these viral genes can function independently in primary or established rodent cells, although E7 generally shows the strongest activity. In primary human genital epithelial cells, a cell type more closely related to the natural target cell of these viruses, cooperation between E6 and E7 is required for efficient immortalization (Hawley-Nelson et al., 1989; Munger el ul., 1989a). This assay is of particular interest since the HPV-immortalized human cells display alterations in growth and differentiation that are ciosely analogous to those seen in cervical intraepithelial neoplasia (McCance el al., 1988; Hudson ef al., 1990) and, like the zn 7~2710 HPV-induced lesions, these cells are not directly tumorigenic (Pirisi et ul., 1988; Kaur and McDougall, 1989). Oncogenic progression can be achieved following the introduction of other oncogenes or exposure to mutagens (DiPaolo et al., 1989; Hurlin et al., 1991; Klingelhutz et al., 1993). This tissue culture system is likely to provide an excellent model in which to identify steps contributing to cervical carcinogenesis. T h e E6 and E7 proteins encoded by the low-risk viruses function only poorly in each o f these immortalization or transformation assays (Schle-
PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSORS
5
gel et al., 1988; Storey et al., 1988; Barbosa et al., 1991) and at least some of the critical differences in the oncogenic activities of the high- and lowrisk viruses lie in the biochemical activities of the E6 and E7 proteins themselves. Much effort has been expended to identify the mechanisms by which E6 and E7 function, with particular emphasis on activities that are absent from or reduced in the low-risk E6 and E7 proteins, to pursue the long-term goal of designing drugs to interfere with the oncogenic activities of the viral proteins. In a series of studies that have drawn heavily on previous analyses of other small DNA tumor viruses such as adenovirus and simian virus 40 (SV40), the mechanism of action of both E6 and E7 was shown to be related to their ability to form complexes with and perturb the normal functions of cell proteins that are involved in regulating cell growth. The intersection of the fields of tumor virology, cell cycle control, and tumor suppression has allowed remarkably rapid progress in our understanding of the significance of these viralhost protein interactions. Ill. Regulation of Cell Growth
A. CONTROL OF
THE
CELLCYCLE
T h e fate of cells within an organism is normally regulated to maintain a balance between cell growth and cell loss. In epithelial tissue, replication is limited to the basal layers. As cells leave these layers they become committed to a pathway of terminal differentiation, resulting ultimately in death. The decision for cell division is regulated by the cellular environment; signals from the extracellular milieu, such as growth factors, are transmitted to the cell nucleus through a complex cascade of signal transduction (Pelech, 1993). Cell proliferation proceeds through a tightly regulated and ordered series of events that constitute the cell cycle. T h e two distinct phases of DNA replication (S phase) and mitotic division (M phase) are separated by the GI and G, periods during which checkpoints and feedback controls operate to monitor the fidelity and completion of the preceding stage and to allow preparation to take place for the next phase of the cell cycle (Murray, 1992). T h e GI period is of particular importance in the interaction between the cell and its environment, since only during this phase is the cell sensitive to inadequacies in nutrient or growth factor levels (Pardee, 1989). Failure to satisfy conditions for progress through the cell cycle at any of the checkpoints results in arrest and possible exit from the cell cycle. Central to this control are the family of cyclin-dependent kinases (cdks), first identified in yeast,
6
KAREN H . VOC'SDEN
which play a pivotal role in regulating progress into both S and M phase (Pines and Hunter, 1991). Active forms of the enzyme consist of a catalytic subunit, encoded by the cdk gene family, associated with a regulatory subunit from the family of cyclins. A growing number of cdks and cyclins are being identified with multiple combinations of catalytic and regulatory domains (Xiong and Beach, 1991; Pines, 1993). It is now clear that, in mammalian cells, the activity of the different members of this family of enzymes is specific to certain stages of the cell cycle. Many of the components involved in transmitting the mitogenic signal from growth factors have been identified as potential oncogenes including growth factors themselves, growth factor receptors, and the cytoplasmic kinases responsible for signal transduction (Cantley et al., 1991). Researchers have determined that growth-factor-mediated sigtials can be received by the cell cycle machinery through the G , cyclins, such as the three D-type cyclins (Sherr, 1993) and not surprisingly, alterations in expression of these cyclins have also been identified during cancer development (Hunter and Pines, 1991). B . TUMOR SUPPRESSOR GENES
Perturbations of any of the normal controls of cell proliferation potentially contribute to malignant conversion, either by gain of inappropriate positive signals, as just described, o r by loss of controlling negative signals. Such negative controls are encoded by tumor suppressor genes, which characteristically suffer a loss-of-function mutation in both alleles during cancer development (Marshall, 1991). T h e paradigm for tumor suppressor genes, the Rb-1 gene originally identified in retinoblastomas (Knudson, 197 l), has now been joined by a growing number of genes whose loss of function contributes to cancer development; subsequently many of these genes have been shown to play a role in preventing or delaying progress through the cell cycle (Knudson, 1993). Although the original identification of tumor suppressor genes was through association of hereditary mutation in one allele with predisposition to certain cancers, subsequent studies have shown that somatic mutation within the tumor suppressor loci frequently contributes to the development of sporadic cancers. Indeed, the most common target for mutation identified in human cancers is the tumor suppressor gene p53 (Hollstein et nl., 1991). Several cell-encoded proteins have been detected in association with E6 and E7 and the identification of some of these as the products of tumor suppressor genes has revealed at least some of the mechanisms of viral oncoprotein function.
PAPILLOMAVIRUS PROTEINS A N D TUMOR SUPPRESSORS
7
IV. Viral/Host Protein Interactions A.
ACTIVITIES OF E7 1 . Interactions of E7 with the pRB Family
The identification of cell proteins that form a complex with the highrisk E7 proteins was greatly aided by the observation that E7 contains a domain with significant sequence similarity to the transforming proteins encoded by two other DNA tumor viruses, adenovirus ElA and SV40 LT (Phelps et al., 1988). This region of each of these proteins is responsible for an interaction with a family of cell proteins that includes the product of the retinoblastoma gene, pRB, and the related p107 and p130 proteins (Munger et al., 198913; Dyson et al., 1992; Davies et al., 1993). Mutational analysis of E7 demonstrated that the ability to bind these cell proteins correlates with transforming and immortalizing activity (Barbosa et al., 1990; Chesters et al., 1990; Phelps et al., 1992), although in the context of the full-length viral genome apparently other viral functions might be substituted for the pRB-binding activity of E7 in the human cell assay (Jewers et al., 1992). The importance of the E7pRB interaction to oncogenic activity is further supported by the reduced affinity of the low-risk E7 proteins for pRB. Substitution of a single amino acid within the pRB-binding region of HPV6 E7 for that found in HPV16 E7 both dramatically increases the affinity of the E7 protein for pRB and also restores transforming activity (Heck et al., 1992; Sang and Barbosa, 1992). Interestingly, in a small sample of cervical carcinoma cell lines, E7 expression could apparently substitute for somatic Rb-1 mutation since these changes were only found in the two HPV-negative cell lines examined (Scheffner et al., 1991). These studies therefore support a role for loss of pRB activity in cervical cancer development and suggest that this loss might be achieved following expression of E7. To test this hypothesis directly, however, an understanding of the normal function of pRB and other related proteins was required. 2 . Normal Functions of the pRB Protein Family pRB, p107, and p130 are related proteins that all play a role in regulating cell growth and progress through the cell cycle. One of the best understood mechanisms by which this family of proteins functions is their interaction with the family of E2F transcription factors (Cobrinik et al., 1993; Zamanian and La Thangue, 1993; Zhu et al., 1993), which in turn participate in the control of expression of several cell genes
8
KAREN H. VOUSDEN
essential for cell cycle progression (Fig. 1; Wiman, 1993). Binding of pRB to E2F negatively regulates the activity of the transcription factor (Heldin et al., 1993), rendering it either inactive or able to function as an inhibitor of transcription from E2F-responsive promoters. For pRB, this activity has been shown to be limited to the under- or hypophosphorylated form of the protein that is present in cells during G , (Nevins, 1992); cumulative phosphorylation of pRB as the cells traverse S and G, correlates with the dissociation of most (although not all) of these complexes. Interestingly, the phosphorylation of pRB can be carried out by the G I specific cdks; overexpression of the cyclin component of these enzymes overcomes the cell cycle arrest induced by pRB (Hinds et al., 1992; Sherr, 1993). The direct association between pRB and cyclins D2 and D3 appears to reflect the ability of the cyclins to participate in the regulation of pRB activity (Ewen et al., 1993; Kato et al., 1993) and evidence exists that both p107 and p130 also interact with the D-type cyclins, suggesting that they may be similarly regulated by phosphorylation (Hannon et al., 1993; Li et al., 1993). p107 and p130 also form complexes with cyclins A and E through a region of similarity in these two proteins that is not shared by pRB (Faha et al., 1992; Lees et al., 1992; Hannon et al., 1993; Li et al., 1993), although rather intriguingly neither cyclin A nor E expression can abrogate the growth-inhibitory effect of p107 (Zhu et al., 1993). Despite the functional similarity shared by the pRB family of proteins, there are also obvious differences with respect to the form of E2F with which they interact and the exact mechanisms by which they regulate the activity of the transcription factor (Cress et al., 1993; Dyson et al., 1993; Zhu et al., 1993). This diversity of function probably reflects the complexity of EPF-dependent transcriptional control and the participation of each of the pRB-related proteins in the regulation of different stages of cell cycle progression. Interactions with E2F factors only represent a facet of the potential pRB activities in regulating cell growth, however. pRB has been shown to complex with several other cell proteins including other transcription factors (Rustgi et al., 1991; Kim et d., 1992; Hageemeier et al., 1993; Wang et al., 1993), the cytoplasmic tyrosine kinase c-Abl (Welch and Wang, 1993), and members of the cyclin D family (Dowdy et al., 1993; Ewen et al., 1993). Although some of these interactions, such as those with cyclins D2 and D3, may reflect control of pRB function, in many cases the converse is likely to be true, that is, pRB is responsible for regulating the activity of the bound protein. Of particular interest in this respect is the possibility that pRB regulates the growth-promoting activity of cyclin D1 (Dowdy et al., 1993).
PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSORS
9
3 . Consequences of the E7IpRB Interaction
Identification of functions for the pRB protein family was rapidly followed by analyses that revealed that E7, which also preferentially binds to underphosphorylated pRB (Imai et al., 1991),could target negatively regulating proteins complexing with E2F and induce expression of E2F-responsive genes (Fig. 1; Phelps et al., 1991; Chellappan et al. 1992; Morris et al., 1993; Lam et al., 1994). Several genes that appear to play important roles in allowing entry into S phase are regulated by E2F and mutational analysis has indicated that the induction of inappropriate E2F-dependent transcription is likely to account for the ability of E7 to induce DNA synthesis in quiescent cells (Banks et al., 1990). This result is consistent with those of studies showing that expression of E2F
A Cell cycle progression Inactivation
of pR6 by phorphorylation
Transcription
G2
I No transcription
B
Inactivation of unphosphorylated pR6
FIG. 1 . Participation of pRB in the control of the E2F transcription factors, which consist of heterodimeric combinations of DP1 and various E2F proteins. (A) Regulation during the G , phase of a normal cell and (B) the consequent disruption of this activity in E7-expressing cells.
10
KAREN H. VOCSDEN
itself is sufficient for entry into S phase (.Johnson et al., 1993). Point mutational analysis of E7 has revealed slightly different sequence requirements for interaction with pRB and p107 (Davies el al., 1993) and E7 mutants that retain plO7-binding activity but fail to interact with pRB have been identified. Such mutants have been useful in analyzing the contribution of pRB and p107 to transcriptional regulation of genes such as B-tnyb, whose expression is repressed during the Go and early G , stages of the cell cycle by E2F/pl07-containing complexes. Activation of the B-myb promoter by E7 correlates with the ability of the viral protein to complex p107 rather than pRB, supporting evidence that pRB is not involved in the regulation of this promoter in mouse cells (Lam et al., 1994). Although E7 clearly disrupts the interaction between pRB and EZF, it can become part of a pl07-E2F-containing complex (Arroyo et ul., 1993; Lam et nl., 1994), further emphasizing the differences among these viral-host protein interactions. The consequence of the presence of E7 in the p 107-E2F complex remains unknown, but such interactions are likely to account for the ability of E7 to associate with a cyclin A-dependent kinase that can phosphorylate p107 and p130 (Davies et at., 1993; Tommasino et al., 1993). Interestingly, the interaction between E7 and the kinase occurs later in the cell cycle, during G,, and may reflect an activity of E7 in overcoming G 2 , as well as G I , blocks in cell growth (Vousden and Jat, 1989). Although analyses of E7 activity have been mostly limited to the effect on E2F regulation, the consequences of the E7 interaction with the pRB family are likely to be manifold. Several of the interactions between pRB and other cell proteins have been shown to be disrupted by E7 in uitro, pointing to extensive pleiotropy of E7 function. Such interactions may result in loss of normal transcriptional control through disruption of pKB complexes with transcription factors and the potential of E7 to release cyclin D 1 from negative regulation by pRB suggests an additional means by which the E7-pRB interaction may affect cell proliferation. Although the consequences of the E7 interaction for many of the pRB complexes have not yet been fully explored, it is already clear that they will vary. Evidence suggests that E7 and E2F d o not bind to identical sites o n pRB (Huang rt al., 1993; Wu et al., 1993a) and it may be possible to disrupt the E7-pKB interaction without significantly affecting normal pRB-E2F complex formation. This will not be true, however, for all the cellular interactions involving pRB. The interaction of pRB with Myc, for example, can be successfully competed using a peptide of E7 containing only the pRB-binding domain (Rustgi et af., 1991); the cyclin D family also appears to complex pRB through the sequence motif found in E7 (Dowdy et al., 1993). Other activities of pRB, for example the
PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSORS
11
recently described regulation of the cytoplasmic tyrosine kinase c-Abl, cannot be perturbed by E7; presumably these functions of pRB remain unaffected in E7-expressing cells (Welch and Wang, 1993). 4 . PRB-Independent Activities of E7
Despite the obvious attraction of assigning many of the biological activities of E7 to the known interactions, mutations in E7 that do not affect any of the known protein interactions can nevertheless also render the E7 protein transformation and immortalization incompetent (Banks et al., 1990; Phelps et al., 1992). Although these additional activities of E7 have not been identified, their importance is graphically demonstrated by the observation that, in the context of the complete viral genome, the pRB-binding activity of E7 is not necessary for keratinocyte immortalization or wart formation (Jewers et al., 1992; Defeo-Jones et al., 1993). Complementation studies have indicated that these additional E7 activities can substitute for pRB-independent activities of E 1A (Davies and Vousden, 1992), although their mechanisms of action do not appear to be identical. B. ACTIVITIES OF E6 1 . Interactions of HPV E 6 with p53
Although there is no strong structural similarity between E6 and oncoproteins encoded by other DNA tumor viruses, a functional parallel exists in the ability of E6, adenovirus ElB, and SV40 LT to target p53, the product of another important tumor suppressor gene (Werness et al., 1990). T h e interaction between the high-risk E6 proteins and p53 results in the rapid degradation of the p53 proteins through ubiquitin-targeted proteolysis (Scheffner et al., 1990). This activity of E6 depends on the interaction of E6 and another cell protein, E6-AP (Huibregtse et al., 1991); the E61E6-AP complex functions as a ubiquitin-protein ligase (Scheffner et al., 1993). Although p53 is normally the target of ubiquitindependent degradation (Ciechanover et al., 199l),whether E6 enhances the normal mechanism of p53 turnover or whether a novel pathway is utilized is not known. T h e ability of E6 to target p53 proteolysis suggests that, like E7, the consequence of the viral-host protein interaction is the inactivation of the cell protein. Advances in our understanding of p53 function have allowed the verification of this hypothesis and there is good evidence that the E6-p53 interaction is an important component of the oncogenic activity of the high-risk HPV types.
12
KAREN H . VOUSDEK
2. Normal Functions of p53 Although loss of wild-type p53 function appears to play a role in most malignancies (Hollstein et al., 1991), there is no clear function for p53 in the proliferation of most cells; mice that lack functional p53 alleles develop normally (Donehower et al., 1992). Inability to express p53 is associated with a high cancer rate in these animals, however, and cells lacking in wild-type p53 display an increased frequency of genetic instability (Livingstone et al., 1992; Yin et al., 1992). Convincing evidence is now available that under some circumstances p53 functions as a checkpoint in the event of DNA damage, after exposure to agents such as ionizing radiation or certain cytotoxic drugs (Lane, 1992). This response is accompanied by a rapid increase in p53 levels due to stabilization of the protein, which results in either arrest at the G, stage of the cell cycle or induction of progranimed cell death. Under normal circumstances, therefore, cells sustaining D N A damage cease proliferation, either temporarily uniil DNA repair has been effected o r permanently through apoptosis. Loss of p53 function can be associated with an inability to undergo G, arrest in the presence of damaged DNA (Kuerbitz et ul., 1992) and the consequent potential to acquire oncogenic genetic lesions is predicted t o contribute to tuniorigenesis. Of course, the cellular responses to DNL4damage are more complex than this and the role of p53 is also less straightforward. This is illustrated by studies indicating that the extremely efficient induction of p53 activity in response to UV daniage is not accompanied by a clear G, arrest, although this response may be related to the activation of proteins that suppress p53 function (Lu and Lane, 1993; Perry et d., 1993). The exact mechanisms by which pi53 functions are not yet clear, although several potentially important activities of p53 have been described and a growing number of inreresting p53-binding proteins have been isolated (Pietenpol and Vogelstein, 1993).Current evidence suggests that the ability of p53 to specifically trans-activate transcription from promoters containing p53-binding sites plays an important role in mediating cell cycle arrest (Fig. 2; Vogelstein and Kinzler, 1992);cell genes that may be transcriptionally regulated by p53 are now being identified (KaStan et nl., 1992; El-Deiry et al., 1993; Wu ei al., 1993b; see Section IV,D). Interestingly, one of these p53-regulated genes, mdm-2, encodes a protein that in turn associates with and negatively regulates the transcriptional activity of p53 in an autoregulatory feedback loop (Momand et al., 1992; Oliner et al., 1993; Wu et al., 1993b). p53 also shows a more general ability to repress transcription of many other cell genes (Ginsberg et al., 1991), particularly those with TATA-containing promoters (Mack et d.,
PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSORS
13
A DNA damage
L!22&& p53 p53
p53 Stabilization
Transcriptional activation
(_> Cell cycle arrest
B DNA damage
p53 Degradation
No transcriptional activation
Cell cycle progression
FIG. 2. (A) Induction of p53 activity following DNA damage in normal cells resulting in the transcriptional activation of cell genes and cell cycle arrest. (B) Targeted degradation of p53 by E6 abrogates the block on cell cycle progression.
1993). This activity may also contribute to the inhibition of cell cycle progression. Evidence for a third activity of p53 in directly interfering with DNA synthesis by binding replication proteins such as RPA (Dutta et al., 1993; He et al., 1993; Li and Botchan, 1993) underscores the important point that regulation of cell proliferation by p53 is likely to be a complex and multifaceted process.
3 . Consequences of the E6lp53 Interaction Initial studies revealed that, although E6 expression in cells results in a reduction of the half-life of the endogenous p53 protein, this effect is not necessarily reflected by a reduction in the total p53 content of E6-expressing cells compared with normal cells (Hubbert et al., 1992;
14
KAREN H. VOUSDEN
Lechner et al., 1992). This result suggests that E6 preferentially targets nascent p53 and implies the existence of a stable pool of p53 within the cell that is not sensitive to E6 although the implications of these observations are not yet understood. T h e identification of the damage-response functions of p53 led to the realization that the E6-p53 interaction may not play an important role in normal cycling cells, which are clearly not growth inhibited by p53. Analyses of the effects of E6 under conditions in which p53 would be expected to induce growth arrest, following DNA damage, have shown that E6-expressing cells do not accumulate p53 protein and subsequently fail to undergo the G, arrest (Fig. 2; Kessis et al., 1993). E6 therefore seems to fulfill its predicted role of inhibiting the growthsuppressing activity of p53. Not surprisingly, expression of E6 can abrogate both the truns-activating and the trans-repressing transcriptional activities of p53 (Lechner et al., 1992; Mietz et al., 1992). Evidence suggests that simply the interaction between E6 and p53 is sufficient for a reduction of p53 activity (Lechner et al., 1992; Crook et al., 1994). E6 has been shown to inhibit p53 DNA binding (M. S. Lechner and L. A. Laimins, personal communication), suggesting at least one mechanism for the abrogation of the transcriptional truns-activation. The transcriptional activity of p53 appears to be mediated or modulated through complex formation with several cell proteins such as TBP, CBF, or WT1 (Seto et al., 1992; Agoff et ul., 1993; Maheswaran et ul., 1993) although the ability of E6 to perturb these interactions remains to be determined. 4 . 156 in Oncogenesis
The significance of the E6-p53 interaction in cancer development is supported by the observation that, unlike many other epithelial tumors, HPV-positive cervical cancers very rarely show evidence of somatic p53 mutations (Crook P t nf., 1991c, 1992; Scheffner et al., 1991; Fujita et al., 1992; Choo and Chong, 1993). The straightforward interpretation of these observations is that expression of E6 in HPV-positive cancers abrogates the tumor suppressor activity of p53 and thus eliminates selection for somatic mutation within the p53 gene itself, a notion supported by the ability of mutant p53 to substitute for E6 in the immortalization of human keratinocytes (Sedman et al., 1992). p53 mutations have been detected in the much rarer HPV-negative cancers (Crook et al., 1991c, 1992; Scheffner et al., 1991), although a significant proportion of these also present without evidence for alterations in the p53 gene (Park et al., 1994). Loss of p53 function through indirect mechanisms is also seen in other types of tumor that display a low incidence of p53 mutation. Sarcomas, for example, frequently demonstrate amplification of the mdm-2 gene (Oliner et a/., 1992); presumably inactivation of p53 in these cancers
PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSORS
15
occurs through interaction with enhanced levels of the mdm2 protein. Preliminary analyses have indicated that mdm-2 is not frequently amplified in HPV-positive cervical cancers (A. Farthing and K. H. Vousden, unpublished observations), consistent with the notion that expression of E6 is sufficient to inactivate p53 and to allow malignant progression. Although E6 expression clearly interferes with the wild-type activity of p53, the p53 gene remains a target for oncogenic mutations even in HPV-positive cells. Evidence exists that some p53 point mutations can induce both loss of wild-type growth-suppressing function and gain of a positive transforming activity (Shaulsky et al., 1991; Sun et al., 1993; Dittmer et al., 1993). The interaction with E6 would be predicted to prevent only the normal function of p53 and expression of mutant p53 might play a role during HPV-associated tumorigenesis, although possibly at a later stage of the oncogenic process (Crook and Vousden, 1992). Importantly, many p53 mutations render the protein insensitive to E6directed degradation (Crook and Vousden, 1992; Scheffner et al., 1992b), thus allowing the expression of a positive transforming function in E6-containing cells. Although the mechanism by which these mutant p53 proteins contribute to malignant progression of HPV-positive cancers is not known, it may be germane to note that in rodent cells strong synergy exists between E7 and mutant forms of p53 in transformation (Peacock et al., 1990; Crook et al., 1991a).
5. $153-IndependentActivities of E 6 Although much emphasis has been placed on the E6-p53 interaction, evidence suggests that some activities of E6, such as the transformation of rodent cells (Sedman et al., 1992), are not dependent on this interaction and that other important functions of E6 remain to be identified. Of particular interest is the observation that the ability of E6 to target proteins for ubiquitination and degradation is not limited to p53 (Scheffner et al., 1992a; Scheffner et al., 1993), raising the possibility that other important regulators of cell growth are also targets of E6-directed degradation. C. FUNCTION OF HPV-ENCODED ONCOPROTEINS IN THE NORMAL VIRALLIFECYCLE
Although the activities of E6 and E7 in abrogating the activities of tumor suppressor gene products can easily be understood in terms of a contribution to tumorigenesis, the importance of these functions to the virus is more likely to be in maintaining cell replication during infection. In the case of E7, perturbation of the control of E2F activity might play a
16
K A R E N H . VOUSDEN
role in maintaining DN'A synthesis is a cell that has embarked on a program of epithelial differentiation and would normally stop dividing. The ability of E7 to interact with p107 is shared by both high- and lowrisk viruses (K.Davies and K. H. \lousden, unpublished observations); the low-risk E7 proteins also retain the ability to activate transcription of EPF-dependent promoters (Storey et al., 1990; Munger et al., 1991). The association o f E7 with pRB, on the other hand, correlates well with the oncogenic activities of the protein in experimental models, suggesting that this interaction does contribute to the malignant potential of the virus. 'l'he low-risk E7 proteins show a much lower affinity for pKB, although they d o retain some binding activity; the relevance of these differences to normal viral replication are not clear. The normal function of E6 may also be in the prevention of cell growth arrest, either following a stress response to viral infection o r during the normal course of epithelial cell differentiation and death. T h e E6 proteins encoded b y the low-risk HPV types interact with p53 much less efficiently than the high-risk proteins (Werness et ul., 1990; Crook et al., 1991b) and, in in zdro assays, are unable to target p53 for degradation (Scheffner ef al., 1990; Crook et ul., 1Wlb), although indirect ejidence suggests that these proteins also retain some degradation activity (Scheffner et al.. 1992a; Band et al., 1993). Corisisterit with these observations is the modest ability of the low-risk E6 proteins to abrogate p53 transcriptional control (Lechner Pt al., 1992; Mietz et al., 1992; Hoppe-Seyler and Butz, 1993; Crook el al., 1994). The weak interactions of the low-risk E6 and E7 proteins with p.53 and pKB may be sufficient to contribute to the replication of these viruses. However, the clearly enhanced efficiency displayed by the high-risk HPV oncoproteins in targeting proteins with an established tumor suppressor activity may be a crucial coniponent contributing to the overall enhanced oncogenic potential displayed by these virus types.
D. E6
AXD
E'~-TARCETING A COMMON PATHWAY?
'l'he gradual expansion of our understanding of various aspects of the regulation of growth control has recently allowed several pieces of the puzzle to be brought together in a pathway involving p53, pRB, and the cdks (Fig. 3 ) . A gene identified as transcriptionally activated in response to p53, called WAFI (El-Deiry et al., 1993), was independently isolated as C I P I , encoding a cdk-interacting protein (Harper et al., 1993); S D I l , a gene active in senescent cells (Noda et al., 1994); and p21, a component of the cyclin-cdk complexes in normal but not transformed cells (Xiong et ul., 1993). The product of this gene, subsequently
PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSORS
17
DNA damage
t
i
I
- A
p53 Stabilization
t
Point of E6 function
Transcriptionalactivation
Kinase inactive
I
CS~Icycle arrest
Point of E7 function
FIG. 3. Model depicting possible functions of E6 and E7 in a common pathway. In a normal cell, activation of p53 following DNA damage results in increased expression of Picl, which inhibits pRB phosphorylation and prevents release of the pRB-mediated block to cell cycle progression. E6 interferes with this pathway by targeting p53 for degradation, thus preventing Picl expression and allowing phosphorylation of pR3. E7 relieves this block by interfering directly with pRB function and may not be expected to prevent the activation of p53 or consequent inactivation of the pRB kinase.
renamed P I C I , negatively regulates the activity of the G,-specific cdks and consequently inhibits entry into DNA synthesis, thus establishing a direct link between p53 activity and regulators of cell cycle progression. A further step can be taken along this pathway, since the GI cdks inhibited by Picl are capable of phosphorylating and inactivating pRB. Transcriptional activation of PIC1 by p53 would therefore be predicted to result in an inability to escape from the pRB-mediated GI arrest of growth. This model is clearly an oversimplification and p53 almost certainly does not function exclusively through Picl. With this caveat in mind, however, it is of interest to consider the potential roles of the HPV oncoproteins in such a pathway. A straightforward corollary of the model is that proteins that inactivate pRB might function downstream of p53 and be capable of overcoming a p53-mediated growth arrest. Identification of SV40 LT as an antagonist of Picl function is complicated by the ability of the viral protein to abrogate the activity of both p53 and pRB, but at least some support for the model is provided by the observation that, in rat cells, expression of either E7 or adenovirus E1A (both pR3binding proteins) can efficiently overcome the growth-inhibitory effects of wild-type p53 (Vousden et al., 1993). It is possible that both E6 and E7 may function independently to overcome DNA-damage-induced cell cycle arrest, E6 functioning by directly inhibiting p53 function and E7
18
KAREN H . VOUSDEN
acting downstream to release the pRB-induced block. A prediction of this model is that expression of E7 alone would not prevent, and may even induce, an efficient, albeit futile, p53 response. It is therefore intriguing to note that human cells expressing E7, but not E6, contain elevated levels of wild-type p53 protein (Demers et nl., 1994). Clearly, p53 and pRB exhibit other important activities; the fact that each of the small DNA tumor viruses has developed mechanisms to interfere with both cell proteins strongly indicates that many of their functions are not equivalent. T h e identification of a pathway potentially linking the activities of these proteins, however, has allowed the first steps toward untangling the complex webs through which positive and negative regulators of growth function. V. HPV Oncoproteins-Tools
and Targets
T h e identification of- the mechanisms by which E6 and E7 function has presented a panoply of potential uses for these viral proteins both in probing the normal regulation of cell growth and in the design of therapeutic drugs to treat cervical disease. The abilities of E6 and E7 to inactivate at least two tumor suppressor gene products have enormous value as tools to investigat.e the normal function of these cell proteins. Differential abilities of E7 mutants to interact with pRB or p107, for example, have also been used to study the independent activities of these cell proteins in regulating transcription. These studies have contributed to the accumulation of evidence that p107 and pRB display distinct, if related, activities. Identification of additional cell proteins that interact with E6 o r E7 will alniost certainly reveal other factors wit.h a role in the regulation of cell growth. Possibly the most exciting consequence of the rapid advance in our understanding of the functions of E6 and E7 at the molecular level, however, is the identilication of viral-host protein interactions as targets for the action of chemotherapeutic drugs. T h e observation that E6 and E7 expression is generally maintained in cervical cancers and cancer cell lines, combined with evidence that continued expression is necessary for tumor cell growth (von Knebel Doeberitz et nl., 1988; Steele et al., 1992; IIwarig et nl., 1993), provides the additional incentive that anti-E6 or -E7 therapies might also be useful for the treatment of advanced stage disease. Small peptides that interfere with the interactions between E7 and cell proteins such as pRB and p107 have been described (Jones et nl., 1990; Davies et nl., 1993), although a biological effect of these peptides on the growth of E7-transformed cells has not yet been identified. T h e
PAPILLOMAVIRUS PROTEINS AND TUMOR SUPPRESSORS
19
possibility remains that they will function as agonists rather than antagonists of E7 function. Despite the obvious problems and caveats, the development of small molecules that target E7 or E6 function holds much promise. The highrisk genital HPVs are the most convincing examples of human tumor viruses, playing a role in the development of the second most common female cancer worldwide. Viral oncoproteins have been identified, and the enormous advances in unraveling their mechanism of action have participated in the convergence of many different areas of research. T h e application of our understanding of the interactions between viral and host proteins directly to treating such a common human disease may be a fitting culmination to these studies.
ACKNOWLEDGMENTS I am extremely grateful to Rachel Davies, Xin Lu, and Roger Watson for their helpful comments and to Lou Laimins for sharing unpublished data. I also apologize to the authors of the many excellent papers that I have been unable to cite.
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Crook, T., Wrede, D., Tidy, J. A , , Mason, W. P., Evans, D. J., and Vousden, K. H. (1992). Lancet 339, 1070- 1073. Crook, T., Fisher, C., Masterson, P., and Vousden, K. H. (1994). Oncogene 9, 1225-1230. Cullen, A. P., Reid, R., Carnpion, M., and Liirincz, A. T. (1991). Analysis of‘the physical state of different human papillomavirus DNAs in intraepithelial and invasive cervical neoplasm. ,J. Virol. 65, 606-612. Das, B. C., Sharrna, J. K., Gopalakrishna, V., and Luthra, U. K. (1992).J . Gnz. Vzrol. 73, 2327-2336. Davies, R. C., and Vousden, K. H. (1992).J. Gen. Virol. 73, 2135-2139. Davies, R., Hicks, R., Crook, T., Morris, .J., and Vousden, K. H. (1993).J. Vzrol. 67, 25212528. Defeo-Jones, D., V~tocolo,G. A,, Haskell, K. M., Hanobok, M. G., Kiefer, D. M., McAvoy, E. M., Ivey-Hoyle, M., Brandsnia, J. L., Oliff, A., and Jones, K. E. ( l Y Y 3 ) . J . Virol. 67, 716-725. Derners, G. W., Halbert, C. L., and Galloway, D. A. (1994). Virology 198, 169-174. d e Sanjose, S., Santarnaria, M., De Ruiz, 1.’ A., Aristizabal, N., Guerrero, E., Castellsague, X., and Bosch, F. X. (1992). “HPV Types in Women with Normal Cervical Cytology.” IARC, Lyon. De Villiers, E. M. (1989).J. Viml. 63, 4898-4903. DiPaolo, J. A,, Woodworth, C. D., Popescu, N. C., Notaario, V., and Doniger, J. (1989). Oncogene 4, 395-399. Dittnier, D., Pati, S., Zambetti, G., Chu, S., Teresky, A. K., Moore, M., Finlay, C., and Levine, A . J. (1993). Nuturu C C I Z P4,~ 42-45. . Dollard, S. C., Wilson, J. L., Derneter, L. M., Uonnez, W., Reichman, R. C., Broker, T. R., and Chow, L. T. (1992). Gu7ir.c Dev. 6, 1131-1 142. Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, <;. A,, Butel, J . S., and Bradley, A. (1992). Nuture (L~ndon)356, 215-221. Dowdy, S. F., Hinds, P. W., Louie, K., Reed, S. I . , Arnold, A., and Weinberg, R. A. (1993). Cl~N73, 499-5 1 1. Dutta, A., Kuppert, J. M., Aster, J. C., and Winchester, E. (1993). Nature ( L O I L ~ O365, U) 79-82. Dyson, N., Guida, P., Munger, K., and Harlow, E. (1992).J. Virol. 66, 6893-6902. Dyson, N., Denibski, M., Fattaey, A , , Ngwu, C., Ewen, M., and Helin, K. (1993).J.Vzrol. 67, 764 1-7647. El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, E., Kinzler, K. W., and Vogelstein, B. (1993). Cull 75, 817-825. Ewen, M. E., Sluss, H. K., Sherr, C. J., Matsushinie, H., Kato, J.. and Livingston, D. M. Faha, B., Ewen, M. E., Tsai, L.-H., Livingston, D. M., and Harlow, E. (1992). Scimcc 255, 87-90. Fujita, M., Inoue, M., Tanizawa, O., Iwamoto, S., and Enomoto, T. (1992). Cancei- Rec. 52, 5323-5328. Ginsberg, D., Mechta, F., Yaniv, M., and Oren, M. (1991). Proc. Natl. Acnd. Sci. USA 88, 9979-9983. Hageemeier, C., Bannister, A . J . , Cook, A,, and Kouzarides, T. (1993).Proc. Nut/. Acnd. Sci. USA 90, 1580-1584. Halbert, C. L., Deniers, G. W., and Galloway, D. A . (1992).J. Virol. 66, 2125-2134. Hannon, G. J., Demetrick, D., and Beach, D. (1993). Gene.\ DPV.7, 2378-2391. Harper, J. W., Adarni, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993).Ce/l75,8058 16.
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THE RETINOBLASTOMA TUMOR SUPPRESSOR PROTEIN Jean Y. J. Wang, Erik S. Knudsen, and Peter J. Welch Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California
I. Overview 11. Mutation of Rb-I in Tumors
111.
IV.
V.
VI.
VII.
A. Biallelic Inactivation of Rb-1 in Human Retinoblastoma B. Inactivation of Rb-I Induces Pituitary Tumor in Mice C. Inactivation of Rb-I Does Not Induce Leukemogenesis D. Mutation of Rb-I in Tumor Progression Growth-Inhibitory Activity of RB A. Inhibition of Tumor Growth in Nude Mice B. Inhibition of Cell Growth in Culture Cell Cycle-Regulated Phosphorylation of RB A. RB as a Substrate of Cyclin-Dependent Protein Kinases B. Dephosphorylation of RB by Phosphatases C. Activation of RB Phosphorylation by Mitogens D. Inactivation of RB Phosphorylation during Differentiation E. Effects of Antimitogens on RB Phosphorylation Protein-Binding Function of RB A. Protein-Binding Domains in RB B. RB-Binding Proteins C. Nuclear Tethering of RB D. Role of RB in Transcription E. RB as a Molecular Matchmaker Regulation of RB Function by Phosphorylation A. Phosphorylation Inactivates Protein Binding Function of RB B. RB Phosphorylation Correlates with Reversal of Growth Suppression C . Phosphorylation Site-Specific Regulation of RB Function Future Prospects References
I. Overview T h e RB protein, first identified as the product of the retinoblastoma susceptibility gene Rb-I, has been the focus of numerous reviews in recent years. A key-word search conducted in November of 1993 on the MEDLINE showed 148 reviews on the retinoblastoma gene and 79 reviews on the retinoblastoma protein. Good reasons support this voluminous output. Rb-1 is the first “tumor suppressor gene” cloned and has
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Copyright 0 3994 by Academic Press, Inc. All rights 01-reproduction in any form reserved.
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growth inhibitory activity. T h e RB protein is a target of viral oncoproteins such as the SV40 T antigen, the adenovirus E1A protein, and the papilloniavirus- 16 protein E7. T h e RB protein is a major substrate of the cyclin-dependent protein kinases (cdks) that drive the progression of the cell division cycle. The RB protein regulates transcription. Homozygous mutation of the Rb gene in mice causes embryonic lethality. Therefore investigators in a wide range of fields from cancer genetics and tumor virology to cell cycle regulation, transcriptional regulation, and developmental biology have become interested in KB. T h e studies of RB have been driven by three hypotheses: ( I ) RB is the suppressor of retinoblastoma and possibly other types of cancer, (2) RB inhibits proliferation by preventing the transcription of genes critical for cell cycle progression, and (3) the inhibitory function of RB can be neutralized by phosphorylation or by the viral oncoproteins. Either phosphorylation or the binding of viral oncoproteins can cause the release of KB-sequestered transcription factors that are critical for activating genes required for cell cycle progression. These three hypotheses provide a conceptual framework for the study of RB. However, they do not satisfactorily explain the phenotypes resulting from Rb-1 mutations in humans and mice, or the binding of-RB to niany proteins other than transcription factors. T h e goal of this chapter is to summarize and evaluate the evidence for the three hypotheses with a focus on the niolecular and cellular function of RB. Because the studies of KK have been reviewed frequently, we will emphasize recent papers and will cite review articles for earlier results. A modified view of the molecular function of RB, based on several recent findings, will be discussed at the end o f t h e chapter.
II. Mutation of Rb-7 in Tumors
A. HIALLELICINACTIVATIONOF Rb-1 I N HUMAN RETINUBLASTOMA T h e existence of a retinoblastonia susceptibility gene was first suggested by .4lfred Knudson (1971) who proposed that this childhood cancer resulted from two mutations in an embryonic retinoblast. In this hypothesis, predisposition to retinoblastoma is caused by the inheritance of one mutation in the germ line. Cytogenetic analysis of retinoblastoma cells showed deletions at chromosome l3q14 in about 10% of the tumors, implicating that chromosomal location as the site of the susceptibility gene. T h e evidence that the two mutations of retinoblastorria
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involved only one gene was obtained by Cavanee and White, who demonstrated a loss of heterozygosity (LOH) at chromosome 13q14 in retinoblastoma cells (Cavanee et al., 1983). The observation of LOH led to the definition of the retinoblastoma susceptibility gene that acts to suppress the oncogenic potential of embryonic retinoblasts. (for reviews see Levine, 1993; Stanbridge, 1990; Weinberg, 1990). T h e cloning of the Rb-I gene was based on its location at chromosome 13q14 and its mutation in retinoblastoma cells (reviewed by Bookstein and Lee, 1991). Analysis of cloned Rb-1 has supported the Knudson hypothesis. First, the RB protein is missing from all the retinoblastomas examined (reviewed by Zacksenhaus et al., 1993b; Hamel et al., 1993). Second, a germ-line mutation at the Rb-1 locus increases the relative risk of developing retinoblastoma to between 30,000 and 40,000 (Hamel et al., 1993). Third, re-introduction of a wild-type Rb-I gene into retinoblastoma cells (WERI-27) could either completely or partially suppress the formation of tumors when the cells were injected subcutaneously or intraocularly into nude mice (Huang et al., 1988; Xu et al., 1991; reviewed by Zacksenhaus et al., 1993b). B. INACTIVATIONOF Rb-1 INDUCESPITUITARY TUMOR IN MICE
The Rb-1 gene has been isolated from several other vertebrate species including mouse, chicken, and frog (Bookstein and Lee, 1991; Goodrich and Lee, 1993; Zacksenhaus et al., 1993b).The mouse Rb is 88%homologous to the human gene and 91 % identical at the amino acid level (Bernards et al., 1989). Mutant mice with a germ-line mutation of Rb have been constructed by targeted gene disruption through homologous recombination. Interestingly, germ-line mutation of Rb in mice does not lead to the development of retinoblastoma (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992). Instead, mice that are heterozygously mutated for Rb, that is, the Rb-tl- mutant, develop pituitary tumors at a high frequency (Jacks et al., 1992). Identical to the human retinoblastoma cells, these mouse pituitary tumor cells show a loss of heterozygosity and are -1- for Rb (T.Jacks, personal communications). Thus, Rb appears to suppress the formation of pituitary tumor, but not retinoblastoma, in mice. Retinoblastoma can be induced by the tissue-specific expression of the SV40 T antigen in transgenic mice (Windle et al., 1990). Thus, mouse retinoblasts do have an oncogenic potential that is kindled by the viral T antigen but is not suppressed by Rb. The tumor suppression function of Rb is therefore observed in different cell types in different organisms.
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C. INACTIVATION OF Rb-I DOESNOT INDUCE LEUKEMOGENESIS Individuals with a gerni-line mutation of Rb-I do not show an increase in the development of leukemias, which are the most prevalent form of childhood cancer (Haniel et al., 1993).T h e Rb-I gene is normally expressed in all cells including those of- the hematopoietic lineages (Bookstein and Lee, 1991). Since the rate of a second Rb-I mutation should be the same in the retinoblasts or the hematopoietic stern cells, the conspicuous absence of leukemias in individuals with a germ-line mutation of Rb-I suggests that the biallelic mutation of that gene does not induce leukemias. ‘Ibexplain the apparent lack of leukernogenesis in humans with a gerni-line mutation of Rh-I, Hamel et al. (1993) proposed that a hematopoietic stein cell lacking Rb-1 may be unable to differentiate and may undergo programmed cell death. In this hypothesis, the loss of Rh-1 would cause uncontrolled proliferation of some stem cells such as embryonic retinoblasts, but would induce the death of other stem cells such as those of‘the hematopoietic lineages (Hamel et al., 1993). If an Rb-/- stem cell dies, there obviously will not be tumor development. ‘l’he idea that Rb may be required for the maintenance o f viability is suggested by the observation that an Rb-/- mouse embryo dies in utero at 12.5-16 days postimplantation (Clarke et al., 1992; Jacks et nl., 1992; Lee et a / . , 1992). Although the gross anatoniy of the dead embryo appears normal, prominent defects are observed in a subset of neurons in the central nervous system and in the red blood cells. Ectopic mitosis and necrosis are observed in the hindbrain, which seems to suggest that the loss of Rh could lead to cell death. Ectopic mitosis and death of the lens epithelial cells have also been observed in Rb-l- embryos (T. Jacks, personal comtnunicatiori). Rb- i - red blood cells are unable to lose their nuclei; these iniinature erythrocytes may be the cause of anemia and, ultimately, of the death of the embryos (Jacks et ul., 1992). The death of specific neurons and lens epithelia1 cells in Rb-/- embryos is consistent with the hypotliesis of Haniel et al. (1993), but does not constitute proof that the loss of’ Rb would also induce the programmed death of the hematopoietic stein cells. Since K h + / - mice also do not develop leukemias, it is possible to test tht. hypothesis of Hamel et ul. in the mouse model. This has been done with a blastocyst complementation system in which Rb-l- or + / - emI q o n i c stein (ES) celis were injected into a RAGP-deficient blastocyst (Chen et ul., 1993). T h e RAG2 gene encodes an activity that is essential for the genomic recombinations that assemble the immunoglobulin or the T-cell antigen-receptor genes (Oettinger et al.. 1990). Mutant mice
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that are homozygous for the RAG2 mutation do not contain any mature B or T lymphocytes because of a complete arrest at an early stage of lymphoid development (Shinkai et al., 1992). However, these RAG2 mutants have no other detectable defects. When either Rb+l- or Rb-lES cells were injected into the RAG2-deficient blastocyst, mature B and T cells were reconstituted (Chen et al., 1993). The B and T cells were shown to be derived from Rb-l- ES cells by the presence of the disrupted Rb allele. The Rb-negative B and T cells that developed in the RAG2-deficient mice are functional, demonstrated by a normal response to lipopolysaccharide and phorbol myristate. These results not only showed that Rb is not required for the development of lymphocytes, but also demonstrated that a hematopoietic stem cell does not undergo programmed cell death in the absence of Rb. This result leads to the inevitable conclusion that Rb-1 is not a suppressor of leukemias. Loss of Rb-1 does not appear to unleash the oncogenic potential of hematopoietic stem cells.
D. MUTATIONOF Rb-1 IN TUMOR PROGRESSION
Although the germ-line mutation of Rb-1 does not enhance the development of leukemias, mutations of Rb-1 have been observed in isolated cases of leukemias and in a wide variety of other tumors including small cell lung carcinoma, breast carcinoma, prostate carcinoma, bladder carcinoma, and osteosarcoma (reviewed by Goodrich and Lee, 1993; Hamel et al., 1993). Unlike retinoblastoma cells, which usually lack any detectable RB protein, these carcinoma and leukemia cells tend to express mutated forms of RB (Horowitz et al., 1990). The detection of Rb-1 mutations in a wide variety of cancer cells has been interpreted as an indication that RB is a general suppressor of tumor formation (Bookstein and Lee, 1991). This interpretation, however, is in contradiction with the results of the genetic analysis, which shows a restricted role of Rb-I in tumor development. Why then is Rb-1 mutated in a wide variety of sporadic cancers? One way of reconciling the two sets of observations is to consider that the tumor suppressor function of Rb-1 is dependent on the cell context, or the “wiring diagram” of a cell. Human retinoblasts are unique because they rely on Rb-1 to suppress their tumorigenic potential. Other stem cells, however, can withstand the loss of Rb-1, possibly because they rely more on other mechanisms of suppression that do not involve RB. Nevertheless, any stem cell may become sensitized to the suppressor function of Rb-1 through other genetic or epigenetic alterations. In
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JEAN Y. .J. WANG E T A L .
other words, Kb-1 may become indispensable when a stem cell is deprived of other recourses to control its tumorigenic potential. The genetic analysis of Rb-l reveals the tumorigenic events that can be initiated by the loss of RB and finds retinoblastoma in humans and pituitary tumor in mice. The survey of cancer cells for Rb-1 mutation, on the other hand, does not distinguish between initiation and progression events. The loss of RB function may not induce leukemogenesis but, when combined with appropriate genetic alterations, may nevertheless contribute to the progression of leukemias. This scenario could also apply to the development of other sporadic tumors. If so, understanding the role of RB in tumor development will require a definition of the cell context that renders Rb-l a necessary suppressor of the tumorigenic potential of a stem cell. Ill. Growth-Inhibitory Activity of RB A. INHIBITIOX OF TUMOR GROWTHI N NUDE MICE
The growth-inhibitory activity of RB was first demonstrated by the reintroduction of wild-type Rb-1 into retinoblastoma cells. In the WERI-27 retinoblastoma line, restoration of RB did not stop cell growth in culture but completely blocked the formation of tumors when the cells were injected subcutaneously into nude mice (Huang et al., 1988). When the RB-reconstituted WERI-27 cells were injected intraocularly, however, tumor formation was observed, although of reduced number and size (Madreperla et al., 1991; Xu et al., 1991). The results obtained with the WERI-27 line could not be generalized because the reconstitution of Rb-1 in two other retinoblastoma lines, WERI-1 and Y-79, failed to suppress growth either in culture or intraocularly in the nude mice (Muncaster Pt al., 1992). The Rb-1 cDNA has also been introduced into other tumor cell lines, for example, DU 145 (prostate carcinoma), HTB9 (bladder carcinoma), and lung carcinoma cell line that express mutated forms of RB. In these three cell lines, reconstituting RB did not affect growth in culture but caused a reduction in the subcutaneous growth of tumors in nude mice (Bookstein and Lee, 1991; Takahashi et al., 1991; Kratzke Pt nl., 1993). Whether these RB-reconstituted cells could grow better in other types of tissue has not been determined. Although RB does not always suppress tumor growth, the accumulated data do indicate that RB has a growth-inhibitory activity that is manifested in certain tumor lines under the right conditions.
RETINOBLASTOMA TUMOR SUPPRESSOR
31
B. INHIBITION OF CELLGROWTH IN CULTURE T h e growth-inhibitory activity of RB can also be demonstrated in culture with the appropriate cell lines, the most often used of which is the osteosarcoma line Saos-2 (Huang et al., 1988; Templeton et al., 1991). Saos-2 cells express a C-terminal truncated RB that is cytoplasmic and has a shorter half-life than the wild-type protein (Shew et al., 1991). When wild-type Rb-I cDNA is expressed in Saos-2 cells, morphologically distinct cells that are unusually large appear in culture (Templeton et al., 1991). These large cells are formed at a frequency of 3-1096 when Saos-2 cells are transfected with an Rb-I cDNA driven by a strong promoter (Hinds et al., 1992). If the Rb-l-transfected cells are examined for DNA content, they are found to be arrested in G, phase of the cell cycle (Ewen et al., 1993a). Thus Rb-1 expression appears to prevent Saos-2 cell entry into S phase. This proliferation block also prevents the loss of the unintegrated Rb-1 DNA, which accounts for the high frequency of large cell formation (Hinds et al., 1992). Since the RB-reconstituted cells continue to increase in size, the RB protein does not appear to inhibit the cytoplasmic growth of Saos-2 cells but does prevent the G,/S transition. T h e G, /S inhibitory activity has also been demonstrated by microinjecting a C-terminal fragment of RB, purified from bacteria, into Saos-2 cells (Goodrich et al., 1991). By micro-injecting at different time points in G,, researchers showed that Saos-2 cells are sensitive to RBmediated inhibition until late G, phase. After entry into S phase, however, the injection of this RB fragment has no effect on DNA synthesis. T w o other RB-deficient tumor lines, TccSUP (prostate carcinoma) and Hs913t (fibrosarcoma), are also sensitive to the growth-inhibitory activity of RB (Fung et al., 1993). T h e RB-mediated cell cycle arrest again cannot be generalized for all RB-deficient cells. For example, expression of wild-type RB does not arrest the growth of retinoblastoma Celts or DU145 in culture, because enough of these RB-reconstituted cells can be grown and then injected into nude mice to assay for tumorigenicity. At least three other RBdeficient tumor lines have been shown to be resistant to the growthinhibitory activity of RB: C33A (cervical carcinoma), 582 (bladder carcinoma), and MD468 (breast carcinoma). However, reports indicate that the expression of wild-type RB in MD468 or a lung carcinoma line can reduce the plating efficiency in soft agar (Lee et al., 1991; Kratzke et al., 1993). In the lung carcinoma line, the suppression effect of RB can be blocked by the addition of Matrigel@,an extract enriched with extracellular matrix (Kratzke et al., 1993). This result is reminiscent of the
results obtained with injection of RB-reconstituted cells into nude mice, in which the sites of injection influenced the growth of the tumor cells. Introduction of' Rh-I cDNA into RB-positive tumor cells usually does not cause any growth perturbation in culture. Among the lines tested are HT1080 (fibrosarcoma), MCF7 (breast carcinoma), T98G (glioblastoma), and MG63 (osteosarcoma) (Fung et al., 1993; Zhu et al., 1993). These observations are consistent with the notion that the growth-inhibitory activity of RB is not absolute but is conditional on the context ofthe cell. Overexpression of RB can inhibit the growth of diploid human fibroblasts or mammary epithelial cells, although whether these cells are arrested at G , / S by the excess RB is not known (Fung et nl., 1993). In a monkey fibroblast cell line that expresses the SV40 '1' antigen, overproduction of wild-type KB at levels between 5- and 200-fold that of endogenous RB was correlated with a cell cycle arrest in the C;, phase (Karantza rt nl., 1993). T h e G., arrest observed in this particular experimental system may be caused by a combination of 'I antigen and excess KB, since this G,/M inhibitory activity of RB has not been observed in other cell types. The gro.rz~h-inhibitoryactivity of RB is also demonstrated at the organisnial level. X 2- to 3-fold overexpression of RB, through extra copies of a wild-type Rb-1 transgene, is found to cause a consistent reduction in the size of transgenic mice (Uignon et al., 1993). Whether the growth reduction observed at the organismal level is an extrapolation of the growth-inhibitory activity of RB observed at the cellular level is not clear. IV. Cell Cycle-Regulated Phosphorylation of RB T h e first clue to a role of RB in cell cycle regulation came from the observation that the phosph"ryl"ti"" of RB showed periodic: oscillation in proliferating cells (Buchkovich el al., 1989; Chen et al., 1989; DeCapri0 et al., 1989). T h e phosphorylation of RB causes a dramatic shift in its electrophoretic mobility. In general, RB migrates as a single tight band in resting cells and a collection of four to five bands in proliferating cells. T h e current consensus is that phosphorylation slows the electrophoretic mobility; thus the upper bands correspond to the phosphorylated forms of RB. When proliferating cells are synchronized, upper bands of RB are found during S, G,, and M phase; a rapid conversion to the lower band is observed as cells complete mitosis (1,udlow et al., 1990). As cells progress through the next G I , the upper bands of RB re-appear as cells approach the G I / S boundary. T h e multiple upper bands may represent several forms of RB that are phosphorylated at different sites or with different stoichiometry (Haniel et ul., 1990; Lin et al., 1991).
RETINOBLASTOMA TUMOR SUPPRESSOR
33
However, the precise differences between these upper bands have not been determine.d. A. RB AS A SUBSTRATE OF CYCLIN-DEPENDENT PROTEIN KINASES Phospho-amino acid and peptide analyses have shown that RB is phosphorylated at multiple serine and threonine sites. No phosphotyrosine has been detected in RB. Inspection of the RB sequence shows a total of 16 Ser/Thr-Pro motifs that are potential sites of phosphorylation by cdc2 and other cdks. Indeed, RB is a good in vitro substrate for activated cdc2, cdk2, or cdk4 (Lees et al., 1991; Lin, et al., 1991; Akiyama et al., 1992; Matsushime et al., 1992; Kato et al., 1993). Comparison of the tryptic peptide maps of phospho-RB isolated from Molt4 or HeLa cells with that of cdc2-phosphorylated RB shows that most of the in vivo sites are phosphorylated by cdc2 in vitro (Lees et al., 1991; Lin et al., 1991; Lin and Wang, 1992). Of the 16 Ser/Thr-Pro motifs, 7 have been shown to be phosphorylated in vim (Fig. 1). Since not all the in vivo phosphopeptides have been identified, RB must be phosphorylated on additional serine and threonine residues. Moreover, the possibility cannot be ruled out at this time that RB is phosphorylated by other types of kinases that do not recognize the Ser/Thr-Pro motif. Although the overall pattern of the in vivo and in vitro tryptic peptide phosphorylation maps is similar, the major in vitro phosphorylation sites do not correspond to the major sites phosphorylated in vivo (Lees et al., 1991; Lin et al., 1991). When intact cells are labeled with 32P, sites in the C-terminal region of RB (Ser 807, 8 11 and T h r 821, 826) are preferentially labeled; however, in vitro phosphorylation by cdc2 favors sites in the N-terminal region of RB (Lin et al., 1991; Lin and Wang, 1992). This result suggests that RB may not be phosphorylated by cdc2 in intact cells, but by related enzymes that have different site preferences. Alternatively, cellular RB may exist in a conformation different from that of purified RB, possibly through association with other proteins, and that conformation may favor the phosphorylation of the C-terminal sites by cdc2 or other cdks. RB can be phosphorylated by a number of cdk-cyclin complexes in different phases of the cell cycle. During metaphase of mitosis, cdc2cyclin B is likely to be the major RB kinase because immunodepletion of cdc2 from the mitotic cell extract leads to a concomitant loss of RBkinase activity (Lin et al., 1991). In mammalian cells, the cdc2 kinase is only activated at G,/M; thus it cannot be responsible for the phosphorylation of RB in interphase cells. During S phase, cyclin A-associated
34
JEAN Y. J . W A N C E T A L .
kinase is activated (reviewed by Sherr, 1993). Micro-injection of rodent fibroblasts with antibodies against cyclin A inhibits DNA synthesis, showing that cyclin 4 is essential for S phase (Girard et al., 1991). I n most cells, the majority of cyclin A is in complex with cdk2 ('I'sai P t al., 1991). The accumulation of cdk2-cyclin A is correlated with the accumulation of RB-kinase activity as cells progress into S phase, supporting the notion that RB is a substrate of this essential kinase (B.-T. Y. Lin, unpublished data). 'I'hus, RB is likely to be phosphorylated by cdk2-cyclin A during S phase. I n uitm, cdc2-cyclin B and cdk2-cyclin A phosphorylate purified RB on identical sites with the same preference (Lin and Wing, 1992). Therefore, cdk2-cyclin A also does not reproduce exactly the phosphorylation pattern observed on RB in intact cells. During G I progression, RB is phosphorylated prior to the detection of the activated cdk2-cyclin A complex. G I phosphorylation of RB is therefore catalyzed by cdk-cyclin complexes other than cdk2-cyclin A. In a variety of cell lines, the kinetics of RB phosphorylation during G, progression are best correlated with the activation of cyclin E-associated kinase (Koff et al., 1992). In most cells, cycliii E preferentially associates with cdk2; thus, cdk2-cyclin E is likely to be an important RB kinase in late G I of the cell cycle (Sherr, 1993). In zlitro, cdk2-cyclin E appears to phosphorylate RB better than histone H 1, a property not found for cdc2-cyclin B (Dulic et al., 1992). T h e phosphopeptides generated by cdk2-cyclin E were not examined in those experiments; thus, it has not been determined whether the more efficient phosphorylation is accompanied by an alteration in the choice of sites. Another group of G , cyclins, the D-type cyclins, may also regulate the phosphorylation of RB. T h e D-type cyclins can bind directly to RB (discussed in Section V). In mammalian cells, D-type cyclins can associate with cdk4 o r cdk6; these active kinase complexes can phosphorylate RB i7i vitm (Meyerson and Harlow, 1993; Kato et al., 1993; Matsushime et ul., 1994). A 21-kDa inhibitor of cdk, CipliWAFI, is found to associate with cyclin D1 in diploid human fibroblasts (Harper et al., 1993; El-Deiry et al., 1993). A cdk4-specific inhibitor of 16 kDa has also been identified (Serrano et d.,1993). The cdk4-cyclin D complexes may phosphorylate RB during G I only in response to specific signals that can inactivate these inhibitors. €5. DEPHOSPHORYLATION OF RB
BY
PHOSPHATASES
I n unsynchronized cells, the half-life of phosphate on RB is about 30 min (Chen et al.. 1989). This rapid turnover indicates that RB is reversibly phosphorylated through the action of kinases and phosphatases.
RETINOBLASTOMA T U M O R SUPPRESSOR
35
T h e rapid dephosphorylation of RB as cells exit mitosis also indicates that the phosphorylation state of RB is regulated by phosphatases as well as kinases. In metaphase cells, either arrested by nocadazole or harvested by mitotic shake-off, all the RB is found to migrate as a single band corresponding to the hyperphosphorylated form (Ludlow et al., 1990; A. 0. Morla, unpublished data). Dephosphorylation of RB is apparent within 90 min of release from the nocadazole block, and RB is completely converted to the unphosphorylated lower band in 3 hr (Ludlow et al., 1993; B. T.-Y. Lin, unpublished data). To determine whether the mitotic dephosphorylation of RB involves the activation of an RB phosphatase, Ludlow et al. measured the dephosphorylation of 32P-labeled RB mediated by crude extracts prepared from various phases of the cell cycle. These researchers found that significant RBphosphatase activity was only detected in a late mitotic extract prepared 40 min after release from nocadazole block (Ludlow et al., 1993). Thus the dephosphorylation of RB late in mitosis is correlated with the activation of an RB phosphatase. The in uitro RB-phosphatase assay used by Ludlow et al. might not be sensitive enough to detect RB-phosphatase activity in extracts from other phases of the cell cycle. The RB phosphatase in mitotic extracts may be a type 1 protein phosphatase (PPl). The RB phosphatase in the late mitotic extracts is not inhibited by okadaic acid (OA) at 2 nM but is completely inhibited at 10 nM (Ludlow et al., 1993). Furthermore, the activity was not affected by EDTA or EGTA, a result consistent with the properties of type 1 but not type 2 phosphatases (reviewed by Fujiki and Suganama, 1993). Indeed, purified PP1 does dephosphorylate 32P-labeled RB in uitro (Alberts et al., 1993; Ludlow et al., 1993). A more compelling piece of evidence, perhaps, is the finding that RB can directly bind to the catalytic subunit of PPl. Using the two-hybrid interaction screen, a novel PP1-a subunit, PP1-132,was cloned on the basis of its interaction with RB (Durfee et al., 1993). Both PPl-a and PPl-a2 proteins can interact with RB in uitro, preferentially binding the hypophosphorylated form. Using a coimmunoprecipitation assay, researchers found that PP 1-13 interacts with RB in a cell-cycle-dependent manner. A complex of PPl-a and RB is detected in Go/G1, mid-G,, and M phase cells (Durfee et al., 1993), suggesting that PP1-a may be important for the maintenance of unphosphorylated RB from late M to mid-G, phase. As discussed earlier, RB is phosphorylated on multiple serine and threonine sites that may not be dephosphorylated by only one phosphatase. By micro-injecting the catalytic subunit of either PP1 or PP2A directly into the nucleus of REF-52 cells, researchers found that both phosphatases could stabilize the tethering of RB to the nucleus (Alberts
JEAN Y. J. WANG E7' A I . .
36
et ul., 1993). Since the unphosphorylated form of RB tethers to the nucleus (Mittnacht and Weinberg, 1991), both PPl and PP2A appeared to maintain RB in the dephosphorylated state. When the two enzymes were injected into the cytoplasm, however, only PP1 could stabilize the tethering of RB (Alberts et ul., 1993). This event was correlated with the nuclear localization o f t h e injected PP1 but not PP2A. Thus, an important regulation of RB dephosphorylation may be the compartmentalization of the phosphatase, and the accessibility of the phosphorylated RB for dephosphorylation by the RB phosphatases. T h e phosphorylation of RB can be influenced by a variety of biologically active molecules including mitogens, antimitogens, and differentiation inducers. In most cases, the effect on RB phosphorylation is the result of cell cycle modulation induced by these agents. In general, mitogens stimulate RB phosphorylation by promoting the entry of quiescent cells into the cell cycle (Table I). Inducers of differentiation arid antimitogens lead t o RB dephosphorylation by promoting the withdrawal of proliferating cells from the cell cycle (Table I). A few o f - these agents, however, may directly activate the dephosphorylation of RB, as discussed subseyuently. C. ACXIVATION OF RB PINISPIIORYLATION BY
MITOGENS
I. Fibroblusts In primary diploid human fibroblasts and immortalized rodent fibroblasts, RB exists solely in the unphosphorylated form in quiescent cells. Stimulation with serum leads to RB phosphorylation that can be detected in 6 hr by labeling with 9'LP.Upper bands of RB also become detectable between 6 and 8 hr and are clearly visible by 10-12 hr after serum addition (DeCaprio ~t al., 1989; Takuwa et ul., 1993; Welch and Wang, 1993; B.-T. Y. Lin, unpublished data). T h e serum-induced phosphorylation of RB is dependent on the ability of serum to drive cells into S phase, as first shown in senescent human fibroblasts. Stimulation of senescent cells with serum does not induce entry into S phase despite the activation of receptor tyrosine kinases and the transcription of c-my- (Stein et ul., 1990). In these senescent cells, RB does not become phosphorylated with addition of serum, suggesting that serum is not sufficient to activate RB phosphorylation. Consistent with this result is the finding that serum does not activate RB phosphorylation when it is given under conditions of low extracellular calcium (Takuwa et al., 1993). Reduction of extracellular calcium blocks the entry
TABLE I FACTORS INFLUENCING RB PHOSPHORYLATION Agent influence RB phosphorylatione
Cell type (growth status)b
Mitogens Serum
PHA Staphylococcus, Cowan
TPA + ionomycin Differentiation inducers TPA Retinoic acid Activin-A TPA Interleukin-6 HMBA NGF Anti-mitogens TGFp TGFp Interferon-a Dibutryl CAMP TPA Calmodulin antagonists (W-7 and CZM) D-Erythro-sphingosine Okadaic acid (10-100 nM) Okadaic acid (100 nh4) .
~~
~~
Effect on RB phosphorylationc
Reference Mihara et al. (1989) Stein et al. (1990) Welch and Wang (1993) Takuwa et al. (1993) Furukawa et al. (1990) Furukawa et al. (1990) Wang et al. (1993)
SW613 (Q) IMR-90 (Q) 3T3 (Q) WI-38 (Q) Human T cells (Q) Human B cells (Q) Human T cells (Q)
Increase Increase Increase Increase Increase Increase Increase
in 6-8 hr in 12-15 hr in 6-8 hr within 13 hr in 24-48 hr in 36-72 hr in 24-48 hr
HL60 (G) HL60 (G) K562 (G) K562 (G) M1 (G) MELC (G) PC 12 (G)
Decrease in 8-12 hr Decrease in 12-24 hr Decrease from 9-24 hr Decrease in 8-24 hr Decrease within 24 hr Decrease from 10- 15 hr Decrease in 72-96 hr
Chen et al. (1989) Mihara et al. (1989) Sehy et al. (1992) Yen et al. (1993) Resnitzky et al. (1992) Richon et al. (1992) Kalman et al. (1993)
MV1-LU (G) JOSK-1 (G) Daudi (G) HL60 (G) WEHI231 (G) WI-38, IMR-90 (Q) Molt-4 (G) Human fibroblasts (G) NIH-3T3 (Q)
Decrease in 6-12 hr Decrease in 18-36 hr Decrease in 4-8 hr Decrease in 48 hr Decrease in 8-15 hr Remains dephosphorylated Decrease in 3-4 hr Increase in 2 hr Remains dephosphorylated
Laiho et al. (1990) Furukawa et al. (1992) Resnitzky et al. (1992) Whyte and Eisenman (1992) Maheswaran et al. (1991) Takuwa et al. (1993) Chao et al. (1992) Yatsunami et al. (1993) Schonthal and Feramisco (1993) ~
~
~~~
A variety of physiological and pharmacological agents have been shown to influence RB phosphorylation. PHA, Phytohernagglutinin; TPA, 12-0-tetradecanoyl phorbol-13-acetate; HMBA, hexamethylene bisacetimide; NGF, nerve growth factor; TGFP, transforming growth factor p; CZM, calmidazolium. * Effects on RB phosphorylation have been observed using quiescent (Q)or proliferating (G) cells. Mitogens stimulate RB phosphorylation as quiescent cclls are stimulated to enter the cell cycle. Differentiation factors and anti-mitogens induce RB dephosphorylation as cell cycle progression becomes inhibited.
38
J E A N Y. J . WANG E T AL.
into S phase and the increase in RB phosphorylation. However, DNA synthesis per se is not necessary for the activation of RB phosphorylation. For example, inhibitors of DNA synthesis such as hydroxyurea and aphidicolin do not inhibit the serum-induced RB phosphorylation. The serum-induced increase in RB phosphorylation is correlated with the activation of cdk-cyclin complexes (Koffet al., 1992).T h e kinetics of cyclin E accumulation best correlate with the appearance of phosphorylated RB in fibroblast cell lines, suggesting that the mitogenmediated activation o f cyclin E-associated kinase may he the cause of increased phosphorylation of RB. In senescent cells or under conditions of calcium deprivation, cyclin E-associated cdk activity is probably not induced. Inhibitors of DNA synthesis do not block the accumulation and activation of cyclin E-cdk2, consistent with the phosphorylation of RB in cells treated with these inhibitors (Koff et a!., 1992).
2. Perifihel-al Lyni~liorytes Peripheral blood lymphocytes are resting cells that contain only the unphosphorylated form of RB. Resting T cells can be activated by phytohemagglutinin (PHA), anti-CD2 antibodies, o r a combination of 12-0tetradecanoyl phorbol- 13-acetate (TPA) and ionomycin to enter into the cell cycle; this results in the phosphorylation of RB (DeCaprio et ul., 1989, 1992; Furukawa et al., 1990; Wang et al., 1993). T h e phosphorylated bands of RB become apparent between 20 and 48 hr after the addition of T-cell mitogens. The slow kinetics may be due to the relatively weak niitogenic activity of these reagents in T cells. In resting T-cell populations, 3 % of the cells are in S phase, as determined by fluorescence-activated cell sorting (FACS) analysis; 48 hr after antibody or PHA treatment., this level only increases to 6%(Furukawa et al., 1990). DIVA synthesis, as assayed by [“Hlthymidine incorporation, is first detectable 31 111. after PHA stimulation (Meyerson and Harlow, 1993), in contrast to fibroblast systems, in which DNA synthesis is first detected within 12 or 18 hr of serum stimulation for rodent o r human cells, respectively. Furthermore, activation of cdk2 activity is first apparent between 6 and 8 hr after serum stimulation of fibroblasts (Akiyama et al., 1992; Tsai el al., 1993), but only apparent 31 hr after PHA treatment of T cells (Meyerson and Harlow, 1993). As in fibroblasts, the phosphorylation of RB is correlated with a commitment to enter S phase but is independent of DNA synthesis. Treatment of PHA-activated T cells with aphidicolin has no effect on the ability of RB to become phosphorylated (Terada et al., 1991). However, deferoxamine, an iron chelator, and cyclosporin A (CsA), an immunosuppressive drug, completely inhibit RB phosphorylation in response to
RETINOBLASTOMA TUMOR SUPPRESSOR
39
PHA treatment (Terada et al., 1991). CsA and deferoxamine block early events in T-cell activation. In the case of CsA, this block is prior to the commitment to proliferate (Kumagai et al., 1988). Less is known about the action of deferoxamine, but it clearly blocks cell cycle progression before aphidicolin (Terada et al., 1991). Release from an aphidicolin block results in rapid entry into S phase, whereas release from a deferoxamine block results in a 14- to 16-hr lag before significant DNA synthesis is observed. Hence, drugs that block the commitment to proliferate inhibit RB phosphorylation in T cells. B lymphocytes isolated from peripheral blood can be induced to undergo a transient proliferation when treated with Staphylococcus aureus. RB phosphorylation becomes apparent, by electrophoretic mobility shift, within 72 hr of treatment, corresponding with an increase in cells entering S phase (Furukawa et al., 1990). As proliferation slows, after 6 days, RB phosphorylation is no longer observed (Furukawa et al., 1990). D. INACTIVATION OF RB PHOSPHORYLATION DURING DIFFERENTIATION Terminal differentiation involves a withdrawal from the cell cycle and a change in gene expression that accounts for the development of the differentiated phenotype. Various in vitro differentiation systems have been utilized to address the regulation of RB phosphorylation in the differentiation process. 1 . HL60 Cells
T h e promyelocytic leukemia cell line HL60 is capable of differentidtion to monocytes or macrophages in response to TPA, retinoic acid (RA), or dimethylsulfoxide (DMSO). These inducers of differentiation have been shown to cause RB dephosphorylation in HL60 cells. In the first two studies (Chen et al., 1989; Mihara et al., 1989), RB dephosphorylation is detected 8-10 hr after the addition of the inducers and RB is completely dephosphorylated by 18-24 hr after treatment. The complete dephosphorylation of RB precedes the development of the differentiated phenotype; only 10-30% of the cells show morphological alteration and are stained positive for acid phosphatase at 18-24 hr (Chen et al., 1989; Mihara et al., 1989). In two other studies, RB dephosphorylation is delayed and only becomes apparent after 48 hr of treatment with inducers (Akiyama and Toyoshima, 1990; Whyte and Eisenman, 1992). Again, the dephosphorylation of RB precedes the appearance of the differentiated phenotype; only 10-20% of the cells stain positive at 48 hr in these studies.
40
JEAN Y. J. WANG El’ A L .
‘The discrepancy between the time course of KB dephosphorylation and the development of the differentiated phenotype in these two sets of studies is probably due to the use of different types of HL60 cells. ‘l’he induced differentiation of HL60 cells normally occurs over two cell cycles, that is, approximately 48 hi- (Yen et al., 1986).In the first cell cycle, a “precommitted state” is developed. If the inducer is withdrawn, these cells will continue to proliferate in the “precommitted state.” In the continued presence of the inducer, these cells will commit to differentiation and become growth arrested in one cell cycle time. T h e observation of complete RB dephosphorylation in 24 hr was probably made with precomniitted H L60 cells, whereas the slower time course was observed with HL.60 cells that had no previous exposure to any inducer. T h e differentiation inducers do not have a direct effect on the dephosphorylation of RB. This characteristic is demonstrated by treating a G I population of HL60 cells, which contain only underphosphorylated RB, with RX (Mihara P& ul., 1989). RA does not block RB phosphorylation since these G , cells progress through the cell cycle. T h e dephosphorylation of RB only becomes apparent at the end of the cell cycle when further proliferation is inhibited by KA. This result suggests that the dephosphorylation of RB is concomitant with the withdrawal of the differentiating cells from the cell cycle.
2. iM1 Cells T h e myeloblastic cell line M I can be induced to differentiate to monocytes by a variety of agents including interleukin 6 (IL-6) (Shabo et nl., 1988). IL-6 causes a cell cycle block that is thought to involve suppression of c-wy expression as well as changes in KB phosphorylation (reviewed b y Kimchi, 1992). In cycling M 1 cells, most of the KB protein exists in the hyperphosphorylated state. On treatment of the cells with IL-6, RB dephosphorylatiun beconies apparent at 24 hr; within 48 hr RB migrates as a single unphosphorylated band (Resnitzky et al., 1992). T h e dephosphorylation of RB at 24 hr occurs prior to any detectable effect on cell cycle distribution. However, the complete dephosphorylation of RB occurs in conjunction with the accumulation of cells with a 2N DNA content (Resnitzky et al., 1992). Ectopic expression of c-myc from the SV40 early promoter is capable of blocking the differentiation and cell cycle arrest induced by IL-6 (Resnitzky et al., 1992). Interestingly, KB still becomes dephosphorylated on treatment with IL-6 in these c-mycexpressing cells. ‘l’his result indicates that IL-6 may directly trigger the dephosphorylation of RB independent of cell cycle arrest. This study also implies that RB dephosphorylation is not sufficient to cause cell
RETINOBLASTOMA TUMOR SUPPRESSOR
41
cycle arrest or cellular differentiation in the presence of ectopic c-myc expression.
3. PC12 Cells Nerve growth factor (NGF) induces differentiation of the rat pheochromocytoma line PC12 (Green and Tischler, 1976). NGF treatment results in a cessation of cell division, as well as morphological and physiological changes toward a neuronal phenotype. The NGF receptor p 140trkcontains a cytoplasmic tyrosine kinase domain that is activated on binding of NGF. p140trk activates the Ras, Raf, and MAP-kinase pathway that is associated with mitogenic stimulation in many cell types but, in the context of a neuronal precursor, gives rise to cell cycle arrest and differentiation (Gomez and Cohen, 1991; Klein et al., 1991; Kremer et al., 1991). T h e cell cycle block brought about by NGF becomes apparent by day 3 of treatment, when asynchronous PC12 cells begin to accumulate in G, (Rudkin et al., 1989). No effect on RB phosphorylation is seen at day 2, but RB is completely dephosphorylated by 4.5 days (Kalman et al., 1993). Therefore, RB dephosphorylation correlates with the accumulation of PC12 cells in G,. Differentiation of PC12 cells causes a decrease in cdk and cyclin-associated kinase activity (Koff et al., 1992). In proliferating PC 12 cells, significant cdc2 and cyclin E-associated histone H1-kinase activity is detected. In cells exposed to NGF for 4-5 days, this activity is reduced to background level, suggesting that RB dephosphorylation may in part be due to down-regulation of cdk-cyclin activity in the differentiated cells. T h e adenovirus ElA protein is capable of blocking NGF-mediated differentiation of PC12 cells (Maruyama et al., 1987). To address which functional domains of E 1A are necessary to block PC 12 differentiation, a series of EIA mutants was micro-injected into PC12 cells and the influence on differentiation was assayed. Researchers found that deletion of the transcription activation domain had no effect on the ability of ElA to antagonize PC 12 differentiation. However, deletions in the two domains of ElA required for binding to RB, the RB-related proteins p107 and p130, or the E1A-associated protein p300 abrogated the ability of EIA to block NGF-induced differentiation in PC12 cells (Kalman et al., 1993). Thus one or more of these four proteins that are sequestered by ElA may be necessary in the differentiation of PC12 cells. 4 . K562 Cells
K562 cells were isolated from a patient with chronic myelogenous leukemia at blast crisis. These cells contain the Philadelphia chromo-
42
J E A N Y. J. WANG ET .4L.
some (Lozzio arid Lozzio, 1975). K562 cells can be induced to differentiate down erythroid, myeloid, or megakaryocytic lineages by a variety of agents (Lozzio t t al., 1981; Villeval et al., 1983). Activin A, a member of the transforming growth factor P (TGFP) family of peptides, was first identified as an activity that stimulated the secretion of follicle stimulating hormone (Ling et al., 1986; Vale et at., 1986). Activin A can induce the erythroid differentiation of K562 cells (Sehy et al., 1992). The effect of activin A is characterized by a lengthening of G I during the first cell cycle. A transient increase in hypophosphorylated RB is observed at 10-24 hr of treatment, corresponding with the prolongation of C , (Sehy et al., 1992).Following this period, the phosphorylation pattern of RB returns to that of untreated cells. This change in RB phosphorylation and the transient arrest in G I precedes the expression of erythrocyte-specific genes, which are first observed in 24 hr and are apparent in all cells within 3 days. The phorbol ester ‘IPA induces megakaryocytic differentiation of K5G2 cells (Villeval et ul., 1983). TPA causes the dephosphorylation of RB, which can be observed within 8 hr of treatment and is almost complete within 24-48 hr. In contrast to the activin A-mediated differentiation, the dephosphorylation of RB is not transient in .IPA-treated K562 cells. T h e dephosphorylation of RB occurs prior to the expression of the megakaryocyte-specific gene CD4 1, which is expressed in 8% of the cells after 24 hr and in 50% after 72 hr (Yen et al., 1993). 5 . MELC Cells
Treatment of MELC, a murine erythroleukemia cell line, with hexamethylenebisacetamide (HMBA) induces erythroid differentiation (reviewed by Marks and Kifkind, 1978). T h e differentiation is characterized by a prolonged G I phase on addition of inducer. In subsequent cell cycles, progressively more cells commit to differentiation. Researchers observed that treatment of GI-enriched MELC cells with HMBA resulted in the prolonged presence of hypophosphorylated RB (Richon et uf., 1992; Kiyokawa et al., 1993). In a population of HMBAtreated G I cells, hypophosphorylated RB is present up to 19 hr after plating. This result is in contrast to control cells or cells treated with aphidicolin, in which RB is completely hyperphosphorylated within 12 hr of plating (Richon et al., 1992; Kiyokawa et ul., 1993). HMBA retards the expression of cyclin A so none is detected 15-19 hr after plating, whereas in control cells cyclin A is readily detectable at these time points (Kiyokawa et at., 1993).This result suggests that the prolonged presence of hypophosphorylated RB may be due to reduced cdk/cyclin activity in HMBA-treated cells.
RETINOBLASTOMA TUMOR SUPPRESSOR
43
Collectively, these results show that RB dephosphorylation is observed in every differentiating cell system that has been examined. In each case, the dephosphorylation of RB is correlated with the cessation of the cell cycle; thus we cannot determine if the dephosphorylation of RB is the cause or the effect of differentiation. Through the construction of Rb-l- mice, researchers have shown that RB does play an important role in the terminal differentiation of some cell types, for example, red blood cells (Jacks et al., 1992; Lee et al., 1992). However, almost all tissues and organs are formed in the complete absence of RB. Redundant genetic functions may exist in the mouse genome to mediate differentiation in the absence of RB. The exact role of RB dephosphorylation in the cellular differentiation systems remains to be determined. E. EFFECTS OF ANTIMITOGENS ON RB PHOSPHORYLATION A wide variety of antimitogenic factors has been identified, including small molecules such as cyclic AMP and peptides such as TGFP and interferon-a (IFNa), that are capable of arresting the growth of specific target cells. Both IFNa and TGFP can affect the phosphorylation of RB. 1. TGFP
In a variety of cell culture systems, TGFP has been shown to have growth-suppressive activity: MV- 1LU, JOSK- 1, human keratinocytes, and others (reviewed by Massague, 1990). In these cells, TGFP induces alteration in gene expression, cell cycle arrest, and dephosphorylation of RB. T h e TGFP-induced cell cycle arrest occurs late in G,. This effect has been demonstrated by adding TGFP at various time points after the induction of a synchronous entry into the cell cycle. Addition of TGFP as late as 12 hr after G, entry can block DNA synthesis. However, after DNA synthesis beings, TGFP no longer has any effect until the next G, phase (Laiho et al., 1990; Howe et al., 1991). Therefore, the execution point of growth inhibition for TGFP is in late G,. T h e biological response to TGFP is mediated by three types of TGFP receptors that form heterodimeric complexes that contain intracellular serinelthreonine kinase domains (reviewed by Massague et al., 1992). An early event triggered by TGFP is the change in the expression of several early response genes, including c-my and junB (Laiho et al., 1991; Zentella et al., 1991). The effect on the early response genes is independent of the cell cycle block and the dephosphorylation of RB, as demonstrated by the expression of SV40 T antigen in MV-1LU cells. T h e T antigen abrogates the growth-inhibitory effect of TGFP, but does not
44
JEAN Y. J . WANG E T At..
stop the induction of the early response genes (Laiho et al., 1991). Another example of this effect is found in DU145 cells from a prostate carcinoma that expresses a mutated RB. TGFP does not affect the cell cycle progression in DU145 cells, but does induce the same early response genes found in MVI-LU cells (Zentella et al., 1991). These observations suggest that induction of the early response genes is not sufficient to cause a growth arrest and that RB may be required for TGFP to inhibit cell cycle progression. RB is dephosphorylated in response to TGFP. I n asynchronous MV- 1LU cells, RB exists predominantly in the hyperphosphorylated form. Treatment with TGFP results in the appearance of unphosphorylated RB within 6-8 hr, concomitant with the accumulation of cells in G1 (Laiho et cil., 1990). Furthermore, in synchronous G 1cells, TGFP inhibits the phosphorylation of RB as it blocks progression into S phase (Laiho et nl., 1990). Interestingly, SV40 ?’ antigen abrogates the growth-inhibitory effect of TGFP but does not restore the phosphorylation of RB (Laiho et nl., 1990). T h e rescue by T antigen appears to occur at a point downstream from the TGFP-induced block of RB phosphorylation. This result is consistent with a hypothesis that the inhibition of RB phosphorylation is critical to the growth-inhibitory effect of TGFP. ‘rantigen can circumvent this block by sequestering the unphosphorylated RB, thereby allowing entry into S phase in the absence of RB phosphorylation. The inhibition of RB phosphorylation is likely due to the inhibition of cdk-cyclin kinase activity observed in TGFQ-treated cells. In synchronous G , cells (MV-ILU), treatment with TGFP does not affect the expression of cyclin E or cdk2. However, cyclin E and cdk2 do not form a stable complex in TGFP-treated cells and there is no cyclin E-associated kinase activity (Koff et al., 1993). Furthermore, TGFP appears to induce an inhibitor of cdk2-cyclin E, p27Kip1, which is found in extracts prepared from TGFP-treated cells and can inhibit a preformed cdk2-cyclin E complex (Koff et al., 1993; Polyak et al., 1994). Some evidence suggests that p27Kipl may act in a stoichiometric manner to block the activation of cdk2-cyclin E and possibly other cdk-cyclin complexes at G,/S. Another target of the ‘I‘GFQ-mediated inhibitory pathway is cdk4. This cyclin-dependent kinase is usually associated with the D-type cyclins and is thought to play a role in G I progression (reviewed by Sherr, 1993). In TGFP-treated cells, the accumulation of cdk4 protein is blocked, possibly because of an inhibition of translation of the cdk4 mRNA (Ewen P t ul., 1993b). TGFP selectively inhibits the expression of cdk4 but not cdk2 during G , . Interestingly, in MV- 1LU clones that ectopically express cdk4, TGFP can no longer inhibit the cdk2 kinase or RB phosphorylation; these cells are no longer blocked in G, by TGFP (Ewen et al.,
RETINOBLASTOMA TUMOR SUPPRESSOR
45
1993b). This observation suggests that the inhibition of cdk4 expression is an important event in TGFP-mediated growth suppression. T h e hypothesis that cdk4 may be required for the activation of cdk2cyclin E is also supported by in nitro evidence. Addition of an active cdk4-cyclin D2 complex, produced from recombinant baculovirus in insect cells, to the extracts of TGFP-treated cells can neutralize the inhibitor of cdk2-cyclin E (Polyak et al., 1994). In these in nitro assays, a kinase-defective cdk4-cyclin D complex can also neutralize the inhibitor found in TGFP-treated cell extracts (Polyak et al., 1994). This result suggests that the cdk4-cyclin D2 complex may restore cdk2-cyclin E activity by sequestering the p27Kipl. Whether a kinase-defective cdk4, when ectopically expressed in MV-1LU cells, can overcome the growthinhibitory effect of TGFP remains to be determined. Although the precise mechanism has not been elucidated, it is very clear that the growth suppression of TGFP is due in part to the inhibition of cdk-cyclin activities in G, sells. This effect provides an explanation for the block in RB phosphorylation. However, whether the persistence of unphosphorylated RB is responsible for the G,/S block is not clear. The rescue by T antigen of the TGFP block suggests that unphosphorylated RB may be responsible. However, since T antigen has many functions, we cannot conclude that the sequestering of RB is the key to the rescue. Whether the persistence of unphosphorylated RB is sufficient to block MV-1LU cells in G, can be tested by the ectopic expression of an RB mutant that cannot be phosphorylated. Another test for the role of RB in TGFP-mediated growth inhibition may be to use the Rb-l- cells derived from knock-out mice. If RB is an essential mediator of TGFP-induced G1 arrest, then Rb-l- cells should be resistant to TGFP whereas the matched control cells of Rb+/+ or Rb+lmice should be sensitive to the T G W effect. Such experiments might shed light on the role of RB in the TGFP signaling pathway. 2. Interferon-a IFN-a is a peptide hormone that regulates the physiology of a variety of cell types (reviewed by Kimchi, 1992). Binding of IFN-a to its receptor leads to the activation of the JAK family of cytoplasmic tyrosine kinases (Velazquez et al., 1992). A major set of substrates for the IFN-activated tyrosine kinases appear to be the STAT (signal transducer and activator of transcription) proteins which, on tyrosine phosphorylation, translocate into the nucleus and activate the early response genes (Fu, 1992; Shuai et al., 1992, 1993). In several cell types, for example, the human Burkitt lymphoma cells Daudi, IFN-a displays a growth-suppressing activity. In Daudi cells, IFN-a leads to a cell cycle block in G,, which is
46
JEAN Y. J . WANG E 7 A L .
apparent by the accumulation of cells with 2N DNA within 24-48 hr of treatment (Einat et al., 1985; Thomas et al., 1991). Cycling Daudi cells contain mainly hyperphosphorylated RB. Within 10 hr of exposure to IFN-a, unphosphorylated RB becomes detectable; after exposure for 48 hr, all the RB is in the unphosphorylated or hypophosphorylated form (Thomas el al., 1991; Resnitzky et al., 1992). Therefore, the complete dephosphorylation of RB correlates with withdrawal from the cell cycle induced by IFN-a. There is no evidence yet that IFN-a can inhibit cdk/cyclin activities. However, treatment with IFN-a would be expected to lead eventually to the inhibition of the G , cdk-cyclin kinases or the activation of RH phosphatases. 3 . Small Molecule Inhibitors a. Okadair Arid. OA was originally identified as a tumor promoter derived from the black sponge Halichondria okadai (Tachibana et al., 1981). This molecule is a potent inhibitor of protein phosphatases 1 and 2A (reviewed by Fujiki and Suganama, 1993). As an inhibitor of protein phosphatases, OA would be expected to lead to hyperphosphorylation of RB. Indeed, treatment of asynchronous human fibroblasts with 1001000 ILV OA for 2 hr can promote the hyperphosphorylation of RB (Yatsunami et al., 1993). In established rodent fibroblasts such as 3T3 and 10T1/2 cells, OA can block cell cycle progression. Exposure of asynchronous 3T3 cells to 100 nM OA leads to a G, arrest in 20 hr (Schonthal and Feramisco, 1993). Furthermore, OA can block the entry of quiescent 3T3 or 10T1/2 cells into S phase (Kim et al., 1993; Schiinthal and Feramisco, 1993). The G, arrest induced by OA is correlated with a block in the phosphorylation of RB (Kim et al., 1993; Schijnthal and Feramisco, 1993). The hyperphosphorylation of RB observed at 2 hr of treatment in human fibroblasts is likely a direct effect of the inhibition of phosphatases by OA. However, the dephosphorylation of RB observed in OAtreated rodent fibroblasts is probably the result of a block in G , progression and down-regulation of cdk-cyclin activity (Schonthal and Feramisco, 1993). In the absence of RB kinases, inhibition of the phosphatases by O A is unlikely to promote the hyperphosphorylation of RB. 6. Sphang-osin,c. D-erylhro-Sphingosine (sphingosine) is a sphingolipid breakdown product that can inhibit hematopoietic cell growth as well as protein kinase C activity (Hannun and Bell, 1989). Treatment of the lymphoblastic leukemia cell line Molt-4 with 500 nM sphingosine results in an accumulation of cells in G, within 24 hr (Chao et al., 1992). When the phosphorylation state of RB was determined, researchers found that
RETINOBLASTOMA TUMOR SUPPRESSOR
47
sphingosine led to dephosphorylation of RB in 3-4 hr (Chao et al., 1992). This rapid dephosphorylation of RB occurs long before the accumulation of cells in G I , suggesting that sphingosine may directly inhibit the RB kinases or activate the RB phosphatases, irrespective of the phases of the cell cycle. c. Calmodulin Antagonists (W-7 and Calmidazolium). The calmodulin antagonists W-7 and calmidazolium (CZM) can block the serum-induced entry of quiescent human diploid fibroblasts into S phase (Takuwa et al., 1993). T h e inability to initiate DNA synthesis is again correlated with the inability to phosphorylate RB in these experiments. Human diploid fibroblasts expressing SV40 T antigen are refractory to the inhibitory effect of W-7 and CZM on G,/S progression and on RB phosphorylation (Takuwa et al., 1993). This result is in contrast to TGF-P-mediated inhibition in which T antigen cannot overcome the block of RB phosphorylation (Laiho et al., 1990), suggesting that T antigen can alleviate a G,/S block that is either upstream or downstream from RB phosphorylation.
d. Cyclic AMP. Increases in the intracellular concentration of cyclic AMP (CAMP)has long been known to have antiproliferative effects, and this may involve an inhibition of the MAP kinase signaling pathway (Wu et al., 1993; Sevetson et al., 1993). In HL60 cells, the addition of dibutryl CAMP causes a cell cycle arrest in G,. Complete dephosphorylation of RB is observed 48 hr after treatment, and is concomitant with cell cycle arrest (Whyte and Eisenman, 1992).
e. Phorbol Ester. In the murine B lymphoma WEHI 23 1, the phorbol ester TPA causes an irreversible cell cycle arrest within 24 hr. Hypophosphorylated RB begins to increase following 15 hr of treatment with TPA, again showing that RB dephosphorylation occurs in conjunction with cell cycle arrest (Maheswaran et al., 1991). As summarized in Table I, factors that stimulate proliferation yield an increase in RB phosphorylation, whereas those that cause growth arrest lead to RB dephosphorylation. These results have reinforced the hypothesis that unphosphorylated RB is the active form in suppressing cell growth. Despite the accumulation of many such observations, it is difficult to determine in these experimental systems whether the change in RB phosphorylation is the cause or the effect of cell cycle arrest. The inevitable question raised from these observations is “Which comes first?” Is the change in the phosphorylation state of RB responsible for the proliferative o r antiproliferative response to the various factors, or is
48
JEAN Y. J . WANG E T AL,.
the change in KB phosphorylation merely a reflection of cell cycle progression or cell cycle block? The relevance of RB in these experimental systems, and the role RB may o r may not be playing in a particular response, has not been elucidated to any great extent. An important control that has been missing from these experiments is a matched cell lacking RB, so that the response to a given factor can be examined in the absence of RB. The use of viral oncoproteins such as E 1A or T antigen does not provide the best control because of the pleiotropic effects these proteins have on many cellular processes. T h e development of specific reagents to inactivate RB, for example antisense oligonucleotides or dominant negative mutants of RB, will facilitate the elucidation of the role of RB in these rriitogenic, differentiation, or antimitogenic responses. Alternatively, cell lines ma): be established from Rb-/- mouse embryos with control cells derived from Rb+/- embryos; these lines could be used to examine the requirement of RB in the response to a variety o f regulators of proliferation and differentiation. V. Protein-Binding Function of RB A. PKOIEIN-BINDING DOMAINS I N RB
The RB protein has been shown to interact physically with a variety of different viral and cellular proteins (Table 11). In ztitro binding studies, using both artificially produced and naturally occurring RB mutations, have defined several distinct protein-binding domains in RB (Fig. 1). T h e first to be described is the A / B pocket, consisting of the A domain (amino acids 379-572) and the B domain (amino acids 646-772), separated by the insert domain (amino acids 573-645). The second is the C, pocket (residing in amino acids 768-928), which can function independently of the A)B pocket. T h e N-terminal region of RB (amino acids 1378) may also contain at least one distinct protein-binding domain. I . AIR Pocket
The first protein-binding domain to be identified in RB consists of two nonconsecutive stretches of amino acids, domain A (amino acids 379-572) and domain B (amino acids 646-772), t.hat are thought to form a protein binding “pocket” termed the A / B pocket. This pocket region was first defined as the binding site for two viral oncoproteins, E l A and large T antigen, based on their ability to bind RB deletion mutants in z d r o ( H u et a/., 1990; Huang et al., 1990; Kaelin et al., 1990). Since then, the A/B pocket has been shown to interact with a number of
TABLE I1 RB-BINDING PROTEINS ~~
Proteina ~
Functionb
~
Binding in RBc
Effect of RB phosphorylation on bindingd
Consequence of bindinge
Reference DeCaprio et al. (1988); Dyson et al. (1989) Dyson et al. (1989); Imai et al. (1991) Whyte et al. (1988) Szekely et al. (1993a) Helin et al. (1992); Kaelin et al. (1992) Qin et al. (1992); Heibert (1993) Gu et al. (1993) Gu et al. (1993) Wang et al. (1993) Hagemeier et al. (1993) Rustgi et al. (199 1) Kim et al. (1992) V. Smith and A. Winoto (personal communication) Welch and Wang (1993) Dowdy et al. (1993); Ewen et al. (1993) Hu et al. (1992); Kitagawa et al. ( 1992) Akiyama et al. (1992)
~~~
Large T Ag
Oncoprotein
AIB
Yes
Contributes to viral transformation
E7
Oncoprotein
AIB
Yes
Contributes to viral transformation
ElA EBNA-5 E2F-1, -2, -3
Oncoprotein Viral replication? Transcription factor
AIB
No ND Yes
Contributes to viral transformation
E2F-DNA MyoD M yogenin Elf- 1 PU. 1 C-MYC ATF-2 GATA-3
Transcription Transcription Transcription Transcription Transcription Transcription Transcription
c-Abl D1, D2, D3
?
AIB
?
Inhibits activity
?
Yes Yes Yes Yes ND ND ND Yes
Activates activity Activates activity
Tyrosine kinase Cyclins
C Large AIB
Yes Yes
Inhibits tyrosine kinase Targets RB for phosphorylation?
cdc2
SerIThr kinase
?
ND
RB phosphorylation?
cdk2
SerIThr kinase
?
ND
RB phosphorylation?
factor factor factor factor factor factor factor
Large AIB B B A/B AIB AIB AIB
? ?
Inhibits activity ? ?
(continued)
~
Kl)hp4N
ti.s.<:. PP- I a2
RBHN KBP-1 RBP-2
Yeast MSI 1 Iionio-
~~
Qian rt ril. (IW3)
Yes
lyl
ND
Protein phtsphiitaw ?
large AIB
YCS
ND ND ND
Nihei rl ul. (IW3)
KH clcpliospliorylation'
Durfee rt (11. (1993) Kayrtul.
>
(IWP)
DefctrJoncs rt al. (19Y1) kfeo-Jones rt a/.(l(391)
" Viral and ccllulnr pnwiiin that haw Iwrn rrpir~cdIO hind WB. Many othcr RB-liiiclingp i ~ ~ ~ e 11;ivc i i i s Inwi i d c n t i f i hut haw not yet k n puldishcd. EBNA-5. Epicin-Barr viriin nticlrnr antipi-5; H.S.C.. Iic;it-nhcrk c i ~ i a t c . b Question marks indicate ftinrtion unkncmm. Mort pmicins bind thc A/t) p k c t of WB. l h c conilrx of EPF and LISA Iiindn thc AIR pxkct p l u ~C-terniinal aniino acids, rcfcrml to i ~ iqh"large All) prka." Tlic c-AM tyrosine kinase Inn& ihc <; p k c t . ,t Phhc~sphtirylaiion of WB appears 10 inhibit the binding of 1 i i c ~ 1 ( pnitriiin. Yes, Pln~sphorylationdirupts roniplcx formation: No. phcaphtirylation dcm no( affec~ romplcx tormation; ND. noi dctcrniincd. * Quation marks inditatc untcminty or a lack of kncmkdw.
51
RETINOBLASTOMA TUMOR SUPPRESSOR
1
379
572
646
772
A: Mapped in vivo phosphorylation sites in RB Ser 249, Thr 252, Thr 373, Ser 807, Ser 811, Thr 821, Thr 826
A : Other potential cdk phosphorylation sites in RB Thr 5, Ser 230, Thr 356, Ser 567, Ser 608, Ser 612,Ser 780, Ser 788, Ser 795 FIG. 1 . Domain structure and phosphorylation sites of RB. The human RB protein contains 928 amino acids that are organized into several domains. The N-terminal region, N (amino acids 1-379), is dispensable for the growth suppression of Saos-2 cells by RB. This region does contain a protein-binding domain that is independent of the C-terminal sequences. The A/B pocket is composed of two noncontiguous domains, A (amino acids 379-572) and B (amino acids 646-772), which form the binding pocket for viral oncoproteins and many cellular proteins (see text). The insert domain, ID (amino acids 572-646), has not been shown to possess protein-binding activity. However, the equivalent domain of another AIB pocket-containing protein, p107, has the ability to bind cyclin A. The Cterminal region, C (amino acids 772-928) contains ( 1 ) amino acids that can contribute to protein binding through the A/B pocket (i.e., to form the large A/B pocket), (2) a C pocket that functions independently of the A/B pocket, and (3) a bipartie nuclear localization signal. There are 16 Ser/Thr-Pro motifs in RB (indicated by arrow heads), which are potential sites of phosphorylation by the cyclin-dependent protein kinases. Indeed, seven of these sites have been shown to be phosphorylated in RB in uiuo (filled arrowheads).
cellular proteins including the transcription factor E2F (Chellappan et al., 1991; Kaelin et al., 1991). Furthermore, in homology studies the A/B pocket is being used to define a family of RB-related proteins, including p107 and p130, that also contain A/B pockets (Ewen et al., 1991; Mayol et al., 1993; Hannon et al., 1994; Li et al., 1994). Interestingly, many of the naturally occurring RB mutations found in tumor cells are ones that disrupt the integrity of the A/B pocket (for review, see Hamel et al., 1993; Zacksenhaus et al., 1993b). A number of these point mutations affect splice donor or acceptor sites, resulting in the deletion of entire exons during RNA splicing. Other point mutants create missense mutations (e.g., C706F; Kaye et al., 1990), which presumably prevent the proper folding of the A/B pocket. A functional A/B pocket is necessary, but not sufficient, for RB to act as a growth suppressor. This requirement can be demonstrated in Saos-2 cells, in which exogenous expression of full-length RB causes G, arrest but expression ofjust the A/B pocket has no effect on cell cycle progression (Qin et al., 1992; Hiebert, 1993). Indeed, RB-dependent arrest of
52
JEAN Y. J. WANG E T AL.
Saos-2 cells requires the A/B pocket as well as C-terminal amino acids, at least through amino acid 870 (Hiebert, 1993). Together these observations indicate that the A/B pocket is necessary but not sufficient for RB function.
2. Insert Domuin Between the A and B domains is a stretch of 75 amino acids that has been termed the insert domain (Hu t=t al., 1990; Huang et al., 1990). Small deletions within the insert domain do not affect KB activity, but deletion of the entire insert domain inactivates the protein-binding function of the A/B pocket (Hu t=tal., 1990; Huang el al., 1990; Qian et nl., 1992). Replacement of the insert domain with a random amino acid sequence of identical length restores A/B pocket function (Hu et d., 1990; H ~ i a ~uti gal., 1990; Qian et a!., 1992). This result suggests that the insert doniain functions as a “spacer” between the A and B domains, providing a physical separat.ion necessary for the formation of the A/B pocket. N o proteins have yet been found to interact with this region of KB. T h e insert domain of another AIB pocket protein, p107, has been shown t o interact with cyclin A (Ewen P t nl., 1992; Faha et d.,1992), suggesting that the RB insert domain may, in time, be shown to have protein-biding activity of its own. 3 . C Pocket
,4mino acids C-terminal to the €3 domain have been shown to contribute to the protein-binding activity of KB. For example, interaction of the D-type cyclins with RB requires not only the A/B pocket but also amino acids in the C: terminus of RR (Ewen et nl., 1993a). Additionally, as discussed subsequently, the formation of a n RBIE‘LFIDNA complex that is stable in electrophoresis also requires C-terminal amino acids in addition to the A / H pocket (Huang et al., 1992; Qin P t a/., 1992; Hiebert, 1993). T h e binding site formed by the A/B pocket plus C-terminal amino acids has been termed the “large A/B pocket.” An additional C-terminal protein-binding activity that is completely independent of the A/B pocket has been identified (Welch and Wang, 1993). This independent C pocket is defined by the interaction of RR with the c-Abl tyrosine kinase (Welch and Wang, 1993). ‘l’he C pocket lies within KB amino acids 768-928; however, the actual size of this pocket is considerably smaller (P. J. Welch, unpublished data). The binding of c-Abl through the C pocket is different from all previously known RB binding mechanism because it is not affected by the viral oncoproteins, which displace proteins bound to the ,4/B pocket or those that bind the “large A/B pocket” (e.g., D-type cyclins). I n addition, a point mutation
RETINOBLASTOMA TUMOR SUPPRESSOR
53
of Cys 706 to phenylalanine, which inactivates large A/B pocket function, does not affect the C pocket activity (Welch and Wang, 1993). An interesting question arises from the identification of the C pocket. Does the C pocket overlap with the C-terminal amino acids in the large A/B pocket? Since viral oncoproteins do not affect the C pocket but do disrupt the interactions mediated through the large A/B pocket, the C pocket and the C-terminal part of the large A/B pocket may not overlap. Thus, the A/B and C pockets of RB may interact with different proteins simultaneously. This possibility is supported by the finding that RB can bind T antigen and c-Abl at the same time (Welch and Wang, 1993). Recent resuIts showed that RB could also bind simultaneously to E2F/ DNA and C-Abl (Welch and Wang, submitted). As mentioned earlier, amino acids in the C terminus are required for RB to function as a growth suppressor (Qin et al., 1992; Hiebert, 1993). In fact, one case of acute lymphocytic leukemia has been described in which only exons 24 and 25, encoding amino acids 831-888, have been disrupted (Hansen et al., 1990). The C-terminal region of RB also contains a bipartite nuclear localization signal (amino acids 860-876, Fig. 1; Zacksenhaus et al., 1993b) as well as a nonspecific DNA-binding activity (Wang et al., 1990). Finally, many of the cell-cycle-regulated phosphorylation sites are located in the C terminus (Fig. 1; Lin and Wang, 1992). Whether the specific disruption of C pocket function alone can inactivate the growth-suppression activity of RB remains to be determined. 4. N-Terminal Domain
Until recently, little attention was focused on the region of RB N-terminal to the A domain (Fig. l), primarily because the N-terminal 378 amino acids of RB, as a whole, at first appeared dispensable for RB growth-suppression activity in Saos-2 cells (Qin et al., 1992). More recent data, however, have shown that artificially generated internal deletions in the N-terminal region can inactivate RB function (Qian et al., 1992). In fact, one retinoblastoma has been described that has an internal deletion of only 40 amino acids (exon 4)in the N-terminal domain of RB (Dryja et al., 1993; Hogg et al., 1993). The N-terminal domain has been implicated in mediating an oligomerization of RB molecules in vitro; however, no evidence is available that RB exists as homo-oligomers in intact cells (Hensey et al., 1994). Using the N-terminal domain as a probe to screen expression libraries through protein-protein interaction, nine nuclear proteins have been identified (J. Horowitz, personal communication). At least two of these proteins are cell-cycle-regulated serine/threonine kinases that can phosphorylate histone H1 and RB. One kinase is associated with RB in G,
54
JEAN Y. .J. WANG E T AL.
and S phase, whereas another is only associated with RB at G2/M (J. Horowitz, personal communication). Neither of these kinases reacts with the available cdk antibodies and neither is co-immunoprecipitated with the known cyclins. Thus, these may represent a new class of RB kinases. ‘The identification of proteins that bind to the N-terminal region indicates that there may be an “N pocket” which could play a role in the regulation of RB phosphorylation. B. RB-BINDING PROTEINS T h e growing number of RB-associated proteins ranges from viral oncoproteins and transcription factors to kinases and phosphatases. A summary of the RB-binding proteins, categorized by their function, the way in which they interact with RB, and the effect of RB on their activities, is given in Table 11. I . Virul Oncoprotem
T h e DNA tumor viruses express proteins that function to activate cellular replication machinery and can drive a quiescent cell into S phase. An important step in this process is the binding of RB and the two other A/B pocket proteins p107 and p130. This activation is carried out by ElA of adenovirus, E7 of human papilloma virus type 16 (HPV16) or large T antigen of SV40 o r polyoma virus (for reviews, see Dyson et al., 1989; Ludlow, 1993; Moran, 1993; Vousden, 1993). Mutations in these oncoproteins that inactivate their ability to bind the A/B pocket also inactivate their ability to stimulate cell proliferation. T h e oncoproteins share three conserved regions termed CR1, CR2, and CR3. Amino acids required for high-affinity RB binding are located in CR2 and contain the sequence motif Leu-X-Cys-X-Glu (LXCXE), which directly interacts with the A/B pocket. Binding of the oncoproteins is thought to release cellular A/B pocket-binding proteins such as E2F, which are then free to activate cell cycle progression. A recent addition to the list of virai oncoproteins that bind RB is the Epstein-Barr virus nuclear antigen EBNA-5 (Szekely et nl., 1993a). EBNA-5 shares sequence homology with the CRl domain of E1A and can be competed off of RB by an LXCXE-containing peptide from E7. Interestingly, however, EBNA-5 does not contain the LXCXE motif. Furthermore, it binds wild-type RB and the point mutant C706F with equal affinity in vatro, suggesting a different mechanism of AIB pocket binding. T h e biological consequences of the interaction of EBNA-5 with RB are currently unclear.
RETINOBLASTOMA TUMOR SUPPRESSOR
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2. E2F An important set of A/B pocket-binding proteins displaced by the viral oncoproteins is the E2F family of transcription factors. E2F was first defined as an activator of transcription that specifically binds to the DNA sequence 5'-TTTCGCGC-3', which is present in the promoter of the E2 gene of adenovirus (for review, see Nevins, 1992). Cellular E2F activity is complexed with A/B pocket proteins such as RB, p107, or p130 and is inactive in the complexed form (Hiebert et al., 1992; Weintraub et al., 1992; Flemington et al., 1993; Helin et al., 1993a). Following adenovirus infection, E1A binds to RB, p107, and p130 to displace E2F, thus allowing activation of the E2 promoter. E2F sites are also found in the promoters of cellular genes required for cell cycle progression, including dihydrofolate reductase (dhfr), cdc2, and c-myc (Nevins, 1992). E1A-mediated release of E2F is likely to be one of the mechanisms used by the virus to activate the cellular replication machinery (Hiebert et al., 1991; Nevins, 1992). The regulated release of E2F from RB has been proposed to be an important step in the normal G,/S transition (Nevins, 1992). a. Cloning of E2F-1. The first cDNA clone that encodes a protein capable of binding to RB and to the specific E2F DNA sequence was isolated by screening an expression cDNA library using the A/B pocket of RB as a probe. T h e protein encoded by this cDNA is now referred to as E2F-1 (Helin et al., 1992; Kaelin et al., 1992; Shan et al., 1992). E2F-1 contains a 102-amino-acid DNA-binding domain that specifically interacts with the E2F DNA sequence. The C-terminal 70 amino acids of E2F-1 constitute an acidic transcriptional activation domain which, when fused to the DNA-binding domain of Ga14, can activate transcription from a Gal4-binding site (Kaelin et al., 1992; Shan et al., 1992). Co-transfection of E2F- 1 cDNA with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing wild-type E2F sites, but not mutant E2F sites, also leads to the activation of transcription (Helin et al., 1992). The in vitro binding of E2F-1 to RB is mediated through an 18-amino-acid segment (amino acids 409-426) located in the C-terminal half of the transactivation domain of E2F-1 (Helin et al., 1992). There is no LXCXE motif in E2F- 1.
b. Regulation of E2F by RB. The hypothesis that RB can regulate E2F activity is supported by two lines of evidence. The first is found in RBnegative cells, in which ectopic expression of RB causes a 5-fold decrease
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in the activity of promoters containing E2F sites whereas an RB mutant that cannot bind E2F has no effect (Weintraub et af., 1992; Hiebert, 1993). The second comes from transactivation studies with Ga14-E2F- 1 fusion proteins. Fusion proteins containing wild-type E2F- 1 are inhibited by coexpression of RB, whereas fusion proteins containing transcriptionally active E2F-1 mutants that cannot bind RB are not affected by c-oexpression of RB (Helin et al., 1993a; Flemington el al., 1993). Interestingly, RB has been shown to increase the half-life of the E2F-DNA complex in gel mobility shift assays, as well as to reduce the amount of DNA bending induced by E2F (Huber et al., 1993b). The release of E2F, mediated either by the viral oncoproteins or by the phosphorylation of RB, is thought to activate G,/S-specific genes (for reviews, see h’evins, 1992; Hamel et af., 1992, 1993). Two lines of evidence support the hypothesis that E2F can promote entry into S phase. T h e first comes from experiments in Saos-2 cells. When wild-type RB is expressed, Saos-2 cells arrest in G I with a dramatic decrease in cellular E2F activity (Qin et al., 1992; Zhu et al., 1993). Coexpression of E2F-1 with RB reverts the G , arrest phenotype, showing that the RBinduced G I block can be overcome by excess E2F-1 (Zhu rt ul., 1993). Another piece of supporting data conies from studies in rat embryo fibroblasts (REF). Overexpression of E2F-1 or micro-injection of E2F- 1 cDNA into quiescent REF is found to stimulate entry into S phase (Johnson el al., 1993). c. E2F-1 i s E2F. Several studies have shown that E2F-1 is only one of several prot.eins that make up the E2F activity detected in nuclear extracts. In fact, E2F activity could be separated into at least five biochemically distinct protein fractions by SDS gel electrophoresis and renaturation. Each of the protein fractions alone had relatively poor DNAbinding activity, but pairwise combinations of the fractions resulted in a 100- to 1000-fold increase in specific DNA-binding activity (Huber et al., 1993a). This result allowed the different E2F fractions to be divided into two “complementation groups.” One of the complementation groups is a family of proteins with properties similar to those of the cloned E2F- 1. Two new members, E2F-2 and E2F-3, have been cloned. These three proteins are 73% identical in the DNA-binding domain and 56% identical in the transactivating and RB-binding domains (Ivey-Hoyle et al., 1993; Lees et ul., 1993). The other complementation group contains DP-1, which was first cloned as DRTF1, a protein that specifically binds the E2F site (Girling et al., 1993). DP-1 shares limited homology with E2F-1 in two regions. The first is the DNA-binding domain, which is 42% identical and 7Oc% similar to that of E2F-1 and may explain why
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both proteins recognize the same DNA sequence (Girling et al., 1993). T h e second is a hydrophobic heptad repeat, resembling a leucine zipper, that is 41% identical to E2F-1. This domain mediates the heterodimerization of DP-1 and E2F-1, resulting in a high-affinity DNA-binding heterodimer (Bandara et al., 1993; Helin et al., 1993b; Krek et al., 1993). Moreover, the E2F-1-DP-1 complex has a higher affinity for RB than does E2F-1 alone (Krek et al., 1993). Unlike E2F-1, however, DP-1 does not have detectable transactivation activity nor does it interact with RB, suggesting that the transcriptional activity and its regulation by RB is carried out by the E2F-1 component of the heterodimer (Helin et al., 1993b). T h e finding of multiple heterodimers that bind the E2F site shows that “E2F” is really a mixture of proteins, reminiscent of the AP1 transcription factor that is made up of heterodimers from members of the Fos and Jun families. The different E2F heterodimers may preferentially interact with a given A/B pocket protein. For example, E2F-1, 2, and 3 do not appear to bind p107, either in vitro or in vivo, indicating that p107 binds other yet unidentified E2F proteins (Bandara et al., 1993; Lees et al., 1993). Viral oncoproteins such as E1A can cause the release of multiple E2Fs because they bind to RB, p107, and p130. The loss of RB, however, may not activate all E2Fs in a cell. A great deal is yet to be learned about the specificity of interaction of the many E2Fs with their target promoters and with the A/B pocket proteins. d. Disruption of E2FIRB by Viral Oncoproteins. The viral oncoproteinmediated release of E2F from RB is an important observation, not only for the regulation of E2F but also for the regulation of other RBbinding proteins. The high-affinity interaction of the viral oncoproteins with the A/B pocket of RB is mediated through the LXCXE motif in the CR2 domain of ElA, E7, and large T antigen (for reviews, see Ludlow, 1993; Moran, 1993; Vousden, 1993). The cloned E2F-1 does not contain the LXCXE motif, yet the complex of E2F-1 with RB can be disrupted by a peptide containing LXCXE (Helin et al., 1992; Kaelin et al., 1992). Whereas the association of E2F-1 with RB can occur with only the A/B pocket, the association of the heterodimeric E2F/DNA and RB requires not only an intact A/B pocket but also amino acids in the C-terminal region of RB (Qian et al., 1992; Qin et al., 1992; Huang et al., 1992; Hiebert, 1993). Although the LXCXE-containing peptide can disrupt RB/E2F-1 it cannot disrupt the RBIEPFIDNA complex (Huang et al., 1993; Ikeda and Nevins, 1993; Wu et al., 1993). How do the viral oncoproteins disrupt the RBIEPFIDNA complex? In the case of E 1A, both CR2 (which has the LXCXE) and the CRl domain are required for this
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disruption (Ikeda and Nevins, 1993). In the case of E7, both CR2 and CR3 are required to disrupt RB/E2F/DNA (Huang et al., 1993; Wu et al., 1993). T h e CRl of ElA, as well as the CR3 of E7, has a weak affinity for RB, approximately 10-fold weaker than that of CR2 (Dyson et al.. 1992). The CRl of E1A binds the AIB pocket while the CR3 of E7 binds amino acids in the C-terminal region of RB (H. Huber, personal communication). Researchers have proposed that the disruption of the RBIEQF complex, by E1A or E7, is a two-step process (Ikeda and Nevins, 1993; Fattaey et al., 1993). First, the LXCXE motif in CR2 allows the viral oncoproteins to target RB through a high-affinity interaction with the A/B pocket. This interaction allows the weaker interaction of E1A CR1 or E7 CR3 to displace E2F from RB (Ikeda and Nevins, 1993; Fattaey et al., 1993). This hypothesis is supported by several pieces of evidence. First, an E1A mutant lacking the CR1 domain does not disrupt RBIE2F but can actually join RB/E2F to form a supercomplex on E2F DNA (Ikeda and Nevins, 1993; Fattaey et al., 1993). Second, a large excess of the ElA CRl peptide alone is sufficient to disrupt RBIEPFIDNA (Ikeda and Ne\rins, 1993). Third, pre-incubation of an RBIE2FIDNA complex with the CR2 peptide of E7 can prevent the full-length E7 protein from disrupting the complex (Huang et al., 1993). An apparent paradox arises from these results with respect to the disruption of RBIE2F-1 and RB/EPF/DNA by the viral oncoproteins. The LXCXE peptide alone can disrupt RBIEPF-1, but both the LXCXE and a second domain are required to disrupt the RB/E2F/DNA complex. One possible answer is that the observation made with the RB/ E2F-1 complex is specific for E2F-1, whereas examination of the RB/ E2F complexes on DNA addresses a diverse pool of E2F, only a small fraction of which contains the E2F-1 protein. This possibility is indicated by the fact that antibodies specific for E2F-1 have very little effect on E2F gel shifts from nuclear extracts (Bandara et al., 1993). A more radical explanation for this paradox would be that E2F interacts with RB in more than one way. In this scenario, the RBIEPF complex formed on DNA adopts a conformation that does not involve the LXCXE-binding region of RB, whereas the complex without DNA is mediated by the LXCXE-binding region in RB.
3. Other Transcription Factors a. ~ ~ ~ ~ I ~ ~ MyoD ~ ~ eand ? myogenin i z r ~ are . muscle-specific transcription factors that are essential to muscle development (for review, see Edmondson and Olson, 1993). When MyoD is expressed in a nonmuscle cell, it induces myogenesis by activating muscle-specific genes.
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Activation of muscle-specific genes by MyoD appears to require a functional RB, as can be demonstrated in the RB-negative osteosarcoma cell line Saos-2. In these cells, exogenous expression of MyoD is not sufficient to induce myogenesis. However, coexpression of RB with MyoD results in the activation of muscle-specific genes (Gu et al., 1993). Interaction between RB and MyoD can be demonstrated in vztro and by coimmunoprecipitation (Gu et al., 1993). The interaction, mediated by the basic helix-loop-helix domain in MyoD, is competed by an LXCXEcontaining peptide, suggesting an A/B pocket interaction (Gu et al., 1993). On further in vitro mapping studies, however, the binding of MyoD does not seem to require the A domain. Instead, the B domain alone is sufficient for binding MyoD in vitro. The RB point mutant C706F, which is located in the B domain, does not bind MyoD. Whether the B domain of RB alone was sufficient to collaborate with MyoD in inducing muscle-specific genes in the Saos-2 cells was not determined. T h e mechanism by which RB promotes the induction of muscle-specific genes is not understood. Muscle development does not appear to be affected in Rb-/- mutant embryos (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992). The result obtained with Saos-2 cells may not be true for normal muscle development. Alternatively, perhaps in the knock-out mice another A/B pocket protein, p107 or p130, is up-regulated and can compensate for the loss of RB function to promote myogenesis. b. Elf-1. Elf- 1 is a lymphoid-specific transcription factor belonging to the Ets protein family (Thompson et al., 1992; for review, see Wasylyk et al., 1993). Elf-1 recognizes the purine-rich Ets DNA consensus sequence 5'-AGGAA-3', which is present in many promoters induced during T-cell activation, including those of granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-2 (IL-2) (Thompson et al., 1992). Researchers have suggested that Elf-1 is responsible for the activation of these genes on T-cell stimulation. The mechanism of regulation of Elf- 1 is not understood, but probably involves post-translational events, because both resting and activated T cells have the same level of intracellular Elf-1 (Wang et al., 1993). T h e Elf-1 protein contains the LXCXE motif, which is located in the acidic transactivation domain and is subsequently found to interact with unphosphorylated RB in resting T cells (Wang et al., 1993). Similar to E2F, the transcriptional activation activity of Elf-1 is inhibited when RB is coexpressed, indicating that RB may regulate Elf-1 activity (Wang et al., 1993). As discussed in Section IV, RB becomes hyperphosphorylated during T-cell activation (DeCaprio et al., 1992). Following T-cell activation, the RB/Elf-1 complex can no longer be detected (Wang et al., 1993).
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Analogous to E2F, Elf-1 is inactive when coniplexed with RB. On T-cell activation, the phosphorylation of RB ma): disrupt the RB/Elf-1 complex and the released Elf-1 may be responsible for the activation of T-cell-specific genes. c. P U . 2 . PU. 1 is another lyniphoid-specific transcription factor containing a n Ets-like DNA-binding domain (Klemsz et al., 1990). PU.l recognizes the purine-rich DKA sequence 5'-GAGGAA-3' (the PU box). Since PU.1 is expressed only in macrophages and B cells, it has been hypothesized that PU. 1 plays a role in the expression of specific genes whose promoters contain PU boxes, for example, the immunoglobulin enhancers (Klemsz et al., 1990; for review, see Wasylyk et al., 1993). Studies have shown that the acidic transactivating domain of PU. 1, which does not contain the LXCXE motif, can bind the A/B pocket of RB in d r o (Hagemeier et al., 1993). T h e existence of an RB/PU.l complex has not been demonstrated in €3 cells. Since immunoglobulin gene expression can occur in Rb-l- cells (Chen et al., 1993), as discussed in Section II,C, the role of the RB1PU.I complex in B cells and macrophages is not understood.
d. ATF-2. T h e ATF proteins constitute an extensive transcription factor family that recognizes the DNA sequence 5'-GCACGTCA-3'. T h e human TGFPP promoter contains an ATF DNA-binding site that is essential for TGFP2 gene expression (Kim et al., 1992b). Interestingly, co-transfection of a CAT reporter driven by the TGFP2 promoter with an Rb cDNA leads to activation of CAT expression. T h e promoter element responsible for this RB-dependent activation is the ATF site (Kim el al., l992b). One ATF family member with high affinity for the TGFP2 ATF site is ATF-2. T h e transactivation activity of a Ga14-ATF-2 fusion protein on a C.4T reporter plasmid containing the Gal4-binding sites is also enhanced by RB. T h e region in ATF-2 that is responsive to RB is the N-terminal transactivation domain. Thus R B appears to enhance the transactivating activity of ATF-2. Moreover, a specific interaction can be shown between the transactivation domain of ATF-2 and the A/B pocket of RB in vitro (Kim et al., 1992b). This result is in direct contrast to the regulation of E2F by RB, in which interaction through the transactivating domain of E2F inhibits the activity. At this point, however, it is unclear whether RB is part of the ATF-2 DNA-binding complex in vivo. Further studies should determine the mechanism by which RB can enhance the activity of ATF-2.
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e. c-Myc. T h e Myc proteins are a family of transcription factors that play an important role in the regulation of cell proliferation and differentiation (for review, see Marcu, 1992; Evan and Littlewood, 1993). An association of c-Myc and n-Myc with the A/B pocket of RB can be demonstrated in vitro; this interaction is disrupted by the viral oncoprotein E7 (Rustgi et al., 1991). Similar to other RB-bound transcription factors, this interaction is mediated through the transactivating domain of the Myc proteins (Rustgi et al., 1991; Cziepluch et al., 1993). Interestingly, however, Myc-dependent transcriptional activity does not appear to be regulated by RB, since Gal4-Myc fusion proteins are not responsive to RB (Cziepluch et al., 1993). Although an RB/Myc complex has not been observed in vivo, an in vivo complex has been demonstrated between c-Myc and the RB-related protein p107, suggesting that p107 may be a partner of c-Myc (Gu et al., 1994). T h e biological significance of the complex between Myc and A/B pocket proteins is unclear at this time.
4 . c-Abl Tyrosine Kinase
T h e c-Abl tyrosine kinase is a ubiquitously expressed proto-oncoprotein that is located in both the cytoplasm and the nucleus of mammalian cells. In the cytoplasm, c-Abl can reside on actin filaments; in the nucleus, c-Abl can bind DNA (Kipreos and Wang, 1992; McWhirter and Wang, 1993; Wang, 1993). The c-Abl protein is differentially modified in the cell cycle, with hyperphosphorylation observed in metaphase cells (Kipreos and Wang, 1990). At least one of the consequences of this mitotic hyperphosphorylation is an inhibition of the DNA-binding activity of c-Abl (Kipreos and Wang, 1992). The cell-cycle-regulated binding of c-Abl tyrosine kinase to DNA indicates that this nuclear tyrosine kinase may play a role in cell cycle regulation. Constitutively activated Abl tyrosine kinase, either the Gag-v-Abl of Abelson murine leukemia virus o r the Bcr-Abl fusion protein of Philadelphia chromosome-positive human leukemias, can stimulate cell proliferation or arrest cells in GI depending on the cell context (Renshaw et al., 1992; Wang, 1993). Interestingly, the nuclear c-Abl forms a complex with RB. This complex is detected in all human and rodent cells that express both proteins, including primary human fibroblasts (Welch and Wang, 1993). T h e RB/c-Abl interaction is mediated by the C pocket of RB and the ATPbinding lobe of the c-Abl tyrosine kinase. Binding of RB results in the inactivation of the tyrosine kinase (Welch and Wang, 1993). Hence, RB is not tyrosine phosphorylated despite its direct association with a tyrosine kinase. As discussed earlier, the RB/c-Abl interaction allowed the definition of the C pocket of RB, an independent protein-binding domain that
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is not affected by the viral oncoproteins (Welch and Wang, 1993). T h e c-Abl/RB complex is detected in quiescent and early G , cells and is disrupted at G,/S when RB becomes phosphorylated. T h e consequence of this event is an inactive c-Abl tyrosine kinase in the nucleus of quiescent and G , cells, followed by an activation of this nuclear tyrosine kinase as cells enter S phase (Welch and Wang, 1993). One potential nuclear substrate for c-Abl is the C-terminal repeated domain (CTD) of mammalian RNA polymerase 11 (Baskaran et al., 1993). Phosphorylation of the CTD is correlated with the transition from initiation to elongation of transcription (Dahmus and Dynan, 1992). Interestingly, c-Abl can be shown to enhance the activity of VP16 in transient co-transfection assays. This activity requires active nuclear c-Abl and can be blocked by coexpression of RB (Welch and Wang, 1993). These results suggest that c-Abl may play an active role in cellcycle-regulated gene expression. In addition, since c-Abl is also located on actin filaments in the cytoplasm, it is possible that c-Abl transduces extracellular signals from actin to the nucleus (Wang, 1993). The interaction of nuclear c-Abl with RB may allow an integration of the cytoskeletal signal with the cell cycle control program. 5 . Cell Cycle Regulatoiy Proteins
a. D-Type Cyclzns. T h e D-type cyclin family contains at least three proteins-D1, D2, and D3-that may play critical roles in the regulation of G , progression (for review, see Sherr, 1993). In most cell types, at least one or more of the D-type cyclin genes is activated as cells enter the cell cycle from quiescence. In general, D-type cyclin genes are induced at an earlier point in G , than the E-type cyclin gene in mammalian cells. The D-type cyclins can interact with cyclin-dependent kinases cdc2, cdk2, cdk4, cdk5, and cdk6. Several lines of evidence suggest an important role for the D-type cyclins in cell cycle regulation. T h e cyclin D1 gene is translocated in parathyroid adenomas and in some B-cell leukemia (reviewed by Motokura and Arnold, 1993); it is also found to be amplified in 20-30% of breast cancers and esophageal cancers (Jiang et al., 1993; Motokura and Arnold, 1993). These observations indicate a potential oncogenic function for cyclin D1. Ectopic expression of D1 in established rodent fibroblasts results in a shortened G , phase; also the cells grow to a higher density (Ewen et al., 1993a; Dowdy et al., 1993). Micro-injection of anticyclin D1 antibodies prevents S phase entry (Baldin et al., 1993). Moreover, coexpression of any of the D-type cyclins can revert the RBinduced G , arrest of Saos-2 cells (Dowdy et al., 1993; Ewen et al., 1993a). Each of the three D-type cyclins contains the LXCXE motif at the N
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terminus, suggesting that they may directly interact with the A/B pocket of RB (Dowdy et al., 1993; Ewen et al., 1993a). Indeed, all three D-type cyclins can interact with RB and p107 (Dowdy et al., 1993; Ewen et al., 1993a; Kato et al., 1993). These complexes are disrupted by the viral oncoproteins and by an LXCXE-containing peptide, as expected (Dowdy et al., 1993; Ewen et al., 1993a). The in vitro binding studies also showed that C-terminal amino acids, in addition to the A/B pocket, are required to bind the D-type cyclins. This result led to the definition of the large A/B pocket (Ewen et al., 1993a). T h e association of the D-type cyclins with RB may be to target RB for cell-cycle-dependent phosphorylation by the cdk-cyclin D complexes (Dowdy et al., 1993; Ewen et al., 1993a). Active cdk4-cyclin D complexes phosphorylate RB very well in vitro. However, coexpression of RB with any of the D-type cyclins in Saos-2 cells does not lead to a significant increase in RB phosphorylation, whereas coexpression with cyclin A or E does promote RB phosphorylation (Hinds et al., 1992; Dowdy et al., 1993; Ewen et al., 1993a). Moreover, cyclin D1 with a mutation in the LXCXE motif, and is unable to bind RB, can nevertheless overcome the RB-induced GI arrest of Saos-2 cells (Dowdy et al., 1993). These observations are at odds with the notion that the interaction between RB and D-type cyclins occurs to target RB for phosphorylation. An alternative model is that RB may be an inhibitor of the D-type cyclins. RB may sequester the D-type cyclins and prevent their interaction with other targets that are important for cell cycle progression. b. cdc2Jcdk.2. Both cdc2 and cdk2 have been detected in immune complexes containing RB. A histone H1-kinase activity is co-immunoprecipitated with RB from cell lysates (Akiyama et al., 1992; Hu et al., 1992; Kitagawa et al., 1992). Furthermore, Western blot analysis of the anti-RB immunoprecipitates indicates the presence of both cdc2 and cdk2 (Hu et al., 1992; Kitagawa et al., 1992). Although cdc2 and cdk2 are found in the RB immunoprecipitates, there is no evidence that these two cyclin-dependent kinases directly bind to RB. Although cdc2 contains an ATP-binding lobe, no interaction between cdc2 and the C pocket of RB can be demonstrated with purified proteins (Welch and Wang, 1993). T h e association of cdc2 and cdk2 in the RB immunoprecipitates is likely to be mediated by an undetermined mechanism. 6 . Other RB-Binding Proteins a. RbAp48. RbAp48 was isolated as a major RB-binding protein in HeLa cell lysates (Qian et al., 1993). This ubiquitously expressed nuclear protein shares 46% homology with the MSI1 protein of Saccharomyces
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cere'e7iisiue. MSII appears to be a negative regulator of the Ras-CAMP
pathway in yeast because overexpression of MSI 1 can suppress the heatshock sensitivity of RAS2Vctl'g and wul mutants (Ruggieri et a[., 1989). Indeed, expression of the human RbAp48 in those mutant yeast strains can also suppress heat-shock sensitivity (Qian et a[., 1993). RbAp48 can interact with a C-terminal 60-kDa fragment of RB zn vitro, and is coimmunoprecipitated with RB from cell lysates. Whether RB can affect the activity of RbAp48 remains unclear, and the biological significance of this interaction is unknown.
6. Heat Sliock C q p a t e (73 kDa). T h e 73-kDa heat shock cognate (73hsc) is a constitutively expressed member of the 70-kDa heat shock family and is Iocalized primarily in the nucleus (Nihei et aE., 1993). A complex containing 73hsc and RB can be detected in human cell lysates b! co-immunoprecipitation using either anti-RB or anti-73hsc antibodies (Nihei et ul., 1993). Interestingly, this complex can be dissociated I)! A'I'P, but not ADP nor ATP-yS. Which domains of RB mediate this interaction, and whether or not the interaction of 73hsc with RB is direct remains unclear. c. Protein Phospfiatase 7jpe 1 - d . A large number of cDNAs encoding potential RB-binding proteins have been isolated using the yeast twohybrid system (Durfee el al., 1993). One such cDNA encodes a unique form of the catalytic subunit of protein phosphatase type 1 (PPl-a2). Since this interaction occurs in yeast, it is likely to be direct and not require any other human proteins. Similar to the D-type cyclins, YP 1-a2 binds only to the large AIB pocket of RB. By co-immunoprecipitation, a complex containing RB and PPI-a2 can be detected during mitosis and early G I . As discussed in Section IV,B, this interaction may maintain RB in its dephosphorylated form during late M and G I phases of the cell cycle.
d. RBP60. Although a stable complex of RBIE'LFIDNA can be detected in crude nuclear extracts, purified RB does not interact with highly purified E2F as determined by an electrophoretic mobility shift assay (EMSA). This result suggests that other cellular proteins may be required to stabilize the RB/E2F/DNA complex. Therefore, a study was conducted to identify such an activity (Ray et al., 1992). By fractionating cell extracts, a partially purified protein fraction was obtained that stabilized the RBIEQFIDNA complex, observed as an RB-dependent supershift of the EPFIDNA complex in EMSA. T h e major protein species in this partially purified fraction was approximately 60 kDa and was named RBP60 (Ray ~t al., 1992). Using far-Western blotting, purified RBP6O
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could be shown to interact with in vitro-translated RB, suggesting a direct interaction between the two proteins. The biological function of RBP6O is presently unknown. e. RBP-1 and RBP-2. Using a purified C-terminal 60-kDa fragment of RB as a probe to screen human cDNA expression libraries, two clones were isolated, named RBP-1 and RBP-2, that encode novel proteins (Defeo-Jones et al., 1991). Both proteins contain the LXCXE motif and their interaction with RB in vitro is disrupted by the viral oncoprotein E7. At this point, however, it is unclear whether either protein is associated with RB zn vivo, the biological functions of RBP-1 and RBP-2 are unknown. Although the list of RB binding proteins given here is long, it is far from complete. Many other RB-binding proteins have already been identified by a number of investigators, but these results have not yet been published. The IE2 protein of human cytomegalovirus, an activator of viral early gene expression, binds RB in vitro (D. Spector, personal communication). The GATA-3 transcription factor also binds RB and its transactivating function is enhanced by RB (V. Smith and A. Winoto, personal communication; see Table 11).The search for RB-binding proteins has been focused on interactions mediated by the A/B pocket. It would not be surprising if the C pocket also binds a number of cellular proteins in addition to the c-Abl tyrosine kinase. Clearly, RB can interact with many cellular proteins, and the cell is unlikely to contain enough RB to bind every molecule of this long list of proteins. Thus, RB is not likely to be a stoichiometric regulator of any of the RB-binding proteins. As discussed subsequently, the role of RB may not be simply to sequester cellular proteins but may instead be to promote the assembly of specific protein complexes through the simultaneous interactions with the different pockets of RB.
C. NUCLEAR TETHERING OF RB Immunofluorescence staining with antibodies against RB shows a punctate pattern indicating a nonhomogeneous distribution of RB in the nucleus (Mittnacht and Weinberg, 1991). The nuclear RB is resistant to nucleases and nonionic detergents, as assayed by immunofluorescence. Thus, researchers have proposed that RB is “tethered” to the nuclear structure (Mittnacht and Weinberg, 1991; Templeton et al., 1991). RB mutants that are defective in viral oncoproteins binding are efficiently translocated to the nucleus but are not tethered there, suggesting that the nuclear tether requires a functional A/B pocket (Mitt-
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nacht and Weinberg, 1991; Templeton et al., 1991). T h e nuclear tethering of RB appears to be regulated by RB phosphorylation, as suggested by three types of observations. First, incubation of purified hypophosphorylated RB with RB-deficient nuclei leads to the association of RB with the nuclear pellet, whereas hyperphosphorylated RB does not become associated with the nuclear pellet in the same zn vitro assay (Templeton, 1992). Second, micro-injection of protein phosphatases into the nucleus results in an increased resistance of RB to extraction of nonionic detergent (Alberts et al., 1993). Third, the nuclei of quiescent and G, cells (which contain only hypophosphorylated RB) are shown to stain very strongly for RB by immunofluorescence after extraction with nonionic detergents, whereas the nuclei of S and G, cells (containing mostly hyperphosphorvlated RB) contain very little detectable nuclear RB after extraction with detergent (Mittnacht and Weinberg, 1991). Indeed, observation of an asynchronous population of detergent-extracted cells by immunofluorescence reveals a mixture of RB-positive and RB-negative nuclei. Researchers have proposed that RB is associated with the nuclear envelope since RB-deficient nuclei, treated with nucleases or high salt, are still capable of binding the purified RB (Templeton, 1992). In vztro, lamins A and C have been shown to interact with RB suggesting RB may be tethered to the nuclear matrix (Mancini et al., 1994). The significance of the tethering of RB is unknown. Note, however, that the distribution of RB in the nuclei of a myotube is not uniform (Szekely et al., 1993b). Proliferating rat myoblasts have a low level of nuclear RB. When they are induced to differentiate and fuse into myotubes, an increase in the level of nuclear RB is observed. T h e nuclei at the two ends of the myotubes are found to contain significantly higher levels of RB than the nuclei in the interior of the myotube. Exogenously expressed RB, translated from a cytoplasmic source of mRNA, is also preferentially retained in the terminal nuclei (Szekely et al., 1993b). T h e uneven distribution of RB in the nuclei of a muscle fiber may be indicative of a regulation in the nuclear tethering of RB.
D. ROLE OF RB
IN
TRANSCRIPTION
Many of the RB-binding proteins described to date are transcription factors (Table 11). Most of these interactions are mediated by the A/B pocket of RB and the transactivation domains of the transcription factors, as has been demonstrated for E2F, Elf-1 , PU. 1, ATF-2, and Myc. It has been noted that considerable homology is found between the A domains of RB and TBP (the TATA-binding protein of TFIID). In fact,
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many transcription factors that bind RB, such as PU.1, c-Myc, ElA, and the IE2 protein of human cytomegalovirus, also bind TBP (Hagemeier et al., 1993; Hateboer et al., 1993; D. Spector, personal communication). In some cases, RB binding and TBP binding have been shown to be mutually exclusive (Hagemeier et al., 1993; Hateboer et al., 1993). These observations have led to the proposal that one function of RB may be to block the communication between transcription factors and TBP (Hateboer et d., 1993). This, however, is not true for all transcription factors, since RB enhances the transactivating function of ATF-2 (Kim et al., 1992b). RB can also affect transcription without directly binding to a transcription factor. One example is the “RB control element” (RCE) located in the promoters of several genes, including c-fos, c-myc, and TGFP (for review, see Horowitz, 1993). Coexpression of RB results in a downregulation of promoters containing RCE sequences in transient transfection assays (Udvadia et al., 1992). However, RB protein is not detected in the three or more nuclear protein complexes that specifically bind to the RCE DNA sequence. Investigators propose that RB may inhibit transcription at RCEs by removing a critical component from the protein/DNA complex (Udvadia et d., 1992). The RCE can be activated by the transcription factor Spl in transient co-transfection assays. In the presence of S p l , RB enhances rather than inhibits RCE-mediated transcription (Kim et al., 1992a; Udvadia et al., 1993). The transactivating activity of a GaH-Spl fusion protein is also enhanced by RB (Kim et al., 1992a). T h e mechanism of RB-mediated activation of Spl function is not understood; no evidence exists for a direct interaction of RB and Spl. I n summary, the RB protein can function either as a positive o r as a negative regulator of transcription. Moreover, the effect of RB can be exerted through direct or indirect interactions with transcription factors. T h e effect of RB on transcription is context dependent, as shown by the regulation of the RCE. T h e context-dependent regulation of transcription by RB is reminiscent of the context-dependent growthsuppression activity of RB, discussed earlier in Sections I1 and 111. E. RB
AS A
MOLECULAR MATCHMAKER
Clearly proteins can bind RB in several ways (see Table 11).The first is the classical A/B pocket binding, defined by the viral oncoproteins and also true for E2F-1 and Elf-1. This type of interaction requires only sequences in the A and B domains. Even less of a recognition site is required by MyoD and myogenin, which appear to bind only within the
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B domain. Another group of RB-binding proteins requires not only the AIB pocket but also amino acids in the C-terminal region. These include the D-type cyclins, the EZF/DNA complex, and protein phosphatase type I-a2. T h e interaction of the c-Abl tyrosine kinase led to the definition of the C pocket, which functions independently of the A/B pocket. Finally, the N-terminal region of RB, outside of the A/B pocket, has also been shown to have protein-binding activity. Since the RB protein contains several independent protein-binding domains, RB may function as a “molecular matchmaker,” bringing together two o r more proteins that bvould not otherwise interact (Fig. 2). Theoretically, proteins could be bound simultaneously to the N-terminal domain, the AIB pocket, and the C pocket of RB, as depicted in Fig. 2. indeed, a trimolecular complex containing c-Abl, RB, and large T antigen can be detected in COS cells (Welch and Wang, 1993). Since such a complex can form with large T, it is reasonable to speculate that cellular A/B pocket-binding proteins could alsojoin into a complex with RB and c-Abl. Likewise, K-terminal-binding proteins could be paired with A/B and/or C pocket-binding proteins, and so on. T h e consideration of RB as a matchmaker is appealing because it explains the seemingly endless finding of KB-binding proteins. In fact, many more RB-binding proteins have been isolated but have not been reported in the literature. If the mission of RB is to match other proteins, it makes sense for RB to bind a large variety of proteins. T h e model of RB as a matchmaker also explains the “context”-dependent functions of RB. According to the availability of its constituents, RB can make different matches and affect different regulations. What could be the biological function for such a matchmaker? Since many RB-binding proteins also bind DNA, the matchmaker may provide a second dimension of specificity for the assembly of protein complexes on specific DNA sequences. ~I’heregulation of gene expression is controlled by the combination of transcription factors assembled on specific DNA sequences in enhancers, silencers, and promoters. T h e study of transcription factors has shown that each specific DNA sequence can be recognized by many proteins of the same family. For example, the E-box sequence CTXXAG is bound by a large number of basic helix-loop-helix proteins; the E2F sequence TTTCGCGC is bound by several E2F-DP heterodimers. T h e specificity in the assembly of- promoter complexes, therefore, must also be dictated by proteinprotein interaction. One level of specificity may be provided by a lateral cooperativity in the binding of transcription factors to DNA. On top of that, :I molecular matchmaker iike RB could further select for a given combination of transcription factors through the fitting of these pro-
QandGl
S and G2
Mitosis
FIG. 2. A model for the molecular function of RB. RB is perceived to function as a molecular matchmaker that brings together proteins that otherwise would not interact, through the simultaneous interaction of RB-binding proteins with the different pockets of RB. In this diagram, RB is depicted as binding to three proteins X, Y , and Z through the N, A/B, and C pockets, respectively. The RB-assembled protein complexes are shown to reside on DNA, because many of the known RB-binding proteins also possess DNAbinding activity. T h e binding to RB and to DNA d o not have to be collinear; RB could conceivably promote the interaction of proteins that are situated at long distances from one another on DNA. In that event, a large loop of DNA would result from the assembly of the RB-mediated complex. Alternatively, the RB-assembled protein complex may reside on nuclear structures, since RB is thought to be “tethered” to some yet undefined component of the nucleus. RB acting as a matchmaker could provide an added specificity in the assembly of protein complexes at specific DNA elements. It could also function to bring together enzymes and substrates, or to allow the compartmentalization of protein complexes at specific nuclear locations. T h e activity of each pocket is proposed to be regulated by the phosphorylation of specific Ser/Thr sites (see text for discussion). The multiple phosphorylated bands of RB, found in S and G2 phase cells, represent forms of RB that have only one (or two, not shown) of the pockets inactivated by phosphorylation. T h e inactivation of a pocket does not necessarily lead to the release of the RB-binding protein in this model, especially if lateral interactions between the RB-binding proteins can be established after the proteins are brought together by RB. Instead, phosphorylation of RB could lead to conformational changes of an RB-binding protein, which might in turn cause a change in the activity of the RB-assembled complex. For example, phosphorylation of specific sites may relieve the inhibition of the c-Abl tyrosine kinase that binds the C pocket, and the activated tyrosine kinase may phosphorylate an A/B pocket-binding protein to modify its function. Viral oncoproteins, by targeting the A/B pocket, can join the RB-assembled complexes and alter their activities. Of course the viral oncoproteins also cause the release of the cellular AIB pocket-binding proteins, such as EZF. T h e RB-assembled complexes are completely disrupted at mitosis when RB is stoichiometrically hyperphosphorylated.
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teins to the fixed geometry of the pockets in RB. Additionally, a matchmaker may allow a close interaction of proteins situated at large distances from one another on DNA, thus adding specificity to long-range interactions as well. Another possible function for a molecular matchmaker may be to bring together kinase and substrate. For example, some A/B pocketbinding proteins may be substrates of the c-Abl tyrosine kinase; these proteins are brought together by RB. Since RB itself is associated with a nuclear tether and is localized in discrete areas of the nucleus, it may function to compartmentalize other nuclear proteins by virtue of its ability to bind more than one protein simultaneously. Finally, a molecular matchmaker may provide a mechanism to integrate a plethora of information that a cell must sample to proliferate or differentiate properly. The progression from G, to S, for example, must be sensitive not only to growth regulatory factors but also to contact information between a cell and its surrounding cells and extracellular matrices. By binding to and bringing together transducers of a host of signals, the matchmaker RB could conceivably allow a cell in a complex organism to integrate the numerous signals that are relevant to the control of cell cycle progression. VI. Regulation of RB Function by Phosphorylation
A. PHOSPHORYLATION INACTIVATES PROTEIN OF RB BINDING FUNCTION The interaction of RB with other proteins is regulated by the phosphorylation of RB. Several methods have been used to demonstrate that RB-binding proteins preferentially interact with the unphosphorylated and hypophosphorylated RB. The first method is to immobilize a known RB-binding protein and then allow it to interact with a whole-cell lysate that contains phosphorylated and unphosphorylated forms of RB. Examination of the electrophoretic mobility of the bound and unbound RB by immunoblotting will then allow a determination of the forms of RB retained by the immobilized protein. This method has been used to show that immobilized T antigen binds to the two lower bands of RB, indicating an interaction with the unphosphorylated and a hypophosphorylated species of RB (Welch and Wang, 1993). T h e C pocketbinding protein c-Abl tyrosine kinase binds the three lower bands of RB, suggesting that c-Abl can interact with the unphosphorylated and at least two hypophosphorylated forms of RB (Welch and Wang, 1993).
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With E2F, however, only a single unphosphorylated RB band appears to be retained by immobilized E2F-1 protein or total E2F from nuclear extracts immobilized on an E2F-DNA affinity matrix (Chellapen et al., 199 1 ; Kaelin et al., 1992). I n the second method, purified RB is phosphorylated by an activated cdk-cyclin in vitro to high stoichiometry using radiolabeled ATP. The phosphorylated RB is mixed with unphosphorylated RB and passed over a column containing an RB-binding protein of interest. The bound and unbound fractions are then analyzed by immunoblotting and autoradiography. Using this method, investigators have shown that hyperphosphorylated RB does not bind to the c-Abl ATP-binding lobe immobilized on glutathione agarose beads (E. S. Knudson, unpublished data). I n addition to the two in vitro methods, co-immunoprecipitation has also been used to demonstrate a selective interaction with RB in vivo. Using antibodies specific for a given RB-binding protein, it is possible to examine the electrophoretic mobility of the co-immunoprecipitated RB on immunoblots and to determine which form(s) of RB is in a stable complex with that protein in the cell lysates. The association of the two lower bands of RB with T antigen has also been shown by this method (Resnick-Silverman et al., 1991). Alternatively, co-immunoprecipitation can also be performed with lysates prepared from cells that are radioactively labeled with 32P. By autoradiography and immunoblotting, researchers can then determine if the 32P-labeled RB is excluded from the immunoprecipitates and is preferentially found in the supernatant. Finally, the co-irnmunoprecipitation experiments can be performed with lysates from synchronized cells. If the complex of RB with a given protein is detected in a cell-cycle-dependent manner, it would suggest a regulation by RB phosphorylation. This evidence alone, however, does not prove that the phosphorylation of RB per se regulates the formation of a given protein complex. Using one or more of these methods, investigators have found that RB phosphorylation regulates the association of many of the RBbinding proteins (see Table 11). Most of the RB-binding proteins identified to date preferentially interact with the hypophosphorylated and unphosphorylated forms of RB (see Table 11).A notable exception is the viral oncoprotein E 1A, which appears to co-immunoprecipitate with unphosphorylated and phosphorylated forms of RB (Whyte et al., 1988). It is not clear whether phosphorylated RB can bind E1A directly or whether it is retained in the E1A immunoprecipitates through other types of interactions. At present, it cannot be ruled out that proteins exist that bind exclusively to the hyperphosphorylated forms of RB.
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B. RB PHOSPHORYLATIOK CORRELATES WITH REVERSAI. OF GROWTH SUPPRESSIOI~ A large body of circumstantial evidence supports the hypothesis that phosphorylation regulates the growth-suppressing activity of-RB, that is, that unphosphorylated RB is the active form which, on phosphorylation, is inactivated, thereby allowing cell cycle progression (reviewed by Hamel et al., 1993; Hollingsworth et al., 1993). This model is primarily based on three types of evidence. First, RB is unphosphorylated in quiescent or arrested cells and is phosphorylated in cycling cells; thus phosphorylation is correlated with growth and dephosphorylation with growth suppression (see Section IV). Second, only the unphosphorylated and hypophosphorylated RB are bound by T antigen. Targeting of these forms of RB by the viral oncoprotein suggests that these are the active forms in growth suppression (I,ivitigston, 1992). Third, the ability of RB to complex with cellular proteins is regulated by phosphorylation. As discussed earlier, most RB-binding proteins interact with the unphosphorylated and hypoI-'hosphoi-ylated KB. Since RB tends to inhibit the function of its associated proteins, the unphosphorylated RB is the active inhibitor. The phosphorylation o f RB has been correlated with the inactivation of its groM,th-suppressing activity in cultured cells. As discussed in Section 111, RB can inhibit the growth of some but not other tumor cell lines in culture. In each case, growth inhibition is observed when the exogenously expressed RB fails to become phosphorylated, as in the Saos-2 cells (Hinds et ~ l . ,1992; Qin et al., 1992; Zhu et al., 1993). T h e lack of growth inhibition, O I ~the other hand, is correlated with the phosphorylatiori of the exogenously expressed KB, as in the WERI-27 retinoblasroma cells and the C33A carcinoma cells (Huang ~t al., 1988; Zhu et at., 1993).These observations are consistent with the model that growth suppression in mediated by the unphosphorylated RB. Co-transttction of Rh-1 cDNA with either cyclin A or cyclin E cDNA into Saos-2 cells leads to a 90-95%', reversal of the RB-induced formation of GI-arrested large cells (Hinds ut al., 1992).T h e inactivation of the RB growth-suppressing function is correlated with the phosphorylation of RB in the presence of exogenously expressed cyclin A or cyclin E. Two mutants of RB-MX496, which contains a 4-amino-acid insertion at position 496 in the A/B pocket, and Di6-302, which lacks most of the N-terminal region-are resistant to reversion by co-transfection with cyclin A or cyclin E (Hinds et ul., 1992). These two mutants retain the C;, arrest function of RB and they are not efficiently phosphorylated. These observations also suggest that the phosphorylation of RB is responsible for the inactivation o f its gr-owth-suppressing activity.
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T h e strong prediction of this hypothesis is that an RB mutant that lacks phosphorylation sites should be a constitutive growth suppressor. This prediction, however, has not been demonstrated experimentally. Hamel et al. (1990, 1992) constructed phosphorylation site mutants of RB that cannot be phosphorylated efficiently in viva The biological effect of such RB mutants in cell cycle progression or cellular differentiation, however, has not been described. C. PHOSPHORYLATION SITE-SPECIFIC REGULATION OF RB FUNCTION
Since the discovery of its cell-cycle-dependent phosphorylation, RB has been viewed as existing in two conformational states, the unphosphorylated form that is active and the phosphorylated form that is inactive. This simplistic view of RB phosphorylation may not be entirely accurate, as demonstrated by several observations. First, RB is phosphorylated on at least seven and possibly more serinelthreonine sites (Fig. 1). The phosphorylated “form” of RB may well be a heterogeneous population of RB phosphorylated at different combinations of sites. Second, the finding of multiple electrophoretic species of phosphorylated RB also suggests that RB can exist in different phosphorylated forms (Fig. 2). Between four and five “phosphorylated” upper bands can be detected in asynchronous cells, depending on the resolution of the SDSpolyacrylamide gels. The electrophoretic mobility is related to the stoichiometry of phosphorylation (Lin et al., 1991), as well as to the phosphorylation of specific sites (Hamel et al., 1992; E. S. Knudsen, unpublished data). The multiple phosphorylated RB species are found during S and G, phases of the cell cycle (Fig. 2). Only in metaphase cells is RB converted to one single phosphorylated species (Fig. 2). Thus, there may be several differently phosphorylated forms of RB in S and G, cells. Third, RB contains at least two if not more independently functional protein-binding domains (Fig. 1). Each of the independent protein-binding pockets may be regulated by a subset of phosphorylation sites, thus accounting for the presence of multiple sites of phosphorylation. Some evidence suggests a phosphorylation-site-specificregulation of RB function. As mentioned earlier (Section VI,A), T antigen, c-Abl, and E2F seem to select different forms of RB found in extracts of asynchronous cells. Both E2F and E2F-1 selects a single band corresponding to the unphosphorylated RB, T antigen selects the two lower bands, and c-Abl selects the three lower bands. All three proteins preferentially bind the unphosphorylated RB, but exhibit differential affinity for the
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different phosphorylated forms of RB. By constructing phosphorylation-site mutants, Hamel et al. (1992) showed that mutations at Ser 800 and 804 of murine RB, which are equivalent to Ser 807 and 8 1 1 of human RB, do not abolish the regulation of T-antigen binding by phosphorylation. This result indicates that the A/B pocket interaction with T antigen is not regulated by phosphorylation at those two serine sites. However, Ser 807 and 81 1 appear to be critical for the regulation of c-Abl binding. A mutant RB lacking these two phosphorylation sites bind c-Abl well even when it is phosphorylated to high stoichiometry on other sites. T h e wild-type RB phosphorylated under identical conditions, however, loses its ability to bind c-Abl (E. S. Knudsen, and J. Y. J. W a g , unpublished results). This result indicates that the C pocket interaction with c-Abl is regulated by phosphorylation at Ser 807 and/or 81 1. Collectively, these observations d o suggest that the A/B and C pockets of RB may be regulated by different phosphorylation sites. T h e phosphorylation-site-specificregulation of RB function, if real, would change the way we view the role of RB during cell cycle progression. As depicted in Fig. 2, the different forms of phosphorylated RB found in S and C;, cells could conceivably assemble protein complexes with one o r more of the binding partners “relieved” through the phosphorylation of specific sites in RB. In this model, phosphorylation of RB does not necessarily induce the “release” of the bound proteins. An RB-binding protein undergoes a conformational change in response to RB phosphorylation but may or may not dissociate from the overall RB-assembled complex. If there is also a lateral interaction between the RB-bound proteins, then the complex would remain but the function of the complex could be altered through the phosphorylation of specific sites on RB. For example, phosphorylation of RB at Ser 807 and/or 81 1 may relieve the inhibition of the c-Abl tyrosine kinase, leading to the tyrosine phosphorylation and regulation of proteins bound to the A/B or N pocket (Fig. 2). In this model, the phosphorylation of RB pockets could modulate the RB-assembled protein complexes in several different ways depending on the combination of phosphorylation sites. The model depicted in Fig. 2 also changes the way RB function is viewed in cells that are no longer proliferative. The continued expression of RB in nonproliferative cells may contribute to the suppression o f cell proliferation, but this may not be the only reason to keep RB around. In nonproliferating cells, RB can still assemble protein complexes and the functions of these complexes can still be regulated by RB phosphorylation mediated by RB kinases other than the cdk/cyclin complexes. The phosphorylation of RB may therefore not be restricted to cell cycle progression.
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A strong prediction of the model is that RB can be phosphorylated at specific sites by different kinases. At present, there is no evidence for the existence of site-specific RB kinases. In vitro, each cdklcyclin complex appears to phosphorylate many sites in RB (discussed in Section IV,A). However, the novel kinases that have been found as binding proteins to the N-terminal region of RB seem to phosphorylate preferentially the serine/threonine sites in the N-terminal region (J. Horowitz, personal communication). Perhaps the different cdk/cyclin complexes could also select specific serine/threonine sites in vivo; this specificity may be dictated by conditions that are lost in the in nitro reactions. In support of this notion is the finding that A/B pocket mutations of RB abolish RB phosphorylation in vivo (Kaye et al., 1990; Lin et al., 1991; Templeton et al., 1991; Hinds et al., 1992), but these mutants are phosphorylated in uitro as efficiently as the wild-type RB by the cdk-cyclin complexes (B. T.-Y. Lin, unpublished data). Apparently there is much to be learned about the regulation of RB phosphorylation in intact cells. VII. Future Prospects
T h e data gathered to date strongly indicate that the tumor suppression function of RB is not absolute but may be dependent on the cell context as well as the tissue milieu that surrounds a cell. The cell context dependence may be due to redundant suppressor functions that are present in stem cells that are resistant to the loss of RB. An alternative possibility, based on the notion that RB is a molecular matchmaker, could be that RB assembles different types of complexes in different stem cells. In the RB-sensitive stem cells, such as the embryonal retinoblasts in humans, some critical regulatory events are placed under the charge of RB through the expression of regulatory proteins that bind to RB. I n the RB-resistant stem cells, such as the hematopoietic stem cells, those regulatory events do not involve RB because a different set of regulatory proteins is used. This hypothesis would predict that not all the RB-binding proteins are important to the tumor suppression function of RB. T h e influence of the extracellular milieu on RB function deserves further attention. T h e environment of a cell does influence the cellular response to RB, as indicated by several observations. RB-reconstituted retinoblastoma cells do not form tumors in the muscle flanks of nude mice but d o grow in the eyes (Zacksenhaus et al., 1993a). Addition of Matrigel to RB-reconstituted lung carcinoma cells appears to counter the suppression activity of RB (Kratzke et al., 1993). Thus, some illdefined extracellular factors could overcome the growth-suppression
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function of RB. On the other hand, extracellular environment also seems able to compensate for the loss of RB. Rb-l- cells created in mice can be rescued by Rh+l+ cells. Although the Rb-l- embryo dies zn utero, a chimera formed between Rb-l- and Rb+l+ embryonal stem cells develops normally. Examination of the mature animals for isozyme markers demonstrates the presence of Rb-l- cells in every tissue, interspersed with the Rb+/+ cells (T.Jacks and A. Berns, personal comniunication). T h e formation of Rb-l- T and B cells in RAG2-deficient blastocysts is another indication of this type of rescue (Chen et al., 1993). These observations suggest that RB function can be replaced if an RBnegative cell is in contact with RB-positive cells o r matrices elaborated by the RB-positive cells. In this respect, note that RB interacts with the c-Abl tyrosine kinase, which can bind to actin filaments in the cytoplasm (McWhirter arid Wang, 1993). The actin cytoskeleton is an important sensor of cell-cell and cell-matrix contact information. Perhaps the RB-c-Abl complex is involved in the integration of cell contact signaling with the regulation of gene expression. T h e role of KB in transcriptional regulation is supported by 5ome evidence. However, RB does not appear to play an essential role in cell cycle progression, as clearly demonstrated by the Rb-l- mutant mice that undergo numerous rounds of cell proliferation to form largely normal embryos in the complete absence of RB. Although RB has been implicated in the regulation of cell-cycie-dependent genes such as DHFR and cdc2 (h’evins, 1992), there is no evidence that these genes become constitutively expressed in RB-negative cells. This result is perhaps not surprising since every known mammalian promoter is regulated by a large number of transcription factors. The loss of RB may not be sufficient to disrupt the expression of genes that promote cell cycle progresSiWl.
RB may be essential for the expression of genes that encode inhibitors of cell cycle progression. This may explain the ectopic mitosis observed in the Rb-l- embryos. In other words, RB may have a “positive” function in activating the expression of growth inhibitory genes. ’That RB could activate transcription has been demonstrated with ATF-2 and Spl (Kim et al., 1992a,b). There is also evidence that RB may activate the expression of cyclin D1 (Muller et al., 1994). T h e RB-mediated activation of growth-inhibitory genes may be triggered by specific extracellular signals, which would account for the f d C f that the loss of RB does not disturb the cell cycle in general. Identification of genes that are “positively” regulated by RB might be a fruitful approach to the study of RB function. A widely accepted view has been that the expression of viral oncopro-
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teins in a ceil is equivalent to the loss of RB function, either by mutations of the Rb-1 gene or through the phosphorylation of RB. This view may not be entirely correct, based on some of the recent findings. First, viral oncoproteins such as E1A bind not only to RB but also to other A/B pocket-containing proteins such as p107 and p130. The expression of E l A in theory could lead to the release of all A/B pocket-binding proteins. However, the loss of RB would only release a subset of these proteins. For example, the p107-E2F complex is readily detected in RBnegative cells but not in cells expressing ElA (Nevins, 1992). Second, viral oncoproteins do not disrupt all the RB-assembled protein complexes. For example, the C pocket binding of c-Abl tyrosine kinase is not affected by T antigen o r E7 (Welch and Wang, 1993). In fact, viral oncoproteins could conceivably join some of the RB-assembled protein complexes, as indicated by the finding of a T antigen-RB-c-Abl complex in COS cells (Welch and Wang, 1993). As a participant, T antigen or E l A may alter the function of certain RB-assembled complexes to disrupt regulation. Hypothetically, by replacing a cellular A/B pocketbinding protein, a viral oncoprotein could change the nature of an RBassembled complex. If a complex containing E1A can no longer activate an important growth-suppressor gene, this effect would contribute to the E1A-induced entry into S phase. Third, the binding of viral oncoproteins to RB may not be equivalent to the phosphorylation of RB as previously thought. As discussed in Section VI,C, RB may exist in one of several phosphorylated forms that are all functionally distinct. If so, targeting of the A/B pocket by the viral oncoproteins would only be equivalent to the phosphorylation of a subset of the sites in RB. The selective disruption of the A/B pockets by viral oncoproteins may be important for the overall strategy of viral replication. The phosphorylation of RB, on the other hand, may be a mechanism for fine-tuning the activity of RB assembled complexes. The loss of RB, above all, would have consequences that are not mimicked either by the expression of the viral oncoproteins or by phosphorylation. Knowing precisely the differences between the biological effects of viral oncoproteins, RB phosphorylation, and RB mutations will be required to fully understand the function of RB. T h e complexity of RB function is illustrated by the large number of proteins interacting with RB through different mechanisms, by the multiple phosphorylation sites in RB, and by the tissue- and cell type-specific response to the loss o r gain of RB. The hypothesis that RB is a matchmaker, containing several pockets that are differentially regulated by specific phosphorylation events is but one way of accommodating the complexity that has emerged from recent findings. This modified view
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of the molecular function of RB, we hope will provoke new directions of‘ investigation that may eventually reduce this currently complex picture to a cogent description of the biological function of RB.
ACKNOWLEDGMENTS We wish to thank Dr. Edward Harlow, Dr. Tyler Jacks, Dr. Anton Berns. Dr. Jonathan Horowitz, Dr. Hans Hubert, Dr. David Heimbrook, Dr. Astar Winoto, and Dr. Deborah Spector for sharing the preprints of their papers or unpublished results. We thank Ms. Lyn Alkan for assistance in the preparation of this manuscript. T h e authors are supported by NCI grants to J. Y. J. \V.
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Shinkai, Y., Kathbun, G.,Lam, K.-P., Oltz, E. M., Stewart, V., Mendelsohn, M., Charron,J., Datta, M., Young, F., Stall, A. M., and Alt, F. (1992). Cell 68, 8.55-867. Shuai, K., Schindler, C., Presiozo, V. R., and Darnell, J . E., Jr. (1992). Science 258, 18081812. Shuai, K., Stark, G. R., Kerr, I. M., and Darnell, J. E., Jr. (1993). Scieirce 2G1, 1744-1746. Stanbridge, E. J. (1990). Arinu. Rev. Cenct 24, 615-657. Stein, G. H., Beeson, M., and Gordon, L. (1990). Scirnce 249, 666-669. Szekely, L., Jin, P., Jiang, W.-Q., Rosen, A,, Wiman, K. G., Klein, G., and Ringertz, N. (1 993a). J . Cell. Physiol. 155, 3 15-322. Szekely, L., Selivanova, G., Magnusson, K. P., Klein, G., and Wiman, K. G. (1993b). Proc. Natl. Acad. Sci. USA 90, .5455-5459. Tachibana, K., Scheuer, P. J., Tsukitani, Y., Kikuchi, H., Van Engen, D., Clardy, J . , Gopichand, Y., and Schniitz, F. J. (1981).J. Am. Clirm. Soc. 103, 2469-2471. Takahashi, R., Hashimoto, T., Xu, H.-J., Hu, S.-X., Matsui, T., Miki, T., Bigo-Marshall, H., Aaronson, S . A,, and Benedict, W. F. (1991). Pmc. Natl. Acnd. Scz. USA 88, 5257-5261. Takuwa, N., Zhou, W., Kuniada, M., and Takuwa, Y. (1993).J. B i d . Cherti. 268, 138-145. Templeton, D. J. (1992). Mol. Cell. Biol. 12, 435-443. Templeton, D. J., Park, S . H., Lanier, L., and Weinberg, R. A . (1991). Proc. Natl. Acad. Sci. USA 88, 3033-3037. Terada, N., Lucas, J. .J., and Gelfand, E. W. (1991).J. Immune/. 147, 698-704. Thomas, N. S . B., Burke, L. C., Bybee, A , , and Linch, D. C. (1991). O r i c o p i e 6, 317-322. Thompson, (1. B., Wang, C.-Y., Ho, I.-(;., Bohjanen, P. R., Petryniak, B., .June, C;. H., Miesfeldt, S . , Zhang, L., Nabel, G.J., Karpinski, B., and Leiden, J. M. (1992).Mol. Cell. Biol. 12, 1043-1053. Tsai, L.-H., Harlow, E., and Meyerson, M. (3991). Nature (London) 353, 174-177. Tsai, L.-H., Lees, E., Faha, B., Harlow, E., and Riabowol, K. (1993). Oitcogeiie 8, 15931602. Udvadia, A. J., Rogers, K. T., and Horowitz, J. M. (1992). Cell Growth Diff: 3, 597-608. Udvadia, A. J.. Rogers, K. T., Higgins, P. D. K., Murata, Y., Martin, K. H., Huniphrey, P. A,, and Horowitz, J. M. (1993). Proc. No//. Actid. Sci. USA 90, 3265-5269. Vale, W., Rivier, J., Vaughan, J., McClintock, R., <:orrigan, A., Woo, W., Karr, D., and Spiess, J. (1986). Nature (Loridon) 321, 776-778. Velazquez, L., Fellous, M., Stark, (;. R., and Pellegrini, S. (1992). Cell 70, 313-322. Villeval,J. L., Pelicci, P. G., Tahilio, A,, Titeux, M., Henri, A , , Houesche, F., Thomopoulos, P., Vainchenker, W., Garbaz, M., Rochant, H., Breton-Gorius, J., Edwards, P. A . W., and Testa, U. (1983). Exp. Cell. Re.ys.146, 428-435. Vousden, K. ( I 993). FASEB J , 7, 872-879. Wang, C-Y., Petryniak. B., Thompson, C. B., Kaelin, W. G.,and Leiden, J. M. (1993). Science 260, 1330- 1335. Wang, J. Y. J. ( 1 993). Ctirr. Oflin. G m e t . Drv. 3, 3.5-43. Wang, N . P.,(;hen, P.-L., Huang, S., Donoso, L. A,, Lee, W.-H., and Lee, E. Y.-H. P. (1990). Cell Grouifh Diff: 1, 233-2 Wasylyk, B., Hahn, S . L., arid Giovane, A. (1993). Eur.1. Hioclieiti. 211, 7-18. Weinberg, R. A. (1990). Trendy Biorhem, Sci. 15, 399-202. Weintraub, s. J., Prater, <;. A,, and Dean, D. C.(1992). Naturr (l.ondon) 358, 259-261. Welch, P. J., and Wang, J. Y. J. (1993). Cell 75, 779-790. Whyte, P., and Eisenman, K. N. (1992). Biorhem. Cell B i d . 70, 1380-1384. Whyte, P., Buchkovich, K . J . , Horowitz,J. M., Friend, S . H., Raybuck, M., Weinberg, K. A., and Harlow, E. ( 1988). Nature (London) 334, 124- 129.
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SH2 AND SH3 DOMAINS IN SIGNAL TRANSDUCTION Tony Pawson Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1x5
1. Protein Tyrosine Kinases and Their Targets
11.
111.
IV.
V.
A. Tyrosine Kinases and Intracellular Signaling Proteins B. Oncogenic Activation of Tyrosine Kinases SH2 Domains A. SH2 Domain Structure and Binding Properties B. Signaling Proteins with SH2 Domains C. Insulin Signaling-IRS-1 D. SHP-Mediated Signaling in T Cells E. Cytokine Signaling-STAT Proteins Regulate Gene Expression F. SH2 Domains Have Multiple Functions SH3 and PH Domains A. SH3 Domain Structure and Binding Properties B. PH Domains-Possible Functions Coupling Tyrosine Kinases to Ras A. T h e Ras Regulatory Cycle B. SH2/SH3 Adaptor Proteins in Ras Activation C. T h e Bcr-Abl Oncoprotein and Ras Activation D. Multiple Grb2 Complexes in Growth Factor-Stimulated Cells SH2-Containing Phosphotyrosine Phosphatases and the Genetics of Signal Transduction A. Phosphotyrosine Phosphatases with SH2 Domains B. T h e motheaten Mouse Mutant References
1. Protein Tyrosine Kinases and Their Targets A. TYROSINE KINASESAND INTRACELLULAR SIGNALING PROTEINS
Many of the polypeptides that control the survival, proliferation, differentiation, and metabolism of embryonic and adult cells bind to cellsurface receptors with protein tyrosine kinase activity (Pawson and Bernstein, 1990; Ullrich and Schlessinger, 1990; Pawson and Schlessinger, 1993). Receptor tyrosine kinases contain an extracellular ligand-binding region, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. Binding of a polypeptide ligand induces receptor clustering, and stimulates the activity of the intracellular tyrosine kinase domain. 87 ADVANCES IN CANCER RESEARCH, VOL. 6 4
Copyright 0 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Activated receptors autophosphorylate by an intermolecular mechanism, primarily at tyrosine residues located within noncatalytic regions juxtaposed to the kinase domain (Fig. 1 ; Pawson and Schlessinger, 1993). Autophosphorylation, in turn, serves as a molecular switch to induce binding of the receptor to cytoplasmic signaling proteins that regulate biochemical pathways controlling gene expression, DNA synthesis, cytoskeletal architecture, protein sorting, and cellular metabolism (Cantley ~t al., 1991; Koch et nf., 1991). These receptor-binding proteins are characterized by one or two copies of the Src homology 2 (SH2) domain (Fig. 2; Sadowski et al., 1986),a protein module that recognizes specilic phosphotyrosine-containing sites on activated receptor tyrosine kinases and cytoplasmic polypeptides ( Pawson and Cish, 1992). Proteins with SH'L domains frequently possess a distinct module, the Src homology 3 (SH3) domain (hlayer ef af., 1988), that also controls protein-protein interactions through recognition of proline-rich motifs (Cicchetti rt al., 1992; Ken ut al., 1993). These results have suggested that intracellular signaling proteins are constructed in a modular fashion of domains that regulate protein-protein interactions. 'I'he activation of intracellular biochemical pathways by receptor tyrosine kinases appears, i n large part, to be regulated by the formation of multiprotein complexes mediated by SH'L and SH3 domains. In addition t o transniembrane receptor tyrosine kinases, mamm a1'ian cells express a large and diverse family of cytoplasmic tyrosine kinases
fi PDGF-R
n
740 751 771
YMDM YV P M YM A P
1021
Y I I P
F I ~ . 1.. SHY-birding sites o n the PDGF-receptor. 'The PDGFK kiriase domain is shdcleti. Receptor aurophosphor) lation sites a r r indicated (P-TYK). Specific SHY-binding sites, ronipsed of the phosphot).rosine aiid the residues at lhe + 1 t o + 3 positions (i.e.. 1,111') art' shown t o the r-ight, and cogiiate SI-lL'-ront;iiniri~ proteins to the left.
S H 2 AND S H 3 IN SIGNAL TRANSDUCTION
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s H 2 m 3 lvoN-cAmm(ADApT0R)rn7EIM
p85 P13K
8
GrbWdr WSem-5 c-Crk Nck
Shc Shb
Grb7 ISGFW (STAT) -SH
Tendn
4
1
ISH3'
FIG.2. Proteins with SH2, SH3, and PH domains. These proteins are divided into those with SH2/SH3 domains and catalytic domains within the same polypeptide chain, and noncatalytic proteins that may function as molecular adaptors. Gly/Pro, Glycinei proline-rich region; Pro, proline-rich region; DBL, region of' homology with the Dbl protein; BCR, region of homology with the BCR protein. The PH domain of PLC-y is split.
that do not cross the membrane, and therefore do not directly bind extracellular signaling molecules. As discussed in this chapter, such cytoplasmic tyrosine kinases apparently play important roles in signaling from multisubunit receptors, including antigen receptors and cytokine receptors. B. ONCOCENIC ACTIVATION OF TYROSINE KINASES
Structural mutations in tyrosine kinase genes can result in the constitutive enzymatic activation of their products, which are thereby en-
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TONY PAWSON
dowed with oncogenic potential. A number of retroviral and cellular oncogenes encode such activated tyrosine kinases. Notably, rearrangement of the BCR and ABL genes in human chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL) results in the production of a hybrid Bcr-Abl protein, thereby activating the intracellular Abl tyrosine kinase (Konopka et al., 1984; Clark et al., 1987). The BCR-ABL oncogene appears central to the development of' these leukemias. Rearrangement of the RET and TRK genes, and consequent activation of their encoded receptor tyrosine kinases, is a frequent event in papillary thyroid carcinomas (Grieco et al., 1990; Pierotti et al., 1992). Furthermore, germ-line mutations within the RET gene are apparently responsible for a dominantly inherited cancer syndronie-multiple endocrine neoplasia type 2A (Mulligan et al., 1993). Amplification and overexpression of the genes encoding Neu and the epidermal growth factor receptor (EGFR) are commonly observed in breast and ovarian cancer (Slamon el ul., 1987,1989); similarly, the EGFR and the platelet-derived growth factor receptor (PDGFR) are overexpressed o r rearranged in glioblastomas (Libermann et al., 1985). These results indicate that tyrosine kinases are important not only in normal growth and development, but also in carcinogenesis. Oncogenic tyrosine kinase variants apparently stimulate DNA synthesis through the Ras pathway (hlulcahy et al., 1985; Smith et al., 1986), which is regulated by proteins containing SH2 and SH3 domains (Shc and Seni-.5/drk/Grb2) (Pawson and Schlessinger, 1993). Hence SH2/ SHS-containing proteins control cellular responses to many normal stimuli, and to transforming variants of tyrosine kinases. Indeed SH2containing proteins such as Shc, Crk, and Nck are themselves transforming when overexpressed, consistent with the notion that these polypeptides might normally be involved in mitogenesis (Mayer et al., 1988; Chou et ul., 1992; Li et ul., 1992; Matsuda P t ul., 1992; Pelicci et al., 1992). II. SH2 Domains
A. SH2 DOMAIN STRVCTURE A N D BINDING PROPER rIES SH2 domains were originally defined as protein modules of approximately 100 amino acids (Sadowski et al., 1986) that bind phosphotyrosinecontaining proteins during signal transduction (Anderson et al., 1990; Matsuda et nl., 1990; Moran et al., 1990; Mayer et al., 1991). In particular, SH2 domains expressed on their own i n bacteria retain the ability to bind with high affinity to autophosphorylated receptor tyrosine kinases
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(Anderson et al., 1990; Margolis et al., 1990; Moran et al., 1990; McGlade et al., 1992a; Kazlauskas et al., 1993). High-affinity binding of an SH2 domain to a phosphotyrosine-containing peptide requires specific amino acids in the three positions immediately C-terminal to phosphotyrosine (the + 1 to + 3 positions) (Figs. 1, 3; Fantl et al., 1992; Songyang et al., 1993). To date, every SH2 domain examined has shown a distinct selectivity for the amino acids C-terminal to phosphotyrosine (Songyang et al., 1993). Hence tyrosine phosphorylation regulates SH2 binding, whereas the nature of the residues C-terminal to the phosphotyrosine determines which SH2 domain will bind preferentially to a specific site. In some cases, specificity may still be determined by residues in the SH2binding site in addition to those found at the + 1 to + 3 sites. In the binding site on the PPDGFR for the Nck SH2 domain, residues N-terminal to the phosphotyrosine appear to be important (Nishimura et al., 1993). SH2 domains can bind to phosphotyrosine-containing peptides with relatively high affinities (Kd = 1-10 nM),provided that the residues surrounding the phosphotyrosine are optimal. Using real-time measurement of kinetic parameters, the association and dissociation rate constants for SH2 domains binding to phosphopeptides have been shown to be relatively high (Felder et al., 1993; Panayotou et al., 1993; Payne et al., 1993). This characteristic may reflect a physiological requirement for rapid turnover of SH2-mediated complexes.
SH2
PEPTIDE LIBRARY
PHYSIOLOGICAL SITES
PI3K p85a-C
P.Y-(M/L/I)-X-M
P.Y-M-D-M P.Y-V-P-M
TYR-740 pPDQ?R TYR-721 CSF-lR
PLCyl-c
P.Y- (V/I)- (I/L)-(P/I) P.Y-I-I-P P.Y-L-I-P
TYR-1021 pPEGFR TYR-992 EGFR
GFtB2
P-Y-X-N-X
P.Y-V-N-V P.Y-V-N-V P.Y-I-N-Q
TYR-317 SHC TYR-177 BCR TYR-1068 EGFR
SYP-N
P.Y- (I/V)-X- (V/I)
P-Y-T-A-V
TYR-1009
src
P.Y-E-E-I
P.Y-A-E-I P.Y-I-Y-V
TYR-372 FAK TYR-759
FIG. 3. Examples of SH2 domain specificity. SH2 domain-binding sites have been identified either by in nitro selection of peptides from a degenerate phosphopeptide library (PEPTIDE LIBRARY) or by identification of in vivo sites (PHYSIOLOGICAL SITES). A good, although not perfect, correlation exists between the optimal sites identified in vitro and those used in viva X, No selection; P.Y, phosphotyrosine.
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TONY PAWSON
Structural analysis of several SH2 domains by X-ray crystallography and nuclear magnetic resonance ( N M R ) spectroscopy has indicated how ligand binding is achieved, and the potential basis for binding specificity. The SH2 domains of the Src, Lck, and Abl non-receptor tyrosine kinases, and of phosphatidylinositol 3'-kinase ( P B K ) , have a conserved structure composed of a central p sheet flanked by two (Y helices. Highresolution structures of the Src and Lck SH2 domains bound to a highaffinity phosphopeptide containing the sequence pTyr-Glu-Glu-Ile (pYEEI) have identified a conserved binding pocket for phosphotvrosine that is lined by basic residues (Eck et al., 1993; Waksman et a,?., 1993). Of these, the most critical is an invariant arginine that forms an ion pair with the phosphate group (Waksman ct al., 1992). Various biochemical and genetic studies have shown that the conserved SH2 residues involved in phosphotyrosine binding are required for association of'SH2 domains with activated growth factor receptors, and for signal tr-ansduction in zliz~o (Marengere and Pawson, 1992; Mayer et al., 1992; Stern et ul., 1993). Although the conserved residues of SH2 domains (Koch et al., 1991) are involved in forming both the hydrophobic core of the module and the phosphotyrosine-binding site, the more variable residues participate in binding the three residues immediately C-terminal to the phosphotvrosine. In particular, the residue at the + 3 position of the bound peptide fits into a hydrophobic pocket in the SH2 domain. Variations in the side chains of the SH2 residues that line this pocket likely determine which amino acid is selected at the + 3 position ofthe ligand. In contrast, the amino acids at the + 1 and + 2 sites contact the SH2 surface (Eck et al., 1993; Waksman et ul., 1993).
B . SIGNALING PROTEINSWITII SH2 DOMAIXS SH2-containing proteins are involved in the control of phospholipid metabolism [phospholipase C (PLC)-y: PI3K], protein phosphorylation and dephosphorylation [Src-like tyrosine kinases; ZAP-7OISyk tyrosine kinases; PTP lC/Syp phosphotyrosine phosphatases (PTP)], Ras-like G'I'Pases [Shc, Grb2, Ras GTPase activating protein (GAP), 012chimerin], cytoskeletal architecture (tensin), and transcription [signal transducer and activator of transcription (STAT) proteins]. These SH'Lcontaining proteins can be roughly divided into t w o classes (Fig. 2). Proteins such as Src, GAP, and PLC-.)Icontain SH2 and catalytic domains within the same polypeptide chain. In contrast, P13K is a heterodimer composed of an 85-kDa subunit ( ~ $ 5 that ) possesses receptor-binding SH2 domains but has no P13K activity (Otsu et al., 1991; Skolnik et al.,
SH2 A N D
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1991; McGlade et al., 1992a) bound to a 110-kDa catalytic subunit (Hiles et al., 1992). A considerable number of SH2-containing proteins are similar to the p85 PI3K subunit in the sense that they have no obvious enzymatic function. We have suggested that, in some cases, these polypeptides serve as adaptors to link receptor tyrosine kinases to catalytic subunits that themselves lack SH2 domains (Koch et al., 1991; Pawson, 1992). As shown in Fig. 2, several of these proteins are composed almost exclusively of SH2 and SH3 domains (i.e., Sem-!i/drk/GrbP, Crk, Nck). In Drosophila and mammalian cells, the Sem-5/drk/Grb2 protein does indeed function as an adaptor to couple tyrosine kinases to Sos, an activator of Ras guanine nucleotide-binding proteins (Pawson and Schlessinger, 1993). Note that there are several SH2-containing proteins whose functions remain obscure, suggesting that the regulation of signal transduction downstream of tyrosine kinases is likely to be more complex than currently thought. Several mechanisms can be envisioned by which cytoplasmic signaling proteins might be activated by binding of their SH2 domains to receptor autophosphorylation sites. Receptor binding may recruit proteins to the plasma membrane, where their substrates are frequently located. Once physically associated with the activated receptor tyrosine kinase, SH2containing signaling proteins can become preferred substrates for phosphorylation on tyrosine (Ellis et al., 1990; Rotin et at., 1992), which in turn can contribute to their activation (Kim et al., 1991). In the case of PLC-yl, for example, it appears that both SH2-mediated binding to activated receptors and subsequent tyrosine phosphorylation of PLC-y 1 are required to stimulate phosphoinositol biphosphate (PIP,) hydrolysis (Kim et al., 1991; Mohammadi et al., 1991; Sultzmann et al., 1991; Vega et al., 1992; Valius et al., 1993). An additional possibility is that the physical association of a protein’s SH2 domains with phosphotyrosine-containing sites may stimulate the protein’s enzymatic activity through an allosteric mechanism. Indeed, the association of PISK with phosphotyrosinecontaining peptides can stimulate its activity 5- to 10-fold in vitro (Backer et at., Carpenter et al., 1992). Consistent with this observation, binding of high-affinity phosphopeptides induces a conformational change in the N-terminal SH2 domain of the p85 PISK subunit, as measured by circular dichroism and NMR spectroscopy (Shoelson et al., 1993). Since the pllO catalytic PISK subunit apparently binds to a region on p85 between the two SH2 domains (Hu et al., 1993; Kiippel et al., 1993), occupancy of the p85 SH2 domains might affect the catalytic activity of the pllO subunit. In a similar vein, a phosphopeptide corresponding to the binding site on the PPDGFR for the SH2 domains of Syp/SH-PTP2 stimulates Syp/SH-PTP2 phosphatase activity in uitro (Lechleider et al.,
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1993). This mode of activation may be especially pertinent for enzymes with two SH2 domains. These and similar findings have led to the (paradoxical) idea that the targets of receptor tyrosine kinases need not necessarily be substrates for tyrosine phosphorylation. T h e notion that SH2-containing signaling proteins bind to specific phosphotyrosine-containing sites has been confirmed by an analysis of autophosphorylation sites on receptors, such as those for macrophage colony stimulating factor (CSF-1) and PDGF (Fantl et al., 1992; Kashishian et al., 1992; Reedijk et ul., 1992; Kazlauskas et al., 1993; Valius and Kazlauskas, 1993). The cytoplasmic portions of the PPDGFR and the CSF- 1R contain three noncatalytic regions-the juxtamembrane domain, the kinase insert (presumably forming a loop on the surface of the kinase domain), and the C-terminal tail (Fig. 1). These areas are not required for enzymatic activity but provide sites for autophosphorylation and hence for SH2 binding. In general, each of these autophosphorylation sites couples to a distinct signaling protein, and thereby controls the activation of a specific biochemical pathway. In the case of the CSF- 1R, phosphorylation of Tyr 72 1 within the kinase insert pro\ides a specific binding site for PI3K; furthermore, the SH2 domains of the p85 subunit expressed in bacteria retain the ability to discriminate between P.Tyr 721 and other autophosphorylation sites (Reedijk et al., 1992). Tyr 721 is followed at the + 3 position by a methionine, which appears to be a crucial component of the PI3K-binding site (Cantley et al., 1991; Songyang et ul., 1993) (Fig. 3). On occasion there may be some overlap in binding sites; for example, the Nck protein is proposed to bind the same PPDGFR autophosphorylation site as PI3K (Nishimura et al., 1993). The PPDGFR binds to Src family kinases through autophosphorylation sites in the juxtamembrane region, and to PI3K and GAP through sites in the kinase insert (Fantl et al., 1992; Kashishian et al., 1992; Mori et al., 1993). Phosphorylation of Tyr 1021 within the PPDGFR tail is required for PLC-y 1 binding and for stimulation of PI-4,5-P2 hydrolysis (Riinnstrand et al., 1992; Larose et al., 1993; Valius et al., 1993). T h e C-terminal PLC-y 1 SH2 domain binds preferentially to phosphotyrosinecontaining sites with proline at the + 3 position (i.e., Pro 1024 in the PPDGFR) (Songyang et al., 1993). Conversion of this + 3 proline to methionine, which is recognized by PI3K SH2 domains, allows binding of PI3K to the Tyr 1021 site of-the PPDGFR (Larose et al., 1993). Phosphorylation of Tyr 1009 within the PPDGFR tail induces specific binding of the Syp/SH-PTP2 SH2-containing phosphotyrosine phosphatase (Kalauskas et al., 1993).
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SIGNALING-IRS-1 C. INSULIN In contrast to other receptor tyrosine kinases, the principal substrate of the insulin receptor is a protein, IRS-1, that has no SH2 domains (Sun et al., 1991). Rather, the phosphorylation of IRS-1 on tyrosine creates specific binding sites for the SH2 domains of signaling proteins such as PISK, Grb2, and Syp/SH-PTP2 (Lavan et al., 1992; Kuhne et al., 1993; Skolnik et al., 1993; Sun et al., 1993). The binding of PI3K to tyrosine phosphorylated IRS-1 in vitro stimulates its enzymatic activity (Backer et al., 1992). IRS-1 therefore serves as a docking site for SH2-containing proteins that are likely to be crucial in the cellular response to insulin. IRS-1 is a large protein with over 20 potential tyrosine phosphorylation sites located in motifs that might form SH2-binding sites. These observations suggest that IRS-1 may be coupled to a large number of SH2regulated biochemical pathways, and thereby may mediate the diverse metabolic and mitogenic effects of insulin on its target cells. Of interest, interleukin (1L)-4 induces the phosphorylation of an IRS-l-like molecule in myeloid cells; expression of IRS-1 in 32D myeloid cells converts this previously nonresponsive cell line to IL-4 sensitivity (Wang et al., 1993). D. SH2-MEDIATED SIGNALING I N T CELLS Binding of antigen to the antigen receptor of T cells activates a signaling pathway leading to expression of the gene for IL-2. The T-cell receptor, in contrast to the relatively simple transmembrane receptor tyrosine kinases, is composed of at least six chains, of which the polymorphic a and p chains recognize antigenic polypeptides bound to a major histocompatability complex molecule. The associated y, 6, and E chains (which together compose CD3) and the 5 chain are responsible for signaling. T h e CD3 and 5 polypeptides contain one to three copies of an 17-amino-acid motif [variously termed the tyrosine-base activation motif (TAM) or antigen activation recognition motif (ARAM)], whose salient feature is the presence of two tyrosines separated by 10 intervening residues, each followed at the + 3 position by a leucine or isoleucine (Reth, 1989). Various genetic and biochemical data have suggested the following scheme to account for the apparent importance of the TAM motifs in T-cell activation (Letourneur and Klausner, 1992; Romeo et al., 1992). Antigen binding to the T-cell receptor stimulates Src-family kinases, notably Lck, which is required for efficient T-cell signaling (Abraham et al., 1991; Strauss and Weiss, 1992; Karnitz et al., 1992).
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Concomitant with Lck activation, the conserved tyrosine residues within the TAM motifs of the CD3 and 5 polypeptides become phosphorylated. Phosphorylated 5, in turn, associates with a distinct cytoplasmic tyrosine kinase, ZAP-70, that possesses two K-terminal SH2 domains linked to a C-terminal tyrosine kinase domain (Chan et al., 1992). This interaction is explained by the finding that tyrosine phosphorylation of the 5 TAM motifs creates binding sites for the ZAP-70 SH2 domains (Weiss, 1993). According to this scheme, the Z.4P-70 tyrosine kinase goes on to activate signaling proteins, such as PLC-y, involved in calcium mobilization and transmission of the signal toward the nucleus (Kolanus et al., 1993). Curiously, the SH2 domain of the Lck tyrosine kinase also appears to participate in T-cell activation (Xu and Littman, 1993). A similar series of events is likely to be important in the response of B cells and mast cells to antigen. The Iga and Igp chains associated with surface immunoglobulin in B cells have TAM motifs, as d o the P and y chains of the high affinity IgE receptor. Mast cells and B cells express a tyrosine kinase with two SH2 domains, Syk, which may perform a role analogous t o that of ZAP-70.
E. CYTOKINE SIGNALINC.-SI’A’I’ PROTEINS REGULATE.GLNEEXPRESSION T h e receptors for interferons (IFN) a l p and y and for a growing number of cytokines (such as erythropoietin, IL-3, GM-CSF), although they are not themselves protein kinases, bind and activate members of the JAK family of cytoplasmic tyrosine kinases (Velazquez et al., 1992; Argetsinger et al., 1993; Muller et al., 1993; Wittuhn et al., 1993). These events lead to the phosphorylation of cytoplasmic proteins (STATs), each of which contains an SH2 and an SH3 domain; tyrosine phosphorylated STAT proteins migrate to the nucleus and bind directly to specific promoter sequences, thereby regulating expression of their target genes (Fu, 1992; Fu eta!., 1992; Fu and Zhang, 1993; Larner et al., 1993; Ruff-Jamison et nl., 1993a; Sadowski et al., 1993; Shuai et al., 1993; Silvennoinen et al., 1993). A preliminary model suggests that these transcription factors may form homodimers through an intermolecular interaction in which the SH2 domain of one molecule binds a tyrosine phosphorylation site on its partner (Sadowski et al., 1993). STAT proteins are also phosphorylated in response to growth factors such as EGF. Interestingly, a promoter element adjacent to the serum response element (SRE) of c-fos (the Sis-inducible element, SIE) binds at least one tyrosine phosphorylated SH2-containing STAT transcription factor following stimulation with EGF (Fu and Zhang, 1993; Sadowski et al.,
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1993). These results suggest that tyrosine kinases can directly regulate gene expression, independently of other signaling pathways, by inducing the phosphorylation of STAT proteins. F. SH2 DOMAINS HAVEMULTIPLEFUNCTIONS The SH2 domains of proteins such as the c-Src cytoplasmic tyrosine kinase and its relatives are involved not only in binding to activated growth factor receptors, but also in a series of intramolecular and intermolecular interactions that regulate c-Src activity and function (Fig. 4). c-Src enzymatic activity is inhibited by phosphorylation of a tyrosine residue (Tyr 527) within the c-Src C-terminal tail by the inhibitory kinase Csk (Cooper et al., 1986). This repression of kinase activity requires an intact SH2 domain, and apparently involves a direct intramolecular interaction between the SH2 domain and the phosphorylated Tyr 527 site (Roussel et al., 1991; Liu et al., 1993a; Superti-Furga et al., 1993). Evidence suggests that not only the SH2 domain, but also the adjacent SH3 domain, is required for inhibition of c-Src kinase activity, possibly through recognition of proline residues in the catalytic core (SupertiFurga et al., 1993). Similar results regarding the importance of the SH2 domain in repressing kinase activity have been obtained for Lck (Veillette et al., 1993). c-Src tyrosine kinase activity can be induced in vitro by incubation with a pYEEI phosphopeptide that binds to the c-Src SH2 domain with high affinity and presumably displaces the SH2 domain from the phosphorylated tail (Liu et al., 1993a). Once Tyr 527 is dephosphorylated in vivo, the kinase domain is activated and the SH2 and
p130 FAK PDGFR
PI 3’-KINASE
PI10
FIG.4. Multiple c-Src SH21SH3 interactions. In this model, c-Src activity is repressed by binding of the SH2 domain to phosphorylated Tyr 527 in the c-Src C-terminal tail, and of the SH3 domain to proline residues in the kinase core. On dephosphorylation of Tyr 527, the SH2 and SH3 domains are free to interact in trans with signaling proteins, as indicated, thereby regulating their activity. FAK, Focal adhesion kinase. p130 and pl10 are SH2- and SH3-binding proteins, respectively.
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SH3 domains are freed to interact in trans with target proteins (Reynolds et al., 1989; Koch et al., 1992; Flynn et al., 1993; Liu et al., 199313).
i l l . SH3 and PH Domains A. SH3 DOMAINSTRUCTURE A N D BINDING
PROPERTIES Many proteins with SH2 domains also possess a distinct sequence of -50 amino acids termed the SH3 domain, which is commonly located
adjacent to the SH2 domain. SH3 domains are protein modules with a conserved three-dimensional structure, as revealed by X-ray crystallography and N M R analysis, that bind to proline-rich motifs found in a variety of intracellular proteins involved in signal transduction (Ren et al., 1993). SH3 domains are present in a wide range of proteins from yeast to humans (Musacchio et al., 1992a), involved in processes as diYerse as cytoskeletal control and bud site selection in yeast, generation of the respiratory burst in human neutrophils, and control of the Ras pathway. Hence, understanding SH3 biological function and binding properties is of considerable interest. ‘The crystal structures of the SH3 domains from the cytoskeletal protein spectrin and the Src-like tyrosine kinase Fyn have been solved, as have the solution structures of the c-Src, PLC-y1, and p85 PI3K subunit SH3 domains (Musacchio et al., 199213; Booker et al., 1993; Kohda et al., 1993; Koyana et al., 1993; Noble et at., 1993; Yu et al., 1992). Despite the fact that the SH3 domains of these proteins have relatively limited sequence identity, their overall structures are very similar. As with SH2 domains, their I S and C termini are in close proximity to one another, indicating that SH3 domains are independent protein modules that can be inserted at different locations within a protein. The SH3 structures contain between five and eight p strands that form a barrel-like structure. SH3 domains contain relatively well-conserved aliphatic and aromatic residues that form a hydrophobic surface to which proline-rich ligands might bind. Conserved carboxlic amino acids are located in loops adjacent to this hydrophobic surface. NMR analysis of the binding of short proline-rich peptides to the c-Src and p85 PI3K SH3 domains is consistent with the notion that the conserved hydrophobic region functions as a binding site for cellular ligands. Sequence variations in the hydrophobic surface and surrounding loops of different SH3 domains may allow for the specificity observed in the binding of SH3 domains to their ligands (Cicchetti et af., 1992).
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Little is known about the physiological ligands for SH3 domains. The v-Src SH3 domain binds to proline-rich sequences within the p85 subunit of PI3K (Liu et al., 1993b), as do the SH3 domains of other Srcfamily kinases such as Fyn and Lck (Prasad et al., 1993; Vogel and Fujita, 1993). As discussed subsequently, the drk/Grb2 SH3 domains bind proline-rich motifs in the tail of Sos. A number of SH3 domains bind in vitro to dynamin, a protein implicated in microtube regulation of vesicle trafficking, and stimulate dynamin GTPase activity (Booker et al., 1993; Gout et al., 1993). T h e N-terminal Crk SH3 domain binds proline-rich sequences in the tail of the Abl tyrosine kinase (D. Baltimore, personal communication). In addition to mediating the formation of cytoplasmic signaling complexes, SH3 domains may be involved in targeting proteins to the microfilament network and to membrane ruffles (Bar Sagi et al., 1993). An especially tantalizing issue involves the frequent juxtaposition of SH2 and SH3 domains, and the possibility that these domains might act synergistically. Indeed, one suggestion is that structures related to SH2 and SH3 domains can be found adjacent to one another in the BirA protein of Escherichia coli (Russel and Barton, 1993), raising the possibility that this is a rather ancient relationship. B. PH DOMAINS-POSSIBLE FUNCTIONS PH domains are sequences of -90 amino acids originally found in the protein kinase C (PKC) substrate pleckstrin and now identified in a variety of signaling proteins, including some that have SH2 and SH3 domains (Haslam et al., 1993; Lefkowitz, 1993; Mayer et al., 1993; Musacchio et al., 1993). By analogy with SH2 and SH3 domains, PH domains are anticipated to be protein modules that regulate specific protein-protein interactions in signal transduction. A consensus PH domain has been noted in the P-adrenergic receptor kinase, encompassing a region previously shown to bind the Ply subunit of the heterotrimeric G, guanine nucleotide-binding protein that is activated by the serpentine P-adrenergic receptor (Lefkowitz, 1993). Hence, one possible role of PH domains is to interact with Ply subunits of large G proteins. Proteins with SH2/SH3 and PH domains could therefore provide a link between signaling pathways regulated by large G protein-coupled receptors and receptor tyrosine kinases. Of particular interest is that a mutation within the PH domain of the Btk tyrosine kinase appears to be responsible for the immunodeficiency observed in XID mice (Musacchio et al., 1993), suggesting that these elements do have a physiological role.
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IV. Coupling Tyrosine Kinases to Ras
A. THERAS REGULATORY CYCLE A specific example of the coordinate use of SH2 and SH3 domains to couple tyrosine kinases to a downstream signaling pathway involves the activation of the Ras guanine nucleotide-binding protein. Ras proteins are inactive when bound to GDP, and are activated by exchange of GDP for GTP in response to a guanine nucleotide-releasing protein (GNRP) (Bourne et al., 3991). Once in the GTP-bound state, Ras interacts with a GAP that greatly accelerates the hydrolysis of bound C'TP to GDP (Fig. 5). C'I'P-bound Ras is also proposed to activate a cascade of protein kinases that may ultimately phosphorylate and regulate transcription factors, and thereby control gene expression.
H. SH'LISH3 ADAPTORPROTEIM IN
RAS A C ~ I V A T I O N
Genetic data from Caeno?hubditiA eLeguns and Drosophzla have implicated a protein with a single SH2 domain and two flanking SH3 domains (Seni-5 in C. clcgaiu; drk in Drosophzla) in coupling receptor tyrosine kinases to the Ras pathwa) (Clark ef al., 1992; Olivier et al., 1993; Simon
QAP Raf FIG. 5. Regulation of Ras activity. Receptor tyrosine kinases couple through GrtWdrk/ Sem-5 t o the Ras-(;NRP Sos, which converts Ras from the inactive GDP-bound state to the active GI'P-bouiid state. Once activated. Ras associates with a G'f'Pase-activating protein (GAP), which stimulates Ras GTPase activity arid converts Ras to the inactive GDP-bound state. GTP-bound Ras also interacts with the Raf serine/threonine protein kinase, and may thereby activate the MAP kinase patliuay.
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et al., 1993; Stern et al., 1993). Ras activation is apparently directly cata-
lyzed by Sos, a protein with a central Ras-GNRP domain related to the Cdc25 protein of Saccharomyces cerevisiae, a C-terminal proline-rich tail, and an N-terminal region containing a PH domain (Fig. 6 ; Simon et al., 1991; Bonfini et al., 1992; Bowtell et al., 1992). Based on a combination of genetic and biochemical data, we have proposed that Drosophila receptor tyrosine kinases such as Sevenless and DER (the Drosophila EGFR homolog) bind to the drk SH2 domain, whereas the drk SH3 domains physically associate with proline-rich motifs in the C-terminal tail of Sos, which in turn stimulates the exchange of GDP for GTP on Ras. In particular, we have shown that substitutions in the drk SH2 domain that impair phosphotyrosine and receptor binding block signaling downstream from the Drosophila Sevenless and DER receptor tyrosine kinases, and that the SH3 domains of drk bind proline motifs in the Sos tail (Olivier et al., 1993). Similar interactions can be detected in mammalian cells, involving the mammalian homologs of Sem-5/drk (Grb2) and Sos (mSos1, mSos2) (Buday and Downward, 1993; Chardin et al., 1993; Egan et al., 1993; Gale et al., 1993; Li et al., 1993; Rozakis-Adcock et al., 1993). A constitutive complex between Grb2 and mSos1 can be detected in unstimulated fibroblasts. On stimulation with a growth factor such as EGF, the Grb2-
FIG.6. Sos structure and function. (A) The SH3 domains of drk/Grb2 bind to prolinerich sequences in the tail of Sos, thereby stimulating the activity of the Cdc25-like RasGNRP domain. The N terminus of Sos has PH- and Dbl-like domains. (B) Proline-rich motifs in the tail of mSos1.
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mSos1 complex associates with the activated receptor (Fig. 7). T h e mechanism by which the formation of these complexes activates mSos1 remains uncertain; however, neither Grb2 or mSos 1 is significantly phosphorylated on tyrosine, suggesting that mSos1 is controlled as a direct result of SH2ISH3-mediated protein-protein interactions.
C. THEBCR-ABLONCOPROTEIN A N D RASACTIVATION A physical complex can also be detected between oncogenic Bcr-Abl tyrosine kinases and Grb2-mSos1 (Pendergast et al., 1993; Puil et al., 1994). Interestingly, autophosphorylation at Tyr 177 within the N-terminal Bcr region of Bcr-Abl creates a specific binding site for the Grb2 SH2 domain, suggesting that one role of Bcr is to provide a site through which Bcr-Abl can couple directly to the Ras pathway (Fig. 8). This interaction is apparently critical for the leukemogenic activity of BcrAbl proteins. T h e Grb2 SH2 domain recognizes phosphotyrosine sites with asparagine at the +2 position (Songyang et al., 1993); binding of Grb2 to Bcr-Abl apparently depends on Asn 179. Inhibiting this association between Grb2 and Bcr-Abl represents a potential therapeutic approach to CML and Philadelphia-chromosome-positive ALL.
FIG. 7. SH2iSH3 interactions in Ras activation. Epidermal growth factor (ECF) stimulation induces EGFR autophosphorylation at a site with a i-2 Asn. Grb2 binds to this site through its SH2 domain, and to proline-rich motifs (Pro) in mSosl through its SH3 domains. mSosl is therefore recruited into a heterotrinieric complex. These interactions are proposed to stimulate mSosI Ras-GNRP activity, and thereby convert Kas from the GDY- to the CTP-bound state.
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FIG. 8. BCR-ABL binds activators of Ras. The juxtaposition of BCR and ABL sequences creates a novel tyrosine phosphorylation site in BCR, which can bind directly to Grb2 and mSos1.
D. MULTIPLE GRB2 COMPLEXES IN GROWTH FACTOR-STIMULATED CELLS Many tyrosine kinases, including the EGFR, the insulin receptor, and Bcr-Abl, phosphorylate the Shc SH2-containing protein at a site that binds specifically to the Grb2 SH2 domain, and hence forms a complex with Grb2-mSos1 (McGlade et al., 1992b; Pelicci et al., 1992; RozakisAdcock et al., 1992; Pronk et al., 1993; Puil et al., 1994; Ruff-Jamison et al., 1993b; Segatto et al., 1993). Overexpression of Shc induces neoplastic transformation of fibroblasts and neurite outgrowth in PC 12 cells, apparently through activation of the Ras pathway (Pelicci et al., 1992; Rozakis-Adcock et al., 1992). The nerve growth factor receptor (Trk) and oncogenic Src variants cannot bind directly to Grb2, but nonetheless induce Shc phosphorylation and the formation of Shc-Grb2-mSos 1 complexes (McGlade et al., 1992; Rozakis-Adcock et al., 1993; Suen et al., 1993). These results indicate that tyrosine kinases may employ multiple mechanisms to couple to Grb2-mSos1, and thereby to the Ras pathway.
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V. SH2-Containing Phosphotyrosine Phosphatases and the Genetics of Signal Transduction A. PHOSPHOTYROSINE PHOSPHATASES W I T H SH2 DObfAIxS
Since tyrosine kinases play such a crucial role in signal transduction, the phosphatases responsible for dephosphorylating tyrosine are also likely to be important. Remarkably, two mammalian PTPs have been identified that each possess two N-terminal SH2 domains, a central phosphatase catalytic domain, and a C-terminal tail. One of these, variously termed Syp, SH-PTP2, PTPlD, and PTP2C, is widely expressed (Fig. 9; Freeman et al., 1992; Ahmad et al., 1993; Feng et ul., 1993; Vogel et ul., 1993). In contrast, the closely related PTPlC phosphatase (Shen et uI., 1991; Plutzky et al., 1992) is primarily expressed in hematopoietic cells. Despite its similarity to PTPlC, Syp/SH-PTP2 is most closely related to the Drosophila gene product corkscrew (Perkins et al., 1992), which is involved in signaling downstream from the Torso receptor tyrosine kinase and, therefore, in formation of the terminal structures of the Drosophila embryo. T h e identification of PTPs with SH2 domains was somewhat surprising, since SH2 domains are implicated in the binding of proteins to activated receptor tyrosine kinases. Indeed, Syp/SH-PTP2 becomes physically associated with the activated EGFR and PDGFR in zGzm (Feng P t al., 1993). In addition, SypISH-PTP2 is highly phosphorylated on tyrosine in cells stimulated with EGF o r PDGF and in v-STCtransformed cells (Feng et al., 1993; Vogel et al., 1993). We have also mapped a specific binding site for the Syp/SH-PTP2 SH2 domains to Tyr 1009 on the PPDGFR, as noted earlier, and have shown that the isolated Syp/SH-PTP2 SH2 domains retain the property of binding specifically to the Tyr 1009 site in vitro (G. S. Feng and T. Pawson, unpublished results). These data indicate that there is a direct physical association between receptor tyrosine kinases and the SyplSH-PTP2 phosphatase, and further indicate that Syp is a substrate for tyrosine kinases. The obvious implication of these data is that Syp is directly involved in dephosphorylating either autophosphorylated receptors o r other receptor substrates.
B. THEMO?'HEATENMOUSE MUTANT Genetic data have suggested a function for the mammalian gene hcph encoding PTPlC. Mice homozygous for loss-of-function mutations in hcph have a phenotype termed motlieaten, in which monocytemacrophages and erythroid cells are overproduced (Kozlowski et al.,
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1993; Schultz et al., 1993; Tsui et al., 1993). Motheaten mice also have defects in lymphoid development, possess elevated levels of CD5f autoreactive B cells, and as heterozygotes are prone to develop lymphomas. These results raise the possibility that PTPlC serves to limit tyrosine kinase mitogenic signaling and, as such, may function as a tumor suppressor gene. Collectively, the results outlined here begin to address the complexities of protein-protein interactions mediated by SH2, SH3, and PH domains during signal transduction. REFERENCES
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ACTIVATION OF THE SRC FAMILY OF TYROSINE KINASES IN MAMMARY TUMORlGENESIS Senthil K. Muthuswamy and William J. Muller institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario LBS 4K1, Canada
I. Introduction 11. Src Family of Protein Tyrosine Kinases A. Structure of the Src Family of Tyrosine Kinases B. Regulation of Src Protein Tyrosine Kinase Activity C. Activation of Src Family Kinases by Other Proteins 111. Elevation of c-Src Kinase Activity in Primary Mammary Tumors and Tumor-Derived Cell Lines IV. Transgenic Mouse Models for Testing the Role of Src Family in Mammary Tumorigenesis A. c-Src Plays an Important Role in PyV Middle T-Mediated Mammary Tumorigenesis B. Involvement of c-Src in Neu-Mediated Mammary Tumors V. Future Prospects References
I. Introduction
Growth and development of any given cell type involves a series of controlled signaling events originating from the cell membrane and terminating in the nucleus. Tumorigenesis is thought to be a consequence of loss of control in one or many of these checkpoints in a signal transduction pathway. For example, constitutive activation of cell-surface receptors, by ligand-dependent or -independent mechanisms, can lead to deregulated cellular proliferation. Alternatively, constitutive activation of downstream intracellular signaling molecules including cytoplasmic proteins (i.e., Src, Ras, Raf) and nuclear transcription factors (i.e., Myc o r Fos) can also lead to deregulated cell proliferation. The expression of certain members of the tyrosine kinase family of oncogenes in the mammary epithelium has been correlated with the induction of breast cancer in humans. For example, amplification and consequent overexpression of the neu (c-erbB-2, HERS) receptor tyrosine kinase (RTK) has been observed in almost one-third of human breast cancers (Slamon et al., 1987,1989)and appears to be inversely correlated with clinical prognosis 111 ADVANCES IN CANCER RESEARCH. VOL. 64
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(Gullick et al., 1991; Paterson et al., 1991). In addition, several reports have suggested that a large proportion of human breast cancers possess elevated p~60'-~71 tyrosine kinase activity (Rosen et al., 1986; OttenhoffKlaff et al., 1992). This review will concentrate on recent work that implicates the activation of Src family kinases as an important event in mammary tumorigenesis.
I!. Src Family of Protein Tyrosine Kinases A. STRUCTURE OF THE SKCFAMILY OF 'IYROSINEKINASES Members of the Src family belong to the non-receptor protein tyrosine kinase (NRPTK) class of signaling molecules. T h e prototype of this class, c-src, was identified as a cellular homolog of the transforming protein v-Src encoded by Rous sarcoma virus (Stehelin et al., 1976; Brugge and Erickson, 1977). T h e subsequent discovery that p p 6 0 ~ - ~ ~ [ possesses intrinsic tyrosine kinase activity provided the first evidence that tyrosine kinases are directly involved in cellular transformation (Hunter and Sefton, 1980). Within two decades of the discovery of c-src, eight additional Src-related genes had been identified, with molecular masses ranging from 55 to 62 kDa [c-yes, fiin, c$q, lyn, lck, hck, 61k (Toyoshima et ol., 1992) and yrk (Sudol et al., 1993j1. The members of the Src family share a common structural organization (Fig. 1). All members of the Src family are associated with the inner face of the cytoplasmic membrane via niyristylation of a glycine residue at position 2 (Schultz et al., 1985; Toyoshinia et al., 1992). Because mutations that interfere with myristylation of pp60\-"' are transformation defective, the membrane \ ~critical ' for its transforming properties (Cross et localization of p ~ 6 0 \ - is al., 1984; Kamps P t al., 1985; Pellman et al., 1985). Following this N-terminal sequence is a region of 50-60 amino acids that is unique to each member of the Src family (Parsons and Weber, 1989). This domain is thought t o play a role in mediating proteinprotein interactions. For example, the interaction between Lck and T-cell surface molecules CD4 and CD8 and the association between Fyn and multiple units of the CDS/TCR complex have been shown to occur through the &-terminal unique region of the Src family members (Shaw et al., 1990; Turner et al., 1990; Gauen et ul., 1992). T h e unique region is followed by three distinct domains of homology termed the Src homology 1 (SHl), Src homology 2 (SH2j, and Src homology 3 (SH3) regions (Sadowski et ul.. 1986). SH1 is the catalytic tyrosine kinase domain that is conserved among all the members of the
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FIG. 1 . Structural organization of the Src family members. This diagram ofc-Src protein tyrosine kinase shows major functional domains of the molecule. The numbers 2, 295, 416, and 527 correspond to the amino acid number of glycine (G), lysine (K), and tyrosine ( Y ) ,respectively (see text for details). Tyr (Y) 416 is not discussed in this chapter. SH represents domains of Src homology.
NRPTK class of molecules including Abl, Fps/Fes, Tyk2, Syk/Zap, Fak, and Csk (Bolen, 1993). Interestingly, the SH2 and SH3 domains are not restricted to the members of NRPTK class but are also present in a variety of other intracellular signaling molecules including PLC-y 1, GTPase activating protein (GAP), the p85 subunit of PIS'K, Shc, Grb2, Vav, Nck, and c-Crk (for a review, see Mayer and Baltimore, 1993). The SH2 domain is a module approximately 100 amino acids in length that plays an important role in mediating protein-protein interactions between tyrosine kinases and their substrates. The SH2 domains bind directly to phosphorylated tyrosine residues embedded in a protein motif (Anderson et al., 1990 Matsuda et al., 1990; Mayer and Hanafusa, 1990; Koch et al., 1991). Although the SH2 domains bind directly to phosphotyrosine residue(s), researchers believe that the amino acids immediately C-terminal to the phosphotyrosine + 1 and + 3 convey specificity and selectivity to the SH2-mediated binding to phosphorylated tyrosine (Songyang et al., 1993). The SH3 domain is about 50 amino acids long and is also involved in mediating protein-protein interactions through a proline-rich motif (Ren et al., 1993). The functional significance of SH2 and the C-terminal regulatory domain (see Fig. 1) in regulating the protein tyrosine kinase activity of the Src family members will be discussed later. B. REGULATION OF SRCPROTEIN TYROSINE KINASEACTIVITY Although v-src was initially identified as a transforming gene, overexpression of its normal cellular homolog c-src is incapable of transforming
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cells in culture (Ibn Pt a/., 1984; Parker d a/., 1881;Shalloway 1'1 id., 1981; 'l'anaka and Fujita, 1986). Interestingly, pp60v-~rcpossesses at least 10fold higher protein kinase activity than pp60'-~rc,as estimated by in vitro kinase assays (Coussens et al., 1985; Iba et a!., 1985). T h e differences in the enzymatic activity of the virally encoded protein and the chicken pp60<-"' correlate with a number mutations in the former (Takeya and Hanafusa, 1983; Hunter, 1987). In particular, a 19-amino-acid sequence 1ocat.ed in the C terminus of pp6WbrTwas replaced by a unique 12amino-acid sequence in pp60v-3rc (Takeya and Hanafusa, 1983; Levy et nl., 1986). Replacing the unique C-terminal 12 amino acids of pp60~-.7rc with the C-terminal l9-amino-acid sequence of pp60c-.\rcresulted in a reduction in the transforming ability of pp60v-'r-c(Iba et al., 1984; Wilkerson ut al., 1985). T h e importance of this region was further highlighted by the observation that two independent avian sarcoma virus isolates, S1 and S2, have deletions involving the C-terminal amino acids (Ikawa et al., 1986). ?'he functional significance of the C: terminus was revealed by the observation that dephosphorylation of a tyrosine residue in the C-terminal half of pp60C-irt leads to an increase in its tyrosine kinase activity (Courtneidge, 1985). This regulatory tyrosine was later identified as Tyr 527 in chicken pp60"-.'p-c(Cooper et d., 1986). Mutation of Tyr 527 to phenylalanine has been shown to increase both the transforming activity and the intrinsic kinase activity of p p 6 0 ~ - \ '(Cartwight ~ et al., 1987; Kmiecik and Shalioway, 1987; Piwnica-Worms et nl., 1987). Phosphorylation of Tyr 527 in p ~ 6 0 ' - \results ~~ in an intramolecular association between the SH2 domain in its K terminus and Tyr 527, perhaps interfering with the catalytic kinase domain (Fig. 2). Indirect support for this model derives from the observation that mutation or deletion of the SHY domain of pp6Wr.' activates its kinase activity and results in cellular transformation (Hirai and Varmus, 1990; O'Brien et al., 1990; Seidel-Dugan et al., 1992). Further evidence supporting this model o f pp(io'-$r(regulation derives from the demonstration that phosphopeptides corresponding to the C terminus of ppGO~-~rr bind to activated but not t o unactivated pp60C-5rc molecules (Roussel et ul., 1991; Cobb and Parsons, 1993). Interestingly, the amino acids around Tyr 527 constitute a low-affinity binding site for the Src SH2 domain (Songyang et al., 1993).Given the low affinity of the Src SH2 domain for the Tyr 527 site, it is conceivable that phosphotyrosine residues located on other signaling molecules can compete with 'Tyr 527 for its interaction with the Src SH2 domain and thus inhibit the C-terminus-mediated repression of ppGOc-\)~ activity.
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FIG.2. A potential model for the regulation of the tyrosine kinase activity of Src family members. The inactive molecule is shown on the left and the active molecule is shown on the right (see text for details). RTK, receptor tyrosine kinase; PDGFR, platelet-derived growth factor receptor; CSF- 1R, colony stimulating factor-] receptor; PTPs, protein tyrosine phosphatases; CSK, C-terminal Src kinase.
C. ACTIVATION OF SRCFAMILY KINASES BY OTHERPROTEINS T h e activity of pp60c-~rchas been shown to be elevated in a majority of colon carcinomas (Bolen et al., 1987; Cartwright et al., 1990; Talamonti et al., 1993). Since activating mutations have not been detected in these tumors, activation of ppGOc-~nis occurring by a process that does not involve somatic mutations (Wang et al., 1991). Considerable evidence suggests that activation of p~60'-~"kinase activity can occur through its interaction with either cellular or viral proteins. For example, polyoma virus middle T antigen (PymT) specifically associates with and activates the tyrosine-specific kinase activity of c-Src family members (c-Src, c-Yes, and Fyn) (Courtneidge and Smith, 1983; Kornbluth et al., 1987; Kypta et al., 1988). Moreover, formation of these complexes appears to be critical for PymT to transform cells (Courtneidge and Smith, 1984; Cheng et al., 1986; Cook et al., 1990). Fine structure mapping of pp6Oc-src and PymT
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interaction indicated that a region bounded by residues Asp 5 18 and Pro 525 in is required for complex formation (Cheng et al., 1988). This association of pp60'-$)-(t o PyniT may facilitate the dephosphorylation of Tyr 527 by preventing the intramolecular interaction between the SH2 domain of ppGO'-o~and Tyr 527. Cellular proteins that belong to the receptor protein tyrosine kinase family such as platelet-derived growth factor (PDGF) receptor (PDGFR), colony-stimulating factor (CSF-1) receptor (CSF-lR), epidermal growth factor (EGF) receptor (EGFRlerhB-I), and Neu (erbB-2) have also been shown to form physical complexes with p p 6 0 ~ - \in) ~u i z w (Kypta et al., 1990; Courtneidge et al., 1993; Luttrell et al., 1994; Muthuswamy et al., 1994). Ligancl stimulation of PDGFR and CSF-1R is shown to result in an increase in the tyrosine kinase activity of pp60'-~~'.1. (Twamely et al., 1992; Cour-tneidge et af., 1993). The importance of the association of pp60'-,\r( with R'I'Ks has been demonstrated by the observation that micro-injection of dominant negative mutants of c-Src into cells ef'fectively ablates PDGF-mediated mitogenesis in fibroblasts ('Iwamely-Stein Y / ul., 1993). Although a functional correlation between ligand stimulation and Src activation has not been shown for the Neu RTK, it has been demonstrated that murine o r human mammary epithelial cells expressing a kinase-active form of Keu also possess elevated pp60~-.\"tyrosine kinase activity (Luttrell et al., 1994; Muthuswamy et al., 1994). However, established cell lines expressing elevated levels of c-src are hyperresponsive to the mitogenic stimulus of EGF (Luttrell et al., 1988). T h e precise mechanism by which receptors such as N e u activate the Src family kinases remains to be elucidated. The observation that kinase-defective pp60'-"~cmutant (Lys295Arg) is also phosphorylated at Tyr 527 (Thomas el al., 1991) suggests that phosphorylatiori of Tyr 527 is riot an autophosphorylation event but is carried out h y a distinct tyrosine kinase. Consistent with this expectation, a C-terminal Src kinase (Csk) that specifically phosphorylates Tyr 527 has been identified (Okada et ul., 1991; Nada el ul., 1991; Sabe el al., 1992). Germ-line inactivation of csk results in an 1 1-fold increase in the activity of the c-Src family of kinases (Imaniato and Soriano, 1993; Nada el d., 1993). However, phosphorylation of the C-terminal tyrosine (I-yr 527) of pp6Oc-,~rtwas still observed in cells lacking a functional Csk, implying that a family o f Csk-related kinases may exist (Imamato and Soriano, 1993; Nada et ul., 1993).Csk has also been shown to be involved in regulating the activity of other members of the Src family (Okada et al., 1991; Bergnian et ul., 1992). Whether Csk plays any role in regulating the kinase activity of Src family members during tumorigenesis remains to be assessed.
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Activation of pp6Oc-5~~ could also occur through activation of specific protein tyrosine phosphatases (PTPs). Overexpression of a receptor-like PTP (PTPor) in rat embryo fibroblasts resulted in dephosphorylation of Tyr 527 and activation of pp6Oc-s~~ tyrosine kinase activity (Zheng et al., 1992). These data suggest that PTPa may be the tyrosine phosphatase responsible for dephosphorylating Tyr 527. However, it is likely that in any given cell type the kinase activity of Src family members is regulated by more than one mechanism. Ill. Elevation of c-Src Kinase Activity in Primary Mammary Tumors and Tumor-Derived Cell Lines
Although expression of c-src at high levels does not induce transformation, the activation of p p 6 0 ~ - is ~ ~thought c to play an important role in mediating cell proliferation (see Sections II,B and 11,C). In fact, a number of primary tumors and tumor-derived cell lines including breast, colon, melanoma, and sarcoma have all been shown to possess elevated ppGOc-5rc tyrosine kinase activity (Jacobs and Rubsamen, 1983; Rosen et al., 1986; Barkenow et al., 1987; O'Connor et al., 1992; Ottenhoff-kalff et al., 1992; Talamonti et al., 1993). Of these tumor types, activation of pp6Oc-s'~tyrosine kinase activity is thought to be a particularly common event in the genesis of breast carcinomas. A major proportion (>80%)of primary breast tumors exhibits elevated p p 6 0 ~ - kinase * ~ ~ activity compared with normal breast tissues (Rosen et al., 1986; Ottenhoff-kalff et al., 1992). In one of these studies, the increase in ppGO~-~rr kinase activity resulted from an increase in the specific activity of c-Src (Rosen et al., 1986). Overexpression of pp60~-"~ in mammary epithelial cells (IM2) results in loss of epithelial characteristics and loss of lactogenic hormoneinduced terminal differentiation. IM2 cells overexpressing pp60c-.~rcalso lose their ability to repress AP1 activity on lactogen-mediated signals to differentiate (Jehn et al., 1992), suggesting that high levels of Src tyrosine kinase activity would repress the differentiation process by promoting proliferative signals. IV. Transgenic Mouse Models for Testing the Role of Src Family in Mammary Tumorigenesis A. C-SRCPLAYSA N IMPORTANT ROLEIN PYV MIDDLET-MEDIATEDMAMMARY TUMORIGENESIS
Further evidence supporting the involvement of c-Src in mammary tumorigenesis derives from observations made with transgenic mice ex-
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pressing the PymT oncogene. Expression of PymT under the control of the mouse mammary tumor virus (MMTV) promoter/enhancer in transgenic mice leads to the development of multifocal mammary tumors that metastasize to the lung with high frequency (Guy et al., 1992a). T h e unexpected finding that mammary-gland-specific expression of PymT induces metastatic disease argues that the signaling pathways activated by PymT are involved in promoting tumor invasion. In fact, these mammary tumors possess elevated pp6O~-~rr and pp62c-y~~ kinase activities because of their association with PymT (Guy et al., 1994). In addition to the ability of PymT to associate with and activate members of the Src family, it is also known to associate with other cytoplasmic signaling molecules such as the p85 subunit of PI3' kinase (Whitman et al., 1985; Courtneidge and Hebner, 1987), protein phosphatase PPPA (Pallas et al., 1990; Walter el al., 1990), and an adapter molecule Shc that links tyrosine kinases to the Ras signaling pathway (Dilworth et al., 1994). Although activation of multiple signal transduction pathways by PymT plays a role in middle T-induced mammary tumorigenesis, the relative contribution of these pathways to the transformed phenotype is unclear. Direct evidence supporting the involvement of p p 6 0 ~ - ~ ~ ~ in PymT-mediated tumorigenesis derives from the results of a study involving interbreeding of the MMTV/PymT strains with mice bearing a germ-line mutation in c-src (Soriano et al., 1991; Guy et al., 1994). Mice homozygous for the c-src mutation are viable and develo p osteopetrosis (Soriano et al., 1991). Expression of PymT in the presence of at least one functional c-src gene (heterozygous, + / - or wildtype, +/+) leads to the development of metastatic mammary tumors with identical kinetics. In contrast, mice expressing PymT in a c-Srcdeficient (- /-) background rarely developed mammary tumors. In fact, only 2 of 31 mice in this genetic background ( M T + I - ; c-STC-/-) developed mammary tumors, and these occurred only after a long latency period (GUYet al., 1994). However, all these Src-deficient animals eventually developed mammary epithelial hyperplasia. These hyperplasias are likely the result of activation of the c-Yes kinase by PymT since elevated pp62c-'e$ kinase activity can be detected in these tissues (Guy et al., 1994). I n contrast to these observations, interbreeding of the MMTV/PymT strains into a c-Yes-deficient background had little effect on tumor formation (Guy et al., 1994). These observations strongly indicate that activation of p p 6 0 ~ - 'by ~
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absence of c-Src contrasts with the observations made in endothelial cells and fibroblast cells (Thomas et al., 1993). In these cell types, expression of PymT is still capable of inducing morphological transformation in the absence of c-Src. Thus, the activation of closely related c-Src kinases may be dispensable for PymT-induced tumorigenesis in cell types other than mammary epithelium. Although these studies strongly suggests the importance of c-Src in mammary tumorigenesis, it is unclear whether activation of c-Src is sufficient for mammary tumorigenesis. The generation of transgenic mice expressing constitutively activated c-Src in the mammary epithelium should allow this question to be addressed. B. INVOLVEMENT OF C-SRCIN NEU-MEDIATED MAMMARY TUMORS Amplification and overexpression of the receptor tyrosine kinase Neu (c-erbB2) is known to occur in 20-30% of human breast tumor cases (King et al., 1985; Yokoto et al., 1986; Slamon et al., 1987,1989; van de Vijer et al., 1987) and is inversely correlated with clinical prognosis (Slamon et al., 1987,1989; Gullick et al., 1991; Paterson et al., 1991). The role of Neu in mammary tumorigenesis was further strengthened by the observation that mammary-gland-specific overexpression of activated Neu (MMTVlneu) results in the induction of metastatic mammary tumors in transgenic mice (Muller et al., 1988; Bouchard et al., 1989; Guy et al., 1992b). Two studies have demonstrated that pp60~-~7c kinase activity is elevated in both human and murine primary mammary tumors expressing high levels of Neu (Luttrell et al., 1994; Muthuswamy et al., 1994). Activation of c-Src tyrosine kinase activity in the mammary tumors is thought to occur as a result physical association between Neu and pp60~-57~, since physical complexes between Neu and pp60c-s7~can be detected both in vitro and in vivo (Luttrell et al., 1994; Muthuswamy et al., 1994). Researchers further established that this complex formation is dependent on tyrosine phosphorylation of Neu (Muthuswamy et al., 1994).Although these observations suggest that ~ p 6 0 c -may s ~ ~play a role in Neu-induced mammary tumorigenesis, future genetic crosses between MMTVlneu mice and Src-deficient mice will provide direct evidence for the role of pp60c-s7c in mammary tumorigenesis. Consistent with the transgenic mammary tumor models, preliminary data suggest that human breast cancers overexpressing Neu also possess elevated c-Src kinase activity (S. K. Muthuswamy, R. D. Cardiff, and W. J. Muller, unpublished observations). Collectively, these observations suggest that pp6Oc-s7~may be playing an important role in human breast cancer.
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V. Future Prospects T h e establishment of transgenic mouse models expressing the various EGFR family members in the mammary epithelium provides a unique system in which to test the involvement of the c-Src family of kinases in mammary tumorigenesis. Studies in both primary tumors and their derived cell lines expressing elevated levels of EGFK or Neu suggest that. pp60'-\,. may be an important downstream effector for these KI'Ks. Future experiments designed to identify the pp60'-\'cbinding site on Neu and EGFR will he crucial in establishing the importance of pp60c-,57( activation in Neu- and EGFR-induced tumorigenesis. In addition, it will be interesting t o determine whether the other members of' the EGFR family (c-urhB-3, c-urhB-4) (Kraus et al., 1989; Plowman et al., 1990,1993) also utilize pp60~ as a downstream effector. These experiments will provide important insight into the mechanism by which these EGFK fBmily niernbers signal cell proliferation through p p 6 0 ~ - \ arid ' ~ , will provide important information for the design of therapeutic strategies to treat human breast cancer. -$'I
We w o t i l d like t o rlianh Peter Siege1 a n d bfarc il'ebster for their comments o n this nunuscript. This work w a s supporled b\ grdnts rcccivcd fium the Medical Research <:ouncil of. Canada. the (Iancer Research Societv, and the National Cancer Institute o f ( h n a d a . W.J.M. is a recipient 0f.a National Cancer Institute Scientist Award and S.K.M. is a Canadian International Developnient Agency (CIDA) scholar.
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ONCOGENIC PROPERTIES OF THE MIDDLE T ANTIGENS OF POLYOMAVIRUSES Friedemann Kiefer,”’ Sara A. Courtneidge,t and Erwin F. Wagner’ ‘Institute of Molecular Pathology, A-1030 Vienna, Austria tEuropean Molecular Biology Laboratory, D-69117 Heidelberg, Germany
I. Introduction A. Murine Polyomavirus B. Biochemical Properties of PymT Antigen C . Hamster Polyomavirus D. Rationale for Studying the Oncogenic Action of PymT in Vzvo 11. Consequences of PymT Expression in Vivo A. Expression of PymT under the Control of the Polyomavirus Early Region or General Promoter Elements B. Expression of PymT under the Control of Tissue-Specific Promoter Elements C. PymT Antigen Expression in Organ Reconstitution Systems 111. Expression of the Hamster Polyomavirus Middle T Antigen in Vivo IV. Analysis of PymT-Transformed Endothelial Cells V. Outlook References
I. Introduction The realization that viruses can cause cancer in humans and animals (Rous, 1911; Shope, 1932; Bittner, 1936; Zilber, 1946) clearly has been one of the biggest milestones in cancer research. Oncogenic viruses for the first time provided reagents that, in contrast to chemical carcinogens, were capable of transforming cells in a relatively small number of defined molecular events. Among the oncogenic viruses, the murine polyomavirus is outstanding in its ability to cause oncogenic transformation of a broad spectrum of cell types, which in its entirety is called the “polyomavirus tumor constellation” (Dawe, 1980). To date, two polyomaviruses have been isolated from their respective hosts, mice and hamsters. Both display a very similar genomic organization consisting of a circular genome; strands encoding early and late gene products are transcribed in opposite directions from a noncoding region that Present Address: Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario, Canada M4X 1K9.
125 ADVANCES IN CANCER RESEARCH, VOL. 64
Copyright 0 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
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functions as a replication origin as well as a transcriptional control region (Delmas P t al., 1985; Fried and Prives, 1986). Polyomaviruses share this general genomic structure with other papovaviruses; therefore, the hamster polyomavirus was initially named hamster papovavirus (Graffi et ul., 1969). In this chapter, we will refer to the murine polyomavirus simply as polyomavirus, whereas w e will always use the explicit designation hamster polyomavirus for the hamster papovavirus. A. MURINEPOLYOMAVIRUS
Polyomavirus was discovered accidentally in 195 1 by Ludwik Gross in cell-free filtrates from Akr mice that had developed spontaneous leukemia (Gross, 1953a,b). When subsequently tested on newborn mice, the preparation consistently induced parotid gland carcinoma and related tumors of the salivary gland. It soon became clear that this relatively narrow oncogenic potential of the virus, which was due to low viral titers of the tissue extracts, could be dramatically enhanced when high-titer virus preparations of polyomavirus grown in tissue culture were inoculated into newborn mice. T h e virus caused, in addition to the consistent appearance of parotid neoplasms, tumors as diverse as submaxillary and sublingual gland neoplasms, thymic epitheliomas, mammary carcinomas, renal and bone sarcomas, epidermoid carcinomas, adrenal medullary tumors, and hemangiomas (Stewart et nl., 1958). However, it never caused leukemias. Because of this ability to induce a wide range of tumors, Stewart and Eddy proposed the name S. E. “polyoma” virus (Stewart and Eddy, 1959). More recently, Dawe et al. (1987) showed that four different wiid-type strains of polyomavirus show markedly different oncogenic properties. Two of the strains are highly oncogenic in vivo and two display a lower oncogenic potential, giving rise only to tumors of rnesenchymal origin that develop with prolonged latency periods. All these strains are highly transforming in zritro. High-titer virus preparations also showed a significantly expanded host range, inducing tumors not only in a variety of different mouse strains but also in Syrian (golden) hamsters, rats, ferrets, guinea pigs, and rabbits (see review by Gross, 1983). In all cases, however, tumors only arose if the host animal was inoculated immediately after birth. Under normal circumstances, polyomavirus does not behave as a tumor virus in its natural host (Rowe, 1961). T h e development of tissue culture systems that allowed the propagation of the virus and the accumulation of high amounts of virus particles led to major breakthroughs in the understanding of the structure and lytic cycle of polyomavirus. However, the basis of the transforming
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events caused by polyomavirus remained largely unclear. Polyomavirustransformed cells always contain a functional copy of the viral genome early region integrated into the host genome. However, it became clear from integration studies that the virally induced transformed phenotype does not depend on a particular integration site or integration into a specific region of the host genome, leading to the activation or inactivation of host genes. Rather, viral transformation is a consequence of the addition of a viral gene to the host genome. i n 1970, Benjamin showed that three proteins of molecular sizes 2 1,55, and 96 kDa could be immunoprecipitated by treatment of extracts from polyomavirus-infected cells with sera from animals bearing polyomavirus-induced tumors. T h e three tumor, or T antigens-which were designated “small” T antigen, “middle” T antigen, and “large” T antigen (Benjamin, 1970)-are located in the early region of the viral genome (Soeda et al., 1979). Studies with mutants of polyomavirus indicated that small and middle T or middle T alone are necessary for the initiation of transformation, whereas large T might be necessary to sustain transformation (reviewed by Topp et al., 1980). Molecular cloning of the individual cDNAs of the polyomavirus early region finally allowed the analysis of the contribution of each of the three T antigens to the transformation process. Treisman et al. (1981) could show that a cDNA encoding the polyomavirus middle T antigen (PymT) is sufficient for the transformation of established rodent cell lines. Therefore PymT was unambiguously identified as the transforming gene of polyomavirus. Although PymT suffices to transform established rodent cell lines, maintenance of the transformed state in primary cells requires the cooperation of polyomavirus large T antigen (Rassoulzadegan et al., 1982; Land et al., 1983). Large T provides an immortalizing function for the establishment of continuously growing cell lines from primary cells, whereas PymT is necessary to sustain the transformed characteristics of these cells (Rassoulzadegan et al., 1983). OF PYMTANTIGEN B. BIOCHEMICAL PROPERTIES
PymT is a membrane-bound protein (It0 et al., 1977) of -55 kDa. It does not carry any intrinsic enzymatic activity, but associates with several cellular enzymes that are crucial regulators of cell proliferation. Therefore, PymT most likely exerts it oncogenic effect by subversion of the intrinsic functions of its cellular association partners. The first activity shown to be associated with PymT was that represented by tyrosine kinases; indeed, this was one of the first descriptions of stable tyrosine phosphorylation. Since the transforming protein of Rous sarcoma virus, pp60v-~”(Brugge and Erikson, 1977), was also demonstrated to be a
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tyrosine-specific protein kinase (Collett et al., 1980; Hunter and Sefton, 1980), it was an exciting thought that these viruses transformed via similar mechanisms. However, the obsei-vation that monomers of Pyiii’l‘ were n o t kinase active raised doubts that the activity was intrinsic to the protein. Indeed, researchers subsequently showed that the cellular tyrosirie kinase c-Src (the cellular hoinolog of pp60\-”()was complexed with Pym-1. (Courtneidge and Smith, 1983); two more Src-related tyrosine kinases, Yes and Fyn, have since also beeii identified in a complex with PymT (Koi-iibluthct ~ l . 1987; , Cheng et al., 1988; Kypta et d., 1988). These three members of the SI-cfamily are thought to account for all the tyrosine kinase activity associated with PymT (Fig. 1). Although association of Src and Yes with PyrnT leads to a marked up-regulation of kinase activity. no stimulaliori of tyrosine kinase activity was detected on binding of Fvn to Yyml~.I n fibroblasts, only a portion of the tyrosine kinases Src, Fyr;, and Qes form a complex with Pyni‘I‘; similarly, only a fraction of 1’ymT is bound to tyi-osine kinases (reviewed b y Kaplan et al., 1989; Brimela ot d., 1994). High activity of the kinase iiioiety of Src is associated with dephosphorylat ion of the C-tei-niinal Tvr 327 and phosphoi-ylation of Tyr 4 16. Kinase-inactive Src molecules display the opposite phosphorylation pattern: high phosphorylat ion of Tyr 527 and low phosphorylation at ’l’yr 4 16. Current evidence suggests that phosphorylation of Tyr 527 is part o f the regulation o f Src kinase activity and results in the intracellutar association of P-’lyr 527 with the Src SH2 domain, leading to the inactivation of the kinase moiety (reviewed by Cooper and Howell, 1993; Superti-Fiirga P t (11.. 1993). Mutant versions of Src in which the tyrosine at positioii 527 is exchanged with phenylalaniiie show an elevated kinase activity and clisplap oncogenic properties in 7 d r o (Cartwright el al., 1987; Kmiecik and Shalloway, 1987; Piwiica-Wornis r t ol., 1987). The Src tiiolecules that i1i-e associated with PymT are not phosphorylated on ‘lyr 527 and therefore are constitutively active (Cartwright Pt d.,1986). Pym?‘ may cause this ef‘fect either by binding to dephosphorylated active SIC and preventing phosphorylation of- Tyi- 327 or hy exposing inactive SIT to ii tyrosine phosphatase and stabilizing this state. The atialysis of niutant versions of PymT that retain the ability to bind and fully activate Src, but are still transformation defective, suggested that additional biochemical activities are complexed to PyrnT and take part in the transformation process (reviewed by Markland and Smith, 1987; Kaplan et nl., 1989). Subsequently, a ptiosphatidylincisitol 3-kinase (PIS-K) activity was shown to associate with PymT (Whitman P t al., 1985; Kaplari P t ul., 1986). T h e presence in PymT complexes of-a protein of 85 kDa u7as shown to correlate with the detection of PIS-K activity (Court-
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NH2
FIG. 1. Schematic representation of a putative PymT protein complex in mouse fibroblasts. The PymT molecule, which is anchored to cellular membranes by its C-terminal sequence, exists as a multimeric complex with several other proteins: p85 and pl10 correspond to the regulatory and catalytic subunits of PIS-kinase; Shc denotes the adaptor protein; A and C represent the accessory and catalytic subunits of PPBA, respectively; Src, Fyn, and Yes refer to the tyrosine kinases; Y refers to tyrosine residues.
neidge and Heber, 1987; Kaplan et al., 1987). Subsequent analysis showed that this was the regulatory subunit of the enzyme. The catalytic subunit of 110 kDa is also present, although it is not phosphorylated on tyrosine. Genetic analysis shows that the ability of PymT to associate with PI3-K is closely correlated with its ability to transform fibroblasts Zn vztro. Most recently, researchers showed that the transforming protein Shc is also capable of binding PymT (Fig. 1; Dilworth et al., 1994). Again a tyrosine phosphorylation site (Tyr 250) in PymT serves as a binding partner for the Shc SH2 domain. As a result of this interaction, Shc itself becomes tyrosine phosphorylated by the kinase present in the complex,
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and is thereby capable of binding the adaptor molecule Grb2, which is likely to result in the activation of the p21?aspathway. Several years ago, biochemical analysis had shown that two other proteins of molecular weights 61 and 37 kDa were also associated with Y y m r. Subsequent cloning showed that these two proteins are the regulatory (A) and catalytic (C) subunits of PP2A, which are phosphoserineand phosphothreonine-specific phosphatases, respectively (Pallas et al., 1990; Walter et al., 1990).Whereas PP2A is normally composed of three subunits called '4,B, and C, PymT seems to compete with the B subunit of the holoenzyme. PymT as well as the C subunit of PP2A bind in a highly cooperative manner to the A subunit (Ruediger et al., 1992).T h e function of PP'LA in poiyomavirus-mediated transformation is not understood. Whether the interaction between PymT and PP2A leads to a stimulation of PP'LA activity, and whether such a stimulation results in the dephosphorylation of specific cellular proteins, is not yet clear. C. HAMSTER POLYOMAVIRUS
In 1967, Graffi and colleagues described a virus associated with skin epithelioma in a Syrian hamster (Graffi et al., 1967,1968).The virus was originally designated hamster papillomavirus, but subsequent analysis showed that the virus was in fact most closely related to the mouse poiyomavirus, so it has been designated the hamster polyomavirus (HaPV) (Delmas et al., 1985).T h e tumors seen in the Berlin-Buch colon); of Syrian hamsters involve hair root keratinocytes, primarily on the head and back. However, when tumor extracts were inoculated into newborn hamsters from a different virus-free colony (from Potsdam), epitheliomas were not detected. Rather, the hamsters developed lymphoma and leukemia with a high incidence and short latency. These tumors, which appear in the liver, thymus, and kidneys, have not been well characterized but serological analyses suggested that both T and B cells were involved (Graffi et al., 1969). The tumor profile of HaPV is very different from that of the mouse polyomavirus, which has never been reported to cause lymphoma, even when expressed under the control of a iymphoid-specific promoter (Rassoulzadegan et al., 1990). T h e restricted tumor profile may in part be dictated by the inability of the virus to replicate in all tissues. However, this does not seem to be the case since viral DNA was found in all organs tested in infected hamsters. In addition, mice transgenic for the HaPV genome have been described; the founders developed both skin epitheliomas and lymphoid tumors (de la Roche et al., 1989). However, the exact nature of the lymphoid tumors was not established.
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The differences in tumor profiles observed suggested that there might be fundamental differences between the mouse and hamster polyomaviruses. However, molecular cloning of the HaPV showed it had a high degree of homology to the mouse polyomavirus (the open reading frames have an average 50% amino acid similarity), including the ability to encode a middle T antigen (Delmas et al., 1985). HaPV is thus only the second example of a middle T antigen-encoding papovavirus. The early region of the HaPV also encodes large and small T antigens. Despite the fact that HaPV causes predominantly lymphoma and leukemia in uiuo, the early region of the virus was shown to be able to transform primary rodent fibroblasts in uitro, although with a somewhat reduced efficiency compared with the mouse polyomavirus early region (Bastien and Feunteun, 1988). Furthermore, researchers also showed that the large T antigen carried the immortalizing properties, whereas the middle T antigen was responsible for the morphological transformation. A clear role for the small T antigen has not been defined. The middle T antigen of the hamster polyomavirus was identified and characterized using NIH 3T3 cells transformed with a cDNA encoding just the middle T antigen (Courtneidge et al., 1991). The use of NIH 3T3 cells also allowed a comparison of its properties with those of the mouse PymT in the same cellular background. The hamster middle T antigen (HamT) was identified as a 45-kDa phosphoprotein conforming to the corresponding predicted molecular weight. The HamT was found to have binding properties very similar to those of PymT; it could associate with cellular tyrosine kinase activity, serine/threonine phosphatase activity, and PIS-K activity. However, a surprising result was obtained when the exact nature of the associated tyrosine kinase was examined-the HamT bound exclusively to Fyn, and was not able to associate with c-Src or c-Yes. This result is in contrast with the binding specificity of PymT, which shows preferential association with c-Src and c-Yes, and only a very low degree of association with Fyn (reviewed by Brizuela et al., 1994). The specificity of association between HamT and Fyn is clearly demonstrated by the experiment shown in Fig. 2. Here, extracts of cells transformed by HamT were depleted of either Src or Fyn using specific antibodies. Subsequent analysis demonstrated that depletion was complete. When such depleted extracts were then examined for HamT-associated kinase activity, removal of Src was found not to affect mT-associated kinase activity whereas depletion of Fyn resulted in a complete loss of kinase activity in m T immunoprecipitates. These results demonstrate that, in fibroblasts at least, no tyrosine kinases other than Fyn are associated with HamT. The Fyn that is associated with HamT is activated in its intrinsic kinase activity compared with unbound
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FIG. 2. T h e association of. Ham?’ ~ i t hFyn. A lysate of HamT-transformed NIH3T3 cells was immunodepleterl using either normal rabbit serum (NKS, lanes 1-3) or antibodies specific for St-c (lanes 4-6) or Fyn (laiies 7-9). T h e depleted extracts were then used for a second round of inimunoprecipitation with antibodies specific for Src (lanes 1, 4,and 7 ) ,Fyn (lanes 2. 5. and 8).o r HamT (TBI-I; lanes 3, 6. and 9). Following washing of the immunocompl~xes,ail zit a h kinase assay was performed. The positions of the HamT (niT), the FVII( ~ 6 0 )and . the 85-kDa subunit of the P I 3 kinase (p85) are shown.
Fyn, although the mechanism of this activation has not been explored. These data therefore provide more evidence for the association of transforming proteins of polyomaviruses with members of the Src family, but suggest that in zjizto these kinases are functionally distinct. Furthermore, the fact that the two polyomaviruses have such distinct tumor profiles leads one to suggest that the ability to bind and activate different members of the Src family leads to this difference in tumor profile. Interestingly, Fyn kinase has recently been shown to be associated with the CDS/T-cell receptor complex in T cells (Samelson et nl., 1990) and has been proposed to play an important role in signaling in lymphocytes following antigen presentation. T h e possibility therefore exists that the introduction of HamT into these cells bypasses the need for antigen presentation and allows deregulated cell growth. If this is the case, the study o f lymphocytes expressing HamT might allow one to dissect the role of Fyn in the signal transduction process.
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D. RATIONALE FOR STUDYING THE ONCOCENIC ACTIONOF PYMTI N VIVO Polyomavirus is one of the most powerful carcinogens known, causing tumors in vivo with latency periods as short as 3-4 wk. In addition, PymT interacts with only few cellular proteins. These unique features give rise to the expectation that PymT-induced tumor formation might involve only a few, if any, unknown secondary genetic alterations and therefore might be amenable to systematic molecular analysis. Studying the oncogenic effect of PymT in transgenic mice in viuo rather than the complete virus has distinct advantages because the ability of the virus to infect or replicate in particular tissues is no longer relevant. Furthermore, possible effects of PymT on cell lineages that are refractory to tissue culture can be assessed; cell lines established at different time points of tumor formation should allow investigation of cellular alterations at different stages of tumor progression. i t is crucial to realize that the synchronous initiation of tumorigenic events in a large number of cells caused by the viral oncogene is essential to these studies and makes them superior to an approach in which, for example, chemical carcinogens are utilized. II. Consequences of PymT Expression in Vivo
The first experiments that aimed to investigate the oncogenic potential of PymT in uiuo were reported by Asselin el al. (1983). Direct subcutaneous injection of DNA encoding PymT did not induce the formation of tumors in newborn Fisher rats. In contrast, co-injection of a plasmid encoding either polyomavirus small or large T antigen led to the formation of tumors. In a second study, the same authors found that in newborn hamsters the direct injection of recombinant plasmids encoding PymT invariably led to the formation of subcutaneous tumors at the site of inoculation, albeit with a 5-10 times longer latency than wild-type polyomavirus DNA (Asselin et al., 1984). However, the tumors that arose in the course of these studies were not analyzed histologically. OF PYMTUNDER THE CONTROL OF A. EXPRESSION THE POLYOMAVIRUS EARLYREGION OR GENERAL PROMOTER ELEMENTS
Several investigators aimed to express polyoma middle T antigen either under its own promoter or under the control of promoter elements that confer expression in a wide number of tissues. In most of
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these studies, PymT caused the formation of endothelial tumors or hemangiomas. Transformation of endothelial cells by PymT zn vzim was first described by Kornbluth et al. (1986). Wing webs of 1-wk-old chickens were inoculated with a replication-competent avian retrovirus that transduced PymT under the control of the Rous sarcoma virus long terminal repeat (LTR). As early as 1.5 w k after the inoculation, endothelial tumors formed that developed into massive cavernous hemangiomas of u p to 1-cm diameter within the next 10 days. Hemangiomas were detected within the skin and muscles as well as in the mesenterium and intestine. Inoculation of the chorioallantoic membrane of 4-day-old eggs led to hemangioma formation within 4 days as well as to a squamous metaplasia of the ectodermal epithelium. In all cases, the provirus was found integrated into the host genome and PymT protein was expressed. T h e study also demonstrated that PymT was capable of transforming nonestablished cell lines In uttro in the absence of complementing genes such as polyomavirus large T or SV40 large T antigen, if overexpressed from a strong retroviral promoter. Bautch ut al. (1987) generated transgenic mice to study the effect of isolated expression of PymT in the natural host of polyomavirus, using a construct in which the PyniT cDNA was under the control of a replication-defective version o f the polyomavirus regulatory early region. Two transgenic sublines that originated from the same founder animal, but differed in the copy number of the transgene, were established; in both lines, all animals that carried the transgene either died or suffered from severe anemia due to the formation of multifocal hemangiomas o f the vascular endothelium. PymT acted as a dominant oncogene leading to the transformation of vascular endothelial cells within 4-10 wk after birth, with 100% penetrance. However, expression of the transgene could only be demonstrated in testis and tumor tissues. Efficient testicular expression was also observed in other transgenic lines carrying polyomavirus large and small T antigens under the control of the virus early regulatory region (Bautch, 1989), as well as in transgenic mice carrying the entire polyomavirus early region (Wang and Bautch, 1991). Interestingly, testicular tissue seems to be refractory t o the turnor-promoting effects of the polyomavirus early gene products. In transgenic mice carrying the entire polyomavirus early region, hemangiomas were not the predominant tumor type. In addition to vascular lesions, bone tumors were observed; individual lines also developed lymphangiomas and fibrosarcomas. Specific RNAs for the small and middle T antigens were detected in tumor tissues as well as in liver, spleen, and kidney. In transgenic mice generated by pronuclear DNA injection, expres-
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sion of the transgene, in this case PymT, often only occurs after birth in various tissues depending on the promoter or regulatory elements used. To elucidate a possible action of PymT on early mouse development, we decided to employ embryonic stem (ES) cell chimeras utilizing PymT-expressing ES cells. Such ES cells were generated by infection with a replication-defective PymT-transducing recombinant retrovirus, N-TKmT, and were selected and analyzed for PymT expression in vitro (Williams et al., 1988). This experimental approach to investigating PymT-induced oncogenesis through the generation of chimeric mice by blastocyst injection of PymT-expressing ES cells was based on the assumption that expression of PymT would be maintained throughout development in a broad range of tissues (reviewed by Wagner et al., 1991). By analogy to the experiments performed by Kornbluth et al. (1986), we had demonstrated the functionality of our recombinant virus, which expressed PymT under the control of the thymidine kinase promoter, by showing that a single injection into newborn mice caused the death of these animals within 2-4 wk because of the formation of cavernous hemangiomas. Surprisingly, expression of PymT had no obvious effect on the growth characteristics of ES cells in vitro, although we noted that the requirement of PymT-expressing ES cells for leukemia inhibitory factor (LIF) was decreased (E. F. Wagner, unpublished observations). Furthermore, the differentiation potential during the implantation stage and the subsequent early stages of mouse development in chimeras in vivo was also not affected. However, at midgestation, PymT exerted a dramatic effect. All chimeric embryos were arrested in development. Vessel formation in the yolk sac was disrupted; instead of primary capillary plexae, blood-filled sac-like structures formed and frequent hemorrhaging into the amniotic fluid caused the death of embryos (Fig. 3). T h e embryos often showed expanded chest cavities, enlarged primitive hearts, and misshaped heads. Injection of chimeric material originating from the embryo proper o r from the yolk sac caused the formation of hemangiomas in syngeneic 129/Sv mice, demonstrating that endothelial cells in extraembryonic as well as in embryonic structures had been transformed. Several transformed endothelial (End) cell lines were established from midgestation embryos as well as from tumor tissue derived from neonatal mice. The analysis of these cell lines will be described later. Although we had used a replication-deficient virus for our study to avoid viremia that could complicate the analysis of an observed phenotype, Fusco et al. (1988) described the development of an acute thrombocythemic myeloproliferative disease after infection of young adult
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NIHiOLAC mice with a recombinant PymT-transducing retrovirus in the presence of helper virus. Within 1-2 wk of intraperitoneal virus injection, the animals developed a profound spleen enlargement; multiple thrombi in skin, muscles, and mesenterium; and hematomas and hemorrhagic effusions. Of the cells in the bone marrow, 10% were identified as megakaryocytes; although the erythroid and lymphoid lineages were decreased, the numbers of myeloid cells of all stages of differentiation were abnormally expanded. The authors found reduced platelet aggregation and ATP secretion in response to aggregating agents. They detected PymT RNA exclusively in the bone marrow and PymT protein by indirect immunofluorescence in only few cells of this organ. Longterm bone marrow cultures obtained from infected mice contained more than 90% megakaryocytes, most of which expressed PymT as evidenced by indirect immunofluorescence. Interestingly, C57BL/6J Fv-2r mice inoculated with the same viral preparation showed no signs of disease. The study by Fusco et al. (1988) represents the only case to date in which an effect of PymT on a tissue type is reported that is not affected by the intact virus. Although the authors detected a profound effect of retrovirally transduced PymT on megakaryocytes and myeloid progenitors, they did not observe the development of hemangiomas. Since the pathology was detected in several animals, it must have been integrationsite independent. On the other hand, we have not detected any hematological disorders caused by PymT, in transgenic mice that expressed the oncogene in myeloid cells or in a bone marrow reconstitution system (discussed subsequently). Therefore, the pathology observed by Fusco et al. was very likely to be peculiar to the combination of the retrovirus and recipient strain used. An alternative explanation is that the clotting defect observed was the consequence of massive hemorrhaging in the animals rather than a specific functional impairment of the megakaryocyte compartment. T h e transgenic and chimeric mouse model systems have clearly demonstrated that expression of PymT is sufficient to induce tumors in specific cell types in vivo (Table I). These results confirm that part of the tumorigenic potential of polyomavirus resides in the viral early region and that PymT-mediated tumorigenesis does not need viral replication or the viral life cycle. Surprisingly, in the transgenic lines harboring the FIG. 3. Hemangiomas develop on the yolk sacs of PyinT ES chimeric embryos. (A) Control day 13 embryo. (B) Multiple hemangiomas are visible in a PymT chimeric embryo. (C) Embryo died prior to analysis by hemorrhaging, presumably as a result of endothelial cell disruptions. Yolk sacs of non-chimeric control (D) and PymT chimeric (E) day 12 embryos are shown.
TABLE I MODELSYSTEMS POK PYMT-MEDIATED TUMORIGENESW Oncogene
-
PpmT
Promoter
Py-early region
Experiniental system Direct DNA injection
Expression ND
Tumor types
+ (ND)”
w
30
References Asselin et al. (1983, 1984)
PymT
KSV-LTK
Retrovirdl infection
Primary CEF
Hemangioma
Kornbluth et al. (1987)
PymT
Py-early region
Trdnsgenics
Tumor tissue, testis
Heniangioma
Bautch et al. (1987)
PymT+ PylT+ PysT
l’y-early region
Transgenics
Tumor tissue
Osteosarcoma, hemangioma, lymphangioma, tibrosarcoma
Wang (1991)
PyniT
TK
Retroviral infection
Tunior-derived cell lines
Heniangioma
Williams et al. (1988)
PymT
TK
<;himeras
ES cells, tuniorderived cell lines
Heniangioma
Williams et al. (1988)
PymT
MLV-LTR
Retroviral infection
BM, megakaryocytes
Myeloproliferative disease
Fusco et al. (1988)
PymT
IgEiPy-early region
Trdnsgenics
Tumor tissue, brain, (spleen, liver) niyeloid cells
Various carcinomas, niammary tumor hemangioma
Kdssoulzadegdn et al. (1990)
-
Ds
PymT
MMTV-LTR
Transgenics
PymT
Rat insulin
P y m T + Pylt/SV40T
Rat insulin
Guy et al. (1992)
Mammary gland, t u m o r tissue, salivary gland, ovary, epidid ymis
M a m m a r y adenocarcinoma
Transgenics
Not detected
-
Bautch (1989)
Double transgenics
T u m o r tissue
p Cell t u m o r <
Bautch (1989)
PymT
TK
Transgenics
CNS t u m o r tissue
Neuroblastoma
Aguzzi et al. (1990)
PymT
TK
Retroviral infection, neuronal transplant
Neuroectoderrnal cells, t u m o r tissue
Hemangioma
Aguzzi et al. (1991)
PymT
TK
Rertroviral infection, b o n e marrow transplant
BM, T-cells, mast cells, H e m a n g i o m a
Retroviral infection
-,I
HamT
TK
myeloid colonies Hemangioma
Abbreviations: EM, bone marrow; CNS, central nervous system; HamT, hamster polyomavirus middle T antigen; IgE, immunoglobulin enhancer; MMTV, mouse mammary tumor virus; MLV, murine leukemia virus; ND, not determined; CEF, chicken embryo fibroblasts; Py-early region, polyomavirus early regulatory region; PylT, polyomavirus large 1 antigen; PysT, polyomavirus small T antigen; RSV, rous sarcoma virus; SV4OIT. SV40 virus large T antigen; TK, thymidine kinase. The authors have only reported the formation of tumors but not the tumor type. c p Cell tumors are caused independently of PymT expression by SV40IT and PylT under the control of the rat insulin promotor. c' Due to the presence of virus-producing cells, expression of HamT could not be detected unambiguously in infected cells. 0
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complete polyomavirus early region generated by Bautch et al., the tumor spectrum caused by PymT was not expanded beyond the tumor spectrum elicited by the intact virus, arguing that viral tropism is, at least in part, based on intracellular signal transduction pathways. Indeed, none of the transgenic models described earlier completely recapitulated the full spectrum of tumors caused by polyomavirus, that is, “the polyonia tumor constellation.” When PymT was expressed under the control of either its own promoter or broad-specificity regulatory elements, hemangiomas were the predominant tumor type observed; expression of’all three -1. antigens caused the formation of bone tumors, rather than the formation of parotid tumors as initially observed by Gross (1953a). T h e rapid kinetics by which endothelial cells are transformed by Pym‘T may account for the fact that mainly this tumor type is observed in PymT transgenic and chimeric mice. Furthermore, the occurrence of lethal hemangiomas in chimeric mice at midgestation precluded obtaining answers to sonie interesting questions. It is not clear if I’vm‘1’-expressing ES cells retain their full developmental potential including colonization of the germ line, or i f they are developmentally compromised. We have recently started to approach this question by generation of chimeric mice in which the embryo proper is completely ES cell derived. Such “ES mice” generated from aggregates of tetraploid niorulae and ES cells (described by Nag)- cf al., 1993) allow1 a precise determination of the developmental potential of the ES cells used; expression of a transgene can be monitored in different tissues throughout development. Preliminary results using PyniT-expressing ES cells indicate that embryonic development ceases as soon as vasculogenesis starts. Histological analysis suggests that the formation of the primary capillary plexus in the yolk sac as well as vessel formation in the embryo proper is substantially impaired. However, these observations have not yet been correlated with a detailed expression analysis of PymT. B.
E X P R E S S I O N OF 1’YM-i- U N D E R ‘THE CONTROL
Ok
TISSUE-SPECIFIC PROMOTER
ELEMENTS
More recently researchers have described the generation of transgenic mice that express constructs in which PymT is under the control of tissue-specific promoter elements. Rassoulzadegan et ul. ( 1990) derived a transgenic strain M‘1’-75 in which PymT was expressed from a chimeric promoter containing the IgE heavy chain enhancer instead of the polyoma enhancer. Although the founder was a phenotypically normal male, following serial breeding most of the females developed tumors, including carcinomas of the salivary and the thyroid glands, mammary tumors,
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adenocarcinomas of unknown tissue origin, and small liver hemangiomas. Several pulmonary metastases were observed. With the exception of one male that developed a salivary tumor, all males were phenotypically normal. Other than in one female that showed PymT expression in spleen and liver, PymT was only detected in tumor tissue and in brain. This expression pattern does not correspond to the pattern observed previously for genes controlled by the IgE enhancer and was most likely caused by cis-acting elements present at the integration site. Investigators had shown that the IgE enhancer can efficiently direct the synthesis of the c-myc oncogene in myeloid cells (Adams et al., 1985). Therefore, we were interested in determining whether PymT was expressed in bone marrow cells of MT-75 mice. We performed in uitro kinase assays on -50 pooled colonies, each of different myeloid lineages, and found PymT expression in erythroid, macrophage, granulocytemacrophage, and mixed colonies (Fig. 4). Despite the expression of PymT in myeloid cells, we did not find any hematological abnormalities in MT-75 mice. Transgenic mice described by Bautch (1989), which carried PymT coding sequences linked to the rat insulin promoter, did not show any tumor formation. Although expression of the transgene could not be demonstrated in this line, PymT was detected in p cell tumors of the pancreas after these mice had been crossed with transgenic strains that expressed either SV40 or polyomavirus large T antigens under the control of the rat insulin promoter. Independent of the presence of PymT, p cell tumors are caused by both SV40 and polyomavirus large T antigens. In the insSV40 x insPymT and insPylT x insPymT transgenic mice, the p cell tumor profile was not altered by the expression of PymT. However, the expansion of the target tissue allowed the detection of the PymT transgene. In contrast to numerous in uitro experiments, these
FIG. 4. PymT expression in various myeloid cells from transgenic mice MT-75. Expression was measured by an in ~ i t r oassay for PymT-associated tyrosine kinase activity. D3.3 are PymT-expressing ES cells; erythroid (Ery),mixed (Mix), macrophage (Mac),and granulocyte/macrophage (GM) colonies were picked from methyl-cellulose medium and assayed for PymT activity.
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double-transgenic mice showed no oncogene cooperativity between the PymT and large T antigens. A transgenic line described by Aguzzi et al. (1990) contained Pym'I' under the control of the thymidine kinase promoter. These mice expressed PymT in a highly tissue-specific manner; expression was only detected in the central nervous system, leading to the development of sympathetic hyperplasia and neuroblastomas with high penetrance. The unusual pattern of transgene expression, the observation that only one of four founder animals gave rise to a line that developed a neurological disorder, and the fact that in other transgenic models PymT expression in the brain did not lead to a neuronal hyperplasia suggest that the chroniosotnal integration site may be a major component of' this malignancy. Inspired by the fact that the inoculation of newborn or immunodeficient mice with polyoniavirus can lead to mammary adenocarcinomas, Guy el nl. (1992) generated several transgenic lines that expressed a PymT fusion gene under the control of the murine mammary tumor virus ( M M T V ) LTR. In all lines and in strict correlation with the onset of transgene expression, which showed a wide variation in levels and temporal pattern, multifocal tumors of the mammary gland appeared. In one transgenic line males also developed mammary tumors, most likely because of the expression of PymT before the normal regression of the male mammary epithelium. Apart from the mammary gland, PymT was detected to a lower degree in the salivary gland, the ovary, and the epididymis. Transgene expression in the lung was due to pulmonary metastases that occurred with a very high frequency in all tumor-bearing mice. Pulmonary metastases were also observed after transplantation of' primary tumors into the mammary fat pads of syngeneic mice. The simultaneous occurrence of multifocal tumors, with a latency period as short as 30 days, suggests that PymT suffices to transform the mammary epithelium in this transgenic model without additional activating events. When the MMTVIPymT strains were crossed with a strain that carried a disrupted c-Src proto-oncogene, the rapid tumor progression was no longer observed (Guy et al., 1994). Only rarely after long latency periods did abnormal hyperplasia of the mammary gland develop. These malignancies were accompanied by elevated expression of PymT and activation of the PymT-associated Yes kinase. However, crosses between MMTVIPyml' transgenic mice and mice harboring a disrupted allele of the Yes tyrosine kinase did not display an altered pattern of tumor formation compared with the parental MMTV/PymT transgenic mice. Expression of PymT in uiuo using tissue-specific promoter elements allowed the investigation of tumor formation in a variety of tissues since
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the lethal phenotype due to rapid transformation of vascular endothelium was no longer observed. The detected tumor types were of mesenchymal and epithelial origin, the same target compartments of the intact virus. Exceptions to this rule are the transgenic mice described by Aguzzi et al. (1990), which developed neuroblastomas. However, in this case the phenotype was most likely caused by the integration site of the transgene, since the same author has demonstrated normal differentiation of neuroectodermal cells after infection with a PymT-transducing retrovirus (Aguzzi et al., 1991; described next). Interestingly a number of tissues including testis, ovary, neuronal cells, and bone marrow cells are apparently refractory to the action of the PymT oncogene. Therefore, they must lack important intracellular signal transduction components that participate in the PymT-mediated transformation process. In contrast, PymT seems to act on endothelial cells and mammary epithelium as a single-step oncogene. The apparent susceptibility of endothelial and epithelial cells is likely to involve specific intracellular components such as Src-related tyrosine kinases. The availability of mouse strains carrying a disrupted allele of a signal transduction molecule makes it finally possible to test for the contribution of the individual components to the transformation process. In contrast to PymT-induced mammary tumorigenesis for which the Src tyrosine kinase seems to be required, we were able to show that in the transformation of endothelial cells the Yes kinase is predominantly involved (see subsequent discussion). Interestingly, the neu oncogene, which also transforms mammary epithelium in an apparently single-step fashion, is a constitutively activated tyrosine kinase. Therefore, protein tyrosine phosphorylation of particular target molecules may be part of the specific action of PymT on endothelium and mammary epithelium. The available data suggest that these targets might be different in endothelial and epithelial cells and that the transforming complex might, at leas: in part, be directed by the tyrosine kinase toward these critical intracellular mediators. Finally, there seems to be a class of tissues in which secondary events are a prerequisite for tumor formation. This is particularly obvious in the transgenic strain generated by Rassoulzadegan et al.’ ( 1990). These mice develop tumors in only a fraction of the tissues that express PymT and show a clear progression from preneoplastic to fully malignant forms of some tumors. Secondary events are obviously necessary in some organs but not in others. A threshold of PymT expression may exist that makes additional genetic modifications necessary in the case of subthreshold expression levels (see also Fig. 6B). Surprisingly no oncogene cooperation is observed in transgenic animals containing PymT in combination with PylT or SV4OlT antigens. In
144
FRIEDEMANN KIEFER E T .*\I..
a great number of other transgenic model systems, oncogene cooperation has been demonstrated (for review, see Hunter, 1991). Cell-cell interactions in the intact tissue may represent additional elements of transformation control that are missing in primary cell cultures in which t.he cooperativity of PyniT arid PylT has mainly been demonstrated. Alternatively, expression levels of PymT may be too low to be effective since, in all cases of double-transgenic mice, expression has only been demonstrated by RKase protection analysis (Bautch, 1989). C;.
PYMTL RECONSTITLTION
4EXPRESSION ~ I N ORGAN ~ ~ SYSTEMS
~
~
~
To express PymT in a particular cell compartment or tissue, organ reconstitution systems were utilized. These systems circumvent problems of inappropriate expression or the lack of suitable tissue-specific promoters encountered during the generation of transgenic mice, by retrovii-a1infection of embryonic or adult tissue h i 7dro and reimplantation of infected selected cells into a mouse. 1. 2Veitm i u I ?inw f ~ ltitut u ion
Aguzzi et d.(1991) described a neuronal transplantation model that mimics the structural and functional properties of the normal rat brain. Embryonic neuronal cells were infected with a replication-deficient PvmT-transducing retrovirus and injected stereotactically into the caudoputamen of adult. rats. O f the recipients, 70%,died between 13 and 50 days after the transplantation from intracerebral and subarachnoidal bleedings. T h e remaining 30%.of the recipients also developed hemangiomas but did not suffer from hemorrhages. PymT R N A was detected by in situ hybridization in neuroectodernial cells with neuronal and glial morpholog);; the highest RKA levels were expressed by vascular tumor tissue. Thus, the tissue-specific transformation of vascular endothelial cells was caused by a differential susceptibility o f t h e infected cells to the action of Pym'I' rather than by selective integration of- the virus. In contrast to all other murine PymT-transformed endothelial cell lines, lines that were derived from neuronal transplants failed to induce hemangiomas in syngeneic mice.
2.Bone M u rroii~Recorzstitution We were interested in investigating possible consequences of Pym?' expression in hematcipoietic cells in more detail. Although we had demonstrated that expression of PymT in four niyeloid lineages of the transgenic line MT-'75 (Rassoulzadegan et al., 1983) did not cause overt hema-
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145
tological alterations, Fusco et al. (1988) had reported a profound effect of PymT expression on megakaryocytes. Previously, researchers had shown that PymT can render the hematopoietic progenitor line FDC-P 1 growth factor independent (Metcalf et al., 1987; Muser et al., 1989) and that the constitutively activated tyrosine kinase v-Src causes a myeloproliferative disease when expressed in hematopoietic cells (Keller and Wagner, 1989). To direct expression of PymT to the hematopoietic system, we chose the well-established retroviral gene transfer protocol (Wagner and Keller, 1992). Primary bone marrow originating from either CBA/J or C57BL/6J mice was exposed to a recombinant replication-defective PymT-transducing retrovirus (Williams et al., 1988), either by cocultivation with a viral producer cell line or by incubation with high-titer virus supernatant. The experimental details of several infections are summarized in Table 11. The efficiency of the retroviral infection was monitored by the growth of G4 18-resistant myeloid colonies in semisolid medium. To detect a possible effect of PymT on hematopoiesis, we performed differential colony counts and established liquid cultures from the infected bone marrow. With the exception of a single experiment (indicated by * in Table 11)that gave rise to an immortalized primitive hematopoietic cell line, we did not observe significant differences between PymT-infected bone marrow and bone marrow that was infected by a retrovirus transducing only the neomycin resistance gene. The singularity of the event as well as the molecular analysis of the immortalized hematopoietic cell line obtained suggested that the retroviral integration, possibly in combination with PymT, caused the immortalization event. Having shown that the retroviral expression system was suitable for expression of PymT in hematopoietic cells, we injected infected bone marrow cells into lethally irradiated syngeneic recipient mice, in which these cells reconstituted a functional hematopoietic system. At various time points after transplantation, individual recipients were sacrificed and their hematopoietic status was assessed. We found G4 1%resistant colony-forming cells in the bone marrow and spleens of 5 of 10 analyzed recipients (data summarized in Table 111).Note that the percentage of G4 1&resistant progenitor cells in the spleen of these mice was consistently higher than in the bone marrow. In two individuals, we demonstrated the presence of PymT-associated kinase activity in bone marrow and spleens as well as in myeloid colonies, T cells, and mast cells derived from these organs. We were not able to detect any hematopoietic alterations in these animals other than mild enlargement of the number of clonogenic progenitors in the spleens of three individuals. We assume
Donor BM 5-FU pretreatment"
Strain
Yes
CBA/J C:BA/J
Yes
CUA/J C.5 7 B IJ6J (257BLJ6J
g m
~~
~
~~~
Yes NO
Yes
C:iSUI/6J
NO
CB7BI/6J
NO
G418-resistant CFC after 24 hr. Manipulation< Infection(' None None Ery-Lys Gradient None Gradient None Ery-Lys
IkS
(37 B I /ti] ~~
~
~
I
I:! 10
44 14 10 >
2 2
cocult vsup
10 0.5
vsup vsup vsup
0.5 6.5
22
(%I
ND 6.5 70 ND ND ND ND 37 48 35 53 .5 9 72
PymT-associated kinasex
Hematological alterations
Bone marrow transfer"
+ + ND +
Yes* NO NO
Yes
N O
Yes
ND ND ND ND
No No No No
Yes Yes
+ + + +
N0
ND
~~
No No No No ~
1'i.iiiiary Imie niarrow ( R h l ) o f t h c indicated n i w s e strain W;IS infccrctl with the l'yni.1-traristlucing recombinant murinc retrovirus I\j-TKniT (Williams a/ nl., 1988) the prrscnce o f intei-lrukin-l a , interleukin-3, and er) thropoietin. ND,N o t determined. Whew i ndia trd. In)ne inarrow w a s harvcstrd from niice that had I~CCIIpi-etrratetl with 9-lluoroitracil (5-FU). the niiiltiplicity of infection. in sonic experiments I)one niiiri-ow w a s fract ion;itctl using a Percoll density gradient to rcniove mature hematopoietic cells, c,xperinirnts ervthrocycs were Iysed using ;inimoniuni clhriclc. Keti-ovii-alintection \ v i i ~;icconiplishecl either b y cociiltiratii)ii with virus-p luring libroblasts (co~ult)or b) incubation in a high titer virus supernatant (vsup). Aftei- 24 h r the percentage o f successfully infected cells wits determined I) I standard nicthyl ccllulose colony asaay in the presence of 1.3 mginll G4 18. / Where indicated, 21 second tolon) assay was pcrlimmd aficr a %clay selcc n period in Ole presence of 1 mg/ml G4 18. x I ' y n i l expr-ession win cicterininetl h y an t i ! 7 r i / , o kinasc assay pel-formed o n p o d s of inyeloid colonies growth in methyl cellulose or in liquid bulk cultures. Wherc. indicated, infected I)onc inarrow w;is ti-anspl;intrcl into syngcncic, lethall) irradiated recipients. (1
in
~~
None Ery-Lys None Ery-Lys
\.sup vsup cocult vsup cocult vsup vsup cocult
(7;
G418-resistant CFC af-ter 3d selection/
'8
I'
TABLE I11 I'YMTEXPRESSION I N BONEMAKROW RE~:ONS.~ITITI.E~ MI(:E~~
Gene transferred
Time after BM graft" (mo)
PymT
2.5
G418-resistant CFC in BM a n d spleen" UM: 65%; Spl: 69%.
DNA analysis(
PyinT expressionf'
int prov
BM, T cells, mast cells
int prov
ND
int prov
ND
(>4000<:F(:/lO(i)
PymT
3
BM: 3%; Spl: 14% (825 CFC/ 10") EM: 1%; Spl: 5%
PymT
4
BM: 23%; Spl: 34%
ND
PymT
7
BM: 10%; Spl: 17% (13.50 C:FC/IW)
int prov
BM, Spl ND
neo
2.5
BM: 65%; Spl: 69% (>200 CFCI 10")
int prov
-
neo
4
BM: ~52%;Spl: 49% (<200 C F U 10")
ND
-
PymT
3
Lethally irradiated ntire werc reconstituted with syngeneic bone marrow that had k e n infected with the PymT transducing virus N-TKniT. ND,Not clctet-mined. At the indicated time point after tile bone inarrow graft, the percentage of.(;4 I8-resist;mt colony-forming cells ( W C ) in bone marrow and splecn wis clcterniinrd. Niinibers in brackets give the trcqucncy of colony-forming cclls found in the spleen per 10" splcnocytes. c The presmcc of the intact provirus (int prov) in spleen and Ixmc inarrow was demonstrated by Southern blot analysis. d Pynil' expression was shown b y is 7 4 ~ kinase 0 assay. BM, Bone niarrow; Spl, spleen. (8
'2
148
FRIEDEMANN KIEFER E7’Al..
that this effect is the result of the disturbance of steady-state hematopoiesis in these animals rather than a speciIic effect of the oncogene. The number of’ progenitor cells in the bone marrow of the same mice was normal, and no progenitor cells were found in the peripheral blood. In this model, PymT was not able to reproducibly imniortalize hematopoietic cells o r to cause gross alterations in the hematopoietic system. However, w e detected the formation of hemangiomas in -30% of our bone marrow-reconstituted mice. Ill. Expression of the Hamster Polyomavirus Middle T Antigen in Vivo We were interested in determining whether HaniT would be capable of transforming target tissues other than endothelial cells. We therefore constructed a recombinant retrovirus containing Ham’l’ sequences, designated N-TKHamT (S. A. Courtneidge, unpublished), the structure of which is analogous to that of the PymT-transducing virus N-TKmT (Williams et d., 1988).When we inoculated newborn mice with this virus w e detected the formation of minute hemangiotnas in 60% of the animals after a latency period of 30 days or longer. An attempt to isolate transformed endothelial cell lines from these lesions was unsuccessful. It was possible to generate more pronounced lesions that developed with the same latency period by injection of virus-producing fibroblasts into newborn mice. Because of the transformed nature of the producer cell line, isolation of transformed endothelial cells was not possible from these lesions. We obtained a very surprising result when viral producer cells secreting &-TKHamT were injected into mutant mice that carried a null allele for h e tyrosine kinase Fyn (Stein et al., 1992). Newborn Fyn-deficient homozygous mice developed hemangiomas after inoculation with N-TKHamT. From these experiments we concluded that, despite their different biochemical properties, both HamT arid PymT can transform target endothelial cells. Furthermore, our results suggest that a tyrosine kinase other than Fyn may associate with HamT in endothelial cells of Fyn-deficient mice, despite the evidence that Fyn preferentially binds to Hanil-. An alternative explanation is that mT-associated kinase activity is not required for heniangioma formation and that one of the other associated proteins causes the transformation. However, this possibility seems unlikely in view of the finding that hemangiomas occur less frequently in Yes-deficient mice (Kiefer el al., 1994).
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IV. Analysis of PymT-Transformed Endothelial Cells Endothelial cells have proven notoriously difficult to culture. Chimeras and young mice provide a convenient source of PymT-transformed endothelial cells (endothelioma, End cells) which, in contrast to primary endothelial cells, grow continuously although they retain important features of differentiated endothelium (for review, see Wagner and Risau, 1994; Fig. 5). Williams et al. (1988,1989) derived highly tumorigenic End cell lines that caused the formation of hemangiomas within 18 hr to 4 days of injection into syngeneic and nonsyngeneic mice, or even into other species such as rat, chicken, and quail. Hemangioma formation was shown to be a specific property of End cells, since PymT-transformed fibroblasts or embryonic carcinoma cells were not capable of eliciting this tumor type. Although syngeneic newborn mice succumbed to the tumors caused by the End cell graft within 1-2 wk, only 50% of the injected adults died. The remaining half formed hemangiomas which, on serial transplantation, lost their potential to induce tumors rapidly. Using an isoenzyme marker as well as [3H]thymidine-labeled, mitomycinC-treated End cells, Williams et al. (1989) showed that the vast majority of tumor tissue was host derived. Some insights into a possible mechanism of hemangioma formation came from in vitro experiments in which researchers showed that all End cell lines examined formed large cystic structures, very reminiscent of hemangiomas in fibrin gels (Montesano et al., 1990). The aberrant morphogenic behavior of End cells was tightly associated with a high proteolytic activity secreted by these cells that was caused by an increased production of urokinase-type plasminogen activator (u-PA) and a reduced synthesis of the plasminogen activator inhibitor PAI-1. Most surprisingly, the addition of exogenous serine protease inhibitors to fibrin gels altered the morphogenic properties of End cells; they now formed asterisk-like structures similar to the tubes formed by primary endothelial cells (Montesano et al., 1990). Several attempts to establish a causal link between proteolysis/u-PA expression and hemangioma formation using retroviral gene transfer of u-PA and PAI-1 into primary endothelial cells and End cells have failed (F. Kiefer and E. F. Wagner, unpublished data). However, we recently succeeded in deriving additional End cell lines that show a significantly reduced net proteolysis and still are able to form hemangiomas in vivo. Independent evidence arguing that u-PA is not solely responsible for hemangioma formation comes from End cell lines that have been established from mutant mice lacking
MIDDLE T ANTIGENS OF POLYOMAVIRUSES
151
a functional u-PA or t-PA gene (in collaboration with P. Carmeliet, Leuven). Preliminary data show that these cell lines are still capable of forming hemangiomas. Analysis of PymT mutants has established the importance of the interaction between PymT and the Src-related tyrosine kinases Src, Fyn, and Yes for PymT-mediated transformation. It has recently been shown that PymT can efficiently induce hemangiomas in Src-deficient mice (Thomas et al., 1993). Although the same observation was made with Fyn-deficient mice, in Yes-deficient mice hemangiomas formed with a lower efficiency and with a significantly longer latency period, arguing that the Yes kinase is particularly important for the transformation of endothelial cells (Kiefer et al., 1994). End cell lines derived from all three types of kinase-deficient mice were indistinguishable from their wildtype counterparts. Biochemical analysis of the transforming complexes in various End cell lines suggested that Yes contributes significantly more to the PymT-associated kinase activity in this cell type than in fibroblasts (Figs. 5 and 6). The availability of PymT mutants such as HamT and of Src kinasedeficient mutant mice allows the possible identification of molecules necessary and sufficient for PymT-mediated tumorigenesis. Clearly tyrosine kinases belong to this group, although there seems to be a threshold level of kinase activity that can be reached by any two of the three ubiquitously expressed kinases Src, Fyn, and Yes (Kiefer et al., 1994; Fig. 6). More interestingly, the observation that HamT is still able to transform endothelial cells in Fyn-deficient mice suggests that a further, perhaps endothelial cell-specific, kinase is involved in this process. Although all End cell lines described here were established by culture of tumor tissue obtained from midgestation embryos or newborn mice, Dubois et al. (1991) described the isolation of one PymT-transformed endothelial cell line from adult transgenic mice of the strain Py-4 (Wang and Bautch, 1991). These mice carry the complete polyoma early region and develop multiple skin hemangiomas at 6-8 wk of age with 100% penetrance. End cells were established from these hemangiomas by labeling the cells with fluorescent acetylated low density lipoprotein (LDL) and two subsequent rounds of cell sorting. The cell lines obtained showed a typical endothelial cobblestone morphology and were tumorigenic in uiuo. In contrast to the End cell lines first isolated by Williams et al. (1988), these endothelioma cells caused hemangiomas with a much FIG. 5. Morphology of End cells (A) and a typical End cell-induced hemangioma (B). (C) PymT-associated tyrosine kinase activity in different kinase-deficient End cell lines (Src, Fyn, Yes). wt, Wild-type End cells.
152
FRIEDEMANN KIEFER E T AL.
k
[PyrnT.Fynl
\ Oncogenic Signal
PymT
3 known kinases
2 known kinases lhreshold lor translormalion
low
Iransformalion efficiency
high
FIG. 6. Proposed model of PymT activity in endothelial cells as a function of its association with the three known Src-family tyrosine kinases Src, Fyn, and Yes. The existence of an as yet unidentified tvrosine kinase (X) cannot be excluded.
longer latency period of 2-4 wk and most likely without the involvement of host cell recruitment. Similar to transformation of endothelial cells by PymT in uiuo and in uitio, which appears to occur as a rapid apparent single-step process, pancreatic ductal adenocarcinomas can be induced by retroviral transduction of Yym'l' into the islets of Langerhans (Yoshida and Hanahan, 1994). Infection of pancreatic islets from juvenile mice yielded cell lines that, after several cycles of single-cell cloning, consisted of two subtypes of cells: one that does not express specific markers and a second that expresses markers of ductal epithelial and islet cells. On re-injection into mice, these cells formed well-differentiated ductal adenocarcinomas.
MIDDLE T ANTIGENS OF POLYOMAVIRUSES
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This model system may provide insights into the naturally occurring islet progenitor cells as well as the target cells in pancreatic cancer, and the molecular changes in the progression of this cancer type.
V. Outlook Tumorigenesis is currently understood as a multistep process that can be elicited by the alteration of a number of pathways and processes. Transgenic and chimeric animals that reproducibly develop tumors as the result of a defined alteration, namely the expression of an isolated viral oncogene, are important tools in developing an understanding of the various steps involved in tumor formation. The existence of different types of tissues that display a differential susceptibility to the action of PymT will ultimately lead to the identification of particular intracellular components that are only present in susceptible cell types and are responsible for the specific action of the oncogene. PymT-transformed cell lines will be crucial tools in these studies. Endothelioma cell lines, for example, should allow the identification of unknown tyrosine kinases involved in the specific transformation of this cell compartment. In addition, the availability of novel strains of mutant mice lacking particular molecules of the PymT complex will allow a genetic analysis of the complex, and will complement the studies that have been performed to date using PymT mutants. Employing PymT mutants, it has not been possible to dissect the individual roles of PIS-K, the tyrosine kinases, or PP2A. For example, all mutant forms of PymT that have lost the capacity to bind PP2A lack associated kinase activity. The availability of mice carrying a targeted allele for the regulatory subunit of PIS-K or the A subunit of PP2A would allow us to address these questions. Substantial progress toward an understanding of how PymT subverts the growth control of cells has already been achieved. We know now that polyomavirus activates several cellular signal transduction molecules such as PIS-K and ShdGrb2, and thereby most likely causes the activation of the subsequent signal transduction pathways (Fig. 7). Therefore, PymT mimics the action of activated tyrosine kinase-associated growth factor receptors, giving us a first clue to an understanding of why certain tissues, such as testes, or cell types, such as hematopoietic cells, are refractory to the action of the oncogene. Either these cells to not contain the appropriate receptor molecules and/or their corresponding signal transduction elements, or a stimulation of these has no consequences because downstream effector molecules such as transcription factors are not active. Exhaustive knowledge of the molecules that interact with PymT will
154
FRIEDEMANN KIEFEK E T AL. PymT proteincomplex
FIG. 7. Schematic view of possible molecular niecliariisms involved in PymT-induced single-step endothelial cell transformation it/ UWO. Py V, Polyomavirus; GF-GFR, hypothetical endotlielial-specific growth factor-growth factor receptor loop. For other abbreviations, see Fig. 1 .
eventually lead to an understanding of how the proliferative signals generated by this oncogene cause the rapid transformation of cells. Most importantly, the lessons learned from studying PymT-mediated viral oncogenesis in uitro and in VZZIO have impressively enhanced our knowledge of growth regulation in normal cells, and have set a paradigm of an apparent single-step rather than multistep model of oncogenesis. ACKNOWLEDGMENTS We would like to thank Denise Barlow and Agi Grigoriadis for critical reading o f t h e manuscript, 1Iannes Tkadletz for photographic assistance, and Irene Acas for helping prepare the manuscript. REFERENCES
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SELECTIVE INVOLVEMENT OF PROTEIN KINASE C ISOZYMES IN DI FFERENTI AT10N AN D NE0PLASTIC TRANSFORMATION JoAnne Goodnight, Harald Mischak,’ and J. Frederic Mushinski Laboratory of Genetics;, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
I. Introduction A. Structure of Protein Kinase C Isozymes 8. Activation of PKC Kinase Activity and Translocation to Membranes C. Down-Regulation D. Induction of Early Genes E. Transduction of Physiological Stimuli on PKC Activation F. Differential Expression and Subcellular Distribution of PKC Isozymes G. PKC Substrates H. Identification of Additional Substrates I. PKC-Associated Proteins 11. PKC Isoforms Involved in Differentiation A. Myeloid Differentiation B. PKC in B and T Lymphocytes C. Keratinocyte Differentiation D. Erythroleukemia Gels E. PKC in Pheochromocytomas F. Neuronal Differentiation and Neurite Outgrowth G. Neuroblastoma Differentiation 111. Involvement of PKC Isoforms in Tumorigenesis A. Tumor-Specific Isozyme Expression B. Correlation of PKC Activation or Depletion with Tumorigenesis C, Changes in PKC Substrates and Tumorigenesis D. Role in Transformation by the ras Oncogenes E. Role in Transformation by Other Oncogenes F. Involvement in Tumor Promotion G. Oncogenic Mutations of PKC H. PKC Isozyme Overexpression and Tumor Induction I. Colon Carcinoma IV. Conclusions References I Present address: Institute for Clinical Molecular Biology and Tumor Genetics, GSF, Marchioninistrasse 25, D-8000 Munich 70, Germany.
159 ADVANCES IN CANCER RESEARCH, VOL. 64
Copyright 0 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
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1. Introduction Protein kinase C (PKC)' denotes a ubiquitous family of protein kinases that specifically phosphorylate serines and threonines in a variety of proteins. PKC was initially described as a serine/threonine protein kinase that required calcium (Inoue et d.,1977) and phospholipid (Takai et al., 1979) for activation of its kinase activity. When PKC was found to be a major receptor for the phorbol esters (Castagna et al., 1982), which were known to be important promoters of carcinogenesis in mouse skin and potent stimuli for certain forms of differentiation, PKC was inferred to be an important mediator of both carcinogenesis and differentiation. PKC is now known to consist of at least 11 different subspecies (a through p,), most of which d o not require calcium for activation (Nishizuka, 1992a; Selbie et al., 1993; Johannes et nl., 1994). All these isozymes have similar kinase regions but exhibit major differences in their developmental expression, tissue distribution, regulatory properties, and susceptibility to activators and substrates. An enormous literature has accumulated concerning PKC. Although one o r more members of this family of enzymes appears to be involved in a variety of signal transduction, differentiation, and transformation pathways, many of the published papers contain apparently contradictory data. In this chapter, we will concentrate on the current literature that implicates PKC in differentiation and neoplastic transformation, when possible emphasizing the data that delineate specific roles for individual PKC isozymes. We will deal only generally with PKC structure and function and normal transduction of mitogenic (growth factor) signals.
A. STRUCI'UKE OF PROTEIN KINASEC ISOZYMES As shown in Fig. 1, the primary structure of all members of the PKC family can be divided into two main sections, a regulatory domain and a catalytic domain. Sequence comparison of all the PKC isoforms reveals four highly conserved regions (Cl-C4) and five variable regions ( V l Abbreviations used: CTL, cytotoxic T lymphocytes (CD8-positive); DAG, diacylglycerol; DMSO, dirnethyl sulfoxide; EGF, epidermal growth factor; GSK-JP, glycogen (a PKC inhihitor); spthase kinase-3P; H7, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine H M B A , N,h"-hexamethylerte bisacetamide; IL-2, interleukin 2; IL-ZR, receptor for IL-2; IP.,. inositol triphosphate; LPS, Iipopolysaccharide; MARCKS, myristoylated alanine-rich (: kinase substrate; MELC, mouse crythroleukemia cells; rnT, middle T antigen of oncogenic DNA viruses; NFG, nerve growth factor; PC, phosphatidyl choline; PDBu, phorb l dibutvrate (a phorbol ester); PI, inositol phospholipids; PKC, protein kinase C; PS, phosphatidyl serine; RA, retinoic acid; RACK, receptor for activated C-kinase; T h , helper T cells (CD4-positive); TPA, 12-O-tetradecanoyl phorbol- 13-acetate (a phorbol ester).
161
PKC IN DIFFERENTIATION AND TRANSFORMATION Regulatory Domain
Catalytic Domain Hinge v3
v1
v5
v4
cPKCs
nPKCs
aPKCs
naPKC
Pseudosubstrate Region
Ifi
DAG/Phorbal Binding
0
Cys-rich Zinc Finger
i
Ca-binding Region
0
ATP-binding Region
0
E
n
FIG. 1 Schematic comparison of the 12 known isozymes of protein kinase C (PKC). Similar isoforms are designated cPKCs (conventional or calcium-requiring), nPKCs (novel, calcium-independent), aPKCs (atypical, lacking one cysteine-rich zinc finger, and phorbol ester unresponsive), and naPKC (novel, atypical, but not lacking two cysteine-rich zinc fingers). PKC-A is the mouse counterpart of human PKC-t. The characteristics of various portions of the proteins are indicated according to the key at the bottom of the drawing. The two boxes at the left end (amino terminus) of PKC-p indicate putative leader (L) and transmembrane (M) sequences (Johannes et al., 1994).
V5). T h e regulatory domain at the N-terminal end (Vl-C2) contains a “pseudosubstrate” region, one or two cysteine-rich regions that are referred to as zinc fingers (Berg, 1990), the diacylglycerol (DAG)-binding site, the phorbol ester-binding site, and the calcium-binding site. The C2 region, which is present only in PKC-a, -f3, and -y, has been implicated in Ca2+ binding because deletion of the C2 region of PKC-P resulted in a Caz+-independent kinase (Kaibuchi et al., 1989). The catalytic domain (C3-V5) contains the ATP-binding site and the substrate-recognition site. T h e V3 region, between C2 and C3, appears to have high flexibility and is thought to function as a hinge region, allowing the regulatory section to swing away from the active sites of the kinase domain (activation) or to swing back and obstruct the kinase section (de-activation). This region is also thought to be accessible to proteolysis when the hinge is unfolded.
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The kinase activity of the PKCs remains silent until activator molecules such as DAG, phorbol ester, and phospholipid are bound to it. T h e physiological consequences of the activation of PKC are determined by the choice of isoform that is activated and by the nature of the substrate that is phosphorylated by this isoform. Serines (S) and threonines (T) in the vicinity of basic amino acids, lysine (K) and arginine (R), have been shown to be targets of PKC phosphorylation (e.g., SIT-X-K/R; K/RX-SIT; KIR-X-X-SIT; KIR-X-SIT-X-SIT-X-KIR; and K/R-XX-SIT-X-KIR; Azzi et a[., 1992). Of the 1 1 PKC isoforms, 10 are encoded at different chromosomal locations (Parker et d.,1986; Huppi et al.,1994) and, therefore, must be encoded by independent genes. T h e exceptions, PKC-PI and -PII, are the products of alternative splicing of a single RNA transcript (Coussens et al., 1987). Based on their biochemical properties, the isozymes can be classified into three major groups designated as classical calciumdependent PKCs (cPKC), novel PKCs (nPKC), and atypical PKCs (aPKC).The cPKCs-PKC-a, -@I, +II, and-y-contain all four constant regions. T h e nPKCs-PKC-6, -E, -q,and -6-lack the C2 region and have substantial differences in the length of the V2 and V3 hinge. The aPKCs-PKC-5 and -A (and its human counterpart, PKC-L)-lack not only the C2 region but also one of the two cysteine-rich motifs. PKC-k, a 115-kDa phosphoprotein, is the largest reported member of the PKC family (Johannes et al., 1994). This isozyme has been called a novel atypical PKC because, like the four nPKCs, it has two cysteine-rich sequences and lacks C2 but it is not clear yet whether PKC-p does bind to and is activated by phorbol esters. The pseudosubstrate region, a short sequence at the N terminus of the C l region, resembles a PKC substrate except for the lack of a serine or threonine to be phosphorylated. When the V3 hinge is closed, the pseudosubstrate region is thought to fold into the kinase region and inhibit the kinase activity of the enzyme (Pears and Parker, 1991). Three observations provide support for this notion: (1) peptides with this sequence inhibit PKC activity; (2) if serine is substituted for Ala 25, the peptide becomes an excellent substrate for all PKC isoforms (House and Kenip, 1987);and (3) a mutation in the pseudosubstrate region leads to a higher constitutive activity of PKC-a (Pears et al., 1990). €3. ACTIVATION OF PKC KINASEACTIVITY AND
TRANSLOCATION TO MEMBRANES Acidic phospholipids, commonly phosphatidylserine (PS), are essential for PKC activation. However, the cPKCs require Ca2+ as well for full
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enzymatic activity. Although PS can fully activate PKC, under physiological conditions an additional requirement for complete activation of the cPKCs and the nPKCs is membrane-bound DAG. Phorboi esters can substitute for DAG and activate all the PKC isoforms except the aPKCs. DAG and phorbol esters are thought to up-regulate the activity by binding to the regulatory domain, which induces the unfolding of the enzyme and exposes the catalytic sites. Exactly how the aPKCs are activated has not been critically assessed. However, PKC activity is enhanced following treatment with arachidonic acid (Wang et al., 1993) or a combination of gamma linolenic acid and PS (Kochs et al., 1993) or phosphatidylinositol-3,4,5-trisphosphate(PIP,) (Nakanishi et al., 1993). 1. PKC Activation by Physiologacal Molecules
Initially investigators thought that the major sources of PKC-activating DAG were inositol phospholipids (PI), which are hydrolyzed by phospholipase C (PLC) to inositol triphosphate (IP,) and DAG. Recent data suggest that PI turnover is only one of several sources for DAG, and that hydrolysis of phosphatidylcholine is also an important physiological source of DAG. For a more detailed review of PKC-activating phospholipid hydrolysis, see Nishizuka (199213). 2 . PKC Activation by Phorbol Esters The interaction with membrane-bound DAG can be obviated by various phorbol esters such as 12-O-tetradecanoyl phorbol- 13-acetate (TPA), which is also known as phorbol-12-myristate- 13-acetate (PMA). Most phorbol esters are potent and direct activators of PKCS kinase activity, even in the absence of CA2+. The mechanism by which TPA activates PKC is similar to that of DAG; these molecules are believed to compete for the same binding sites (Sharkey and Blumberg, 1985). A more detailed review of the modes of PKC activation by lipids is given by Bell and Burns (1991). 3. PKC Translocation to Membranes
In resting cells, PKC is predominantly localized in an inactive form in the cytosol. On activation in vivo, most PKCs translocate from the cytosol to cellular membranes, resulting in their appearance in insoluble fractions generated by homogenization and centrifugation (Gopalakrishna et al., 1986). Translocation from soluble cytoplasmic fractions to insoluble membrane fractions is essentially complete following activation by phorbol esters. More physiological activation of PKC by stimuli that generate DAG, on the other hand, typically leads to only partial
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translocation, suggesting that these physiological signals may activate only a subset of the PKC isoforms. 4 . Other Consaderatzolzs 'This simplified model of PKC activation becomes more complicated upon closer examination. 1. Other activators and modulators of PKC kinase activity have been described including unsaturated fatty acids (Murakami and Routtenberg, 1985; Shinomura et al., 199l), phosphatidylinositol 4,5-diphosphate (O'Brian et al., 198'7).mezerein (Kiley et al., 1992; and Kazanietz et al., 1993), bryostatin (Kraft et al., 1986). The different activators and modulators used to stimulate PKCs have been shown to alter PKC substrate specificity 112 uitro and in uivo (Kreutter et al., 1985; Omri et al., 1987; Ways P t al., 1987; Robinson, 1992b). Robinson (1992a) suggested that PKC substrates could be subdivided into at least three groups: (1) substrates that are phosphorylated maximally only in the presence of Ca2+ and PS, for example, histone and casein; (2) substrates that are phosphorylated to a greater extent in the presence of PS and ' U A , for example, the very abundant PKC substrate (MARCKS) rnyristoylated alanine-rich C kinase substrate (see subsequent discussion) and myelin basic protein (MBP); and (3) substrates that are phosphorylated in an activatorindependent manner, for example, protamine. In one study, Leventhal and Bertics (1'393) demonstrated that PKC is activated by poly-Larginine and that this activation is independent of any additional cofactor. Hence, arginine-rich proteins such as several of the cytoskeletal proteins may be able to activate PKC directly without Ca*+ or phospholipids. 2. PKC translocation that is induced by phorbol ester stimulation is a two-step process (Bazzi and Nelsestuen, 1991; Nelsestuen and Bazzi, 1991; KaLanietz Pf a/., 1992). T h e first step seems to be dependent on the presence of low concentrations of Cay+ for the cPKCs and can be reversed on Ca2+ chelation. T h e second, an irreversible step, confers on PKC the characteristics of an integral membrane protein. In this stage, PKC is constitutively activated, and its kinase activity is independent of activators such as Ca2+ o r phorbol esters. 3. In contrast to stimulation by the phorbol esters, stimulation by DAG (undoubtedly more physiological) leads to a transient and shortlived activation and translocation of PKC (Drust and Martin, 1985). 4. Activation by another class of compounds, the bryostatins, is different still. These PKC activators aroused interest when they demonstrated antineoplastic activity toward the P388 mouse leukemia cell line. They are currently being evaluated as candidate chemotherapeutic
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agents (Blumberg and Pettit, 1992). Bryostatin compounds bind to PKC with subnanomolar affinity (DeVries et al., 1988; SzalIasi et al., 1993). However, this form of PKC activation induces only a subset of the responses induced by TPA in some cell types. Several studies have shown that in certain biological systems in which TPA acts as a differentiating agent, bryostatin not only lacks this activity but even blocks the effects of TPA. For example, bryostatin-1 fails to induce differentiation in HL-60 promyelocytes and inhibits TPA-induced differentiation in HL-60 cells, erythroleukemia cells, and primary mouse keratinocytes (Kraft et al., 1986, 1988; Berkow et al., 1993; Szallasi et al., 1993). Similar divergent effects of these two PKC activators were observed in differentiation of K562, a human erythroleukemia cell line (Hocevar et al., 1992), in which TPA induces differentiation whereas bryostatin-1 blocks the effects of TPA. Furthermore, these divergent responses in K562 cells corresponded to differential activation of two isoforms, PKC-a and PKC-PII, in response to TPA and bryostatin- 1. TPA activation caused translocation of PKC-a to the plasma membrane, whereas PKC-PI1 remained cytosolic. Bryostatin activation translocated both PKC-a and -PI1 to the plasma membrane and PKC-PI1 to the nuclear membrane. Szallasi et al. (1993) provide evidence for differential regulation of PKC-a, -6, and -E by bryostatin-1 in NIH 3T3 cells, whereas TPA distinguished only weakly among these isozymes. Furthermore, these authors extended the finding that TPA and bryostatin regulated PKC by very different mechanisms by demonstrating a biphasic dose response for down-regulation of PKC-S and inhibition of down-regulation of this isoform by TPA at high bryostatin concentrations. 5. Although some data on PKC activation frequently appear contradictory, the discord may be explained by the different cell types, different activators, and variable conditions used in the assays reported by different laboratories. All these reports underscore the need for standardized PKC assays that would enable one to compare the different data reported in the literature on PKC isozymes and activity. C.
DOWN-REGULATION
1 . Consequence of Prolonged Stimulation In addition to translocation of most PKCs from the cytosol in resting cells to insoluble membranous structures during stimulation, activation of PKC also leads to an increased sensitivity of the enzyme to proteolytic degradation. Treatment of cells with TPA for 18 hr (overnight) often results in the complete disappearance of immunologically detectable
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PKC. This phenomenon is frequently referred to as down-regulation. Proteolytic down-regulation appears to be a consequence of an altered PKC conformation induced by kinase activation. This change probably takes the form of an opening up of the molecule at the V 3 hinge, which occurs when PKC binds TPA in solution or DAG in membranes. Proteolysis by the calcium-dependent proteases calpains I and I1 has been shown to yield two PKC fragments: (1) the regulatory domain and (2) the catalytic domain. T h e latter, a -50-kDa phospholipid- and calcium-independent kinase sometimes referred to as M kinase or PKM, can frequently be detected in the cytoplasm after prolonged TPA treatment (Kishimoto et al., 1983). PKM may be only a degradation product without any function, although Pollock and Snow (1991) showed by in uitro phosphorylation assays that PKM may be involved in a signaling pathway that leads to c-myc induction in B lymphocytes. The fate of the regulatory domain after proteolytic cleavage of PKC is unknown. Further studies on the consequences, if any, of the proteolytic fragments are in order. 2. Crzt ique
Prolonged (overnight) treatment of cells with TPA has been widely accepted as a means of complete down-regulation of PKC and, by definition, elimination of PKC from any processes that are still functional after this treatment. For several reasons, this is a potentially misleading overinterpretation. (1) Usually this method of down-regulation does not lead to the complete absence of PKC activity. Certainly for some isoLymes immunologically detectable amounts are eliminated, but this test is relatively insensitive and is critically dependent on the availability of isozyme-specific antibodies for all isoforms. (2) PKC mRNA is, in general, still present (Rose-John et al., 1988) unless translation is inhibited; thus PKC protein can be synthesized continuously. (3) T h e efficiency of down-regulation is isozyme, cell, and activator dependent. (Blackshear et al., 1991; Arnold P t al., 1993). In hematopoietic cells, PKC-a, -p, -y, -E, and -q generally seem to be quite susceptible to down-regulation by TPA treatment, whereas while PKC-6 and -2; seem to be less affected by this maneuver (Mischak et al., 1993b). The PKC-2; results are not surprising, since PKC-2; does not bind TPA and, hence, should neither be activated nor down-regulated by phorbol esters. These data suggest that a reevaluation is needed for the experiments that ruled out a role for PKC in certain signal transduction pathways based solely on evidence derived following TPA-induced down-regulation. More specific down-regulation of PKC isozyrnes by other approaches may now be possible, since PKCspecific inhibitors are available and isozyme-specific antisense oligonu-
PKC I N DIFFERENTIATION AND TRANSFORMATION
cleotides have been shown to be effective for PKC-a,
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+,and -E (Farese et
at., 1991; Baxter et al., 1992; Murray et al., 1993).
3 . Use of Specific Inhibitors A more reliable approach to eliminating PKC activity would be the use of PKC-specific inhibitors. Early attempts at PKC inhibition used inhibitors such as staurosporine or l-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), which proved to be cytotoxic and largely nonselective since they inhibit PKC through binding to the catalytic domain and therefore inhibit other kinases with similar catalytic domains (Hidaka et al., 1984). More recent reports, however, suggest that bisindolylmaleimides such as GF 109203X (Toullec et al., 1991) exhibit very high PKC specificity and very low toxicity. These inhibitors do not show any cytotoxic effects on mouse hematopoietic cells, keratinocytes, or fibroblasts and, with the exception of PKC-%,seem to inhibit all PKC isozymes completely at concentrations between 1 and 10 pM (Martiny-Baron et al., 1993; Mischak et al., 1993b). Calphostin C, a light-dependent kinase inhibitor, interacts with the regulatory domain. This inhibitor inhibits (PDBu) phorbol dibutyrate binding and inhibits PKC-a, -p, and -y, but it is somewhat cytotoxic to human tumor cells (Bruns et al., 1991). Another inhibitor, chelerythrine chloride, seems to have a high specificity for PKC but unfortunately is quite toxic to most cells (Herbert et al., 1990). To elucidate the physiological roles of the PKC isozymes fully, much effort needs to be expended to find PKC inhibitors that have high potency, isozyme specificity, and low cytotoxicity. D. INDUCTION OF EARLY GENES
TPA treatment of cells results in expression of a specific set of transcriptionally induced genes including c-fos, cjun, collagenase, and metallothionein IIA (Treisman, 1986; Angel et al., 1987, 1988; Imbra and Karin, 1987; Hass et al., 1991a,b),as well as a number of other genes that encode proteins involved in differentiation of monocytes, including c-fm (Sariban et al., 1985), platelet-derived growth factor (PDGF) (Sariban and Kufe, 1988), and tumor necrosis factor (TNF) (Horiguchi et al., 1989). Some of these TPA-responsive genes are known to share a common cis-acting element referred to as the TPA-response element (TRE), which binds the fos/jun AP- 1 transcription factor heterodimer (Angel et al., 1987; Chiu et al., 1988; Ryder et al., 1988; Hirai et al., 1989; Zerial et al., 1989). Hata et al. (1993) demonstrated that overexpression of PKC-a, +II, or -E in 3Y1 rat fibroblast cells enhanced transcriptional activation [as seen by increased levels of TPA-responsive chloramphenicol
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acetyltransferase (CAT) expression through the TRE as well as expression of the c-jun gene, which contains a TRE in its promoter region (Halazonetis et at., 1988). PKC-y, however, only induced transcriptional activation of the serum response element (SRE), another kind of TPAresponsive gene, which binds the serum response factor (Treisman, 1987; Norman et at., 1988; Schonthal, 1990). Further, these researchers showed that overexpression of PKC-a, +XI, -y, and -E enhanced TPA induction of TRE-binding nuclear factors, suggesting that transcriptional activation through TRE requires an additional step that is not activated by PKC-y. The differences among PKC family members in the enhancement of the TPA response may be mediated, in part, by alterations in gene expression through the TRE and SRE, structural differences (PKC-y has a unique 5-amino-acid-shorter C3 region), or differences in subcellular localization of individual PKC isoforms. OF PHYSIOLOGICAL E. TRANSDUCTION O N PKC ACTIVATION STIMULI
1, insulin
T h e importance of PKC in insulin signaling, especially in insulinstimulated glucose transport, has been controversial in recent years. Each of the opposing models, one that implicates PKC in transduction of insulin signals (Cherqui et al., 1990; Egan et al., 1990; Heidenreich et al., 1990; Messina et al., 1992) and the other that denies a critical role for PKC (Blackshear et al., 1991), has supportive data. Unfortunately, the experiments are not directly comparable; therefore, they need not be considered contradictory. To evaluate the role of PKC in insulin signaling, Blackshear ei al. (1991) investigated insulin-stimulated MARCKS phosphorylation and found only a weak increase in one of the five different cell lines tested. Additional evidence against PKC’s participation in transduction of insulin signals was that TPA-induced down-regulation of PKC did not influence insulin signaling as measured by S6 kinase activation. Raf- 1 phosphorylation, induction of ornithine decarboxylase (ODC), and induction of c-fos. As mentioned earlier, TPA treatment does not reliably deplete all PKC isoforms, and these experiments did not employ specific PKC inhibitors to accomplish this goal. The opposing conclusion, namely that PKC was important in insulin action, was supported by evidence that PKC was activated by insulin stimulation of BCSH-1 myocytes (Farese et al., 1989; Cooper et al., 1990). These authors also demonstrated that insulin-stimulated glucose transport was dependent on PKC, since it could be prevented by specific PKC inhibi-
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tors and by antisense oligonucleotides against PKC-a and -p. Further, glucose transport could be restored by reconstitution of cells with purified PKC. To resolve the discordant results, direct experimental approaches must be carried out on identical cell lines in the different laboratories. Farese et al. (1992) determined the effects of insulin and TPA on the subcellular distribution of immunoreactive PKC-a, -p, -6, -6, and -5 in rat adipocytes. TPA induced complete translocation of PKC-a to the plasma membrane, but was less effective in inducing translocation of PKC-E or -5. Both insulin and TPA translocated PKC-P to the endoplasmic reticulum; however, only insulin could translocate PKC-P to the plasma membrane. These finding indicate that insulin and TPA have overlapping as well as distinct effects on subcellular redistribution of specific PKC isozymes in rat adipocytes, which may help explain the similarities and differences in the effects that these two agonists have on biological processes. 2. Other Growth Factors
T h e series of proliferation and differentiation steps that hematopoietic cells undergo are governed, in part, by the interaction of immature cells with stroma cells in the bone marrow or thymic epithelium (Fearon et al., 1986; Sawyers et al., 1991) and by the presence of cytokines such as interleukin (1L)-2, IL-3, macrophage colony-stimulating factor (M-CSF), granulocyte CSF (G-CSF), and granulocyte-macrophage CSF (GM-CSF) (Devalia et al., 1992; Nishimura et al., 1992). Signaling by these growth factors also may be mediated by PKC. Whetton et al. (1988) showed that IL-3 stimulation of hematopoietic cells activates PKC; Imamura et al. (1990) demonstrated that M-CSF activates PKC in monocytes. Further, Nishimura et al. (1992) reported that GM-CSF has a stimulatory effect on PI turnover, leading initially to PKC activation in HL-60 cells and subsequently to cell differentiation. Unfortunately, most of these reports are almost exclusively descriptive, and any biological relevance of PKC activation via these growth factors must be further explored. In fact, data suggest that PKC activation may not be essential for growth factor signaling. For example, overexpression of PKC isozymes in different IL-3-dependent cell lines could not abrogate factor dependence, that is, high amounts of activated PKC do not influence the sensitivity of the examined cell lines to IL-3 deprivation, nor do they lower the amounts of IL-3 required for cell survival (Kraft et al., 1990; Mischak et al., 1993c). A direct way to perform experiments that would enable one to answer the question of whether PKC activation is, in fact, necessary for signaling
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would be to use specific PKC inhibitors. To test the involvement of PKC in CSF- l-induced macrophage differentiation, we examined 32D cells that overexpress either PKC-6 (32D-PKC-6; Mischak et al., 1993c) or CSF-1 receptor (32D-CSF-1R; Pierce et al., 1990). Each cell line can be induced to differentiate into macrophages by treatment with TPA or CSF-1, respectively. Using the PKC inhibitor GF 109203X, w e could completely block the TPA-induced differentiation of 32D-PKC-6, but the inhibitor had no effect on CSF-l-induced differentiation of 32DCSF-1R (H. Mischak, unpublished data). These results suggest that, although CSF-1 might activate PKC, this activation is not essential for CSF-l-induced myeloid differentiation. F. DIFFERENTIAL EXPRESSION A N D SUBCELLULAR OF PKC ISOZYMES DISTRIBCTION Extensive surveys of different cell lines and tissues have revealed that the members of the PKC gene family are differentially expressed. The pattern of isozyme expression not only varies among different cell types, but quite frequently also changes during differentiation. Many of these studies have been compromised by the lack of isozyme-specific antibodies and cDNA probes. Antibodies that were thought to be specific for one isozyme often cross-reacted with other PKCs as well as with other unrelated proteins. This cross-reactivity sometimes was not appreciated until additional isoforms were isolated and purified. Thus, data on PKC isozyme distribution that rely exclusively on the use of isoform-specific antisera must be interpreted with caution. In general, more reliable results can be obtained using cDNA probes on Northern blots. However, cDNA probes are not without their own specificity problems. To be completely reliable, mRNA expression studies should be supported by Western blot analysis and vice versa. Since a complete summary of PKC isoform expression in normal cells and tissues is beyond the scope of this chapter, we refer the interested reader to various published reports (Sposi et al., 1989; Mischak et al., 1991b; Dlugosz et al., 1992; Osada et al., 1993). Although it is difficult to generalize about PKC isozyme expression, brain is usually the organ with the highest levels of mRNA and protein for most PKC isotypes. Some tissue-specific patterns of isozyme expression in other organs seem to be emerging. Of the cPKCs, PKC-a seems to be the most widely expressed isoform, and can be detected in most but not all cells [it is absent in most mouse myeloid cell lines (Mischak et al., 199lb)l. In contrast, expression of PKC-y seems to be restricted to the brain. Although PKC-y expression has been described in other tissues
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(Strulovici et al., 1989; Nishikawa et al., 1990), these reports relied on the use of antibodies that may have been cross-reactive and therefore must be interpreted with caution. The most widely expressed nPKC is PKC-6 (Mischak et al., 1991a), which has been found in high quantities in almost every tissue examined to date. PKC-E and -g, although not totally restricted to the brain, are expressed at highest levels there, but these isoforms have also been detected at low levels in some other tissues (Goodnight et al., 1992; Wetsel et al., 1992). The data on PKC-q and -0 are quite limited to date. High expression of PKC-q has been observed in skin and T cells (Osada et al., 1990; Mischak et al., 1991b), whereas PKC-0 is highly expressed in skeletal muscle and T cells (Osada et al., 1992; Mischak et al., 1993a). The consistent finding that most tissues contain more than one PKC isoform and that the pattern of expression is tissue specific implies that different PKC isozymes fulfill different function in vivo. Unfortunately very few results have identified the specific functions that are served by particular isozymes. Extrapolation of tissue-specific expression patterns to surmise specific biological effects that can be attributed to individual members of the PKC gene family is complicated by the fact that several different isozymes are often coexpressed in a single cell type. One cannot simply attribute PKC-dependent effects observed in a particular cell line to the most abundant isozyme; instead, all the enzymes present must be considered. This task could be somewhat eased as isozyme-specific activators and inhibitors become available. Initial reports have suggested that thymeleatoxin specifically activates PKC-a, -PI, and -7 but not PKC-6 and -E (Ryves et al., 1991) and that -6976 specifically inhibits PKC-a, -P and -y (Martiny-Baron et al., 1993). T h e usefulness of these activators and inhibitors remains to be determined in different cell systems. G. PKC SUBSTRATES T h e physiological targets that are naturally phosphorylated by each of the PKC isozymes are of considerable interest in understanding how this large family of protein kinases functions. A large number of proteins have been shown to be phosphorylated in uitro by PKC isoforms. Only a few proteins have been demonstrated by in vivo studies to be physiological substrates for PKC. To avoid naming hundreds of proteins that have been described to be phosphorylated by PKC, we have concentrated on substrates that have been shown to be phosphorylated by PKC in vivo for which phosphorylation has been shown to have some biological effects: (1) MARCKS and
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actin; (2) cytoskeletal proteins; (3) receptors; and (4) intracellular signal transducers. 1 . MARCKS and Actin
On activation of PKC, phosphorylation of an 80 to 87-kDa protein becomes prominent in several studies. This protein has been called MARCKS: myristoylated alanine-rich C kinase substrate. More detailed reviews of MARCKS and the related proteins MacMARCKS (F52 or MRP), neurogranin, and GAP-43 (neuromodulin) have been published by Adereni (1992) and Blackshear (1993). These proteins seem to be quite similar with respect to structure and function; hence, only the best described member of this family, MARCKS, will be discussed in any detail. MARCKS protein has been dest:ribetf and its cDNA cloned from mouse, rat, human, cow, and chicken (Aderem, 1992; Blackshear, 1993). cDNA cloning and sequencing revealed that the expected molecular weight of the protein is between 27 and 32 kDa. The anomalously high molecular weight of 80-87 kDa exhibited on electrophoresis on SDS gels seems to be due to the rigid and rod-like shape of the protein. This protein is very acidic (calculated PIS are between 4.1 and 4.5), but it is also notable for its very high alanine content and a very basic 25-aminoacid domain that represents the site of PKC phosphorylation. The dephosphorylated form of MARCKS has a high Caz+-dependent affinity (Kd = 2-5 nM) for calmodulin (Mcllroy ef al., 1991). The calmodulinbinding site seems to be identical to the phosphorylation site because, on phosphorylation, the affinity of MARCKS for calmodulin decreases about 200-fold. Researchers have suggested, but not entirely proven, that MARCKS serves as a carrier or reservoir for calmodulin (Blackshear, 1993), which is released on PKC activation. The same domain of MAKCKS that binds calrnodulin also interacts with actin. Hartwig et al. (1992) demonstrated that dephosphorylated MARCKS binds to and cross-links actin in vitru. These cross-linking data suggest that unphosphorylated MARCKS may dimerize with itself or may have t w o actin-binding sites. On phosphorylation, MARCKS still binds actin, but with reduced affinity, and is unable to cross-link actin. These results are in accordance with studies reported earlier on the localization of MARCKS Zn u i m Rosen et al. (1990) reported that MARCKS in mouse macrophages colocalizes with vinculin and talin. On activation of PKC, MARCKS is released from the plasma membrane and the typical punctate staining pattern disappears. As Hartwig et al. (1992) also demonstrated, binding of MARCKS to actin can also be competed with Caz+-calmodulin, as expected from the fact that both ligands bind to
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the same domain. These data have generated speculation that MARCKS serves as a mediator for PKC-controlled cell motility and secretion. 2 . Other Cytoskeletul Proteins Other cytoskeletal proteins that are phosphorylated by PKC include lamin B, vinculin, and tau (Werth et al., 1983; Cooper et al., 1989). As described in a study by Hocevar et al. (1993), activation of PKC-PI1 by bryostatin in HL-60 cells leads to its translocation to the nucleus, followed by nuclear envelope breakdown. Further, in vitro experiments with purified activated PKC-PI1 and isolated nuclear envelopes demonstrated that phosphorylation of lamin B leads to solubilization of the nuclear envelope protein, an indication that phosphorylation by PKC can induce nuclear envelope breakdown. Correas at al. (1992) showed that PKC, as well as other kinases, phosphorylate the microtubule-associated tau proteins. Not too surprisingly, the sites phosphorylated by the different kinases are not identical. The sites phosphorylated by PKC could be located within the tubulin-binding domain. Since PKC phosphorylates a synthetic peptide that corresponds to this region, and such phosphorylation reduces the capacity of the peptide to promote tubulin assembly, the authors speculate that PKC might be involved in microtubule destabilization secondary to agents such as neurotransmitters, hence implicating PKC in very complex mechanisms such as rearrangements of neural connections. This suggestion is also supported by observations made by Kose et al. (1990), who found that PKC colocalized with microtubules in pyramidal neurons.
3. Receptors Several proteins involved in signal transduction have been shown to be substrates for PKC. Hunter et al. (1984) and Downward et al. (1985) reported that PKC phosphorylates the EGF receptor on Ser 654, inducing its internalization and, hence, down-regulating the receptor. However, an EGF receptor mutant (Ser 654 to alanine did not influence down-regulation (Morrison et ul., 1993). Therefore, the critical site within the EGF receptor that is responsible for down-regulation by phosphorylation still must be mapped. More recently, the PKC phosphorylation sites of the human insulin receptor were described by two independent groups (Ahn et al., 1993; Chin et al., 1993) using in vivo or in vitro studies, respectively. Some of the phosphorylation sites identified in the two studies differ, probably because of differential accessibility of the substrate in vitro and in vivo. Chin et al. (1993) suggested that the physiological consequence of insulin receptor phosphorylation by PKC might be a decreased signaling
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capacity. This suggestion is based on the observation that the kinase activity of the receptor remains unchanged after PKC phosphorylation, but its ability to phosphorylate tyrosines on PI3 kinase is reduced. Several of the G protein-coupled receptors are phosphorylated by PKC (Houslay, 199 1). Phosphorylation of the acetylcholine receptor by PKC (Hopfield et al., 1988) results in an increased rate of desensitization. Other related membrane proteins that are phosphorylated by PKC under physiological conditions include the photoreceptor (Hardie et al., 1993) and transporters such as the N a + / K + transporter and the Ca2+ATPase (Wang et al., 1991). In the latter, the calmodulin-binding domain is at least one target of PKC phosphorylation, leading to either inhibition or stimulation of CaZ+-ATPase activity, depending on the source of YKC. Phosphorylation of calmodulin-binding domains, which results in modulation or inhibition of calmodulin binding and, hence, the modulation of activation of the targeted calmodulin-binding proteins by PKC, seems to be evolving as a common phenomenon. Waxham and Aronowski (1993) reported that PKC also phosphorylates CaM kinase at T h r 286. Although this phosphorylation does not appear to result in any changes of the Ca2+-calmodulin requirement of CaM kinase, it is still possible that the phosphorylation modulates CaM kinase activity or its cofactor requirements. 4 . Intracellular Signal Transducers
Several signal transduction pathways in which PKC is involved have been described. T h e first report of a direct link between PKC activation and nuclear signaling came from Goode et al. (1992). These authors demonstrated in vitro that PKC-a, -PI, and -y efficiently phosphorylated glycogen synthase kinase-3P (GSK-3P), inactivating it and thereby precluding GSK-3P from inactivating c-jun by phosphorylation, permitting c-jm to dimerize with c-fos and form an active AP-1 transcription activator. PKC-PI1 was much less efficient and PKC-E did not phosphorylate GSK-3P at all. This report was also the first of a PKC substrate that was phosphorylated by only a limited number of PKC isozymes. These experiments must be repeated in uiuo to establish their relevance to physiological situations. A second signal transduction pathway activated by PKC has been delineated more recently. Raf-1, which activates MAP kinase kinase, was found to be activated by phosphorylation by PKC. Sozeri et al. (1992) showed, using baculovirus-synthesized recombinant proteins, that phosphorylation of Raf-1 by PKC activates the kinase activity of Raf-1. A more detailed study (Kolch et al., 1993) demonstrated that Raf-1 activation occurs when PKC-a phosphorylates Ser 499 and Ser 259, both in
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vitro and in vivo. This pathway has ramifications for carcinogenesis because overexpression of both PKC-a and Raf-1 in NIH 3T3 fibroblasts permits anchorage-independent growth, a sign of malignant transformation, whereas overexpression of either gene alone does not produce this effect. Several other molecules that are known to be involved in signal transduction have been reported to be PKC substrates. Unfortunately, however, the physiological relevance of all these phosphorylations has not yet been critically assessed. Baudier et al. (1992) found that p53, an anti-oncogene that is also an important molecule in signal transduction, is a PKC substrate. Preliminary data suggest that the PKC phosphorylation site is within the region of p53 that is important in oligomerization, nuclear transport, and DNA binding. This region shows substantial homology to MARCKS. These authors also showed that SlOOb, a member of the S 100 family of proteins that is involved in cell cycle progression, binds to the MARCKS-like region of p53 and to MARCKS itself. This binding inhibits both PKC phosphorylation and p53 oligomerization. This interaction is a potentially important regulatory mechanism, analogous in several ways to the MARCKS-calmodulin interaction. H. IDENTIFICATION OF ADDITIONAL SUBSTRATES
A very promising approach to identifying additional new PKC substrates was published by Chapline et al. (1993). These authors screened a cDNA expression library by overlaying filter lifts of the cDNA plaques with partially purified PKC obtained from rat brain. After visualization of PKC-a bound to the filter lifts using an isozyme-specific monoclonal antibody, cDNA clones that encoded at least two different PKC substrates were identified. Although this method is limited to PKC substrates that stably interact with PKC, it might provide a promising approach for rapid screening for PKC substrates in different tissues. I. PKC-ASSOCIATED PROTEINS
As described extensively already, PKC translocates to insoluble membranous structures within the cell on activation of its kinase activity. In vitro data suggest that PKC more or less randomly integrates into lipid bilayers. This result is in contrast to observations made on the translocation of PKC in vivo, in which accumulation of PKC at distinct structure, particularly the cytoskeleton, has been observed. Mochly-Rosen et al. (1991b) therefore postulated the existence of receptors for activated C
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kinases (RACKs) that should have the following properties: (1) be present in the particulate fraction, (2) bind in an activator-dependent manner, and (3) bind saturably and specifically. Mochly-Rosen et al. (1991a) could identify at least two, and in some experiments up to five, proteins that fit all these criteria. Since binding of PKC to the RACKs was not inhibited by PKC substrates, evidently a site other than the substratebinding site is responsible for this interaction. These data might also shed new light on the substrate specificity of PKC in uivo. In uitro both PKC and substrate are in close proximity, but this might not be the case in 7 1 i v 0 , since different PKC isozymes could interact with different RACKs and, hence, be located in different subcellular compartments. II. PKC lsoforms Involved in Differentiation
PKC has been shown to play a role in differentiation that can be induced in several cell culture systems (Nishizuka, 1986; Berry and Nishimka, 1990; Hashimoto uf al., 1990; McSwine-Kennick et al., 1991), but data implicating PKC in differentiation that occurs zn uzuv are scarce. 'The earliest studies were mostly based on observations that phorbol esters can induce differentiation of diverse cell types in 7&0. Researchers presumed that these effects were mediated by PKC, but few details are known about the mechanisms involved. Furthermore, the discovery of multiple PKC isozymes has contributed more complexity and confusion than revelation. Since most cells express multiple PKC isoforms that may be individually regulated during differentiation, it has been difficult to discern a coherent picture of PKC's role in differentiation. T h e studies have, in general, employed one of three strategies: ( 1) monitoring the changes in expression, subcellular distribution, or activity of PKC isoforms during chemically induced differentiation; (2) generating cell variants that are refractory to these differentiation signals, followed by comparative analysis of PKC expression; and (3) examining the changes in differentiating systems that accompany overexpression of distinct PKC isoforms. These investigations have been predominantly descriptive in nature, and sometimes yielded contradictory results, iiiost likely because of clonal variation in cell lines and methodological differences. Resolution of these discrepancies must await more mechanistic studies that utilize overexpression of distinct PKC isozymes andlor analysis of PKC in lower organisms, such as yeast or Drosophzla, which are amenable to genetic manipulation. The following discussion of differentiation in a selection of cell systems, therefore, is not intended to be exhaustive, but is intended to provide the reader with an introductory survey of this controversial field.
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A. MYELOIDDIFFERENTIATION 1. HL-60
HL-60, a human promyelocytic cell line, has been a useful and very widely used model for studying mechanisms of cell differentiation. These cells can be induced to differentiate terminally either to a neutrophil phenotype with retinoic acid (RA) (Breitman et al., 1980) or dimethylsulfoxide (DMSO) (Collins et al., 1978) or to a monocytic or macrophage phenotype with vitamin D, (McCarthy et al., 1983). Activators of PKC such as TPA, teleocidin, and dioctanoylglycerol also induce HL-60 cells to differentiate, but only along the monocyte/macrophage lineage (Castagna et al., 1982; Ebeling et al., 1985; Homma et al., 1988). Predictably, several PKC inhibitors are able to suppress induction of macrophage-like differentiation of HL-60 (Feuerstein et al., 1984; Merrill and Stevens, 1989; Lee et al., 1991; Xun et al., 1991). Thus PKC is thought to play a central role in regulating the TPA-induced differentiation into macrophages, and one or more PKC isozymes may also take part in myeloid differentiation induced in HL-60 by other agents. Whereas several earlier studies suggested that, after initial activation by TPA, PKC is down-regulated during differentiation (Zylber-Katz and Glazer, 1985; Homma et al., 1986; Perrella et al., 1986; Shoji et al., 1986), more recent studies showed that sustained activation (Aihara et al., 1991) o r prolonged up-regulation (Edashige et al., 1992) of PKC, particularly of the a isoform, correlates with TPA-induced differentiation of HL-60 cells into macrophages. PKC appears to play a critical role in both the proliferation and the differentiation that are sequentially induced in HL-60 by TPA. On the other hand, RA- or DMSO-induced differentiation of HL-60 cells into neutrophils does not seem to be directly linked to PKC (Durham et al., 1985; Zylber-Katz and Glazer, 1985). Therefore, this event will not be discussed exhaustively in this chapter. However, note that RA increases PKC activity of a and p isoforms as well as of a third unidentified isoform that is immunologically distinct from PKC-a, -p, or -y (Tanaka et al., 1992). Other attempts have been made to determine which PKC isoforms are expressed in neutrophils at different stages of differentiation, but not all the expression data are in agreement (Makowske et al., 1988; Hashimoto et al., 1990; Pontremoli et al., 1990; Stasia et al., 1990). The need for specific assays to differentiate between closely related members of the PKC family cannot be overemphasized. Devalia et al., (1992) used a highly specific RNase protection assay to quantify mRNA expression of human PKC isoforms a, PI, PII, and y to examine differential expression of these isoforms during differentiation of HL-60
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cells. These authors found that PKC-a mRNA is specifically downregulated during human neutrophil differentiation but not during monocyte differentiation. However, despite a decrease in PKC-a mRNA, PKC-a protein actually increases during early stages of RAinduced differentiation, indicating that the levels of PKC-a protein are also regulated post-transcriptionally. T h e availability of HL-60-derived cell lines that are susceptible and resistant to TPA-induced differentiation may permit determination of which PKC isozynies are involved in the signal transduction pathway that leads to TPA-induced differentiation. HL-525 and HL-534 are HL-60 variants that are resistant to TPA-induced differentiation but susceptible to other inducers of differentiation (Tonetti et al., 1992). Both these cell lines are deficient in PKC-P R N A and protein. HL-205 is another HL-60 derivative that is clonally related to HL-525 and HL-534 but, unlike either of them, is susceptible to TPA-induced differentiation like the parental line. Tonetti et al. (1992) examined the expression of PKC and early response genes and demonstrated that expression of PKC-P, and possibly a PKC-&like gene, is associated with susceptibility of these human promyelocytic leukemia cells to TPA-induced differentiation. McSwine-Kennick et al. (1991) treated HL-60 cells and another TPAresistant HL-60 variant, PR17, with 2 pA4 TPA and observed an increased rate of transcription for PKC-PI and PKC-PI1 mRNA in wildtype HL-60 cells but no change in transcriptional rates in TPA-resistant cells. Although the mechanism responsible for the increased intracellular concentrations of PKC-P mRNA in TPA-treated cells remains unclear, McSwine-Kennick and colleagues ( 1991) proposed that specific changes in levels of inRKA of PKC-P, or perhaps of other genes that are regulated by this PKC isozynie, may explain TPA-mediated induction of leukemia cell differentiation. To determine unambiguously whether resistance is due to a defect in PKC or to some other element further downstream, the different PKC isozymes must be overexpressed in resistant HL-60 cells.
2. 320 We have performed an analogous experiment on the mouse promyelocytic cell line 32D. These cells can be induced to differentiate into mature macrophages by a combination of CSF- 1 receptor overexpression and CSF-I treatment (Pierce et al., 1990), but they normally d o not differentiate in response to TPA. Overexpression of PKC-a or -6 enabled 32D cells to differentiate into mature macrophages when treated with TPA, whereas overexpression of PKC-PII, -y, -E, -q,or -5 did not
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have this effect (Mischak et al., 1993b). This result indicates that the a and/or 6 isoforms mediate TPA-induced myeloid differentiation. Although the macrophage differentiation of HL-60 cells becomes irreversible after 18-hr exposure to TPA, maturation of the PKC-or- o r -8-overexpressing 32D cells is completely reversible when TPA is removed. T h e PKC inhibitor GF109203X could completely block the TPAmediated differentiation of the PKC-b-overexpressing 32D cells, confirming the involvement of PKC in the differentiation of these cells. 3 . u-937
U-937 is another human myeloid leukemia cell line that differentiates along the monocytic lineage following TPA treatment (Lotem and Sachs, 1979; Hass et al., 1991b). This treatment also causes induction of immediate early genes of proliferation (Mitchell et al., 1985; Muller et al., 1985; Sherman et al., 1990) and expression of the differentiation antigens CD 14, CD 11, and CD 18 (Pedrinaci et al., 1990). Hass et al. ( I 99 1b) found that prolonged culturing of TPA-differentiated U-937 cells results in retrodifferentiation in which the cells become detached and resemble uninduced promyelocytic cells. This result suggests that U-937 cells also require continuous PKC activation to remain differentiated and that down-regulation, due to prolonged TPA treatment in this case, depletes the cells of required kinase activity. The PKC isoforms a,P, E, and 5 are present in U-937 cells (Ways et al., 1988), but which isoform(s) participates in this process is unknown. B. PKC
IN
B
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T LYMPHOCYTES
PKC activation by TPA has a wide array of biological effects on lymphocytic cells including inhibition of B-cell maturation into plasma cells (Hawrylowicz and Klaus, 1984; Isakson and Simpson, 1984; Marquez et al., 1989, 1991) and regulation of T-lymphocytic responses such as activation, proliferation, and differentiation (Isakov and Altman, 1985; Truneh et al., 1985; Favero et al., 1989; Kim et al., 1989). For example, increased PKC activity promotes T-cell proliferation (Kim et al., 1989) as well as CD25 and IL-2 expression in T lymphocytes (Ando et al., 1985; Isakov et al., 1985; Weiss et al., 1987; Berry et al., 1989). I . B Lymphocytes
Determining the role that second messengers play in B-cell differentiation has been complicated by the presence of contaminating T cells and monocytes in assay systems and by the stimulatory effects of other mitogens or lymphokines. Direct proof that PKC plays an important role
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in lipopolysaccharide (LPS)-induced B-cell proliferation arid differentiation has not been demonstrated. Goldstein et al. (1990) have shown, using the cloned IL-6-responsive B-cell line SKW6.4, that IL-6 does not use PKC to stimulate IgM production but is dependent on a protein kinase A-dependent pathway that is inhibitable by H7. Marquez et al. (1991) characterized the early events that occurred after activation of B lymphocytes by LPS, anti-IgM antibodies, and phorbol esters. Using cytofluoronietrir analyses of intact cells, these investigators demonstrated that PKC-PI and -PI1 are preferentially activated in response to LPS treatment, whereas anti-IgM antibodies and phorbol esters activated all PKC isozymes present in B cells. We have examined the expression of PKC-a, -p, -y, -6, -e, -5 and -q mRNA in mouse cell lines that are representative of the several stages of maturation in the B-lymphocytic lineage (Mischak et al., 1991b), and observed differential expression of the isoforms during B-cell maturation. Our data suggest that d rentiation of B cells into antibodysecreting plasma cells is associated with an increase in PKC-a and a loss of PKC-P mRN A expression. PKC-6 mRNA was abundantly expressed through all stages of B-cell differentiation, whereas PKC-E was rarely detectable. B-cell tumors of virtually all stages of differentiation express only moderate levels of PKC-q mRNA and significant levels of PKC-5 mRNA. Cohen and Rothstein (1991) studied the role of PKC in TPA stimulation of peritoneal B cells and splenic B cells, and reported that these two different lymphoid populations differed in their pattern of PKC i s o y i e protein expression and total PKC activity. Immunoblot analysis revealed that peritoneal cells expressed more PKC-a than splenic B cells. Baseline lebels of PKC activity were significantly higher in peritoneal B cells than in splenic €3 cells. Peritoneal B-cell PKC was more resistant to phorbol ester-mediated down-regulation than splenic B-cell PKC; however, measurements of PKC activity of cells treated concomitantly with cyclohexamide and PDUu indicated that the relative resistance of peritoneal B-cell PKC was not due to enhanced protein synthesis.
2. T 1 . y I l p I l O c y P J Since TPA alone is capable of inducing many differentiated functions of r lymphocytes, it is evident that PKC plays an important role in the activation of T cells (Cantrell et a/., 1985; Imboden and Stobo, 1985; Truneh et al., 1985; Acres et al., 1986; Shackelford and Trowbridge, 1986; M’eiss et nl., 1986; Krangel, 1987; Manger et al., 1987; Abraham et a/., 1988). We refer to Berry and Nishizuka (1990) for a more extensive consideration of PKC and T-cell activation. I n this chapter, we present a
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brief summary of the main events during T-cell activation, but more emphasis will be placed on summarizing recent reports concerning differential regulation and expression of the known PKC isozymes in T lymphocytes, since isozyme-specific reagents have only recently become available. Even before isoform-specific cDNAs and antibodies were available, it appeared likely that PKC played a role in some aspects of T-cell activation. More recent studies have attempted to determine which isoforms are involved in this process. Berry et al. (1989) showed by immunocytochemical staining of a population of human T cells (>99% pure) that, on TPA treatment, PKC-a, +I, and -PI1 are redistributed to discrete focal areas within the cell membrane whereas untreated cells showed diffuse cytoplasmic staining throughout the cell, with a more intense staining at the nuclear periphery. Freire-Moar et al. (199 1) studied mRNA expression of the a, P, y, 6, E, and { isoforms in mouse thymocytes and detected, by message amplification phenotyping (MAPE, and {, but not PKC-y or -6, mRNAs are exPing), that PKC-a, pressed in all unstimulated thymocyte populations. Although it was not possible to correlate the pattern of PKC isozyme expression with thymocyte phenotype, levels of mRNA expression of certain PKC isoforms were uniquely regulated by different stimuli. Clearly different isozymes and isozyme-specific substrates are differentially expressed and differentially activated in T cells. Northern blots were used to study the steady-state mRNA levels of eight PKC isoforms in mouse T cells and T-cell lines; these investigators detected mRNA for PKC-a, -p, -6, -5, -q,and -8 (Mischak et al., 1991b, 1993a). PKC-q was the most abundantly expressed isoform in T cells. PKC-8 expression is also very high in T-lymphocytic cell lines and normal lymph node T lymphocytes. PKC-6 is expressed in all T cells, and PKC-P is expressed as a pattern of different-sized transcripts, ranging in size from 10 kb to 2.5 kb (Mischak et al., 1991b). Although these data confirm and extend the findings of Koretzky et al. (1989), they did not confirm reports (FreireMoar et al., 1991; Strulovici et al., 1991) that PKC-E is a major PKC isoform in thymocytes. This discrepancy could be due to cross-reactivity of putative PKC-E antisera with PKC-q in the latter reports, since the protein sequences of these two isoforms are similar and PKC-q is abundantly expressed in thymus, normal T cells, and T-cell lines. Determining which isoforms are critical to T-cell function is not yet definitive. Koretzky et al. (1989) used PKC-P-negative T-cell lines to show that the expression of PKC-f3 is not required for certain T-cell functions such as TPA-induced CD-3 or CD-4 internalization, IL-2 production, or IL-2R expression. Although PKC-6 mRNA was not detected
+,
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in unstimulated thymocytes, expression of this isoform is induced by exposure of the cells to TPA or concanavalin A (Freire-Moar et al., 1991). Calcium ionophore has been shown to block the TPA-induced upregulation of PKC-6 expression, suggesting (1) that activation of one PKC isozyme can control expression of another isozyme and (2) that intracellular Ca2+ may regulate PKC-6 expression (Freire-Moar et al., 1991). C.
KEHATIKOCYTE
DIFFERENTIATION
Substantial evidence implicates PKC in the signal transduction pathway during differentiation of keratinocytes, the most prevalent cell type in epidermis (Toftgard et al., 1985; Dlugosz and Yuspa, 1991, 1993; Dlugosz et al., 1992). An increase in Ca'+ concentration is known to trigger differentiation in cultured keratinocytes (Hennings et al., 1980; Shipley and Pittelkow, 1987; Pillai et al., 1988; Wilke et al., 1988). Early reports showed that Ca'+ induces changes in subcellular distribution of keratinocyte PKC (Isseroff at al., 1989). Further, TPA induces expression of epidermal transglutaminase and accelerates the formation of a cornified envelope, two markers for the final stage of keratinocyte differentiation (Yuspa et al., 1982; Parkinson et al., 1984; Lichti and Yuspa, 1988), and inhibits expression of early markers, K1 and K10, both in uatro and zn uzuo (Toftgard et al., 1985; Roop et al., 1987). Dlugosz and Yuspa (1993) showed that PKC activation alters keratinocyte gene expression at transcriptional and post-transcriptional levels, acting both as a positive regulator by enhancing transcription of late-state differentiation markers and as a negative regulator by repressing transcription and accelerating mRNA degradation of early markers such as K1 and K10. Studies of expression of individual PKC isozymes in keratinocyte differentiation have indicated that different patterns of isoform expression can be seen in keratinocytes of different animal species. mRNA and protein of PKC-a, -6, -E, -5, and -11 are present in cultured mouse keratinocytes (Dlugosz et al., 1992; Dlugosz and Yuspa, 1993); however, Koyama et a1. (1990) were unable to detect PKC-a expression in intact mouse epidermis and Gherzi et al. (1992) found expression of PKC-a, -6, and -q but not PKC-E or -5 mRNA in cultured human keratinocytes. Moreover, Gherzi et al., ( 1992) also found that keratinocyte differentiation is accompanied by a decrease in the mRNA levels of PKC-a and -6 and an increase in that of PKC-q. The role that the individual isozymes play in keratinocyte differentiation has not been fully defined; however, these findings further support the hypothesis that different isozymes perform different functions in the same cell type.
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Several studies have demonstrated that TPA- and Ca2+-induced keratinocyte differentiation is accompanied by increases in total PKC activity (William et al., 1990; Gherzi et al., 1992). The mechanism(s) by which this occurs may involve the increased levels of DAG that occur in Ca*+induced differentiation (Yuspa and Lee, 1991). D. ERYTHROLEUKEMIA CELLS
1. K562 The human erythroleukemia cell line K562 is a model that has been used widely in studies of both cell proliferation and differentiation, since these cells can be induced to cease proliferation and to differentiate into megakaryocytes on TPA treatment. Hocevar et al. (1992) and Murray et al. (1993) examined the expression of PKC isoforms in undifferentiated and TPA-induced differentiated K562 cells and found that proliferating cells express PKC-a, -PII, and -5 proteins but no detectable PKC-PI, -y, or -E. TPA treatment led to an increase in PKC-a and -5 but a decrease in PKC-PI1 protein levels. Furthermore, Hocevar et al. (1992) showed by several criteria that PKC-PI1 is essential for K562 cell proliferation, whereas PKC-a is involved in megakaryocyte differentiation. This study is similar to studies by Borner et al. (1991) that suggest that growth regulation in fibroblasts is selectively controlled by PKC-P but not PKC-a. The differences in translocation, substrate specificities, and activator response of PKC-a and -6 reinforce the emerging consensus opinion that, in several systems, the activation and translocation patterns of these two cPKC isoforms determine how the cell responds between the alternatives of differentiation and proliferation. 2. MELC
Friend virus-transformed murine erythroleukemia cells (MELCs) provide another useful model for studying processes involved in cellular differentiation, since they can be induced to cease proliferation and undergo terminal erythroid differentiation by exposure to N,N’-hexamethylene bisacetamide (HMBA) or DMSO. MELCs have a biphasic response to these compounds; during the first phase the cells are not irreversibly committed to differentiation, but in the second phase the maturation process cannot be reversed (Fibach et al., 1977; Bridges et al., 1981). During the first phase, many changes occur including expression of the immediate early response proto-oncogenes including c-myb, c-myc, c-fos, and $53 (Lachman and Skoultchi, 1984; Kirsch et al., 1986; Ramsay et al., 1986; Todokoro and Ikawa, 1986; Marks and Rifkind, 1990); reduction
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of cell voluine (Gazitt et nl., 1987); and reduction in the level of PKC activity (Balazovich et al., 1987; Melloni et al., 1987). Other evidence that PKC plays a role in these early stages is the transient increase in membraneassociated PKC activity seen in the early stage and the finding that longterm TPA treatment depletes cellular PKC activity and inhibi'ts HMBAmediated MELC differentiation (Powell et al., 1992). Removal of the phorbol ester permits restoration of PKC activity and the ability of MELC differentiation to be induced by HMBA (Balazovich et al., 1987). Although PKC seems to be required for commitment of MELCs to differentiate, there is a progressive decline in total PKC-f3 activity during maturation (Melloni et al., 1989). Moreover, the rate of differentiation is accelerated if exogenous PKC-P (but not PKC-a) is incorporated into permeabilized MELCs (Melloni et a/., 1990). GuptaRoy and Cohen ( 1 992) showed that, although there is a steady decline of PKC-P, a certain threshold of PKC activity must remain for maturation of committed MELCs. Exactly what function the PKC isozymes have in MELC maturation is still not well defined. One possibility that GuptaRoy and Cohen (1992) suggest is that a continuous source of PKC is required to achieve and maintain down-regulation of transcription factors NF-E 1 or NF-E2, analogous to the down-regulation of the erythroid-specific transcription factors that Mignotte et al. (1990) found after TPA treatment of the human erythroleukemia line K562. Several reports suggest that HMBA and DMSO exert different effects on PKC distribution during MELC differentiation. Several groups reported the appearance of PKC activity in the cytosol on HMBAmediated differentiation (Melloni et al., 1987; Beckman et al., 1990; Sprott at nl., i Y Y l ) , whereas others observed translocation of PKC to the particulate fraction on DMSO-mediated differentiation (Balazovich et nl., 1987). Redistribution of PKC isozymes to specific different subcellular compartments may be one way in which PKC controls its many growth- and differentiation-related functions in MELCs.
E. PKC
IN
PHEOCHROMOCYTOMAS
PC12 is a line of rat pheochromocytonia cells that is widely used to study neuronal differentiation (Greene et al., 1982), since these cells do not require nerve growth factor (NGF) for survival but can be induced to differentiate into mature sympathetic neurons by NGF (Yankner and Shooter, 1982). The mechanisms that mediate the effects of NGF in PC 12 cells are not well defined, but several groups have shown that some of the mechanisms may be mediated by PKC (Hama et al., 1986; Cremins ut al., 1986; Kalnian et al., 1990; Wooten, 1992; Wooten et al., 1992).
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Glowacka and Wagner (1990) demonstrated that TPA activation of PKC stimulates formation of NGF-dependent nerve cell processes whereas an inactive phorbol ester, 4a-PDD, has no effect on process formation. Hall et al. (1988) showed that sphingosine, a PKC inhibitor, inhibits NGFinduced neurite outgrowth. These results suggest that PKC may play a second messenger role in NGF-dependent neurite formation. Other data suggest the involvement of multiple kinases in the NGF pathway and that PKC may potentiate different NGF-associated events in this pathway. For example, Machida et al. (1991) determined that TPA activation of PKC augmented NGF induction of a late metalloproteinase gene product, transin/stromelysin, whereas staurosporine but not other kinase inhibitors blocked NGF induction of transin. Similarly K-252a, another protein kinase inhibitor, blocked NGF-dependent neurite extension in PC12 cells (Hashimoto, 1988; Koizumi et al., 1988). One cannot rule out the possibility that the inhibitors are affecting protein kinases other than PKC that may be specifically involved in neurite outgrowth. T h e contribution of individual isoforms in NGF-mediated neural differentiation has not been fully delineated; however, Wooten et al. (1992) suggest that PKC-PI1 may play a role in maintenance of neuronal morphology through phosphorylation of a specific substrate. The substrate GAP43/B50 has been shown to be a preferred substrate of PKC-PI1 (Sheu et al., 1990; Shinomura et al., 1991). Increased levels of PKC-PI1 have also been demonstrated during the differentiation of other cells such as MELCs (Melloni et al., 1990), neuronal NTl/Dl cells (Abraham et al., 1991), and human pheochromocytomas (Koda et al., 1991). F. NEURONAL DIFFERENTIATION AND NEURITE OUTGROWTH PKC has been shown to be involved in regulation of neuronal differentiation (Lacal et al., 1990; Abraham et al., 1991; Shimohama et al., 1991), which is not surprising since the brain is the richest source for all PKC isoforms. Shimohama et al. (1991) observed that PKC isozymes are differentially expressed during neuronal development, that is, PKC-PI1 is expressed abundantly during early and late stages while PKC-a, -PI, and -y are expressed only during late stages. Moreover, in uiuo, PKC isozymes are localized in distinct subcellular compartments and PKC-y clearly demonstrates nuclear localization. PKC-a is found in stellate and basket cells; PKC-PI1 and -y are found in the Purkinje cell layer; PKC-PI and -PI1 but not PKC-a and -y are detected in granule and Golgi cells. Several other groups have also reported differential expression of PKC isoforms in certain neuronal compartments, suggesting that there must
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be certain mechanisms that regulate expression of YKC isoforms at these sites (Kitano et al., 1987; Hidaka et al., 1988; Huang et al., 1988; Shimohama et a/., 1990).
G.
hTEUKOBLASI'OMA DIFFEKENTIAI'ION
Several studies have shown that neuroblastoma cells can be induced to differentiate into several phenotypes on exposure to differentiating agents such as RA (Ross ef al., 1983; Ross and Biedler, 1985; Sidell et al., 1986; Tsokos et al., 1987). T h e wealth of data that implicates PKC in cellular differentiation (see preceding sections) has prompted studies that examined the role of PKC in this model of neuronal differentiation (Prasad, 1975; Pahlman et al., 1990). SH-SY5Y, a human neuroblastoma cell line, resembles immature sympathetic neuroblasts and has been shown to exhibit TPA-induced (presumably PKC-mediated) signs of differentiation such as decreased proliferation and expression of neuronal markers (Pahlman ut al., 1981; Heikkila el al., 1989). These cells express PKC-a, -@, -y, -6, and -E mRKA (Leli et al., 1993). Within hours o f TPA treatment, these cells differentiate along a neuronal noradrenergic lineage (Scott et a/., 1986; Bjelfman et al., 1990; Pahlman et al., 1990), concomitant with down-regulation of c-nijc expression and increased c-fos expression (Yahlman el a/., 1983; Jalava et al., 1988; Leli et al., l992b). Leli el (11. (1992a) showed that down-regulation of PKC by long-term exposure t o ?'PA or inhibition of PKC by H7 or staurosporine decreased PKC-a and -E expression but had no influence on expression of PKC-0, -y, or -6 during neuronal differentiation in human neuroblastoma cell lines. Although down-regulation of these two isoforms could be the result of a phenomenon secondary to differentiation, Leli et al. (1992b) suggested that these two isoforms may selectively control steps during rieuroblastonia differentiation, since staurosporine and chronic TPA treatment caused down-regulation of the same PKC isoforms. The intracellular delivery of PKC-a and -E isofornr-specific antibodies, but not PKC-P, -7, or 6 antibodies, induced differentiation of SH-SY5Y cells; simultaneous treatment with anti-PKC-a and -E produced an additive effect (Leli et al., 1992b). Other groups have shown similar decreases in mRNA of PKC-a and -E in a mouse neuroblastoma cell line, neuro-2a, during RA-induced differentiation (Wada et al., 1989; Tonini et al., 199l), causing speculation that these two isoforms are particularly involved in the transduction of RA signals in neurons. Two main characteristics of differentiating neural cells are the segregation of neurites (1) into tubuloplasts, which are rich in microtubules
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and intermediate filaments, and (2) into actinoplastic cones, which are abundant in actin and microfilaments (Vasiliev, 1987; Danowski and Harris, 1988; Lyass et al., 1988; Bershadsky et al., 1990; Tint et al., 1991). Tint et al. (1992) showed that TPA-induced activation of PKC in neuroblastoma cells resulted in reversible reorganization of neurite extensions and growth cones by inducing formation of lateral outgrowths. Furthermore, Lederhendler et al. (1990) demonstrated that structural modifications of synaptic connections in neurons are dependent on PKC activation. These findings implicate TPA-induced (PKC-mediated) segregation of tubuloblasts and actinoplasts as an important mechanism in cellular reorganization during development of the nervous system. Further exploration of the system to discover which PKC isoforms are involved in this process should be the next step in completing an understanding of the role of PKC in nervous system differentiation. Ill. Involvement of PKC lsoforms in Tumorigenesis
Researchers have surmised for many years, even before they knew that PKC was a family of enzymes, that PKC or one or more of the PKC isoforms was likely to be involved in an important way in at least some forms of carcinogenesis. Several reasons supported this suspicion. First, 10 yrs ago researchers discovered that PKC was the major high-affinity intracellular receptor for phorbol ester (Ashendel et al., 1983; Leach et al., 1983; Niedel et al., 1983; Sando and Young, 1983; Parker et al., 1984), the classic promoter of mouse skin carcinogenesis (Boutwell, 1974). Second, many protein kinases of both the tyrosine- and the serine/ threonine-specific varieties have been shown to be proto-oncogenes that can be activated to oncogenic form by mutation of overexpression. Thus, investigators expected that at least some of the PKCs, all of which are serinelthreonine-specific protein kinases, would eventually be shown to be proto-oncogenes. A. TUMOR-SPECIFIC ISOZYME EXPRESSION To look for clues that PKC was involved in tumorigenesis, the expression of PKC activity in tumor cells was compared with that in the normal tissue counterparts. Later this comparison was done, with greater success, for individual PKC isozymes. Becker et al. (1990) determined that normal human melanocytes do not express PKC-a, -p, or -y whereas primary and metastatic melanomas express PKC-a, suggesting a connection between this isoform and transformation. Benzil et al. (1992) determined the expression pattern of the cPKCs in human astrocytomas.
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They found that PKC-a is expressed abundantly in well-differentiated (Grade 1) astrocytomas, is moderately expressed in anaplastic (Grade 2) astrocytomas, but is barely detectable in the most malignant and dedifferentiated form, gliohlastoma multiforme (Grade 3 astrocytomas). PKC-f.3 was not detected in any of the astrocytomas, and PKC-y was detected in only one Grade 2 tumor. These results were interpreted as evidence that expression of PKC-a contributed more to the expansion of low-grade astrocytonia cells than to progression of tumor cells to more malignant phenotypes. R6 rat embryo fibroblasts normally express PKC-a, -6, -E, and -5 (Borner et uE., 1992a), but transformation by specific oncogenes led to significant alterations in the expression of some of these PKC isoforms (Borner et nl., 1992b). c-H-rus-transformed R6 cells showed a markedly increased expression of PKC-a and -6, a decreased expression of PKC-E, and unaltered expression o f PKC-1;. To a lesser extent, similar changes accompanied v-fos-infected cells. Transformation of K 6 cells by m y , neulerh-B2, or mos oncogenes, however, induced no significant changes in levels of PKC mRNA or protein expression, suggesting that only some of the oncogenes affect PKC expression and that individual PKC isozymes play different roles in mediating cellular transformation by these oncogenes. In hematopoietic cells and tumors, the PKC isozymes are expressed in similar amounts in normal and malignant cells, with t.he exception of PKC-1;. In B- and 3-lymphocytic neoplasms, this isozyme is consistently expressed at much higher levels than in normal lymphocytic organs or purified B and T lymphocytes (J. Goodnight, 1994). Although these results suggest that PKC-1; is involved in oncogenic transformation in these cells, this theory has yet to be proven directly, for example, by infecting cell suspensions or intact animals with PKC-G-overexpressing recombinant retroF'Iiruses.
B. CORRELATION OF PKC ACTIVATION OR DEPLETION WITH TCMORIGENESIS Down-regulation of PKC in cells treated with TPA is thought by many researchers to be correlated with the transformation that can be induced by TPA or oncogenes. For example, Kischel et al. (1989) showed that PKC is depleted in JB6 epidermal cells during tumor promotion. Weyman ~t a/. (1988) demonstrated decreased PKC activity and protein levels in CSH 10T1/2 fibroblasts following transfection with the H-rus oncogene. Further, Hansen et nl. (1990) showed that depletion of PKC activity in CD-1 mouse skin cells permits epidermal hyperplasia and
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tumor promotion. Wu et al. (1992) used C3H10T1/2 cells that were “partially promoted” with TPA and showed that the rate of proteolytic down-regulation of PKC is enhanced in cells that represent early stages of tumor promotion. These investigators also showed that c-fos and c-jun genes were differentially induced, and AP-1 activity was increased, in TPA-treated cells that represented early and late stages of tumor promotion. On the other hand, several studies have demonstrated a positive correlation between high PKC activity and metastasis. For example, Liu et al. (1992) demonstrated that cells from a highly metastatic subpopulation of the amelanotic melanoma cell line B 16a exhibited higher levels of membrane-bound PKC than a less metastatic subpopulation of cells. Further, calphostin C, a PKC inhibitor, reduced lung colonization, supporting the notion that PKC is an important regulator in metastasis of these cells. There are also examples of inhibition of metastasis by PKC inhibitors in Lewis lung carcinoma (Takenaga and Takahaski, 1986; Grossi et al., 1989), W256 cells (Liu et al., 1991), and human bladder carcinoma cells (Schwartz et al., 1990). Since gross changes in PKC expression in both directions have been associated with malignant transformation, careful isoform-specific studies are likely to be required to clarify these apparently contradictory data. C. CHANCESIN PKC SUBSTRATES AND TUMORICENESIS Changes in PKC function can sometimes be detected by an indirect approach, that is, by observing alterations in proteins that are phosphorylated by PKC. For example, treatment of human and mouse fibroblasts with phorbol esters or other tumor promoters caused increased phosphorylation of several proteins, but the predominating one was the markediy acidic 80-kDa phosphoprotein known as MARCKS (see section I , G , l ; Rozengurt et al., 1983; Feuerstein et al., 1984; Blackshear et al., 1986; Smith and Colburn, 1988). Chicken MARCKS, with an apparent mass of 67 kDa, was found to be elevated in cells that had been transformed by Rous sarcoma virus, suggesting that transformation might be mediated by increased phosphorylation of this protein, perhaps by PKC (Sagara et al., 1986). In sharp contrast, studies of several rus- and src-transformed mouse fibroblast systems showed that MARCKS was down-regulated in transformed cells (Simek et al., 1989; Otsuka and Yang, 1991; Reed et al., 1991). There were no detectable differences in PKC activity to account for the alterations in MARCKS levels, so these
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data suggest that the levels of this substrate may also be relevant to the tumor promotion component of transformation. Since they found that MARCKS was switched off in transformed cells, Simek et al. (1989) speculated that MARCKS may function as a suppresser of neoplastic transformation. A more reasonable hypothesis, espoused by Reed et al. (199 l), was that the low levels of MARCKS might contribute to the transformed phenotype by compromising the normal organization of the membrane cytoskeleton [actin being normally cross-linked by MARCKS molecules (Rosen et al., 1990)]. In this way, abnormally shaped, poorly adherent cells could arise. D. ROLE I N TRANSFORMATION BY THE ?-asONCOGENES Initial studies found no difference between the PKC proteins expressed in normal NIH 3T3 fibroblasts and in those that had been transformed by v-H-rus; both were found to contain predominantly the 01 isoform (McCaffrey and Rosner, 1987). However, in the first of many reports that linked ras transformation and PKC expression, Lacal ut al. (1987) showed that both H-tas p21 (p2lraf)and PKC proteins were required to produce the maximum mitogenic response that occurs when NIH 3T3 cells are infected by Harvey rat sarcoma virus. This result was consistent with reports (Wolfman and Macara, 1987) that rastransformed cells had elevated levels of DAG, which would be expected to activate endogenous PKC. Haliotis et al. (1990) used an inducible TUS expression vector to show that PKC was, indeed, initially activated by p2 1 is but that prolonged p2 1 expression led to PKC down-regulation. Significant, but not complete, PKC down-regulation in ras-transformed cells was confirmed by several other laboratories in other fibroblast systems (Huang et al., 1988; Weyman et al., 1988; Polverino et al., 1990; Fu et al., 1991). Down-regulation of PKA, the CAMP-dependent protein kinase, was also induced by p2 1 ~ 1so s which protein kinase might be important in rus transformation remains unclear. '1-he degree of involvement of PKC in events that are triggered by oncogenic pp2 lras also turned out to be quite complex; some of the dramatic events that are triggered by ray, namely morphological transformation and myc induction, appear to be independent of PKC (Lloyd et al., 1989; Trotta et al., 1990; LitzJackson et al., 1992; Krook et al., 1993), yet PKC may well phosphorylate p21ras itself (Jeng el al., 1987). Similar studies were also done on cells other than fibroblasts. A murine niast-cell line, PB-3c, was infected with normal c-H-rav- and oncogenic v-H-ras-expressing retroviruses. After both cell lines were stimulated with TPA, only the oncogenic r'as led to increased levels of PKC-P
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and slower than usual down-regulation of PKC-a. These alterations in PKC isoform expression were proposed as the mechanism by which ras reduced the IL-3 dependence of PB-3c cells and rendered them tumorigenic (Imber and Fabbro, 1991). Since the other isoforms of PKC were not examined, and v-abl also can abrogate mast-cell dependence on IL-3 (Pierce et al., 1985), this interpretation cannot be considered definitive. Similar alterations in PKC expression patterns in H209, a cultured line of human small cell lung cancer cells, were seen when myc and rus genes were introduced. Exogenous myc expression caused the upregulation of PKC-P expression, but addition of exogenous ras added a down-regulation of PKC-a in cell membranes (Barr et al., 1991). Thus, investigators proposed that PKC alterations may represent a significant portion of the contribution that rm makes when it collaborates with myc in transformation. T h e current consensus is that the stimulation of DNA synthesis induced by p2lras is mediated by PKC via the activation of c-fos and c-my. However, there probably is a separate pathway through which p2 l r a s causes morphological transformation; this second pathway appears to be PKC independent. An important piece of evidence that PKC can participate in multistage carcinogenesis was the demonstration of a synergistic effect between the H-rm oncogene and overproduction of PKC (Hsiao et d., 1989). Rat liver epithelial cells and R6-PKC3 cells, a rat fibroblast line that stably overproduces PKC-PI, were reported to have an increased susceptibility to transformation by an oncogenic H-rus. R6-PKC3 cells that had been given an expression vector that overexpressed v-H-ras displayed a highly transformed morphology, were tumorigenic in nude mice, and yielded a 10-fold increase in the formation of large colonies in soft agar compared with the control line. These findings are consistent with the earlier report that flat revertants of rm-transformed cells have subnormal levels of PKC (Kamata et al., 1987), but the exact mechanism by which the rm and PKC synergism acts is still not known. E. ROLEIN TRANSFORMATION BY OTHER ONCOGENES
I. DNA Virus Oncogenes PKC is thought to be important in mitogenesis in a wide variety of normal cells, for example, B and T lymphocytes, and in certain lymphocytic cell lines because TPA is required for maximal stimulation of proliferation. Further, TPA treatment markedly increases the transforming
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ability (assessed by anchorage-independent growth) of pol yonia virus middle ‘I’antigen on rat F1 11 fibroblasts conconiitant with an increase in membrane-associated PKC (Raptis and Whitfield, 1986). Such was not the case in NIH 3T3 cells, so this aspect of transformation is not universally or inextricably bound with PKC translocation to membranes (Raptis et al., 1993). There remains a suspicion that PKC may function indirectly in these systems, for example, via stimulation of the tyrosine kiriase activity of pp60’-5~’(Raptis et ul., 1991). However, polyoma virus middle T antigen renders another cell line, Mel-ab (an immortal murine melanocyte cell line), mitogenic and tumorigenic in the absence of TPA and even after TPA-induced down-regulation of PKC, also indicating that polyoma virus middle T antigen does not exert its transforming effect by direct action on PKC (Dooley Pt al., 1988). Similarly, down(which regulation of PKC does not block the mitogenic effect of pp60\,-.57~ frequently complexes with polyoma virus middle T antigen) on BALB/c 3’1‘3 cells (Han et al., 1990), suggesting that any PKC-related effects observed earlier were probably indireci and not essential for transformation. Direct cooperation between PKC and genes from DNA viruses in neoplastic transformation was demonstrated when PKC-PI and adenovirus type 5 E1A were coexpressed in rat embryo fibroblasts (Su et ul., 1991), but the biochemical basis for this cooperation is still not known. 2 . Retrozrirul Oncogenes
Phosphorylation and dephosphorylation of key regulatory molecules are known to be important mechanisms of activating and deactivating these molecules. PKC has been thought to play an important role in carcinogenesis by activating some of the known oncogenes such as fm and .m, but the data implicating these particular oncogenes are not convincing. f m s is only phosphorylaied by PKC in uitro (Tamura et al., 1986), an unreliable test for function of PKC in ztivo, and the serine residue (amino acid 12) in pp60.- that is phosphorylated by PKC is not involved in any of the transforming activities of src (Yaciuk et al., 1989). NIH 3T3 cell lines that are transformed by sh, src, and abl display constitutively increased DAG levels, much like those reported for rustransformed lines (Wolfman and Macara, 1987; Chiarugi et al., 1989; Lopez-Barahona et al., 1990). Diaz-Laviada ef ul. (1990) confirmed that src- and rus-transformed NIH 3’13 cells had elevated levels of DAG, but they detected no down-regulation of PKC, determined by immunoblotting. Instead, they were able to demonstrate a “permanent” translocation of PKC to the plasma membrane using imniunofluorescence. Longterm TPA stimulation, on the other hand, did show down-regulation of PKC by both immunofluorescence and immunoblotting. Chiarugi et al. 5 7 ~
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(1990), trying to reconcile the rather contradictory reports that dispute whether oncogenes down-regulate PKC or induce its translocation, suggested that oncogenic transformation subverts PKC translocation from physiological targets in membranes to other targets in the nucleus. This intriguing notion awaits experimental documentation. T h e amplification of the N-myc gene is closely correlated with increased metastatic ability of neuroblastomas, possibly because of a dramatic reduction in expression of neural cell adhesion molecules that is induced by the transfection of N-myc into a neuroblastoma cell line (Akeson and Bernards, 1990). This induced overexpression of N-myc has been shown to cause suppression of PKC-6 and induction of PKC-5 (Bernards, 1991), which might play a role in the other changes that result in increased malignancy. F. INVOLVEMENT IN TUMOR PROMOTION
Many phorbol esters are “tumor promoters,” agents that are not carcinogenic alone but cause the appearance of tumors in a tissue that has previously been exposed to a carcinogen (the “tumor initiator”) (Blumberg, 1980, 1981). Tumor promotion has been demonstrated experimentally in mouse skin, rat liver, rat esophagus, rat colon, rat bladder, rat breast, mouse and rat stomach, rat trachea, and mouse lung (Yuspa and Poirier, 1988). T h e case has been made for analogous two-step carcinogenesis in vitro (Wu et al., 1992), but this in vitro model of tumor promotion has not been universally accepted. T h e most extensively studied system of tumor promotion has utilized phorbol ester treatment of mouse skin or cultured skin cells. Studies with these agents have formed the basis for most of the current hypotheses on the molecular mechanism of tumor promotion. Phorbol esters are not mutagenic in bacterial or mammalian test systems, their effects are reversible, and tumor production requires repeated administration, suggesting an epigenetic rather than a genetic mechanism. Nonetheless, these agents have strict structure requirements, are frequently tissue specific, and are effective at very low concentrations, suggesting that phorbol esters function via activation of a specific receptor. Since the chief receptors for the phorbol esters are the PKCs (excluding the aPKC isoforms), researchers have postulated for at least 10 y r that PKC plays an important role in cancer promotion. This notion has received support from data that demonstrate that different phorbol esters bind to PKC and stimulate its kinase activity with affinities that parallel their promoting action (Blumberg et al., 1984). Potent non-phorbol tumor promoters, for example, teleocidin and aplysiatoxin, also bind
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strongly to PKC and activate its kinase function (Fujiki and Sugimura, 1987). The biology of skin tumor promotion is inseparable from that of keratinocyte differentiation (see Section 11,C). One of the first signs of transformation of epidermal cells is the loss of their ability to respond to differentiation signals, that is, Cay+ (the physiological signal) or phorbol ester (a pharmacological signal) (Yuspa and Morgan, 1980; Hennings et al., 1987). 'The refractoriness of transformed keratinocytes to differentiation when treated with Cay+ or TPA is additional, if indirect, evidence that PKC is involved in the transformation process. A model of skin cancer promotion by phorbol esters may be summarized as follows. In a treated area of skin, phorbol esters accelerate differentiation (Reiners and Slaga, 1983; Toftgard et al., 1985) and mature cells are rapidly lost from the differentiating area of skin. This area undergoes, in effect, a regenerative epidermal hyperplasia in which cells are refractory to this signal to differentiate, for example, initiated epidermal cells, are selectively expanded. 'I'his notion has received some indirect support from an analytical study in which investigators demonstrated that the levels of PKC-5 and -q niRNA were modulated in tumor cells (Dlugosz et al., 1992). Direct proof of the involvement of these PKC isoforms in tumor promotion o r initiation requires introduction of overexpressed isoforms into keratinocytes or selective reduction of particular isoforms by gene knock-out or antisense expression.
G. ONCOGENIC ML-rA-rIms
OF
PKC
Proto-oncogenes can be activated to oncogenes by structural mutations such as truncations (abl, tnyb, re1, and r a f ) or point mutations ( r u ~ ) , so the report that point mutations in PKC-a might be oncogenic when tested in BALB/c 3T3 fibroblasts was not unexpected (Megidish and Mazurek, 1989).Unfortunately this result did not prove to be reproducible in other laboratories (Burner et al., 1991; Mischak and Goodnight, unpublished data), so it must be assumed that the mutations described in this experiment d o not confer oncogenicity on PKC-a.
H . PKC ISOZYME OVEREXPRESSION A N D TUMOR INDUCTION Initial experiments designed to test the involvement of PKC in malignant transformation directly were perforrned by Housey et al. (1988) and Persons et al. (1988), who overexpressed PKC-(3 or PKC-y, respectively, in fibroblasts. T h e overproducing cells revealed subtle changes in
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morphology and would grow in soft agar, but only in the presence of TPA, and they only sporadically formed tumors in nude mice. Hence, overexpression of these two cPKCs in fibroblast lines may be transforming but not tumorigenic. Overexpression in fibroblasts of two nPKCs, PKC-6 and -E, has been described more recently. Overexpression of PKC-6 seems to inhibit cellular growth and to block the cell cycle at G,/M (Watanabe et al., 1992; Mischak et al., 1993b). On the other hand, overexpression of PKC-E leads to transformation and tumorigenesis in two lines of fibroblasts, mouse NIH 3T3 and rat 6 (Mischak et al., 1993b; Cacace et al., 1993). The PKC-eoverexpressing cells are able to grow in soft agar in the absence of activators such as TPA, and they form tumors in nude mice with 100% incidence. Preliminary data suggest that transformation may be due to the secretion of transforming growth factor (TGFa)-like growth factors, since supernatant of PKC-E overexpressers is capable of inducing wild-type NIH 3T3 to grow in soft agar (M. Ueffing and A. Cacace, personal communication). The results obtained from the cell lines that overexpress the PKC isoforms suggest that some of the PKC isozymes are involved in malignant processes. Since the data have been obtained only from fibroblasts, PKC isozymes that have not been found to be transforming in fibroblasts may reveal tumorigenic potential in other cell types. I. COLONCARCINOMA Colorectal cancer is one of the most common malignant tumors, affecting 130,000 people in the United States each year (Baron et al., 1990), and is known to develop through a series of preneoplastic cellular changes (Vogelstein and Kinzler, 1993) including alterations in the oncogene rm and the anti-oncogene p53, both of which are thought to be substrates of PKC (Jeng et al., 1987; Baudier et al., 1992). Preneoplastic cells are converted to benign neoplasms, called adenomas, which progress through multiple stages to a final stage of frank malignancy. Genetic changes that convert normal colonic cells into premalignant cells are thought to also confer susceptibility to proliferate in response to tumor promoters such as TPA (Friedman et al., 1989). For example, TPA stimulates proliferation of preneoplastic cells of familial polyposis patients as well as premalignant tubular adenoma cells. Furthermore, TPA increases the level of urokinase from carcinomas and adenomas, which may contribute to an increased metastatic potential. Since PKC is the receptor for TPA and is activated by DAG,Friedman et al. (1989) examined whether diglycerides present in the colon lumen could directly activate PKC. Their data imply that diglycerides act as
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endogenous niitogens in the colon to induce growth of benign colonic tumors and some carcinomas, although they have no effect on normal cell proliferation. TPA has been shown to induce changes in some human colon carcinoma cells that mimic terminal differentiation, that is, accelerated proteoglycan synthesis and rapid release of cell-surface proteoglycans (Friedman, 19981; Friedman et d.,1984; Baron et al., 1990; McBain et uf., 1990; McBain and Mueller, 1992). Furthermore, PKC inhibitors had considerable success in blocking this TPA-induced differentiation of colon cancer cells, which led McBain and Mueller (1992) to propose that such differentiation is regulated by PKC activation of a membrane-associated protease that cleaves lipophilic anchor peptides. The situation is complicated because evidence correlates increased PKC activity with both cell growth, for example, in human epithelial tumors (Rvdell et al., 1988), human gastric carcinomas (Yasui et al., 1985),and thyroid tumors (Omri and Padovic-Hournac, 1985), and differentiation and cessation of growth (Rydell et al., 1990). Researchers know that different isozymes of PKC participate in competing processes, that is, PKC-6 overexpression stops NIH 3T3 growth whereas PKC-E overexpression induces transformation and tuinorigenesis in NIH 3T3 cells (Mischak et a/., 1993b) and in other mammalian cell lines (Watanabe et al., 1992; Cacace al., 1993). Since colon cancer, like most other malignancies, arises through a series of progressive changes, one or more isoforrns o f PKC may be involved in both progression and regression steps that make up this malignant process. (11
IV. Conclusions Despite the enormous literature, it is still not possible to define a unified view or a unanimous consensus on the role of each PKC isoform in clifferentiation and neoplastic transformation. This is still a worthy aim. T h e exact function of the different isozymes will only become clear when their physiological substrates are identified and characterized. Despite the considerable lack of unanimity, several conclusions can be drawn from the data assembled in this chapter. 1. It is by now well established that expression of the 1 1 known PKC isozymes is often tissue specific and differentiation-stage specific. Thus, it is no longer justified to use TPA stimulation or TPA-induced downregulation to rule in or out “PKC” involvement in a particular phenomenon. T h e availability of cDNAs for all isozymes, recombinant isoenzyme proteins, isoforni-specific antibodies, antisense oligonucleotides, and PKC-specific inhibitors should make it possible to identify which PKC isoforms are involved in particular physiological phenomena.
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2. TPA and other phorbol esters cause myeloid, megakaryocyte, neuronal, and keratinocyte differentiation, but this is only indirect evidence of PKC involvement in these processes. The key isozymes involved have been identified in only a few cases. PKC-a or -6 appears to be the important isoform in myeloid differentiation, based on restoration of this function by overexpressing these isoforms in cells that do not differentiate in response to TPA treatment. Studies of the other differentiation systems have not progressed to this stage, but several experimental systems, for example, erythroleukemia cells, provide clues that differentiation seems to be regulated by the relative concentrations of the two principal cPKCs, PKC-a and -P. Keratinocyte differentiation is associated with an increase in PKC-r) and a decrease in PKC-a and -6, whereas TPA-induced differentiation of K562 erythroleukemia cells results in increased PKC-a and -5 but decreased PKC-PII protein levels. 3. Although progress has been made in dissecting the contribution of PKC isoforms to the multiple processes that constitute transformation, it is not yet clear exactly how PKC is involved in skin cancer promotion. A clue may be that levels of PKC-IJand -r) are different in normal and skin cancer cells. 4. There appears to be a special synergistic relationship between the rus oncogenes and PKC because PKC-P, by some as yet unknown mechanism, increases fibroblast sensitivity to transformation by v-H-rus. 5. Overexpression studies seem to provide the most direct evidence that PKCs can behave as oncogenes. When overexpressed in murine fibroblasts, PKC-P and -y yield apparently transformed cells in vztro, but these cells are not tumorigenic in vivo. Two independent laboratories, however, have shown that overexpression of PKC-Ein rodent fibroblasts is both transforming in vitro and tumorigenic in vivo, making this isoform of nPKC an undoubted proto-oncogene. Interestingly, another nPKC, PKC-6, seems to have the opposite effect, namely growth inhibition, when overexpressed in fibroblasts or Chinese hamster ovary (CHO) cells. 6. Considerable evidence implicates the PKC family in complex signal transduction processes. The contribution of each isoform may be inhibitory or stimulatory, but the operational word is complex. The immediate future holds promise for the sorting out of these details, because most of the tools are now at hand. ACKNOWLEDGMENTS We are grateful to Drs. Stuart Yuspa, Peter Blumberg, Walter Kolch, Denise Cooper, Zoltan Szallasi, and Silvio Gutkind for thoughtful and critical readings of the manuscript.
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We also thank Dr. ,lonathan Ashwell for the prepublication use of Bookends Pro for rnanipulating the references.
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Fcy RECEPTORS IN MALIGNANCIES: FRIENDS OR ENEMIES? Janos Gergely and Gabriella Sarmay Department of Immunology, E6hr6s Lorand University, 2131 GW, Hungary and Vienna International Research Cooperation Center, 1230 Vienna, Austria
I. Introduction 11. Structural Features of FcyRs A. FcyRI B. FcyRII C. FcyRIII 111. Ligand Binding and FcyR Binding Sites A. Localization of Interacting Sites for FcyRs B. Binding Sites on FcyRs IV. Functions Mediated by Membrane-Bound FcyRs V. Signal Transduction Mediated by FcyRs VI. Expression of FcyRs A. Effect of Immunoglobulins on FcR Expression B. Modulation of FcyR Expression by Cytokines C. Cell Activation and FcyR Expression VII. Soluble Fcy Receptors VIII. Mechanisms of sFcyR (IgG-BF) Production A. Shedding of FcyRs B. Production of sFcyRs by Proteolytic Cleavage IX. Regulatory Role of Membrane Bound and Soluble FcyRs A. Regulation of Antibody Production on B Cell Level by FcyRII B. B Cell Cycle and FcyRII Release X. Expression of FcRs on Tumor Cells XI. sFcRs in Malignancies XII. Biological Role of FcR-Mediated Functions in Malignancies XIII. Conclusions References
I. Introduction
T h e observations drawing the attention to the possible relationship between immunoglobulin-binding structures and tumors date back to the early 1970s when the presence of immunoglobulins on human and animal nonlymphoid tumor tissues were first observed (Ran and Witz, 1970; Ran et al., 1978). Since no antibody activity could be detected in these molecules, researchers proposed that they were bound via the Fc portion. Indeed, Fc receptors (FcRs) were demonstrated on the surface of tumor cells (Tonder and Thunold, 1973). 21 1 ADVANCES IN CANCER RESEARCH, VOL. 64
Copyright Q 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Several observations suggested that the FcRs play a role in the hostrumor relationship. FcR-expressing imniunocytes increase under cancer and precattcer conditions (Fujimoto et al., 1976), and soluble FcKs were shown to be released from FcR-expressing tumor cells (Schirrmdcher and Jacobs, 1979; Ran et al., 1988). Soluble FcRs suppress immunoglobulin secretion and may facilitate the escape of tumor cells from immune surveillance. Cytokines [e.g., tumor necrosis factor (TNF)]were shown to influence FcR expression and release (Debets et a/., 1988). Researchers also observed that metastasizing tumor lines contained high proportions of FcR+ cells (Schirrnmacher and Jacobs, 1979). T h e molecular structure and function of FcRs has been widely investigated. These receptors couple antigen-specific recognition and Fcdependent immunological effector functions. In addition, FcRs expressed on nonimmunocompetent cells can connect antibodies with other physiological systems. Immunoglobulin-binding structures (FcRs and FcR-like molecules) are expressed on some bacteria or parasites and enable interaction with the immune system before development of specific immune responses. FcRs can play a role in cell damage, endocytosis of opsonized particles, secretion of soluble mediators, and regulation of antibody responses (reviewed by Anderson and Looney, 1986; Gergely and Sarniay, 1990; Ravetch and Kinet, 1990; Gergely et al., 1992). In this chapter w e deal only with the IgG-binding FcRs (FcyR). We sumniarke briefly the structural features of their membrane-bound and soluble forms, the factors influencing receptor expression, and the possible mechanisms of receptor release. Moreover, we touch on the importance of various forms of FcyRs in the regulation of humoral responses. Finally, we speculate on the significance of FcyRs in antitumor effector functions.
II. Structural Features of FcyRs FcyRs (a subgroup within a larger group of immunoglobulin-binding molecules) belong to the immunoglobulin (Ig) gene superfamily. With one exception (FcyRIII- 1, CD 16-I), they are integral membrane proteins with a leader peptide, an extracellular glycosylated portion, a single transmembrane segment, and a C-terminal cytoplasmic tail. T h e Nterminal extracellular portion contains Ig-related domains that show significant homology with the C2 set of Ig domains. A wide range of cells express FcRs; these are mainly part of the immune system, but FcRs are present in membranes of other cell types as well. Molecules within the family of FcRs are heterogeneous in molecular weight, binding affinity, and cellular distribution.
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Three classes of human IgG-binding FcRs have been defined with monoclonal antibodies recognizing FcR epitopes (Anderson and Looney, 1986). These are expressed on different and partly overlapping cell populations. A. FcyRI FcyRI (CD64) is a 72-kDa glycoprotein (Anderson, 1982) expressed mainly on mononuclear phagocytes. FcyRI has relatively high affinity (K, 10s-109 M-I), and is the only human FcyR that binds monomeric IgG effectively. The rank order of affinity of FcyRI for IgG isotypes is IgGl = IgG3 > IgG4. FcyRI does not interact with IgG2. The receptor is trypsin resistant. The core protein has an M, of 40,000 (Frey and Engelhardt, 1987). Molecular cloning studies have shown that the extracellular part of FcyRI contains three Ig-like domains (Allen and Seed, 1989). T h e first two bear homology to the low-affinity FcyRs whereas the third domain is unique. Three genes (A, B, and C) encoding human FcyRI have been identified (van de Winkel et al., 1991; Ernst et al., 1992). The genes are made u p of six exons, two of which encode the signal peptide, one of which encodes each of the Ig-like domains, and another of which encodes the transmembrane/cytoplasmic region. The FcyRIA-derived transcript encodes a three-domain transmembrane receptor. The third extracellular domain-encoding exon of the FcyRIB and C genes contains stop codons. The transcripts derived from gene B may be alternatively spliced products encoding a two-domain transmembrane receptor. Transcripts derived from genes B and C may encode soluble FcyRs.
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B. FcyRII FcyRII (CD32) is a protein of 40,000 M, which is expressed on monocytes, platelets, neutrophils, and B cells. This receptor has low affinity (Looney et al., 1986); the equilibrium affinity constants (K,) are less than 1 0 7 M-1. Hence, it requires multiple Fc interactions (aggregated IgG). It binds IgGl and IgG3 equally well, but binds the other immunoglobulin subclasses less readily. Several reports deal with the structural heterogeneity and genomic organization of the human FcyRII genes (Lewis et al., 1986; Stuart et al., 1987; Allen and Seed, 1988; Stengelin et al., 1988; Brooks et al., 1989; Seki, 1989; Ravetch and Anderson, 1990; Engelhardt et al., 1991; Ierino et al., 1993).
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Human FcyRIIs are encoded by three genes (FcyRIIA, -B, and -C) that result in six transcripts (FcyRIIal and -a2; FcyRIIbl, -b2, and -b3; and FcyRIIc), all generated by differential RNA splicing. The heterogeneity is further increased by four allelic variants of the FcyRIIa isoform. The genes are composed of eight exons. T w o exons encode the signal peptide, one encodes each of the Ig-like domains, another one encodes the transmembrane region of the receptor, and finally three are cytoplasmic region-encoding exons (Qiu et al., 1990). Transcription of the human FcyRIIA gene yields a 1.4- and a 2.4-kb mRNA as a result of polyadenylation (Stengelin et al., 1988). T h e FcyRIIB and C both give rise to a 1.5-kb mRNA (Stuart et al., 1987). T h e genes contain various exons encoding transmembrane and cytoplasmic regions. Alternative splicing was described for a human FcyRIIA product, in which the cDNA lacks information for the transmembrane region; this may explain the existence of a soluble form of the FcyRIIa (Warmerdam et al., 1990). The human FcyRIIA gene shows allelic variation, which results in high-responder hFcyRIIAHR and low-responder hFcyRIIALR molecules. These allelic variants differ in their ability to bind mouse IgCl and human IgG2 complexes. T h e receptor derived from the HR allele binds mouse IgCl, in contrast to hFcyRIIaLRmolecules, which d o not (Tax et al., 1983; Warmerdam et al., 1993). C. FcyRIII
T h e low-affinity FcyRIII (CDl6) expressed primarily on neutrophils, natural killer (NK), and killer (K) cells, and at low levels on monocytes and macrophages (Perussia et al., 1983), has an M, of 50-80 kDa. FcyRIII on neutrophils differs from that on tissue macrophages (Clarkson and Ory, 1988; Lanier et al., 1988), perhaps because of differences in Nlinked glycosylation. FcyRIII binds preferentially to IgCl and IgC3. I n comparison to the other classes of FcyRs, its amount on cell membranes is high; for example, 103 FcyRII molecules are present on platelets (Karas et al., 1982), whereas a few hundred thousand copies of FcyRIII are detected on neutrophils (Fleit et al., 1982). T h e low-affinity FcyRIII exists in two distinct forms (Warmerdam et al., 1990). On neutrophils it is anchored to the outer leaflet of the plasma membrane by a glycosylphosphatidylinositol moiety [phosphatidylinositol (PI) glycan-linked membrane protein, FcyRIII- 1; Huizinga et al., 1988; Selvaraj et al., 19881. On N K cells, macrophages, and cultured monocytes these receptors are transmembrane proteins (FcyRI11-2; Ravetch and Perussia, 1989). On macrophages FcyRIII is associated with
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the y chain of the FceRI; on NK cells both this y chain and a 5 chain of the TCR-CD3 complex associate with FcyRIII. The 5 chain is required for plasma membrane expression of this receptor (Lanier et al., 1389a; Ra et al., 1989; Anderson et al., 1990). Two different genes (A and B) consisting of five exons encode the human FcyRIII. Two exons encode the signal peptide, another one encodes each of the Ig-like domains, and a single exon encodes the transmembrane/cytoplasmic region. The genes are homologous, differing only in a few nucleotides (Ra et al., 1989). The difference between cDNAs corresponding to FcyRIII- 1 and FcyRIII-2 was characterized by single nucleotide substitutions. The most significant difference between the products of the two FcyRIII genes was discovered at amino acid position 203. I n FcyRIIIb, a serine determines the PI anchoring whereas in FcyRIIIa a phenylalanine was found to specify the transmembrane/ cytoplasmic region of the receptor (Simmons and Seed, 1988; Peltz et aE., 1989; Perussia et aL., 1989; Scallon et al., 1989; Lanier at aL., 1991). An alloantigen (NA) recognized by autoantibodies was detected on human FcyRIII (Werner et al., 1988). The NA polymorphism is only expressed in the FcyRIIIB gene product and is restricted to polymorphonuclear leukocytes (PMNs). It is not present on NK cells or monocytes (Edberg et al., 1989; Huizinga et al., 1990). NA1 and NA2 antigens were reported to be neutrophil specific. Nucleotide dissimilarities between NA- 1 and NA-2 were described, and the allelic differences were assigned to Ser 65 and Val 106 (Tetteroo et al., 1987). Ill. Ligand Binding and FcyR Binding Sites
As already mentioned, a wide range of cells express FcRs. The family of these molecules is heterogeneous in molecular weight, binding affinity, and cellular distribution. The functional versatility of FcRs is, in part, the consequence of the structural heterogeneity and partly due to various types of cooperation with other cell-surface components. The ligand-binding capacity of various forms of FcRs differs also, and is probably influenced by the conformation of the molecule. We mentioned earlier that the different FcyRs bind the immunoglobulin isotypes with different affinity. Because of differences in the cytoplasmic tails and/or associated chains, even FcyRs with identical extracellular domains (i.e., same ligand-binding capacity) transfer different signals. Finally, note that the density of the ligands and the conformation of the immunoglobulin molecules determine their interaction with the FcRs. In this respect, the identification and topographical mapping of groups on the IgG recognized by the FcyR molecules is very important.
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A. LOCALIZATION OF INTERACTING SITESFOR FcyRs Despite the structural homology of the extracellular domains, the binding sites of the various FcyR types recognize different groups on IgG Fc. From the functional point of view, it is important that the binding of FcyRs to the corresponding binding sites on the IgG does not interfere with the interaction of other Fc-binding structures such as Clq and some rheumatoid factor type autoantibodies (Gergely et al., 1992). T w o regions of sequence located close to the hinge (Woof et al., 1986), forming a continuous protein surface on the CH2 domain of IgG, seem to be important in the interaction with FcyRI of monocytes (Nik Jafar et ul., 1982, 1984; Bruggemann et af., 1987; Jefferis et af., 1990). Note that although IgGl and IgG3 have identical sequences at the critical residues 233-236, IgG2 is different. This difference explains the lack of binding of IgG2 to FcyRI. On the other hand, because of the extended rigid structure of the hinge region in IgG3 (Gergely rt al., 1992), the accesGbility of its interacting site, compared with IgGI, is better. T h e two immunoglobulin-like extracellular domains of FcyRI form one active binding site interacting with a region in IgGl or IgG3, located at the K-proximal end of their CH2 domain (a different pattern was obtained, however, in the case of FcyRIII and FcyRII). T h e high-affinity interaction of FcyRI may be supported by the steric effect of the non-Ig-like third extracellular domain of the receptor. T h e fine specificity and signal-inducing capacity of FcyRIII was studied in experiments on the lytic activity of K cells (Sarmay et al., 1984, 1985). Fcy RIIIs involved in antibody-dependent cellular cytotoxicity (ADCC) were shown to possess two active binding sites; efficient lysis depended on the simultaneous interaction of these sites with the CH2 and CH3 domains. T h e groups that react with the CH2 domairi-specific binding site may involve the region of residues Lys 274-Arg 301 and the lower hinge as well, whereas the interacting groups within the CH3 domain have been localized to the region Ser 408-Arg 4 16. The two binding sites seem to have different functions. Lytic signals are mediated only by the CH2 domain binding site of FcR, whereas CH3 domain binding contributes only to increasing the binding affinity (Erdei et al., 1984; Sarmay et af., 1986, 1992). Although the relevant sites on the Fc domains are not yet localized, FcyRII molecules expressed on resting human B cells were also shown to possess two binding sites: one specific for CH2 and another specific for the CH3 IgG domains (Sarmay et al., 1985).
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SITESON FcyRs B. BINDING Based on investigation of genetic polymorphism and direct structurefunction studies using chimeric receptors, the immunoglobulin-binding region of the extracellular domains of FcyRs have been identified (Hulett et al., 1991; Hogarth et al., 1992). The immunoglobulin-binding region of the FcyRs seems to be located in the second extracellular domain, although the possible functional role of the first domain is still not clear. T h e second domain is likely to be responsible for binding whereas the first one mediates signals to the cell (Gergely and Sarmay, 1990). IV. Functions Mediated by Membrane-Bound FcyRS
FcyRs are involved in cell-mediated effector functions. Antigenantibody complexes trigger effector cells for phagocytosis or killing via their FcRs (Walter, 1977; Fanger et al., 1989). All three types of FcyRs can induce superoxide generation (Huizinga et al., 1989; CrockettTorabi and Fantone, 1990). T h e expression of various types and isoforms of FcyRs on different cell types seems to be strictly regulated; each receptor type apparently mediates well defined functions. The functional consequences of structural differences can be exemplified by the isoforms of FcyRII (Miettinen et al., 1989, 1992). The murine macrophage isoform (FcyRIIb2) is identical to the lymphocyte isoform (FcyRIIbl) except for an in-frame insertion in the cytoplasmic tail of FcyRIIbl that increases its length from 47 to 94 amino acids. Researchers found that, whereas FcyRIIb2 mediated efficient ligand uptake and delivery to lysosomes via internalization in coated pits, the cytoplasmic tail insertion characteristic for the lymphocyte isoform (FcyRIIbl) reduced the ability of the receptor to mediate ligand uptake and degradation. In other words, the 47-aminoacid insertion in the FcyRIIbl cytoplasmic tail disrupts the capability to localize in clathrin-coated pits. ADCC is a typical FcR-mediated function. Various cell types and several forms of FcR can mediate ADCC. In FcyRIII-mediated lysis, the cytotoxicity is accomplished mainly by TCR-/CD3- FcyRIII(CD 16)+ NK cells. TCR aB+/CD3+ cells usually do not express FcyR and have no ADCC activity. In contrast, TCRyti+/CD3+ cytotoxic T lymphocytes (CTLs) express FcyRIII and can function in ADCC. Researchers also showed that CD3 and CD8 molecules play a regulatory role in CD16mediated triggering of CTLs (Oshimi et al., 1990). CD3- NK cells express the { chain in association with higher molecular weight structures
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whose expression differs among individual N K cell clones. Since in both clonal and polyclonal populations of CD3- N K cells a coordinate downmodulation of both CD16 and { chain molecules was found, FcyRIII may be included in the 5 N K complex that may play an important role in N K activation. The cross-linking of FcRs with each other and/or with other membrane constituents induces cell-mediated functions as well as secondary events such as release of cytokines and other biologically active mediators. Cross-linking of FcyR on human monocytes with IgC induces secretion of tunior necrosis factor (‘INF). Only the selective cross-linking of FcyRI triggers T N F release. However, after treatment of monocytes with proteases or with neuraminidase, T N F secretion was induced by cross-linking of FcyRII as well (Debets et al., 1988, 1990). Cross-linking of FcyRI on nionocytes triggers interleukin (IL,)-6 production. Increased amounts of IL-6 were detected in supernatants of anti-CD3treated mononuclear blood cells, the prerequisite for which was FcyRI but not FcyRII cross-linking. The binding of the Fc part of antLCD3 to FcyRI ma): generate an activation signal for the monocyte accessory cell, leading to the production and secretion of monocyte IL-6 (Krutmann et al., 1990). Cross-linking of FcyRs modulates the production of other interleukins as well. Interaction of CD 16 with immunoglobulins induces the transcription of IL-2 receptor and lymphokine genes and increases the expression of their products in human NK cells (Anegon et al., 1988). Mast cells and basophils express both high- and low-affinity FcyRs. Similar to cross-linking of FceRI, when IgC immune complexes crossbind FcyR, the mast cells degranulate (Daeron et al., 1980) and release histamine and serotonin. Rat basophilic RBL-2H3 cells transfected with cDNAs encoding FcyRIIhl, FcyRIIb2, and FcyRIIIa release mediators after cross-linking of these receptors. Murine FcyRIII but not FcyRIIbl or FcyRIIb2 induces serotonin and TNFa release when aggregated by (2.4G2-MAR) F(ab’), complexes. We may conclude that the same signal, mediated by FcyRIII, can induce the release of preformed mediators or the production of cytokines requiring de noim RNA and protein synthesis (Daeron et al., 1992; Fridman et al., 1992). These findings also underline the importance of accessory chains in receptor functions. The y chains of FceRI are riot only required for the expression of the FcyRIII (Kurosaki and Ravetch, 1989), but participate in transferring signals by this receptor. Since some of the FcyRs mediate endocytosis of immune complexes, they are likely to play a role in antigen presentation as well especially
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since uptake of exogenous antigens precedes their processing. B lymphocytes are highly efficient antigen-presenting cells since they concentrate exogenous antigens on their surface (Lanzavecchia, 1987). Since B cells express FcyRIIbl, which does not mediate endocytosis, FcyRI cannot be involved in the antigen processing. Indeed, researchers showed that binding of IgG complexes to macrophages (Manca et al., 1991) but not to B cells (Roosnek and Lanzavecchia, 1991) increased significantly the efficiency of antigen presentation. Experiments with B lymphoma cells transfected with FcyRIIbl, FcyRIIb2, or FcyRIII have also shown no increase in the efficiency of antigen presentation for FcyRIIbl-expressing transfectants (Amigorena et al., 1992). Note that FceRII on the B cell occupied by antigen-specific IgE enhanced antigen presentation in a ligand- and receptor-specific manner, and mediated antigen focusing as effectively as mIgM. IgGl antibody-antigen complexes containing a high ratio of IgGl were presented 10-fold less effectively than uncomplexed antigen. Thus, binding of IgGl antibodyantigen complexes to FcyR on B cells not only fails to promote antigen presentation, but is inhibitory for T-cell activation (Kehry and Yamashita, 1989, 1990). V. Signal Transduction Mediated by FcyRs
Specific binding of immune complexes to FcyRs of phagocytes triggers as variety of cellular and biochemical events including phagocytosis, respiratory burst, releases of arachidonic acid and lysosomal enzymes, activation of PI turnover, and increase in intracellular Ca2+ concentration ([Ca2+ji). Cross-linking FcyRI and FcyRII on human monocytes results in activation of the Ca2+-PI signal transduction pathway. Phosphorylation of phospholipase C (PLC)-y1 on tyrosine residues activates its enzymatic activity in cells. These changes indicate that signaling is mediated at least in part by activation of PI-specific PLC (Macintyre et al., 1988; van de Winkel et al., 1990). Independent cross-linking of either FcyRI or FcyRII leads to protein tyrosine phosphorylation in the human monocyte cell line THP-1. T h e inhibitory effect of herbimycin A on cellular Ca2+ flux suggests that tyrosine phosphorylation may be important in regulating FcyR-mediated activation of PLC (Scholl et al., 1992). Thus, FcyRI and FcyRII appear to be functionally coupled to a non-receptor tyrosine kinase that phosphorylates PLC-y 1 after receptor cross-linking, thereby causing activation of PLC-y1 (Liao et al., 1992). FcyR-mediated phagocytosis involves activation of serine/threonine protein kinase C in macrophages (Brozna et al., 1988). Engagement of
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human FcyRIIA causes protein tyrosine phosphorylation of several substrates including FcyRIIA itself in platelets and in FcyRIIA-transfected COS cells (Huang et al., 1992). Arachidonic acid generation induced by FcyR-mediated phagocytosis can be blocked by inhibitors of phospholipase A,, indicating the role of this eruyme in signaling. T h e inhibitory effect of pertussis toxin on FcyRII- and FcyRIIIB-induced superoxide generation in PMNs points to the involvement of G proteins (CrocketTorabi ef al., 1990). FcyRIII, FceRI, TCR, and mIg share a common capacity to transfer signals mediated by associated molecules. T h e y chain of the FcyRIII, the chain of the TCR, and the mIg a and P chains show significant structural homology and possess a similar peptide motif (Wegener el al., 1992). Protein tyrosine kinase activation was shown to occur on FcyRIII engagement in NK cells. Tyrosine phosphorylation of substrates including the 5 chain was induced by cross-linking of FcyRIIIA (Einspahr et al., 1991; O’Shea et al., 1991). T h e signal transduction capabilities ofwildtype and mutant forms of FcyRIIIA and FcyRIIIB were analyzed in transfected lymphoid, myeloid, and fibroblast cell lines. FcyRIIIA generated both proximal and distal responses typical of those seen in N K cells and macrophages on receptor activation. In contrast, FcyRIIIB was incapable of transducing signals. After cross-linking, FcyRIIIA signaling was dependent only on the y chain. FcyRIIIA chimeras in which the a-subunit transmembrane and cytoplasmic domains were substituted with the corresponding y-chain sequences functioned as wild-type hetero-oligomeric receptors. Thus, the capacity of the FcyRIIIA complex to activate pathways for cell activation is cell-type restricted and independent of the transmembrane and cytoplasmic domains of the 01 subunit; the y chain seems to be responsible for signal transduction (Wirthmueller et al., 1992). Investigators also showed that a tyrosinecontaining motif, present in the cytoplasmic domain of the associated y chain, is necessary and sufficient to trigger cell activation via FcyRIII (Bonnerot et al., 1992). Signals mediated by FcyRs may be influenced by cooperation between the various types of FcyRs expressed on the same cell. Studies using hybrid mouse monoclonal antibodies suggested that FcyRII regulates FcyRI-triggered signaling in U-937 cells. O n PMN, FcyRII seemed to be responsible for IgG-mediated activation whereas FcyRIIIB served as a trap to hold the IgG-coated particles in place on the cell surface. T h e treatment of PMNs with FcyRII-specific monoclonal antibody resulted in the abrogation of [Ca*+],signals induced by aggregated IgG or FcyRIIIspecific monoclonal antibody, suggesting regulation of FcyRIIIB signals by FcyRII. Treatment of PMNs with protein tyrosine kinase inhibitors
22 1
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abrogated the [Ca*+Iisignals elicited by both receptors, suggesting that tyrosine kinase enzymes associated with these receptors may be crucial for positive or negative signals triggered by FcyRII and FcyRIIIB (Naziruddin et at., 1992). VI. Expression of FcyRs
Cells can express simultaneously FcyRs specific for each class of immunoglobulins (Daeron et al., 1985). The constitutive expression of FcRs on B cells is developmentally regulated, the time windows being different for each class of FcR. IgE FcRs appear in late stages of B-lymphocyte development. FcyR has the broadest developmental window: it appears on the earliest pre-B cells and persists through the stage of the mature mIgD+ mIgM+ B cell. Neither IgM nor IgE FcRs are expressed on postswitched B cells (Waldschmidt et al., 1988). T h e three types of FcyRs are expressed on different populations of immune competent cells (Table I). The differential expression of FcyRs is regulated by genetic and environmental factors. The developmental expression of FcyRs depends in part on selective demethylation of DNA sequences in the a and p genes (Bonnerot et at., 1988; Daeron et al., TABLE I EXPRESSION OF Fcy RECEPTORS
FcyR
FcyRI (CD64) FcyRIa FcyRII (CD32) FcyRIIa
Cell Monocytes, macrophages
IFN-y, G-CSF, IL-4, IFN-y (eosinophils)
Monocytes, macrophages, platelets, neutrophils, placental endothelium
IL-4
FcyRIIb
Monocytes, B cells, macrophages
FcyRIIc
Monocytes, macrophages, neutrophils
FcyRIII (CD16) FcyRI I Ia
FcyRIIIb
Cytokines (effect on expression)
Macrophages, NK cells, T cells, monocytes (small subset)
TGF-P, TNF-a
Neutrophils
IFN-y (eosinophils)
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1990). In addition to genetic factors, the differential expression of FcRs is regulated by several others including the ligand itself and various cytokines; it also depends on the activation state of the cell. This subject has been reviewed elsewhere (Lynch et al., 1990). A. EFFECTOF IMMUNOGLOBULINS ON FcR EXPRESSION T h e effect of immunoglobulins on FcR expression was first suggested when elevated levels of IgE accompanied by increased numbers of FceR+ lymphocytes were observed (Spiegelberg et al., 1979). A similar phenomenon was found in hosts carrying plasmacytomas: gammopathies often go together with the development of a large number of T cells that express FcRs specific for the isotypes of the corresponding myeloma protein (Hoover et al., 1981a,b; Daeron et al., 1985, 1988). This event is associated with high serum levels of the corresponding isotype and does not appear in mice bearing variant tumors that do not secrete monoclonal immunoglobulin (Mathur and Lynch, 1986). After repeated infusions of ascites fluid rich in monoclonal immunoglobulin, mice develop enhanced expression of the corresponding T-cell FcR (Hoover et al., 1981a,b). T h e relationship between immunoglobulins and FcRs expressed on lymphocytes was also demonstrated in vitro. FcRs of various isotypes were induced on T and B cells when these cells were cultured in the presence of the corresponding immunoglobulin isotypes. Such upregulation of FcRs was also found on T hybridoma cells (Yodoi et al., 1983; Fridman et al., 1984; Fridman, 1991). A striking finding was that, when E6.'1'2D4 cells were passaged in vivo, the cells expressed FcRs that were not detectable on cultured cells, showing that the expression of the receptors may depend o n environmental factors. In this type of FcR induction, the ligand itself may play a role (Daeron et al., 1990). Both in uitro- and in vivo-induced receptors have identical specificity. A remarkable difference, however, between constitutive FcRs and receptors detectable only after induction is that the latter are short-lived. T h e mechanism involved in immunoglobulininduced up-regulation of FcR expression includes increased rate of receptor synthesis (Hoover et al., 1981a,b; Yodoi et al., 1982), avidity maturation of the receptor (Sandor et ul., 1990), and decreased rate of FcR turnover (Lee et al., 1987). These observations show that several mechanisms can influence FcR expression simultaneously. Certain results point to the possibility that, in addition to the immunoglobulins, other factors may also be important in regulation of FcR expression. Note that, in contrast to the consequences of repeated infu-
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sion of IgE-rich ascitic fluid, large amounts of heat-aggregated monoclonal IgE do not change the expression of FcrR on lymphocytes. This result suggests that high immunoglobulin concentrations are not sufficient to induce FcR expression. B. MODULATION OF FcyR EXPRESSION BY CYTOKINES Because of the pleiotropic effect of cytokines, the heterogeneity of FcRs, and their diversified cellular distribution, cytokines are likely to modulate FcR expression through several mechanisms. FcyRI expression was shown to be up-regulated on monocytes, monocyte lineages, and neutrophils by long-term exposure to interferon (1FN)-y (Perussia et al., 1983a,b, 1987; Pan et al., 1990). The FcyRIs expressed on PMNs are involved in cytotoxicity and account for the IFN-y-induced enhancement of ADCC function (Shen et al., 1984, 1986; van Schie et al., 1991). Macrophages stimulated in uiuo by rIFN-y were found to be highly efficient in FcyRI-mediated cytolysis because of an altered cytolytic mechanism and enhanced FcyRI density (van Schie et al., 1992). IFN-y does not augment FcyRII or FcyRIII expression on monocytes (Liesveld et al., 1988; Fanger et al., 1989), but up-regulates FcyRIII on neutrophils (Buckle and Hogg, 1989).Other cytokines, including IL-1, IL-2, IL-3, IL-4, IL-6, G-colony-stimulating factor (G-CSF), GM-CSF, M-CSF, and TNF did not up-regulate FcyRI expression. On the TNFtreated PMNs, FcyRIII expression decreased (Shen et al., 1987). IFN-y treatment resulted in expression of FcyRI and FcyRIII, as well as in upregulation of FcyRII in eosinophils (Hartnell et al., 1992). Note that IFN-y modulates the expression of other immunoglobulinbinding molecules as well. For example, it is a potent inducer of FcrRII on macrophages and on the monocytic cell line U-937 (Naray-Fejes-Toth and Guyre, 1984; Boltz-Nitulescu et al., 1988). The cooperation of various cytokines in regulation of FcR expression was shown in experiments with rIL-4, which synergized with rIL-6 and IFN-y in the increase of FceRII expression on U-937 cells whereas rIFN-y and rIL-6 had additive effects (Willheim et al., 1991). (Modulation of FcrRII by cytokines is reviewed by Lynch, R. G. et al., 1992.) Interestingly, natural antibodies against IFN-y had no inhibitory effect on the antiviral activity of IFN-y but inhibited the IFN-y-induced expression of FcyR and HLA-DR antigens in U-937 cells (Turano et al., 1992). Human blood monocytes express FcyRI and FcyRII whereas FcyRIII appears only during maturation into tissue macrophages or in inflammation
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(Clarkson and Ory, 1988). Transforming growth factor (TGF)-P seems to affect the appearance of FcyRIII on monocytes, whereas other cytokines such as GM-CSF and IL-3 do not modify the expression of FcyRIII (Welch ef al., 1990). IL-4 antagonizes the TGF-P-induced FcyRIII (Wong et al., 1991).
C. CELLACTIVATION A N D FcyR EXPRESSION The functional relationship between FcRs and the antigen receptors is of great importance because they have regulatory role in immune responses. Cross-linking of the cognate receptors for antigen on T and B cells modulates the expression of FcRs (reviewed by Bonnerot et ul., 1988). Expression of FcRs on subsets of activated murine T cells was thoroughly studied (Sandor et al., 1990a). FcR was detected on activated CD4+/Th2 but none, o r small amounts, was detected on activated CD4+/Th1cells. T h e CD4+/Th2cells expressed one or several classes of FcR (some clones even had all five classes). T h e mechanism of the differential induction of FcRs on the two subpopulations of helper T cells is not clarified yet. Activated CD8+ T lymphocytes can also express FcRs (Hoover et ul., 198 lb). In growing plasmacytomas, the infiltrating CD8+ T lymphocytes were found to be activated and expressed multiple classes of FcRs. However, only the class that matched the monoclonal immunoglobulin persisted on the cells. On murine 'T'CR y/6+ T lymphocytes, the transition from the resting to the activated state (triggered via the T3Ti TCR complex) was accompanied by appearance of surface membrane receptors specific for immunoglobulin heavy (H) chain isotypes (Sandor et al., 1992). FcyRII is constitutively expressed on B cells. T h e number of receptors, their binding capacity, and the release of-FcyRII molecules change during the various phases of B-cell development. Murine B cells express only FcyRIIbl; therefore, the genetic and environmental factors seem to affect first the number of functionally active FcyRIIs on the cell membrane. Resting and activated B cells express predominantly the P l transcript. On lipopolysaccharide (LPS)-activation, a significant increase in the P but not the a mRNA is induced. T h e increase of FcyRII on activated B cells results from P l mRNA induction, since (32 transcripts are barely detectable. FcRII expression occurs in the late G I phase of the cell cycle (Amigorena et ul., 1989). The modulatory effect of IL-4 seems to be important in the induction of a functionally active form of FcyRII on the B-cell membrane. IL-4 has been shown to induce loss of FcyRII ligand-binding capacity on murine B cells (Laszlo and Dickler, 1988),
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and can reverse FcyRII-mediated inhibition of B-lymphocyte activation (O’Garra et al., 1987). Kinetic studies on FcyRII expression and release show that, on LPSactivated murine B cells, the level of FcyRIIBl mRNA and the expression of FcyRII in the G,, phase of the cell cycle increase (Amigorena et al., 1989). Moreover, a new epitope of FcyR appears that correlates with the release of soluble FcyRs (Pure et al., 1984). During the activation of human B lymphocytes, the expression of FcyRII shows a biphasic time course. A transient increase of FcyRII expression is shown 10 min after stimulation, with simultaneously decreased ligand binding. Later, after 3-24 hr, the number of FcyRII bearing cells decreases. FcyRII expression increases 48 hr later, mainly on blast cells. At the same time, soluble fragments (33 kDa) of FcyRII with the ability to bind to human IgGFc are released (Sarmay et al., 1990b, 1991). To identify the cell cycle phase in which the activated B cells express high number of FcyRII, the effect of cell cycle blockers was studied (Gergely and Sarmay, 1992). FcyRII expression increases in the G,, phase. T h e release of receptors occurs in the early G, phase. Later, before entrance into S phase, receptor release is accompanied by the enhancement of FcyRII expression. These observations suggest that, on both murine and human B cells, the expression, binding capacity, and release of FcR molecules change according to stages in the cell cycle.
VII. Soluble Fcy Receptors Ehrlich’s prkdiction (Ehrlich, 1900) concerning the dual function of antibodies turned out to be true for cell-membrane structures as well. Originally the proposed dual function of antibodies implied that the same structure could have a cognitive function as a receptor on the cell membrane, but also as a soluble molecule. Ehrlichs prediction was proven when surface immunoglobulins were detected on B cells (Moller, 1961). Since then, membrane-bound and soluble forms of several other molecules including cytokine receptors and various isotypes of FcyRs, have been identified. However, the main message of Ehrlich’s hypothesis -that receptors on the cell surface exist independently from exposure to their ligands and that the soluble (antibody) and membrane-bound (antigen receptor) forms are identical and are produced by the same cells-calls for some modifications. In most cases, the soluble molecule is a truncated form of the membranebound receptor. Usually, the ligand-binding parts are identical whereas
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their C-terminal segments are different. For example, the membranebound and secreted immunoglobulins produced by the same B cell have identical light (L) chains and comparable H chains except, for a short part at the C terminus. The membrane-bound H chain has a stretch of hydrophobic amino acids representing the transmembrane region of the receptor, followed by a short cytoplasmic region. In the secreted immunoglobulins, this region is replaced by a short sequence of hydrophilic amino acids. T h e transition from membrane to secreted H chains occurs at the level of mRNA processing: alternative processing of a primary RNA transcript leads to the corresponding different mRNAs. The membrane-bound immunoglobulin (mIg) was found in a noncovalent complex with a disulfide-linked heterodimer of glycosylated transmembrane proteins, Iga-Igp. Iga and Igp are required for surface expression of all classes of mIg. T h e membrane-bound forms of p, and 6 chains have only 3-amino-acid cytoplasmic regions. The cytoplasmic regions of Iga and Igp consist of 61 and 48 amino acids, respectively, and could physically couple mIg to intracellular effector molecules. Iga and Igp resemble the 5 chain of TCR and the y chain of some FcRs (Desiderio, 1992). FcRs appear both in membrane-bound and soluble forms. However, in contrast to the Ig molecules, in addition to alternative splicing, other mechanisms can also lead to their release (details will be discussed later). Soluble inimunoglobulin-binding factors in cell-free supernatants of activated lymphocytes were first described by Fridman and Golstein ( 1974). Since then, such immunoglobulin-binding factors (IBFs) specific for each immunoglobulin isotype have been demonstrated (reviewed by Fridman and Sautes, 1990a). Researchers showed that T cells expressing FcyRs for a given isotype spontaneously produce IBFs for the same isotype. T h e production of IBFs was enhanced in activated T cells (Le Thi Bich Thuy et al., 1980). Incubation of activated T lymphocytes in serum-free medium resulted in disappearance of FcyRs from the T cells and in the appearance of IgG-BFs in the supernatants (Neauport-Sautes et ul., 1975). T h e released soluble FcRs have functional activities because they modulate the zn uitro synthesis of immunoglobulins and regulate antibody production in an isotype-specific manner. In addition to T lymphocytes, other FcyR-positive cells such as B lymphocytes (Pure et al., 1984), macrophages (Calvo et al., 1986), and granulocytes (Le Thi Bich Thuy, 1982) can produce IBFs. This production is regulated by various cytokines (Fridman, 1989) and by interaction of the cells with immunoglobulin molecules (Daeron et al., 1985). IBFs were detected in both mouse and human serum (Khayat et al., 1984, 1987; Sarfati et al., 1988).
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For a long time, it was not clear whether IBFs are related to FcRs. Recent results, however, suggest that this is the case (Neauport-Sautes et al., 1975). Investigators showed that only FcyR+ hybridoma cells produced IgG-BF whereas FcyR- clones did not. Similarly, only the activated Fcy R-bearing but not the receptor-negative T cells produced IgGBF (Fridman et al., 1981). Researchers also showed that the mouse FcyRII-specific monoclonal antibody 2.4G2 recognized the IgG-BF produced by FcyRII-bearing cells (Daeron et al., 1986). Moreover, L cells transfected with a cDNA encoding FcyRII express FcyR (56 kDa) and produce a glycoprotein (38 kDa) that binds this reagent (Sautes et al., 1990). Site-directed mutagenesis of FcyRIIPl cDNA was used to convert the integral membrane form of FcyRII into a 174-amino-acid soluble form containing only the extracellular portion. L cells were transfected with this mutated cDNA inserted into a eukaryotic expression vector. Researchers found that sFcyRII isolated from the culture medium of the cell line (CulB3) can inhibit, similar to native IgG-BF, secondary and primary in uitro antibody responses (Sautes et al., 1992). T h e assumption that IBFs represent the soluble form of FcRs was verified for IgE-BF as well. The identification of CD23 as FceRII and the cloning of its gene have shown that IgE-BF derives from FceRII (Delespesse et al., 1989). Both the expression of FcyRs and the production of sFcyRs are dependent on environmental factors. Factors that influence the expression of FcRs seem to affect the production of soluble receptors as well. Immunoglobulins (Lowy et al., 1983), IFNs (Fridman et al., 1980), and IL-2 (Daeron et al., 1990) up-regulate FcyRII and/or FcyRIII expression and, at the same time, sFcyR production, whereas IL-4 decreases FcyRII expression (Waldschmidt et al., 1989). Analysis of the cellular mechanisms involved in antigen-induced IgBF production revealed that antigen-primed helper T cells released lymphokines that stimulate unprimed T cells to produce IBFs. Researchers suggested that IBF production may be involved in the antibody-response-enhancing effect of IL-4 (Adachi et al., 1989). FcRs are constitutively produced by various T cell hybridomas. The incubation of these cells with rIL-2 increased the release of IBF and reduced the expression of the corresponding FcRs (Amin et al., 1988,1990; Tamma and Coico, 1992). VIII. Mechanisms of sFcyR (IgG-BF) Production IgG-BF can be generated by two different mechanisms. One produces different molecules through alternative splicing of mRNA. The
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other mechanism generates IgG-BF by proteolytic cleavage from the membrane-bound FcyRII. However, other mechanisms may also exist. Generally, IgG-BF seems to be a fragment of membrane-bound FcyRs with preserved IgG-binding capacity. A. SHEDDING OF FcyRs
Macromolecules can be shed from the surface of viable cells. A subset of FcyR+ human blood lymphocytes releases their FcyR when incubated at 37°C in serurn-free medium; these functionally active molecules can be isolated from the supernatants (reviewed by Gergely, 1988). The released FcyRs are monomeric, have IgG-BF activity, and interact with the CH3 domain of IgG (Sandor el al., 1986). Another subpopulation of FcyR+ cells does not shed the receptors under similar conditions (Sarmay et nl., 1978; Sandor et al., 1978). Such stable receptors are the FcyRIII molecules of N K and K cells and the FcyRII molecules of resting B cells (Sarmay et nl., 1984, 1990b). These receptors have two binding sites for IgCFc, one for the CH2 and another one for the CH3 domain. FcR shedding may be the consequence of membrane reordering and/or alterations in membrane fluidity. [It must be nientioned that similar temperature shift-induced shedding (Krawinkel and Rajewsky, 1976) was used to release other membrane constituents as well.] Blebbing and shedding of membrane vesicles from P8 15 mastocytoma cells could be induced by exposure to low temperature, which disrupts microtubules (Liepins, 1983). Note that the temperature-induced FcR shedding is not accompanied by cell damage, and lymphocytes retain their functional potentials (Tamma and Coico, 1992).
B. PRODUCTION OF sFcyRs CLEAVAGE
BY
PROTEOLYTIC
We referred earlier to the existence of transcripts of alternatively spliced products encoding a two-domain (truncated) receptor. 'The possibility was raised that such transcripts encode the soluble FcyRs (Qiu et al., 1990; van de M'inkel et nl., 1991; Ernst et al., 1992). However, in addition, proteolytic cleavage may also act in receptor release. Acti~atedbut not resting B cells were found to release FcyRII (Sarmay et nl., 1990b, 199 1). This feature coincided with the appearance of a trypsin-like serine protease activity on the cell surfaces (Biro et al., 1992). Furthermore, specific serine protease blockers inhibited the release of E'cyRII from B cells. Analysis of the predicted amino acid sequence of FcyRII showed that at least t w o sites could be potential targets of
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trypsin-like serine proteases. The cleaved products could be held together by disulfide bridges, allowing maintenance of the ligand-binding capacity of the released 33-kDa fragment. Various membrane-associated proteins are phosphorylated in activated B cells. One of these tightly associated to FcyRII was identified as the Fyn tyrosine protein kinase. Cross-linking of mIgM activates this tyrosine kinase, which in turn induces the phosphorylation of other molecules. The serine phosphorylation of FcyRII may be the consequence of activation of serinelthreonine kinases induced by activated Fyn (Sarmay et al., 1990a, 1994). T h e alteration in ligand-binding capacity and the proteolytic cleavage of FcyRII on activated B cells may be connected with the conformation of the phosphorylated receptor (Gergely and Sarmay, 1992). Several other findings support the view that FcyR release is the consequence of proteolysis. T h e phosphatidylinositol-glycan linkage of FcyRIII- 1 expressed on neutrophils is susceptible to cleavage by phosphatidylinositol-specific phospholipase C (PIPLC) whereas the treatment of NK cells with PIPLC did not cleave CD16 from the cell surface. However, in culture, NK cells spontaneously released CD16; this molecule was smaller than the membrane-associated one (Lanier et al., 1989b). The spontaneous and phorbol- 12-myristate- 13-acetate (PMA)-induced release of CD16-I1 from the cell membrane seemed to be the consequence of proteolytic cleavage performed by a metalloprotease (Harrison et d., 1991). Investigators have shown that murine sFcyRII and sFcyRIII can be produced by cleavage of the two extracellular domains of the receptors between amino acids 165 and 180 (Daeron et al., 1989; Sautes et al., 1991; Fridman et al., 1992). By transfection of a fibroblast line with a mutated FcyRIIbl cDNAa, a 174-amino-acid recombinant murine sFcyR was produced that had the functional features of IgG-BF: it inhibited IgG production in vitro (Varin et al., 1989). T h e regulation of B cells by low affinity FceRII and IgE-BF has been thoroughly studied (reviewed by Gordon et al., 1989). The mechanisms involved in IgG-BF and IgE-BF production are very similar. CD23 is a type I1 integral membrane protein of M , 45 kDa with a predicted length of 321 amino acids. The cDNA for human FccRII has been cloned. Although a significant portion of its extracellular domain bears a marked homology to C-type animal lectins and contains an inverse “RGD” sequence, neither of these regions appears to be involved in low-affinity IgE binding (Daeron et al., 1989). Resting B cells express CD23 at a very low level; its expression can be up-regulated by phorbol esters, IL-2, and IL-4 (Gordon et al., 1986; Hivroz et al., 1989). Activated B cells release soluble CD23 derived from the membrane-bound protein
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through proteolysis: 35-kDa fragments are first produced that rapidly degrade to the more stable 25-kDa form, which can bind to IgE (Letellier et al., 1989). (A long-lived 14-kDa product has no IgE-binding capacity.) A thiolesterase (SH-protease) seems to be involved in this proteolytic process. However, more than one enzyme may be responsible in the generation of IgE-BFs of different sizes. T h e enzyme that cleaves the 33,000-37,000 IgE-BF precursors seems to be present in the plasma membrane of all FceRII-positive cells. IX. Regulatory Role of Membrane-Bound and Soluble FcyRs
Immune complexes were found to inhibit antibody responses (reviewed by Uhr and Moller, 1968). This effect required the availability of the Fc part of the antibody (Abrahms et al., 1973). Detailed analysis of the phenomenon suggested that immune complexes interact with both the mlg and the FcyRs and mediate a blocking signal for B cells. A. REGULATION OF ANTIBODY PRODUCTION ON B CELLLEVEL BY FcyRII Immobilized but not soluble immune complexes were found to inhibit activation of murine B cells (Ryan and Henkart, 1976). T h e results were interpreted to show that the immobilized complexes bind to the mIg and FcRs and trigger a central “off” signal. Other observations (Kolsch et al., 1980) confirmed that the linkage of mIg and FcyRs inhibited B cell function directly; the requirement for generation of the “off” signal was the simultaneous ligation of both receptors. This view was supported by the demonstration that monoclonal anti-FcR antibody could reverse the inhibition mediated by an anti-p, antibody (Phillips and Parker, 1984). Resting B cells that had not encountered antigen were highly susceptible to the signal induced by cross-linking of mIg and FcyRs (Uher and Dickler, 1985). T h e biochemical basis of the inhibitory effect of rabbit antiimmunoglobulin has been identified. Activation of B cells via mIg was shown to involve a guanine-nucleotide regulatory protein (Gp) that couples mIg to the PLC-mediated hydrolysis of phosphatidylinositol4,5biphosphate (PIP,) to generate intracellular second messengers (Bijsterbosch and Klaus, 1985; Harnett and Rigley, 1992). Co-cross-linkage of mIg and FcyRs with intact antibody on B cells uncoupled the antigen receptors from Gp, but did not affect Gp/PPI-phosphodiesterase (PDE) coupling (Rigley et al., 1989). These findings suggest that the control of
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the level of PPI-PDE activity may play a role in antigen-induced B-cell regulation. Note that one important function of FcyRII is the “fine tuning” of B-cell responses to antigen. Remarkably, however, some IgG-containing immune complexes enhance immune responses. Trinitrophenyl (TNP)-specific monoclonal IgG antibodies suppressed the antibody response to particulate antigens but enhanced the response to soluble antigens (Wiersma et al., 1989). The enhancing effect of IgG was coupled to complement, and therefore may depend on the interaction of the complexes with FcRs and complement receptors expressed on B Cells. The interaction with the complement receptor may directly stimulate B cells (Heyman, 1990). These observations suggest that both FcyRII and CR2 participate in the regulation of antibody production at the B-cell level. T h e role of membrane-bound FcyRII in regulation of antibody responses is now generally accepted. We can pose the question of whether FcyR fragments released from the cell membrane with maintained Fcbinding capacity (IBFs) can also be involved in the regulation. IBFs released from various FcyR+ cells were shown to modulate the production of the corresponding immunoglobulin isotype. IgG-BFs inhibited the immunoglobulin secretion of fully differentiated B cells, the in uiuo IgM primary responses to sheep red blood cells (SRBC), and the T-dependent and T-independent in vitro responses to antigens. Most of the studies concerning the regulatory role of IgG-BFs are related to T-cell products. However, the regulatory pathways of the various IgG-BFs are due to their similar binding properties. The finding that FcRs and IBFs for various isotypes are induced by the corresponding immunoglobulins underlines that immunoglobulins and membrane-bound and soluble FcRs are integral components of a regulatory network. Based on the interactions of immunoglobulins and various immunoglobulin-binding structures, a formal and functional basis for an isotypic regulatory network was proposed including the dual function of antibodies and its link to the idiotypic network, particularly to components outside the immune system (Jerne, 1984; Fridman et al., 1986). T h e networks formed by functional interactions of idiotopes and paratopes on the one hand, and IgGFc and Fc-binding structures on the other, maintain a steady state that returns to equilibrium after external or internal perturbations. The first step in the immunoglobulin-induced FcR up-regulation and IBF release seems to be the aggregation of FcRs on the cell surface. Since the mRNA content in immunoglobulin-treated cells was not changed, the effect on FcR and IBF is at the post-transcriptional level (Hoover et al., 1981a). IgG-BF inhibits the production of IgM in primary and IgG in
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secondary it1 rutro antibody responses. T h e molecular mechanisms of the suppression have not yet been clarified. Evidence was obtained that inhibition of the IgC production of B-cell hybridomas by FcyRII+ IgC-BFproducing ‘r cells was mediated by soluble factors (Brunati et al., 1990). T h e inhibition affected both H and L chain synthesis and the H- and L-chain-encoding mRNA steady state. Since a complete IgG molecule was required on the B-cell hybridoma to effect the blockade exerted by the IgG-BF-producing T hybridoma, the inhibition may be mediated by interactions between the released IgG-BF and mIgC. Since mIg molecules are the targets of IgG-BFs, pre-€3 cells and plasma cells devoid of mIg are insensitive to the regulatory signals of IgG-BFs. IBFs may inhibit IgG production through interaction with some other surface molecules o r through homophilic binding to FcyR itself. Therefore crosslinking of mIg with FcR through IgG-BF is likely to trigger a cascade of‘ events that gives rise to the inhibitory ef-fects (Teillaud et al., 1990). T h e possibility cannot be excluded that, in addition to IgC-BF, other soluble factors acting synergistically may be involved in the suppression of antibody production. However, IgC-BF alone can mediate this effect. Other studies (Daeron et al., 1989) have shown that recombinant soluble FcyRII inhibits antibody production in zutro. In these investigations, soluble recombinant FcyRII containing only the two extracytoplasmic domains of the molecule was used. L cells were transfected with the mutated cDNA inserted into an expression vector. The soluble FcyRII isolated from the culture medium of the resultant cell line inhibited primary and secondary in mtro antibody responses. B. B CELLCYCLEA N D FcyRII RELEASE We mentioned several lines of evidence showing that both membranebound and released FcyRs (IBFs) play decisive roles in the regulation of humoral immune responses by acting directly on B cells. Based on the correlation between the B-cell cycle, FcyRII expression, and phosphorylation on the one hand and activation of proteolytic enzymes and receptor release on the other, we suggested the following scenario (Gergely and Sarniay, l99Ob, 1992). Resting B cells express both niIg and FcyRII. Constitutive phosphorylation of the low-affinity FcyRII on resting B cells is negligible. Neither activation of trypsin-like serine esterases nor release of FcyRII occurs in the Go phase. During the very early stage of B-cell activation, the FcyRIIs are phosphorylated, which leads to their conformational altersiion.
This event can be regarded as “functional down-regulation,” mean-
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ing that while the FcyRII molecules are present in the cell membrane, they are functionally inadequate and cannot be cross-linked with mIg by antigen-complexed IgG. Consequently, inhibitory signals via FcyRII are not induced, and antibody production during the early phase of B-cell activation is favored. T h e conformational alteration of FcyRII may facilitate its proteolytic cleavage as well. Since activation of proteolytic enzymes was detected simultaneously with the phosphorylation of FcyRII, proteolytic fragmentation of the receptor (IgG-BF production) and its functional downregulation may be simultaneous events. After a transient decrease, a significant increase of FcyRII expression and release occurs in the early G, phase. This “functional upregulation” (increased expression of functional receptors) allows the cross-linking of mIg and FcyRII by antigen-IgG complexes, which transmits the “off” signal resulting in the gradual decline of antibody production. Simultaneously, the proteolytic cleavage of FcyRII provides IgG-BF molecules that contribute to the suppression. T h e mechanism by which IgG-BF inhibits the production of the given immunoglobulin isotype is unknown. However, since the activated B cell can be regarded as the IgG-BF source, it may represent an autocrine regulatory system. Since IgG-BF can interact with membrane-bound IgG molecules (Fridman et al., 1986), its inhibitory effect may be mediated by this direct interaction. In this respect, IgG-BF may behave like the rheumatoid factor (Gergely et al., 1992). On the other hand, researchers showed that T-cell-derived IBFs inhibit antibody production, that is, the involvement of T cells in this regulatory mechanism cannot be excluded.
X. Expression of FcRs on Tumor Cells In vivo localization of immunoglobulins within nonlymphoid tumor tissue suggested that tumor cells may express FcRs (Ran and Witz, 1970; Tonder and Thunold, 1973; Braslawsky et al., 1976; Tonder et al., 19’76; Ran et al., 1978, 1984; Tonder and Matre, 1979). However, at the same time the possibility could not be excluded that the immunoglobulin bound to the FcRs of the infiltrating host cells. In fact, both in cancer and precancerous conditions, an increase in FcyR-expressing immunocytes including suppressor T cells was demonstrated (Fujimoto et aE., 1976). Because of the intimate relationship between immunoglobulins and FcRs expressed on lymphocytes, a large number of FcyR+ T cells expressing receptors for the isotype of the monoclonal immunoglobulin
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can be regularly found in gammopathies. Simultaneously, the serum sFcyR levels are also elevated (Hoover et al., 1981a,b; Mathur and Lynch, 1986; Daeron et al., 1988). This condition was described for IgA- (Hoover et al., 1981), IgC-, and IgM-secreting plasmacytomas (Mathur and Lynch, 1986; Uher et al., 1987), and IRE-secreting hybridomas (Mathur et al., 1986). In mice bearing tumors that secrete IgC, IgA, IgM, or IgE, the FcR+ T cells are mainly CD8+ whereas in tumors secreting IgD the FcR-bearing cells are predominantly CD4+ (Coico et al., 1985). The expansion of FcR+ T cells in the tumor-bearing mice and in patients with multiple myeloma may represent exaggerated (but otherwise normal) immunoregulatory mechanisms (Hunley-Hyde and Lynch, 1986). T h e increased expression of FcRs in gammopathies has far-reaching consequences. One such example is the immunodeficiency that develops in mice bearing immunoglobulin-secreting tumors (Ullrich and ZollaPazner, 1982). Investigators showed that the development of L3T4 Ly2+ FcrR+ T cells in mice bearing IgE-secreting hybridoma was followed by a progressive inhibition of IgE production (Mathur et al., 1986). Another regulatory pathway was shown on B cells in mice bearing plasmacytomas in which 'TGF-P produced by plasmacytoma cells and host macrophages mediated a significant decrease in FcrRII expression (Berg and Lynch, 1991). Certain data show that FcR+ tumor cells may have a growth advantage in uiuo. This effect was shown with a transplanted polyoma-virusinduced anaplastic carcinoma (SEYF-a tumor) (Ran et al., 1984). SEYF-a cells are composed of a major FcR- and a minor FcR+ subset. However, in contrast to the in uzuo conditions, in zritio the expression of FcR successively decreases. When reimplanted into mice, FcR was again expressed, as detected by reactivity with a monoclonal antibody against FcyRs. Therefore, the expression of FcyR on tumor cells seems to depend on a factor provided in aivo. In recent experiments, polyoma-virus-transformedBALB/c 3T3 cells were cloned and passaged once in syngeneic hosts. Thereafter the cells were returned to culture. These (CTC) were compared with the in vitro maintained clones (C). C cells did not, whereas a subset of CTC cells did express FcyRII. Tumors analyzed soon after grafting were entirely FcyRII-, whereas in later stages the number of FcyRII+ cells increased. FcyRII message was detected in CTC cells derived after long, but not after short, latency periods and was not detected in C cells. FcyRII expression was down-regulated in CTC cells when explanted into culture, but this change could be avoided and FcyRII could be induced on C cells when the medium was supplemented with mouse serum or rIFN (Ran et al., 1988, 1991). After a single in zjivo passage, the cells grew and
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metastasized more readily than the in nitro maintained C cells. These results were substantiated by transfection of the FcyRIIPl gene, which increased both the tumorigenicity and the metastatic capacity of the cells (Langer et al., 1992). These results show that “ectopic” expression of FcyRII can lead to elevated malignancy and therefore can be regarded as a step in progression of tumors. Xi. sFcRs in Malignancies Soluble FcRs of different isotypes have been detected in biological fluids under normal and pathological conditions. However, the data usually refer to IgG-binding receptors (Sautes et al., 1992). Although sFcyRs are present in murine serum (e.g., 100 ng/ml in BALB/c mice), the levels are elevated in tumor-bearing mice. A 5- to 10-fold increase was detectable in mice with IgG2a-, IgG2b-, or IgG3-producing tumors. The increase was less pronounced (2- to %fold) in nude mice bearing IgGsecreting tumors. T h e levels in BALB/c mice with non-IgG-producing tumors were slightly higher (Sautes et al., 1990). Interestingly, the serum levels of soluble FcR increased concomitantly with the IgG2a levels in mice carrying IgGPa-secreting tumor cells (Lynch, A. et al., 1992). Therefore, the serum levels of sFcyRs seem to depend on the presence of activated cells of the immune system, including malignant B cells, and the increase is T-cell dependent. A correlation between B cell cycle and FcyRII expression and release was observed in human cells as well (Gergely and Sarmay, 1992), but the serum levels of IgG-BF derived from FcyRIII in patients with multiple myeloma and other related malignant or benign gammopathies were highly variable. Furthermore, patients staged as Grade I11 according to Durie-Salmon staging had very low levels of IgG-BF (Brunati et al., 1990). The relatively low levels of IgG-BF can be attributed in part to the methods of detection (monoclonal antibodies) and in part to the heterogeneity of sFcyRs (various isotypes and/or isoforms could be released, but only one was investigated). I n addition, factors (cytokines) that modulate the expression and release of FcRs may also differ in the patients. Soluble FcRs may be released from tumor cells through proteolytic cleavage of their membrane receptors (Sarmay et al., 1990b; Sautes et al., 1991; Gergely and Sarmay, 1992), but they may also be products of the FcyRII genes that lack transmembrane sequences (Fridman et al., 1992). T h e results obtained with certain well-characterized B-cell lines have shown a correlation between the phenotype and expression of trypsinlike serine protease enzymes of the cell lines. An Epstein-Barr virus (EBV)-negative Burkitt lymphoma line with resting phenotype has low
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activity, whereas its EBV-genome-carrying convertants with the phenotype of activated B cells have shown high proteolytic activity (Biro et al., 1992).These results point to the possibility that soluble receptors may be released from (some) malignant cells through proteolytic cleavage. However, the receptors in the serum of tumor-bearing mice are probably derived from the cells of the host because no direct relationship between the expression of FcyR on the tumors and the increase in serum sFcyR levels was observed. Furthermore, mice with tumors with deleted FcyRII genes also had elevated sFcyR levels (Lynch, A. et al., 1992). As mentioned earlier, monoclonal immunoglobulins play an important role in the release of sFcR. We must emphasize again that immunoglobulins, FcRs, and IBFs are constituents of the same immunoregulatory mechanism. We expect that this regulatory circuit is influenced in patients with B-cell malignancies and mice grafted with irnmunoglobulin-producing tumors. sFcRs released from tumor cells can interfere with the balance between immunoglobulins and sFcRs of the corresponding isotypes. Cytokines can affect the events at different steps; they can modulate both FcR expression and its release. T h e FcR+ T cells are essential parts of the regulation, since they can produce both cytokines and IBFs (Ran et nl., 1988; Fridnian and Sautes, 1990a).
XII. Biological Role of FcR-Mediated Functions
in Malignancies Tumor cells may produce factors that modify FcR expression on immunocompetent cells. Because of the release of FcRs from these host cells, the levels of soluble FcRs can be increased. T h e functional relationship between immunoglobulins and menibrane-bound and soluble FcRs must also be taken into consideration. All these factors are o f great importance in immune phenomena that accompany tumor growth. In addition, the expression of FcRs on tumor cells may facilitate their growth. T h e majority of observations refer to FcyRs. Therefore we consider here only the role of this type of immunoglobulin-binding structure. FcR-mediated functions can influence the proliferation of tumor cells through several mechanisms. Secretion of the cytotoxic and imniunoregulatory cytokine T K F by monocytes can be induced by crosslinking of FcRs. Researchers showed that T N F is secreted in the course of ADCC against tumor cells. Moreover, malignancy may trigger an ongoing immune response that leads, through the interaction of opsonized tumor cells and monocyte/macrophage FcRs, to a continuous
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release of TNF, which can cause metabolic disorder and cachexia (Debets et al., 1988, 1990). FcyR-carrying tumor cells can bind tumor-specific antibodies via their Fc part. By this means, the receptors counteract antibody-dependent effector functions such as complement-mediated lysis or ADCC, and thus protect the tumor cells. FcR-bound specific antibody molecules may even mask antigens sterically, and thus prevent the antigen-specific immune functions. In this way, FcyR expression endows tumor cells with the ability to escape immune mechanisms. Furthermore, FcR+ tumor cells can serve as “sponges” for IgG and thereby lower the level of circulating total IgG or specific isotypes (Langer et d., 1992), which in turn could up-regulate IgG production. This up-regulation may elicit the increase in FcyR+ mononuclear blood cells shown to occur in cancer patients (Ilfeld et al., 1986). The recruitment of Fcy R-expressing tumor infiltrating macrophages (Evans, 1972) and/or T cells (Galili et al., 1979) has also been described. Release of FcRs by these cells can lead to further increases of 1 6 - B F levels in the serum (Ran et al., 1988; Witz and Ran, 1992). Thus the cascade of events that is initiated by the FcyRs facilitates the escape of tumor cells from immune surveillance. The increased number of FcR-expressing T cells in cancer and precancer conditions suggests the role of T cells in these events (Lynch et al., 1990; Mathur and Lynch, 1986). Indeed, IgG-BF-producing FcyRII+ T hybridomas were shown to inhibit the production of IgG by malignant B cells through noncognate interactions (Brunati, et al., 1990). FcR+ T cells and IgG-BF seem to act on B cells and to suppress immunoglobulin synthesis. XIII. Conclusions
T h e interaction of FcRs with the immunoglobulin molecules provides a link between specific antigen recognition and effector cells. Thus, these receptors play important roles in the defense against infection. The FcRs of B lymphocytes (FcyRII and FceRII) are involved in the regulation of antibody production. Their release from activated B cells is the consequence of proteolytic cleavage or of alternative splicing. The soluble “truncated” receptor molecules (IBFs) maintain the immunoglobulinbinding domains and, as soluble factors, regulate the humoral immune response. They inhibit immunoglobulin production in an isotype-specific manner. As components of a regulatory circuit (together with immunoglobulin molecules and membrane-bound FcRs), IBFs also serve immune surveillance. Therefore, under physiological circumstances, due
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to their participation in effector and regulatory functions, both the membrane-bound and the soluble FcRs are important elements of immune defense. Expressed on the tumors cells, however, these molecules may behave as “traitors” and counteract the activities of the immune system: they can thus be deleterious to the host response and facilitate the escape of the malignant cells. Evidence suggests that similar FcR-mediated “self-defense” mechanisms exist in parasites and virus-infected cells that can bind the Fc portion of IgG (Johansson et al., 1985).Several bacteria express FcR-like immunoglobulin-binding molecules (Stenberg et al., 1992). Therefore, FcRs or FcR-like molecules on tumor and virus-infected cells and on bacteria may be used as efficient “survival kits” exploiting the Fc-binding capacity of these molecules. How do FcRs help tumor growth and the escape from immune recognition? 1. FcRs expressed on tumor cells bind (tumor-specific) antibodies via the Fc portion, and thereby divert the antibody-induced effector mechanisms. 2. sFcRs released from FcR-carrying tumor cells and infiltrating T lymphocytes interfere with circulating regulatory IBF, or influence in situ localized T cells to suppress antibody production. 3. Immunoglobulins secreted by tumors stimulate FcR expression and release, which perturbs the regulatory network including immunoglobulins, IBF, and FcR. 4. Cytokines produced by tumor cells and by cells of the stimulated immune system modulate FcR expression and release.
Although many details of the FcR-dependent mechanisms are still missing, our current knowledge is sufficient to speculate on the FcRdependent negative control mechanisms and to try to formulate strategies that could inhibit their undesired effects in malignancies. ACKNOWLEDGMENT The authors thank Eva Klein for her critical review of-this manuscript.
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DISSECTING MOLECULAR CARCINOGENESIS: DEVELOPMENT OF TRANSGENIC MOUSE MODELS BY EPIDERMAL GENE TARGETING David A. Greenhalgh and Dennis R. Roop Departments of Cell Biology and Dermatology, Baylor College of Medicine, Houston, Texas 77030
I. Introduction 11. Development of Single Transgenic Genotypes
A. Design of the Epidermal Targeting Vector B. Targeting the Activated Oncogenes Ha-rm andfos C. Targeting Transforming Growth Factor a D. Targeting the Viral Oncogenes of Human Papillomavirus 18 111. Development of Multiple Transgenic Genotypes A. Cooperation between Ha-ras,fos, and TGFa B. Effect of p53 Tumor Suppressor Gene Loss IV. Development of a Rapid Screening System for Tumor Promoters and Chemical Carcinogens V. Summary and Future Prospects References
I. Introduction
It is now well established that carcinogenesis proceeds via the accumulation of a series of discrete, irreversible, complementary events that convert a normal cell, through a series of benign phenotypes, to malignancy (Cairns, 1975; Bishop, 1991). The tremendous advances in molecular biology over the past decades support the idea that these changes occur at the genetic level, and have led to a working hypothesis that cancer develops via an interactive, cooperative venture between maverick but once normal genes (Knudson, 1986; Weinberg, 1989; Bishop, 1991). In the most simplified model, these genes operate within a twofold mechanism: oncogenes that function in a positive manner to accelerate carcinogenesis and tumor suppressor genes that fail in their negative modulation of growth or induction of apoptosis (Bishop, 1991; Hunter, 1991; Marshall, 1991). These genetic changes are often associated with failures in the inhibitory or proliferative signaling pathways, and provide examples of growth factors, receptors, membrane signaling systems, and transcriptional regulators (Knudson, 1986; Bishop, 199 1 ; Hunter, 1991; Marshall, 1991). Any one of these members concerned 247 ADVANCES IN CANCER RESEARCH. VOL. 64
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with activation or inactivation of cell proliferative pathways is a potential target for environmental carcinogens and for the oncoproteins of' canrer-associated viruses (e.g., human papillomaviruses; Howley, 1991). Our understanding of the events underlying molecular carcinogenesis has been greatly enhanced by the development of a variety of in zdro and in zGzfo model systems. One of the most successful experimental systems employed to date to study multistage carcinogenesis is the mouse skin model of chemical carcinogenesis, which identified the stages of initial ion, promotion, and malignant conversion (Balmain and Brown, 1988; E'uspa and Pourier, 1988). T h e end point of initiation and promotion i n mouse skin is a benign tumor, the syuarnous papilloma, which can persist, regress, or convert t o malignancy (albeit at low frequency) (Hennings et nl., 1985). This inherent multistage nature of tumor progression in mouse skin makes the epidermis a very attractive target tissue for the development of transgenic models of carcinogenesis. K o t only does tumorigenesis proceed via benign papillonias with low frequencies of malignant conversion, but its accessibility is a major advantage; lesions can be easily detected and observed arid biopsies do not require animal sacrifice. Furthermore, the discrete tumor pathologies associated with the skin system make it ideally suited to developing a transgenic model designed to identify the genetic changes t.hat underlie carcinogenesis. T h e probability of developing a functional transgenic mouse model for multistage carcinogenesis has been greatly enhanced by employing the appropriate regulatory elements (Section I1,A) to target gene expression t o a specific tissue (Hanahan, 1988). I n addition to the obvious advantages of an in z k w transgenic mouse approach-for exarnple, assessment o f the influence of factors such as blood supply, an intact immune system, hunioral and cell-mediated growth controls, and physical barriers on disease progression (Hanahan, 1988)-targeted gene expression allows the design o f specific transgenic genotypes that directly test putative molecular mechanisms of carcinogenesis in the tissue suspected of being the target of such genes. Moreover, by breeding separate strains of transgenic mice, the effects of transgene synergism can be explored in the context of the whole animal (Hanahan, 1988). One important conclusion from such transgenic studies is that two cooperating oncogenes (e.g., Ha-ras, my)are insufficient to impart full malignancy; other events are necessary, consistent with the multistage nature of the disease in humans (Sinn et af., 1987). This chapter will focus on our attempts t o target gene expression to the epidermis and to develop the models that will, ideally, be relevant to both skin carcinogenesis in particular and epithelial carcinogenesis in
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general. This chapter is not intended to be an exhaustive review of numerous excellent transgenic models, but will highlight the power of gene targeting to study carcinogenesis in viuo, as well as the occasional surprising results obtained (e.g., Section 111,B). Since the frequency of skin cancer approaches that of all others combined (Glass and Hoover, 1989), and may increase significantly in view of the potential for ozone depletion (Van der Leun, 1988; Kelfkers et al., 1990; Regan, 1991), the need for such animal model systems to assess directly both the molecular mechanisms of carcinogenesis and the influence of environmental factors (carcinogens/promoters) is becoming increasingly apparent (Tennant, 1987; Tennant and Zeiger, 1993). In addition, development of transgenic mice with predictable tumorigenesis kinetics would be a valuable asset in the design and testing of novel therapeutic approaches. Efficacy assessments of therapeutic agents are often restricted to in vitro models, which allow neither evaluation of delivery routes nor assessment of other factors known to affect disease processes in vivo. Furthermore, the prospects for utilizing gene therapy to treat human diseases are coming closer to reality (Anderson, 1992); thus an in vivo test system would be a prerequisite for evaluation of adverse side effects, delivery systems, gene expression, and efficacy. We have developed an epidermal targeting vector and established transgenic mice which express Hams, fos, transforming growth factor (Y (TGFa),' and the E6/E7 transforming genes of human papillomavirus type 18 (HPV-18) exclusively in the epidermis (Section 11). Although the individual genes have distinct phenotypic characteristics, the stability of the preneoplastic and premalignant phenotypes produced by each line over extended time periods is consistent. This stability indicates not only the necessity of secondary events for progression, but also demonstrates that this transgenic mouse model appears to be ideally qualified to assess the nature of these events. Thus, in Section 111, in uivo cooperation experiments are described that assess the consequences of acquisition of an additional genetic event. In Section IV, potential application of these HK 1 transgenic mice as environmental carcinogen/promoter test systems is evaluated. 1 Abbreviations used: HPV, human papilloma virus; ORF, open reading frames; TGFa, transforming growth factor a; H K l , expression vector containing Ha-rm, fos, TGFa or HPVl8E6iE7; HKI-fosiras, transgenic coexpressors of HK1-ras and HK1-fos; ibid fos/TGFa, ras/TGFa; MT, metallothionein promoter; DMBA, dimethylbenzanthracene; TPA, 12-O-tetradecanyl phorbol- 13-acetate; Rb, retinoblastoma; EGF, epidermal growth factor; EGFR, EGF receptor; PDGFR, platelet-derived growth factor receptor; NGFR, nerve growth factor receptor; MAP, mitogen activated protein; MMTV, mouse mammary tumor virus, SCC, squamous cell carcinoma.
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II. Development of Single Transgenic Genotypes A. DESIGN OF THE EPIDERMAL TARGETING VECTOR
1-0 develop a transgenic mouse model for skin carcinogenesis, a prerequisite is the ability to target high-level expression of a transgene to the epidermis. At first sight, the regulatory elements of several genes encoding the major differentiation products of the epidermis appear to be good candidates. These genes include the keratins K1, K5, K14, and K10, filaggrin, and loricriri, and are expressed at high levels correlated with the differentiation state of epidermal cells (Steinert and Roop, 1988). Proliferative basal kei-atinocytes express keratins K5 and K 14 and, on commencement of' terminal differentiation, begin to express K 1, then K 10. Following migration from the basal layer, cells progress through the spinous and granular layers in which filaggrin and loricrin are exbressed; differentiation terminates with the formation of mature epidermal cells (squames) in the cornified layer. This brief description reveals man): candidate genes from which to choose for targeting the epidermis; the keratin genes in particular have been employed as useful targeting vectors. T h e regulatory elements of bovine K 10 were combined with activated human 1-24 (EJ) c-Ha-rns (Bailleul et al., 1990); K14 was employed t o target TGFa to the epidermis (Vassar and Fuchs, 1991); and K 6 has been used to express the HPV early region in the epidermis (Tinsle): et ul., 1992). Each construct elicited an epidermal phenotype. However, these keratins are also expressed at other tissue sites (Steinert and Koop, 1988), potentially inducing disease that may compromise transgenic mice, particularly in experiments designed to assess the cooperation between several oncogenes. An ideal vector should have the properties of exclusive epidermal expression, high expression levels in basal cells (the presumed target for carcinogens) and in differentiated cells (to ilSSeSS the consequences of postmitotic expression), expression at a late stage in development (to avoid potential lethality in uteru), and continued expression in the later stages of progression, including malignancy. We have discovered that a truncated form of the human keratin K1 gene (HK1) possesses the characteristics of exclusive epidermal expi-ession at the correct late stage of development and has efficient expression in approximately 20--30%) of proliferative basal cells (Chung at a/.,1994). Also, unlike the endogenous mouse K 1 gene, the truncated HK 1 gene is efficiently expressed in keratinocytes transtormed by infection in tritro with Ha-rus murine sarcoma virus (Kosenthal et ul., 1991). To create the targeting vector, the coding sequences of the truncated H K 1 gene were removed but the first intron was retained because of its importance for message stability (Nishi
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et ul., 1988); a polylinker sequence was inserted 3’ to the intron to facilitate insertion of the target transgene. Initially, to assess the properties of the HK1 targeting vector, the P-galactosidase reporter gene (P-gal) was employed; analysis of H K l-P-gal transgenic mice confirmed that the vector retained expression characteristics identical to those of the parental truncated HKl gene.
ACTIVATED ONCOGENES B. TARGETING HA-RASAND FOS 1 . Targeting Activated Ha-ras
T h e wealth of data that has accumulated over the past decade on the rus gene family (Ha-rm, Ki-rm, and N-ras) and their functions in normal and oncogenic mechanisms (Barbacid, 1986), made Ha-rm an obvious choice for an initial target transgene. The Ha-rm gene protein product p21 (RasHa) has been shown to be a membrane-bound serinelthreonine kinase. Recently, its putative function in membrane signaling has become clear (Lowy and Willumsen, 1993); RasHa functions as a “molecular turnstile” to funnel the mitogenic signals from tyrosine kinase receptors such as, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and nerve growth factor receptor (NGFR) to the nucleus via a series of downstream kinase cascades (Buday and Downward, 1993; Li et al., 1993; Suen et al., 1993), including the RaflMAP kinase system that interfaces with transcription factors such asfos and myc (Lange-Carter et at., 1993; Levin and Errede, 1993). T h e mechanism of the RasHa-mediated switch involves positive regulation by GDP/GTP exchange factors which, on stimulation of receptor tyrosine kinases, convert inactive GDP-Ras to the active GTP-Ras form (Chardin et al., 1993; Gale et al., 1993). Negative regulation occurs when the GTPase activating proteins (GAP) induce the intrinsic GTPase activity of RasHa, which then returns to its GDP-bound resting form (Bollag and McCormick, 1992; Lowy and Willumsen, 1993). This critical function in integrating tyrosine kinase signaling is probably one reason why the Ha-ras gene is a prime target for carcinogens, and why Ha-ras activation has been implicated in all stages of neoplastic development from initiation to metastasis (Bos, 1988). In virtually every case simple point mutations, predominantly at codons 12, 13, and 61, convert the normal c-Ha-ras gene into an active oncogene (Barbacid, 1986; Lacal and Tronick, 1988). These mutations are thought to maintain the Ras protein in its GTP-bound form, that is, continually “on” regardless of upstream tyrosine kinase signaling. Furthermore, the
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point mutation nature of Ha-rus activation is especially significant for the etiology of human squamous cell carcinomas (SCC) at sun-exposed sites, where the generation of UV-induced pyrimidine dirners culminates in activating muratioris (Pierceall et ul., 1991a). The finding that Ha-rus mutations exist in benign regression-prone keratoacanthomas (Coroniinas et N / . , 1989)supports the notion that, for cert.ain human skin tumors, Ha-ra.r activation can be an early, or even the initiating, event. In mouse skin tumors, an early role for Ha-rus activation is also well established froni both chemical and in zlitro carcinogenesis studies. In two-stage chemical carcinogenesis experiments employing dimethylbenzanthracene (DMBA) initiation and 12-O-tetradecanayl phorbol-13acetate (TPA) promotion, the c-Ha-rus gene was activated in benign papillomas (Quintanilla rt al., 1986; Brown et ul., 1990). Sensitive polymerase chain reaction (PCX) sequencing techniques have found activated Ha-rns in DhfBA-initiated skin (Nelson et al., 1992). Furthermore, introductioii of' the v-Ha-ra.s oncogene into normal primary keratinocytes resulted in benign tumor formation when grafted onto nude mice (Roop ei ul., 1986), and wild-type Harvey niuriiie sarcoma virus (HaMuSV) could be substituted for an initiating agent in two-stage chemical carcinogenesis experiments (Brown e f ( I / . , IYS(5). Given that Ha-ru.s could play an early role, what are the additional genetic or epigerietic requirements for carcinogenesis? One clear epigenetic event, promotion, is a prerequisite for tumor formation tdoiving DhIBA initiation (Quintmilla et a[., 1986) or topical HaMuSV infection (Brown et ul., 1986). and cannot be avoided in nude mouse graft assays because of wounding (Roop et al., 1986). At the genetic level on conversion to iiialignancy, DMBA/TPA tumors denionstrate trisomy of chromosomes 6 and 7 (Aldaz et al., 1989) and mutations in the pS3 tumor suppressor gene (Burns et ul., 1991; Ruggei-i et (I/., 199I). Altei-natively, the Ha-rus oncogene can be involved in the later stages of carcinogenesis, in malignant conversion of papilloma cells (Harper et d . , 1986), or in carcinoma progression to a more aggressive state (Greeiihalgh e f al., 1989). Often, the role of Ha-rus in tumor progression has centered on amplification of the mutant Ha-rus allele (Quintanilla et al., 1986; Biichinan et al., 1991) and/or the loss of the normal proto-Ha-ras allele (Quintanilla et al., 1986; Greenhalgh et d.,1989). This latter point suggests that presence of normal Ras protein has an antagonistic effect on mutant Ras that is important in progi-ession. However, if Ha-rus activation can be a later event, what cooperative event(s) occurs earlier? To begin to answer these questions, an activated Ha-rus gene was expressed in the epidermis to explore the neoplastic roles of Ha-ras activation in the context of a whole animal. T h e activated v-Ha-rus on-
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cogene (Velu et al., 1988) was inserted into the HK1 vector to create the HK1-ras transgene (Greenhalgh et al., 1993a). As shown in Fig. 1, injection of this HK1-ras transgene resulted in immediate phenotypic offspring (Fig. 1A) characterized by a small size, a thickened wrinkled skin, and a massive hyperplastic epidermal histotype (Fig. 1C). The newborn hyperplastic histotype progressed to a massive hyperkeratosis (Fig. ID), resulting in a gross scaly/keratotic appearance in juveniles by 10 days (Fig. 1B). These clinically nonneoplastic histotypes of newborn hyperplasia and juvenile hyperkeratosis are consistent with an early role for Ha-ras activation in epidermal carcinogenesis (Greenhalgh et al., 1993a), and with previous studies employing an activated Ha-ras transgene that resulted in hyperplasia of the Harderian gland from mouse mammary tumor virus (MMTV)-controlled Ha-ras expression (Sinn et al., 1987) o r pancreatic hyperplasia prior to tumor development from elastasecontrolled Ha-ras expression (Quaife et al., 1987). Since hyperplasia is the resultant histotype, the proliferative basal cells that express the v-Ha-ras transgene may have become unresponsive to inducers of differentiation. This conclusion is also supported by the observation that primary keratinocytes infected with v-Ha-ras i n vitro are resistant to calcium-induced terminal differentiation (Yuspa et al., 1985). With respect to this latter point, note that the differentiation state of the keratinocyte at which the Ha-ras transgene is expressed (i.e., vector design) is probably an important factor for the resultant histotype, since Balmain and co-workers, employing a similar strategy but using bovine K10 (BKlO) to target EJ Ha-ras, found hyperkeratosis only, suggesting an increase in differentiation rate rather than proliferation (Bailleul et al., 1990). This difference probably lies in the regulatory elements employed. In the HK1 transgenic study, a vector was employed that allows expression in approximately 20-30% of basal cells, whereas the BKlO vector was expressed predominantly in the nonproliferative suprabasal layers of the epidemis (Bailleul et al., 1990). However, the BK10-ras result is noteworthy because activated Ha-ras expression in supposedly postmitotic; differentiated cells can elicit benign tumors (Bailleul et al., 1990). In HK1-ras mice, the newborn preneoplastic phenotypes did not persist into adulthood; by day 2 1, the epidermis became indistinguishable from that of a normal adult, although the transgene was expressed (Greenhalgh et al., 1993a). Thus, activated Ha-ras can elicit phenotypes in a newborn skin environment that are quite different from those seen in an adult. This difference may center on the different developmental and differentiation-specific factors appearing in newborn and adult skin. One idea was that HK1-ras may elicit these early phenotypes
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through overexpression of TGFa. Since TGFa binds to the EGFR (Massague, 1983), which is subsequently down-regulated in adult epidermis (Green et al., 1983), the loss of phenotype with time would be explained. However, sensitive PCR amplification of TGFa cDNA from HK1-ras hyperplastic newborn epidermis or normal adult epidermis revealed no obvious changes in the low TGFa expression levels (D. A. Greenhalgh et al., unpublished data). Thus, other growth factors may be involved, or phenotypic loss may center on differences in RasHa protein function in newborn epidermis which is naturally mildly hyperplastic (Fig. 1E) and normal nonhyperplastic adult skin (Fig. 5C). While becoming grossly indistinguishable from normal adults, HK 1ras transgenic mice developed tumors at approximately 3 mo of age in the majority of cases, and occasionally as early as 4-5 wk for severe homozygous phenotypes (Fig. 2A; Greenhalgh et al., 1993a). The tumors possessed a typical squamous cell papilloma histotype (Fig. 2B) and seldom grew overlarge. Although all tumors expressed the v-Ha-ras transgene, expression levels did not correlate with tumor numbers or appearance. Also, papillomas appeared at single sites and in few numbers, suggesting the necessity of secondary events as prerequisites for tumor formation. Moreover, tumorigenesis fell into two distinct categories: some lines developed tumors at obvious wound sites (e.g., ear tag, biting; Fig. 2A) whereas other lines appeared to be relatively unresponsive to the promotion stimulus of wounding (although wounding cannot be completely ruled out in the etiology of some papillomas, the ear tag wound did not produce papillomas; Greenhalgh et al., 1993a). This result suggests that wound-independent papillomas may arise because of an additional genetic hit. In two-stage chemical carcinogenesis, a similar phenomenon occurs. Two distinct papilloma types appear: one set, termed TPA independent, are assumed to have events additional to FIG. 1. Preneoplastic phenotypes in HK1-ras transgenic mice. (A) Newborn HKI-ras mice show a distinct wrinkled skin at 48 hr and are smaller than litter mates. (B) Juvenile HKI-ras transgenic mice exhibit a progressive keratinization which peaks at 14 days. SubsequentIy, the phenotype diminishes and by day 28 HK1-ras mice look normal. (C) Histotype of severely phenotypic newborn HK1-ras skin reveals a massive epidermal hyperplasia, with up to a 20-fold thickening of the epidermis. (D) The earlier hyperplasia histotype progresses to a massive hyperkeratosis by day 14. Note also a thin underlying dermis. Both C and D histotypes are preneoplastic, papillomatous (folded/convoluted), and nondysplastic and exhibit few appendages. (E) Normal newborn skin for comparison. Essentially, newborn HKI-TGFa mice exhibited an identical phenotype to that of' HKIras mice, with slight variations depending on expression levels, particularly a persisting adult phenotype (see Section II,C and Fig. 5). Reproduced with permission from Greenhalgh et al. (1995).
FIG. 2. 'Tumorigenesis in HK1-ras mice. (A) Typical example of papillomas arising on the wound-sensitive line 1205. Note the large ear tumor and numerous other tumors appearing singly at sites of scratching and biting. This tumorigenesis was also typical of HKI-TGFa mice. HK1-ras tumors were prone to regress over a 3 to 4 mo period. (B) Histotype of a typical tumor is a well-differentiated squamous cell papilloma, occasionally possessing mild dysplasia but no signs of malignant progression or carcinoma in situ (magnification, 5iOx). A distinctly more keratotic squamous cell papilloma was also produced after long latency in the axilla region of HK1-fos animals.
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Ha-rus activation, negating the requirement for a promotion stimulus. The second, termed TPA dependent, are thought to have a single Ha-rus activation event and remain dependent on the promotion stimulus (Hennings et al., 1983, 1985). Since these TPA-independent papillomas are more prone to conversion (Hennings et al., 1985), note that our single example of malignant conversion to date (in an HK1-ras animal at 16 mo of age) derived from what appeared to be a persistent nonwound-associated papilloma (with a similar histotype to that shown in Fig. 2B). However, although the HK1-ras transgene was still expressed, unlike in chemical carcinogenesis experiments neither p53 (Burns et al., 1991; Ruggeri et ul., 1991) nor the normal endogenous c-Ha-rus alleles became lost or mutated (D. A. Greenhalgh et ul., unpublished data). The association of tumorigenesis in transgenic mice with the promotion stimulus from wounding has been documented previously in mice expressing activated EJ-Ha-rus and TGFa in the epidermis (Bailleul et ul., 1990; Vassar and Fuchs, 1991). This result is also consistent with the requirement for TPA promotion in chemically initiated or virally infected skin (Brown et ul., 1986; Quintanilla et al., 1986). In HK1-ras mice, we noted that wound-associated papillomas regressed more rapidly than their non-wound-associated counterparts (although significant overlap existed). Whether papilloma regression is a consequence of the actions of a fully intact immune system or the influence of tissue barriers o r blood supply is unclear. In addition, the differentiation state of the cell type may be of significance. Consideration should be given to the possibility that some papillomas arise from a subpopulation of keratinocytes that have only limited growth potential. Several conclusions can be drawn from the H K 1-ras transgenic lines. HK 1-ras mice exhibit v-Ha-rus-induced hyperplasia over the entire skin,thus directly confirming an initiating and proliferative role for Ha-ras in the early stages of skin carcinogenesis. However, since tumors appear focally from an apparently normal adult epidermis after 10-12 wk, expression of Ha-ras alone in the epidermis of transgenic mice may be insufficient to elicit even a benign tumor. A promoting stimulus, either wounding or TPA promotion (see Section IV) in the case of the sensitive lines or perhaps a genetic event in lines that develop sporadic tumors, is required for overt tumor appearance. Furthermore, both types of Ha-rus papillomas appear prone to regression; therefore, to achieve an autonomous papilloma phenotype, at least one further epigenetic or genetic event is required. In this scenario, the most logical secondary (tertiary?) event is acquisition of an additional genetic hit@) that achieves the papilloma autonomous growth stage and, later, acquisition of another for malignant conversion. This step may involve
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aclditional oncogene or growth factor cooperation (Section III,A), mut m t gene dosage, o r one of the specific chromosomal changes identified i n papilloma progression that results in the loss of putative tumor suppressor genes on chromosomes 6 and 7 (Aldaz et al., 1989; Bianchi et al., 1991; Kenip PI ol., 199%). T h e interactions between these molecular participants may also underlie the type of papilloma produced (wound associatediwound independent). These possibilities, coupled with assessment of the role of the p53 tumor suppressor gene (Section III,B), the loss of which is associated with malignant conversion in many natural arid experimental tuniors (Hollstein vt a!., 199l), are currently under insestigation. 2. Targeti~igActiuated fos
Considering the multistage nature of carcinogenesis and anticipation that Ha-ras alone would be insufficient for complete carcinogenesis, the role of'fos activation and deregulation \vas investigated in our transgenic system. This stucly was prompted bv several observations that suggested that the c-[os proto-oncogene could play a role in epidermal carcinogenesis and nornial epidermal differentiation. T h e initial identification of \--fo.~ as the transforming component of Finkel-Biskis-Jenkins (FBJ) osteosarcoma sirus demonstrated the oncogenic capacity of an activated fi1.5 proto-oncogene (Curran and Teich, 1982). A role for.fos in epidermal carcinogenesis was suggested by TPA induction of c+s in cultured keratinocytes (Dotto et ul., 1986), through the TPA-responsive element (Hayes rt d., 1987), and was further strengthened by the finding that c-/o,s was transiently overexpressed in classical two-stage chemical carcinogenesis experiments in zjivo (Rose-John et al., 1988). Based on the observation that injection of activated RasHi) protein induced c-fos expression (Stacey et al.: 1987), a direct role for fvs in epidermal carcinogenesis was demonstrated using a nude mouse graft assay in which v-fos or activated c-f.5 was shown to induce malignant conversion of murine papilloma cell'lines or primary keratinocytes that expressed an activated Ha-rcw. oncogene (Greenhaigh and Yuspa, 1988; Greenhalgh et al., 1990). This result was the first indication of direct cooperation between Ha-ras arid fos in multistage carcinogenesis, and is consistent with our current knowledge of tyrosine kinase signaling via the MAP kinase cascade which links Ha-rus tofos (see Section 1I.A). I n human epidermis, immunofluorescence studies have detected Fos expression in proliferative and differentiated cells, suggesting a role for c,[& in normal keratinocyte differentiation (Basset-Seguin et al., 1990). An immunohistochemical approach on mouse skin localized this expression further, finding Fos expression predominantly in a specific subset of
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granular cells (Fisher et al., 1991). A subsequent transgenic study using a Lac Z-fos fusion transgene to identify areas of fos expression also detected preferential Fos expression in the differentiated layers and hair follicles (Smeyne et al., 1992). The finding that Fos was expressed in granular cells just prior to cornification suggests a role for fos in the process of cornification (Fisher et al., 1991) and is consistent with the finding that constitutive c-fos expression precedes programmed cell death (apoptosis) in certain cells (Smeyne et al., 1993). These observations were novel, because previouslyfos expression was more often associated with a rapid response to external stimuli that resulted in cellular proliferation (e.g., serum; Treisman, 1986). This apparent paradox can be resolved if fos expression is considered to be a “harbinger of change” (Smeyne et al., 1993). Thus, the protein product Fos is thought to function in the early response of a cell to external stimuli, coupling shortterm signals into long-term alterations in cellular phenotypes (Curran, 1988; Morgan and Curran, 1991). Given this important role, fos is under exquisite regulation (Cohen and Curran, 1989). Through a variety of regulatory elements in both coding and noncoding sequences, the message and protein are rapidly earmarked for removal (Shyu et al., 1989; Wilson and Treisman, 1988; Lamb et al., 1990). The importance of these regulatory elements within fos to normal cellular function is realized when they are removed. Enhanced expression of a deregulated c-fos is transforming in uitro (Miller et al., 1984) and cooperates with Ha-ras in malignant conversion of papilloma cell lines (Greenhalgh and Yuspa, 1988); both FBJ and FBR forms of the sarcoma viruses have lost or mutated these regions (Curran and Teich, 1982; Curran and Verma, 1984). Indeed, evidence suggests that v-fos can interfere with the rapid shut-down of c-fos by proteins that bind to the 3’ noncoding elements, further amplifying anomalous transcriptional control by fos-mediated mechanisms (Shyu et al., 1989). Fos achieves transcriptional control by forming a heterodimer with members of thejun family in the formation of the AP-1 transcription factor complex (Curran and Franza, 1988). Furthermore, by complexing with alternative jun members, the Fos protein can achieve a high degree of flexibility in transcriptional control and, by regulating different target genes within host cells via alternative AP-1 complexes, the specificity of the response (proliferation, differentiation, or apoptosis) can be determined. Thus, the actual mechanism which deregulated c-fos or v-fos achieves transformation is likely to center on anomalous expression of fundamental AP- 1 -regulated genes. To provide functional data for the role offos in epidermal differentiation, and to assess whether fos deregulation alone can induce neoplasia
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in vivo, the highly transforming chimeric FBJ/R form of v-fos (Miller et al., 1985) was targeted to the epidermis. Four lines of HK1-fos expressors were established that were indistinguishable from normal mice until 4-5 mo of age, when a very specific ear phenotype developed (Greenhalgh et al., 1993b). HK1-fos mice developed a thickened keratotic ear [initially in the tagged (wounded) ear, and later bilaterally] that progressed to form distinct columns of keratin (Fig. 3A). The histotype of these ear lesions was preneoplastic, with a mild area of hyperplasia but a massive degree of hyperkeratosis and usually a very prominent stratum granulosum (Fig. 3B). Phenotypic severity correlated with expression levels (Greenhalgh et al., 1993b); in severe phenotypes, the keratotic columns fused to form a structure grossly resembling a keratoacanthoma, but without a keratoacanthoma histotype. Although they were not prone to regression, these lesions did not convert to malignancy. In addition, older HKI-fos mice (>12 mo) exhibited alopecia (hair loss) on the stomach and chest and hyperkeratosis in the axilla and inguinal areas, all areas prone to mechanical irritation and friction. After long latency, tiny tumors appeared at these keratotic axilla and inguinal sites, which possessed a histotype similar to that of the typical squamous papilloma shown in Fig. 2B. T h e majority of these papillomas remained benign, as determined by histotype and expression of specific keratin markers K1, K6, and K13 (Nischt et al., 1988; Roop et al., 1988). However, one of these papillomas, occurring in an 18-mo-old F, mouse, became obviously malignant (Fig. 4A). The squamous cell carcinoma histotype (Fig. 4B) was confirmed by uniform K13 expression (Nischt et al., 1988) and complete loss of K1 expression (Roop et al., 1988) but, like the malignant conversion in the HK1-ras animals (Section II,B, l), this HK1-fos carcinoma did not possess mutations in the endogenous
FIG. 3 . Phenotype induced in HK1-fos transgenic mice. (A) Typical example of a niultif6llicular, highly keratotic ear lesion(s) in an HK1-fos 488 F, mouse (#3493) at 6-8 mo of age. This particular phenotype arose on the inner surface of the ear and presents as columns of keratinized cells. Eventually, these keratin columns fuse to form an obvious tumor with a gross appearance of a keratoacanthoma produced in chemical carcinogenesis, but without its typical histotype and no sign of regression. (B) Histotype of A reveals a massive hyperkeratosis, with avenues of' severely hyperplastic cells producing cup-like structures with a very prominent stratum granulosum. As these lesions progress to an overt tumor, the histotype remains very keratotic with a large increase in the degree of hyperplasia. Even large examples have a well-organized benign histotype, again with no evidence of malignant conversion. A type of tumor with a distinctly different etiology arises in the axilla from sites of preexisting hyperplasia, and possess a histotype typical of the squamous cell papilloma shown in Fig. 2. (C) Normal ear for comparison (magnification, 150X).
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Ha-ras o r p5? genes (D. A. Greenhalgh et al., unpublished data). This result suggests that malignant conversion in HK1-fos or HK1-ras animals may follow a completely different molecular pathway than that observed in DMBA-initiated chemical carcinogenesis. These observations confirmed a fundamental role forfos in epidermal differentiation and showed that deregulated fos expression can induce preneoplastic disease. Newborn animals were indistinguishable from normal, but later exhibited distinct phenotypes to provide in vivo functional data on the consequences of fos deregulation in neoplasia and possibly cornification. The lack of a neonatal phenotype is unclear, but other studies onfos by both knock-out experiments (Johnson et al., 1992) and untargeted transgenic approaches (Ruther et al., 1989) also observed phenotypic development only after long latency. For the null fos mice, the data clearly demonstrate the redundancies in these systems and are similar to data from a variety of knock-out experiments that show surprisingly few phenotypes (e.g., TGFa; Mann et al., 1993). For the untargeted c-fos transgenics (Ruther, et al., 1989), latency of phenotype may have been the consequence of influence of endogenous factors on the transgene. In our fos mice, in a similar fashion to the Ha-ras animals described, we see a clear influence of wounding. This event was also a prerequisite for tumor etiology in vjun transgenic mice (Shuh et al., 1990). Moreover, consistent with the requirement for wounding was the novel finding that HK l-fos transgenic mice were sensitive to TPA promotion, which is known to activate components of the wound response (Argyris, 1982). Thus, HK1-fos mice may also be a useful system in which to assess novel initiating and promoting agents (Section IV). The phenotypes exhibited in HK1-fos mice may be consequences of a twofold role forfos. First, since wounding was clearly responsible for the onset of the initial hyperplasia, and since friction is associated with appearance of the axillary hyperplasia, in this instance we may observe f o s in its well-characterized role as an early response gene to external stimuli. Since wounds mobilize a wide variety of cytokines and growth factors, and since the HK1-fos transgene is expressed at significant levels in the proliferative basal cells, HK1-fos expression may modify the normal regulatory elements and conscript the constituents of wound repair into an accelerated development of HK1-fos-induced hyperplasia. Second, the observed massive hyperkeratosis is consistent with the proposed role for fos in cornification. As outlined earlier, v-fos can interfere with cTfosdown-regulation (Shyu et al., 1989) and can potentially conscript endogenous c-fos expression into an amplified role. Therefore, since a prominent stratum granulosum is always observed in phenotypic epidermis, and there is a distinct tendency for hyperkeratosis to dominate
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massively over hvperplasia (giving the highly keratotic appearance to ear lesions), the activated HKI-fos transgene may be interfering with the normal role for c-fos in the stratum granulosum in the process of cornification. In agreement with this idea was the observation that c-Fos protein accumulated in a specific subset of granular cells .just prior to cornification (Fisher Pi al., 1991). Currently, experiments employing HK 1-fos mice involve assessment of oncogene cooperation and loss of the p53 tumor suppressor gene, assessment of the role that growth factors such as TGFa play in etiology of the wound-associated H K1 -fos phenotypes (Section I II), and assessment of the role oftuinor promoters (Section IV) and the possibility that U V light ma!. mediate carcinogenesis in part by a pathway involving,fos (Shah et nl., 1993). C . TARGETING TRANSFORMING GROWTHFACTORa
'The anomalous production of growth factors has long been associated with transformation and the uncontrolled cell growth that underlies carcinogenesis (C;i.oss arid Dexter, 1991). In particular, the role of 'IGFu was investigated because of its association with hyperproliferative skin diseases such as psoriasis (Elder rt al., 1989) and the finding that '1'C;F-a is thought to play pivotal roles in epidermal carcinogenesis (Derynck, 1988). Also, the association of TGFa overexpression with wounding (Schreiber ot ul., 1986) may be one of the important facets of the woundpromotion stinitilus observed in carcinogenesis (Argyris, 1982; Furstenhurger t t ( I / . , 1989; DiChvanni, 1992). Since wounding appears to be an important epigenetic event in the etiology of HK1-ras and HK1-fos phenotypes, T G F o l is an attractive target gene. Moreover, TGFa is considered t o be the major autocrine growth factor for keratinocyte growth regulation, being more potent than EGF in stimulating proliferation and migration (Rarrandon and Green, 198'7). Initially described a s a transforming agent in the niedia of retrovirally transformed fibroblasts, 'I'GFcu studies have shown that this potent mitogen is structurally related to the EGF family of proteins (reviewed by Dei-ynck, 1988). T G F a is produced as a glycosylated and palmitoylated transnienibrane precursor that undergoes cleavage, giving 5- to 20-kDa glycosylated fornis that are secreted into the extracellular domain (Bringiiian r t al., 1987). TGFa shares approximately 30% structural homology \zith EGE', and both precursor and mature TGFol species bind to the EGFR to activate the tyrosine kinase pathway (Brachman et al., 1989; see Section II,A,2). I n skin, a low level of TGFa expression is found
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throughout the epidermis. This pattern of TGFa expression in the epidermis correlates to EGFR distribution because expression is greatest in the basal and immediate suprabasal layers (Nanney et al., 1984; Finzi et al., 1991). With respect to a role in neoplasia, elevated levels of TGFa have been associated with transformation of cells in culture and in a variety of naturally occurring human tumor types, including human squamous cell carcinomas (Derynck et al., 1987; Gottleib et al., 1988). In the mouse skin model, although unable to induce neoplasia directly when introduced into primary keratinocytes by recombinant retroviral infection, introduction of TGFa into papilloma cells and grafting onto nude mouse skin resulted in papillomas of a greater size by both autocrine and paracrine mechanisms, but failed to induce malignant conversion in this model (Finzi et al., 1988). Furthermore, the fact that these papilloma cells expressed a DMBA-induced activated Ha-ras (Strickland et al., 1988), and the observation that primary keratinocytes infected with v-Ha-ras overexpress TGFa up to fivefold, suggests that synergism may exist between these two genes (Glick et al., 1991). In chemical carcinogenesis experiments, TGFa expression was induced by TPA both in vitro, via activation of the protein kinase C (PKC) pathway (Pittelkow et al., 1989), and in vivo, again by autocrine and paracrine mechanisms (Imamot0 et al., 1991). Thus, TGFa overexpression was associated with the proliferative promotion phase (DiGiovanni, 1992), consistent with its role in wounding (Schreiber et al., 1986; Furstenburger et at., 1989) and with the failure of TGFa to induce neoplasia when introduced into primary keratinocytes (Finzi et al., 1988). Conversely, in transgenic mouse models in which TGFa was targeted to mammary gland, pancreas, o r liver, hyperplasia resulted, suggesting that deregulated TGFa may play an earlier initiating role in carcinogenesis (Jhappan et al., 1990; Matsui et al., 1990; Sandgren et al., 1990). Therefore, to assess the role of TGFa in epidermal differentiation and the consequences of TGFa overexpression in neoplasia, the human TGFa cDNA was inserted into the expression vector to create HK1TGFa transgenic mice (Dominey et al., 1993). T h e gross appearance of HKI-TGFa newborn mice was virtually identical to that of the HKlras mice shown in Fig. 1. The histology was also similar, demonstrating hyperplasia followed by hyperkeratosis, but without the massive hyperkeratosis shown in Fig. 1C. Unlike in HK1-ras mice, in HKl-TGFa mice phenotypic severity correlated to expression levels. Also novel was the retention of the hyperplastic/hyperkeratotic newborn phenotype into adulthood (Fig. 5) in high-expresser lines, whereas lower expressers
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FIG. i. Phenotypes of aclult HKI-TGFa transgenic mice. (A) Unlike HK1-ras animals. high expressoi-s of HI( 1-'T'GFu have a persistent phenotype in adiilts. Typically this feature was niost pi-ominent on the genital anti abdominal areas. (B) Histotype of persistent hvperplasiaihsperkeratosis in adult high HKI-1'GFu expressors, similar to that of newborn H K l-'T(;Fa epidermis. '-1 similar persistent historype occurred in adult HIS 1 -fos/ras and €IKI-fos/TCFy expressors. (C) Normal adult skin tor comparison shows the cellophane-like nature of adult mouse epicleimiis (magnification, 130~).
gradually lost this phenotype. A similar study employing K14 to target TGFa expression also produced hyperplasticihyperkeratotic K 14T G F a mice that gradually lost their phenotypes (Vassar and Fuchs,
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1991). Acid extraction of TGFa protein revealed a fivefold higher TGFa expression level in HK1-TGFa mice than in K14-TGFa, suggesting that a threshold level of TGFa is required to maintain the hyperplasia/hyperkeratosis phenotype in adults. A second factor to account for this discrepancy may be the fact that K14 is expressed only in proliferative basal cells, whereas the truncated HK1 vector is expressed in both basal and superbasal cells (Chung et al., 1994). This reduction of phenotype severity with time may be a consequence of the reduction in EGFR levels, so lower levels of TGFa can no longer exert autocrine or paracrine growth stimulation effects (Green et al., 1983; Massague, 1983). At 10-14 wk of age, several of the HKl-TGFa lines (particularly high expressers) developed tumors at sites associated with scratching or biting. T h e histotype confirmed a squamous papilloma (indistinguishable from HKI-ras papillomas shown in Fig. 2B). Note that these papillomas were more prone to regression than similar HKI -ras tumors. Unlike HK1-ras and HK1-fos animals, to date no malignant conversion has been observed in HK1-TGFa animals. The majority of HKITGFa papillomas arose at wound sites (Dominey et al., 1993), again demonstrating the importance of the promotion stimulus derived from wounding (Argyris, 1982). This event was also a prerequisite for papillomatogenesis in K14-TGFa mice (Vassar and Fuchs, 1991), and is consistent with the activation of the TGFa autocrine loop by wounding (Furstenberger et al., 1989) and induction of TGFa by TPA promotion, which activates components of the wound response milieu (DiGiovanni, 1992). In addition, however, in high HK1-TGFa expressers spontaneous papillomas appeared in adults in regions that had retained the juvenile hyperkeratotic/hyperplastic phenotype, suggesting the possibility that an additional synergistic genetic event may have occurred in the etiology of these particular papillomas. Considering the potential for synergism between TGFa and Ha-ras outlined earlier, coupled with the potential for a later role for Ha-ras in epidermal carcinogenesis (Harper et al., 1986; Bremner and Balmain, 1990; Buchman et ul., 1991), spontaneous HK 1-TGFa papillomas arising from pre-existing phenotypic epidermis were assessed for endogenous Ha-ras activation. However, no mutations in c-Ha-ras were detected, nor was c-Hams overexpressed in this class of papillomas (Wang et at., 1994). These data therefore support an early role for TGFa in skin carcinogenesis, since clearly other events are required prior to overt tumor appearance. The promotion stimulus from wounding appears to be particularly important in HK1-TGFa mice and prompted a series of TPA studies which demonstrated that overexpression of TGFa could substitute for
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an initiating event in two-stage chemical carcinogenesis (Section IV; Wang et al., 1994). Also, the data suggest that TGFa can mediate these early stages by a mechanism independent of Ha-rus activation or over expression. If TGFa can truly replace Ha-rus activation or overexpressiori in papilloma formation, recent reports on Ha-ras function in membrane signaling may provide clues to understanding the mechanism. KasHa has been identified as an important molecular turnstile through which tyrosine kinase receptors such as EGFK transmit mitogenic signals to the nucleus (Section II,B, 1). Thus, when TGFa binds to the EGFK, the receptor dimerizes and, following autophosphorylation, a binding site for the Grb:! adaptor protein is created. This complex then recruits the Rds activator protein, mammalian son-of-sevenless (MSOS), which then functions as a GTP/CI)P exchange factor to convert inactive Ras-GDP t o the active GI’P-bound form (Li rt al., 1993; Buday and r)ou,nwatd, 1993). Therefore, autocrine ‘I‘GFa expression in HK 1TGFa transgenic mice may substitute for c-Ha-rus activation by maintaining the Ras protein in its active GTP-bound conformation, which is known to induce transformation (Chang et al., 1983). ‘This hypothesis has also been proposed by Fuchs and co-workers (Vassar et al., 1992). In support of t.his idea, note that HK1-TGFa mice are phenotypically very similar t o HK 1 -ras mice. Mitogenic T G F a signal transduction also occurs independently of the Ha-)-us-mediated pathway, as demonstrated by the recently identified pY 1 transcription factor, which directly links the EGFR tofos and jun (Fu and Zhang, 1!)93). T h e presence of such alternative pathways may account for the ability of TGFa to act as an early, possibly initiating, agent in transgenic models, arid yet be associated with a promotion role in wounding (Furstenbei-ger et ai., 1989) or treatment with ’TPA (DiGiovanni, 1992). T h e fact that nude mouse grafting systems failed to demonstrate an early role for TGFa (Finzi et al., 1988) could be explained by the unavoidablc wounding stimulus at the graft site, which produces hyperplasia with normal keratinocytes arid may mask any early hyperproliferative effects of TGFa overexpression (Finzi et al., 1988). Clearly, however, TGFa appears to act as a downstream promoter for Ha-rusexpressing papillonia cells (Finzi et ul.. 1988). While their derivation from DMBA-treated skin (Strickland et al., 1988) cannot exclude TGFa synergism with other DMBA-mutated genes, a synergism exists between Ha-m.s activation and TGFa overexpression in Ha-ras-infected keratinocyte papillomas (Glick ct al., 1991). In an attempt to clarify this apparent dual role for TGFa in carcinogenesis, mat.ing experiment.s of HK 1TGFa transgenic mice with HK1 -fos and HK 1-ras animals have been initiated (see Sections I I I , A , l and 2).
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D. TARGETING THE VIRALONCOGENES OF HUMAN PAPILLOMAVIRUS 18 HPVs have been implicated in the etiology of a wide variety of squamous epithelial tumors in humans (zur Hausen, 1988; Broker et al., 1989; Howley, 1991). In particular, HPVs are seen to be a significant problem in cervical carcinogenesis because of permissive infection of cervical tissue and the potential for subsequent neoplastic progression. Since one of our goals is to employ the epidermal transgenic model as a system applicable to epithelial carcinogenesis in general, we assessed whether mouse epidermis could represent a relevant in uiuo model system in which to analyze the interaction between HPV and cellular genes in neoplasia. To date, the development of a successful transgenic mouse model has been hindered by the regulatory elements within the viral genomes that are attuned to the differentiation state of squamous epithelia (Broker et al., 1989). The life cycle of the virus is so tightly linked to all stages of differentiation of squamous epithelial cells that establishment of successful culture systems has also been difficult. To complicate matters further, HPVs have a specific tropism for squamous epithelial cells, and different types of HPV have specificity for the anatomic sites that they infect (Broker et al., 1989). In essence, HPVs are a family of related double-stranded DNA viruses with circular genomes of approximately 7900 bp (reviewed by Taichman and Laporta, 1989). The organization of their genome is similar, having early expressed and late expressed genes encoded by a single DNA strand. The long control region (LCR) is located upstream from the early genes and contains transcriptional enhancer elements, promoters, and DNA replication control sequences. Among the downstream early genes, the E l gene encodes a transacting factor required for regulated extra-chromosomal replication, the E2 gene encodes a transacting factor than can activate and repress transcription, and the E6 and E7 genes have been shown to participate in the transformation of cells (Howley and Schlegel, 1988), and thus can be considered oncogenes. Although there are over 60 HPV strains, malignant progression is associated with only a specific subset. Of these, HPV16 and HPV18 account for approximately 70% of HPV-positive cervical carcinomas (de Villiers, 1989) and exhibit different biological properties from other HPVs that infect the anogenital tract. Unlike DNA from HPV6 and HPVl1, which induce benign genital lesions, DNA from HPV16 and HPV 18 can immortalize primary keratinocytes in culture. This difference in transformation ability appears to be due to biological differences in their E6 and E7 genes (Munger et al., 1989). Tumor progression is also
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associated with integration of the viral genome, which usually exists as an episonie (Broker et al., 1989). Integration appears to be random with respect to the host genome; however, it is highly specific with respect to the virus, occurring in the E1/E2 regions (Baker et al., 1987; Broker et ni., 1989). 'I-he consequence of integration at this site is the eliniination of E2 expression and, consequently, deregulated expression of the E6 and E7 oncogenes. T h e oncogenicity of the E6 and E7 proteins of malignant-associated HPV but not benign-associated HYV may be derived from their ability to inactivate the tumor suppressor proteins of p5? arid Rb respectively (Dyson et ul., 1989; Werness et al., 1990). A recent study also identified an alternative splice site in HPV 16 and HPV 18 that is not found in benignociated subtypes, which was geared to the efficient production of E7 at the expense of E6 and produced a nonfunctional E6* protein (Sedman et ul., 1991). However, the precise advantage gained by the virus from this increase in E7 production remains unclear. Both E7 and E6 can cooperate with an activated Ha-rus oncogene; E7 appears capable of cooperation to achieve a matignant phenotype in vitro (Phelps et al., 19238; Crook Pt al., 1989), whereas E6 cooperates with Ha-rus in the immortalization of primary epithelial cells (Storey and Banks, 1993) and anchorage-independent growth of 3T3 cells (Sedman et ul., 1991). Differences also exist between the maligriant-associated subtypes; HPV 18 is 50-fold more active in immortalizing keratinocytes in uitro than HPVl6 (Barbosa and Schlegel, 1989).These differences map predominantly to the viral enhancer regions (Villa and Schlegel, 199 1). Furthermore, HPV 18-immortalized keratinocytes have been shown to spontaneously progress to malignancy (Hurlin et al., 1991). Since HPV18 exhibits the highest degree of in uitro transforming activity, and appears to be associated with more aggressive clinical lesions, it appeared to be a highly desirable subtype with which to develop a transgenic niodel of HPVinduced neoplasia and malignancy. Previously, the transgenic approach has employed either intact. genomes (Lacey et al., 1986) or the E6 and E7 open reading frames (ORFs) under coritrol of the mouse mammary tumor virus (MMTV) long terniinal repeat (Kondoh et al., 1991). Although effects were observed in the appropriate tissue for the bovine papilloma virus (Lacey et al., 1986), this was not the case for the latter study which, using HPV16, found only seminomagenesis (germ cell tumorigenesis) and no pathology in squamous cell epithelia (Kondoh et ai., 1991). In addition, transgenic mice expressing the SV40 T antigen fused to the LCR of HPV18 exhibited low! levels of transcription, with a resultant failure to produce pathology
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27 1
in appropriate squamous epithelia (Choo et al., 1992). Thus, to produce a functional transgenic model for HPV disease, it appears necessary to target HPV gene expression to an appropriate squamous epithelium and to remove the HPV regulatory mechanisms inherent in the LCR and E2 genes (Broker et al., 1989; Choo et al., 1992). Two groups have achieved targeted expression. Initially, the HPVl early region was targeted to the epidermis by employing the regulatory elements of keratin K6; these mice exhibited verrucae (Tinsley et al., 1992). Later, by employing an a-crystallin promoter, the E6 and E7 genes of HPV16 were successfully expressed in the lens, which then developed neoplasia (Griep et al., 1993). Since the epidermis is a stratified epithelium, we envisioned that it would be a permissive targe‘t tissue for HPV16- or -18induced disease. In support of this idea, the E6 and E7 genes of both HPV subtypes are capable of transforming epidermal keratinocytes in vitro, and human SCC have been found to contain HPV16 sequences (Hawley-Nelson et al., 1989; Munger et al., 1989; Pierceall et al., 1991b), suggesting that other tissues in addition to the anogenital areas are potential targets for malignant-associated HPV infections (Pierceall et al., 1991b). Thus, it was envisaged that targeting expression of the E6 and E7 genes from HPV18 to the epidermis, and later performing mating experiments with HK 1 transgenic mice, would initiate some of the events that ultimately appear to occur in HPV-associated malignancies. Micro-injection of the HKl-E6/E7 construct generated three founder lines that expressed the transgene but failed to exhibit any obvious phenotype until approximately 9-10 mo of age (Fig. 6A), when F, founder mice exhibited tiny pinhead lesions (Greenhalgh et al., 1994). The histotype was typical of the lesions induced by HPV, exhibiting a prominent stratum granulosum, being hyperplastic and hyperkeratotic, and having a distinct verrucous appendage descending from a keratin plug (Fig. 6B). These verrucous lesions, which appeared on the dorsal surface and were initially identified as a roughness to the skin, persisted for only 2-3 mo and then regressed. In old animals (18-20 mo), a second type of tumor appeared with the gross appearance and histotype typical of a squamous papilloma (similar to Fig. 2B). This was a very rare event; to date only four squamous papilloma-bearing animals in two lines have been detected (Greenhalgh et al., 1994). Also, unlike HK1ras, HKl-fos, and HK1-TGFa animals, the HKl-E6/E7 animals had lesions that did not appear to be associated with a wound promotion stimulus (e.g., ear tag), nor were they sensitive to TPA promotion (D. A. Greenhalgh, unpublished data). This result is in contrast to a further
FIG. 6. Phenotype and histotype of HKI-EfiiE’i lesions. ( A ) HKI-EWE7 F, mouse (#9626) exhibits subtle skin lesions at 3 nio, characterized by skin rigidity, thickening, and roughness untlcrlying the fur. which progresses t o a wart-like structure by 12 nio. (B) Histotype of a wartlike lesion exhillits hyperplasia, hyperkeratosis, and [lie verrucous appendages typical o f HPV-induced disease.
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characterization study of transgenic animals expressing the E6 and E7 genes of HPV16 from an a-crystallin promoter (Greip et al., 1993). These animals developed squamous cell carcinomas in the skin, an unexpected result given the promoter employed (Lambert et al., 1993). These animals were sensitive to a wound stimulus; this result may center not only on differences in the targeting vector but on the fact that both E6 and E7 were expressed. In HKl-EGIE7 mice, initial concerns over the latency and low lesion frequency, coupled with alternative splicing available to the HPV18 E6/E7 region (Sedman et al., 1991), prompted an analysis of the transcripts produced by HK 1-E6/E7 transgene expression. Little full-length E6 message was made in HKl-E6/E7 epidermis; instead, the nonfunctional E6* transcript predominated (Greenhalgh et ul., 1994). Thus, HKl-E6/E7 mice were essentially E7 alone, that is, HK l-E6*/E7. The full-length E6 transcript remained barely detectable in phenotypically normal skin, verrucous lesions, or squamous cell papillomas (Greenhalgh et al., 1994). Thus, the low frequency and long latency for HKl-E6/E7 pathology may be a consequence of low-level full-length E6 expression. T h e fulllength E6, in addition to cooperating with E7 in immortalizing human keratinocytes, is capable of transforming murine NIH 3T3 cells in vitro (Sedman et al., 1991), and the HPV6 or BPVl E6 gene can immortalize human epithelial cells (Band et al., 1993). Also, the HPV16 E6 gene was shown to cooperate with H a m s to immortalize keratinocytes apparently independently of E7 (Storey and Banks, 1993). Furthermore, epidemiological studies on HPV in humans show that a long latency exists between presumed infection and onset of overt disease (zur Hausen, 1988). Nonetheless, these lesions express not only the E6 and E7 genes, but also the additional E5 oncoprotein (Schiller et al., 1986) and the E4 gene, which has been found to interfere with the keratinocyte cytoskeleton network (Doorbar et al., 1991). Thus, it is likely that a combination of HPV genes is required to increase lesion frequency and progression in our HKl transgenic mice. An immediate goal, therefore, is to develop these HPV transgenic genotypes to assess their roles and interaction with ras, fos, and TGFa. Since our mice were predominantly E7 only, and since E7 classically cooperates with Ha-ras in two-stage transformation (Phelps et al., 1988; Crook et al., 1989), we analyzed HKl-E6/E7 spontaneous squamous papillomas for endogenous Ha-ras mutations, since papilloma etiology was consistent with acquisition of an additional genetic event. This proved to be the case; endogenous Ha-ras mutations at both codon 61 (A + T transversions) and codon 13 (A + G transitions) were detected.
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These mutations were not detectable in the verrucous lesions, suggesting that squamous papillomas develop from a cooperation between HK 1 -E6*/E7 and the spontaneously activated endogenous c-Ha-rus oncogene. This result is of significance given the high frequencies of HPV infection in humans (zur Hausen, 1988) and the high incidence of UVinduced Ha-rcw mutations in human skin (Pierceall et ul., l99la). T h e ability to target HPV-E6/E7 specifically to a squanious epithelium has produced a transgenic mouse model that closely mimics the molecular events and epidemiology of HPV-induced disease in hunians (i.e., long latency, oncogene activation). By removing the endogenous HPV control elements, the viral expression restrictions have been overcome to create a general model for HYV 18-associated epithelial carcinogenesis and a specific model for nialignant-associated subtypes in cutaneous carcinomas (Pierceall et d.,1991b). Although at this juncture the transgenic model is at an early stage in development, it is envisioned to become a powerful tool wit.h which to analyze the genetic and epigenetic events associated uith HPV-induced disease.
I l l . Development of Multiple Transgenic Genotypes T h e multistage nature of carcinogenesis is well established and for squanious cell carcinoma of the skin a recent mathematical study based on human epidemiological data predicts the requirement for a niinimum of seven separate, synergist.ic events (Renan, 1993). This appears to be the case for colon cancer also, where six distinct genetic events have been associated with carcinogenesis (Fearon and Jones, 1992). T h e appearance of preneoplast ic or regression-prone benign lesions in our transgenic models outlined in Section II,B is also consistent with this requirement for multiple events. Moreover, the mating experiments documented next, while demonstrating transgene cooperation and benign tumor pi-ogression, show a stability of phenotype absent in previous cooperative st utlies using transgenic niice-for example, q~arid rus cooperation, or q r and TGFa-in \sliich progression to malignancy rapidly oc(:ui.red over a f e w months (Sinn at ul., 1987; Murakaini P t at., 1993). 'I'hus, these data suggest that a transgenic system has been developed with the phenotypic stability necessary to assess multiple genetic insults. A. C ~ O P E R A T I O N
BETWEEN
HA-NAS,FOS,
AND
TGFa
I . Cooperution between Ha-rrzs and Jos Consistently researchers have observed, both in vitro and in vivo, that particular classes of oncogenes can cooperate with each other to impart a
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progressive transformation (Land et al., 1983; Ruley, 1983; Sinn et aE., 1987; Murakami et al., 1993). In mouse skin system, cooperation was observed between Ha-ras and fos (Greenhalgh and Yuspa, 1988) since coexpression of v-fos and v-Ha-ras in primary keratinocytes resulted in highly aggressive, metastatic squamous cell carcinomas whereas expression of v-a-ras alone elicited benign papillomas when grafted onto nude mice (Greenhalgh et al., 1990). This result was in agreement with previous studies implicating Ha-ras activation as an early or initiating event in mouse skin (Balmain and Brown, 1988), producing benign tumors (papillomas) which activatedfos expression could then convert. In transgenic mating experiments, a similar synergistic response has been achieved for a variety of combinations, including Ha-ras and myc or m9c and TGFol (Sinn et al., 1987; Murakami et al., 1993). However, in transgenic cooperation experiments involving Ha-ras and my, the resultant carcinomas were shown to be clonal in origin, consistent with the acquisition of additional events for malignant conversion (Sinn et al., 1987). Therefore, to assess whether ras-fos cooperation could achieve malignancy in immunocompetent nonwounded (graft) transgenic mice, HK 1ras and HK1-fos animals were mated. Regardless of the phenotypic severity of the parental lines, HK1-fos/ras progeny exhibited a greater severity in neonatal juvenile phenotypes (hyperplasia/hyperkeratosis) than HK1-ras siblings (Fig. 7A; Greenhalgh et al., 1993c) and the preneoplastic juvenile phenotypes persisted throughout the HK1-fos/ras adult life-span (Fig. 7). HK1-fos/ras mice also exhibited the early onset of tumorigenesis, with lesions visible in the axilla or inguinal areas at 2128 days. By 6 wk of age, these lesions had grown aggressively but remained pedunculated, and numerous other lesions appeared over the entire surface of the animal (Fig. 7B). The HK1-ras sibling control at this time was free of any obvious phenotypes (Fig. 7B), whereas HK1fos sibling controls remained free of tumors for 6-7 mo or more; significantly, however, tumors did eventually appear at axilla and inguinal sites of pre-existing hyperplasia. In most pairings, by 10-12 wk tumor burden necessitated sacrifice of all but the progeny of the mildest phenotypic pairings. Whereas HK l -fos/ras tumors were large and aggressive and did not regress, their histotype remained that of a typical squamous papilloma, even those biopsied after longer periods (Fig. 7C and D). HK 1-fos/ras papillomas possessed larger areas of dysplasia than other papillomas, but no carcinoma in situ; immunofluorescence experiments confirmed the benign nature by the retention of K 1 expression and only focal K13 expression (Greenhalgh et al., 1993~).This result shows the remarkable stability of even aggressive papillomas in our system; the papilloma phenotype persisted for up to 12 mo in some cases.
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FIG. 7. Phenotype and histot!pe of' HKI-foslras transgenic mice. (A) Comparison of phenotypic juvenile HKI-ras and HKl-foslras mice at I4 days. Note the increase in phenoty-pe of' the HK 1-foslras example compared with HR 1-ras sibling. For certain tnatings. this increase in phcnotypr severity proved lethal at 1 0 days due to massive hyperkeratosis. (a) .$I 6 w k of age, Hlil-ras niuuse exhibits no phenotype whereas the HKIf'os/rasmouse already has large tumors at the axilla and inguinal areas, and retains the earlier juvenile skin phenotypes. (C, D) Hematoxylin and cosin stain [magnification, 50x ((1) and ISOX (D)] of-a large 10-wk axilla tumor reveal a benign papilloma histotype with areas of dysplasia, but no evidence of niicroinvasion or other indications of malignant conversion. Similar results were ubserved in H K 1-fosia transgenic mice (see Section III.A.2). Reproduced with permission from Greenhalgh pt a/.(1993~).
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These results demonstrate that in transgenic mice, Ha-ras and fos can cooperate in papillomatogenesis to achieve an autonomous growth phenotype, but appear to require additional oncogene/tumor suppressor gene involvement for malignant conversion. The apparent discrepancy between the lack of malignant conversion in HK l-fos/ras mice to the nude mouse fos/ras grafting studies (Greenhalgh et al., 1990) may center on the inherent differences within the two systems employed, that is, the wounding at the graft site in nude mice, the minimal immunocompetence of nude mice, and the multiple insertional mutagenes is provided by retroviral integration. This latter point of multiple viral copies may hold the key, as very high Ha-rus and fos expression levels could have contributed to progression. In support of this idea, recently the level of Ha-rus expression produced in this grafting system was shown to correlate to malignant potential (Brisette et al., 1993). Considering the requirements for multiple events in carcinogenesis, this HK 1 transgenic model system can mimic those genetic events necessary to achieve an autonomous latestage (but stable) papilloma. Ha-ras activation appears to be an initiation event, producing immediate preneoplastic phenotypes (hyperplasia/ hyperkeratosis) and predominantly regressing papillomas, whereas fos deregulation in our model produces phenotypes that require a long latency period and are dependent on a wound promotion stimulus. Together, fos deregulation amplifies the Ha-ras-induced phenotypes. T h e actual mechanism by which this is achieved is unknown, but it is likely to center on anomalies in the signaling pathway mediated by Ha-rus activation, which culminates in further anomalous transcriptional control of target genes by activated v-fos. Moreover, that fos deregulation appears to amplify the Ha-rus phenotypes may be of significance in view of the fact that TPA promotion induces c-fos expression in vivo (Rose-John et ul., 1988). Perhaps constitutive HKI-fos expression provides a facet of autonomous promotion creating the transgenic equivalent of TPA-independent tumors observed in chemical carcinogenesis (Hennings et al., 1985). Also, considerations should be given to the role played byjun in this system, since fos cannot bind to DNA at AP-1 sites without complexing with jun family members (Curran and Franza, 1988). Thus, these animals may provide an in vivo opportunity to dissect further the interactions between Ha-rus and fos in the signaling pathways that underlie differentiation and carcinogenesis. The next logical step is to identify the cooperative events that would allow malignant conversion of the HK l-fos/ras autonomous papillomas. Therefore, these cooperation experiments will be expanded (Section II1,B) to include matings of HKl-fodras mice, mice expressing TGFa and mice null by virtue of a
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knock-out experiment for the tumor suppressor gene p53 (Donehower et nl., 1992).
2. Cooperation between TGFa and fos Given the evidence outlined earlier that directly links TGFa to Ha-ras in signal transduction (Sections I l , B , l and 11,C) and the cooperation hetween HKI-ras and HK1-fos, it seemed logical that a synergism might exist between ‘fGFa and fu.s, testable by mating HKl-’I’GFa and HK1-fos mice. In support of this idea, evidence from in uitro studies had already linked these two genes, with t h e finding that TGFa could induce fos expression zn zlitro (Cutry et nl., 1988; Sagar et al., 1991) and that TPA promotion induces both TGFa and f o s expression in uiuo (Rose-John et al., 1988; Imamoto at al., 1991). This latter study associated TGFa and fos expression with the promotion phase, which may be consistent with the results obtained in HK1-fos animals in which phenotypes were produced only after long latency. that is, they required an init.iat.ing(genetic) event. However, as outlined earlier, TGFa appeared able to replace Ha-ras in the early stages of carcinogenesis. Therefore, by mating HK1fos and HKl-TGFa mice, t,he potential existed to assess further the role of TGFa as an initiator. In addition, a comparison of phenotypes produced in HKI -fos/ras mice and HK1-fos/TGFa mice could potentially identify alternative pathways of TGFa- and fos-mediated carcinogenesis that have been highlighted in chemical carcinogenesis by use of different initiators (Brown et nl., 1990). Preliminary data on the H K 1-fos/TGFa genotypes suggests that, overall, t.hese mice were very similar to their HK1-fos/ras counterparts detailed in Section I I I , A , I . There was an increase in neonatal and juvenile phenotype severity and, for low TGFa expressers, the coexpression of TGFa and v-fos now resulted in newborn phenotype persistence into adulthood. HK 1-fos/TGFa papillonias also arose earlier than those of HKI-TGFa controls and, to date, did not appear to regress. However, there were some distinct qualitative differences between HK1fos/TGFa and HK1 -fos/ras mice. T h e HK 1-fos/TGFa phenotypes were not as severe as the mildest of the HK1-ras/HKl-fos cross and, unlike HK 1-fos/ras expressers in which the rns-associated phenotypes were accelerated, HK 1-fos/TGFa mice showed a rapid acceleration in the fos-associated phenotypes, that is, rapid appearance of ear hyperplasia by 8-10 wk versus 5 mo in HKI-fos controls and rapid progression of these preneoplastic ear phenotypes to tumors over a further 2 to 3-mo period as opposed to a 5 to 6-mo period in HK1-fos controls. Additionally, newborn HK1-fos/TGFa mice exhibited a separation of
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the stratum corneum, resulting in a peeling skin phenotype; early anomalous expression of loricrin and filaggrin in proliferative cells; and novel focal expression of K13 in preneoplastic hyperplastic epidermis, in a manner more characteristic of an overt benign papilloma (GiminezConti et al., 1990; Greenhalgh et al., 1993a-c). These results support a massive increase in the growth and differentiation rate of HK 1-fos/TGFa epidermis [confirmed by bromodeoxyuridine (BrdU) analysis]. This dramatic reprogramming of the differentiation characteristics, that is, anomalous expression of loricrin, filaggrin, and K13, is consistent with the important regulatory roles assigned to TGFa andfos in normal epidermal differentiation (Derynck, 1988; Fisher et al., 1991; Smeyne et at., 1992, 1993; Basset-Seguin et al., 1994). Since HK1-TGFa sibling controls do not exhibit changes in loricrin or filaggrin expression in newborn hyperplastic epidermis, fos may be important in regulation of these genes in the later stages of differentiation; under conditions of TGFa-induced hyperplasia, v-fos may induce their early anomalous expression together with the appearance of focal K13 expression. Identical results have also been obtained in a preliminary analysis of HK 1-fos/ras epidermis, further demonstrating that anomalous keratinocyte differentiation is specific to v-fos. These data highlight the potential dual role for TGFa in epidermal carcinogenesis. First, as an early event, TGFa can replace Ha-ras activation, with which HK1-fos then cooperates. This relationship assumes that activatedfos expression acts as a promoter, resulting in autonomous papillomas. Second, the observation in this cooperation experiment that TGFa accelerated the HK 1-fos-associated phenotypes supports a promotion role for TGFa, and is consistent with the requirement for wounding prior to onset of HK1-fos phenotypes. This promotion role appears to be independent of Ha-rar since in HK1-foslras mice no effect on ear lesion etiology was observed. This raises the intriguing possibility that the TGFa acceleration of HK1-fos phenotypes is mediated by a pathway separate from the EGFRIHa-raslMAP kinase cascade. Such a pathway has been recently identified in which the p91 transcription factor STAT (signal transducer and activator of transcription; Shuai et al., 1993) directly interacts with the EGFR and, following activation by a tyrosine kinase which induces rapid nuclear translocation, activates transcription at the SIE (c-sis-inducibleelement) of c-fos (Fu and Zhang, 1993; Shuai et al., 1993). Whether a particular pathway is exclusively involved with either an early role o r a later promotion role or in malignant conversion probably depends on the complemeutary carcinogenic insults, but our data to date suggest that the role of TGFa over-expression
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associated with promotion, absent in HK l-raslfos mice, may involve this p9 1 pathway. Although the results show that TGFa andfos can cooperate to achieve an autonomous papilloma phenotype and, considering the similarities with HKI-fos/ras mice, can d o so via predominantly the same pathway, once again other events are required for malignant conversion. One hypothesis of a mechanism for malignant conversion suggests that an important event is loss of the normal Ha-ras allele and, thus, loss of the antagonistic normalizing effect of normal Ras on mutated Ras proteins (Quintanilla et a/., 1986; Greenhalgh et al., 1989; Buchnian et al., 1991). 'Ib test this hypothesis, experiments are planned that create the triple genotype of HK1-ras/fos/TGFa and constitutively lower the levels of quiescant GDP/cRas protein via overexpression of TGFa. Thus, the ratio of transforming Ras to normal Ras would increase; this has been shown in in iutro nude mouse grafts to lead to malignant conversion (Greenhalgh et al., 1989; Brisette et al., 1993). Considering the requirement for seven events, even this combination may be insufficient to achieve conversion. Thus, these experiments will also be performed in a null p53 background (Section 111,B).
3. Cooperation between TGFa und Hu-ras The links between Ha-rus and TGFa in membrane tyrosine kinase signal transduction are consistent with our observations that these genes produce similar phenotypes alone or in cooperation with .fos. Both appear to be capable of providing the early initiating events of carcinogenesis, Ha-rus via mutations to provide a constitutive mitogenic signal and TGFa via a similar pathway presumably through maintaining high levels of the GTP-bound form of the endogenous RasHaprotein. Alternatively, as detailed earlier, TGFol has been closely associated with a promotion role. 'l(;Fa expression is induced by wounding or TPA promotion, whereas introduction of Ha-ras into keratinocytes induces TGFa expression in grafted papillomas and introduction of TGFa into papilloma cell lines results in bigger tumors but no conversion (Finzi et al., 1988; Furstenburger et ul., 1989; Glick et al., 1991; DiGiovanni, 1992). Moreover, in transgenic mice expressing constitutive TGFa from the mouse metallothionein ( M T ) promoter (MT-TGFa), overexpression of TGFa could substitute for TPA promotion when MT-TGFa mouse skin was initiated with DMBA (Jhappan et al., 1994). Since DMBA induces Ha-ras activation (Quintanilla et ul., 1986). the latter study on MT-TGFa mice would again suggest that TGFa act.s as a downstream promoter of Ha-rus. This MT-TGFa study predicts that in the HK1 transgenic sys-
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tem constitutive overexpression of HK1-TGFa might be an autonomous promoter for HKl-ras and could result in generation of numerous autonomous papillomas, similar to the effects of HK 1 -fos/TGFa and HK1-fos/ras cooperation (Section IILA, 1 and 2). However, this hypothesis has not been supported by initial HK1-ras and HK1-TGFa mating experiments. Very preliminary data on the HK 1-ras/TGFa genotype indicate that a relatively subtle synergism exists between these two genes, since only a mild increase in hyperplasia was observed in newborn mice, similar to that exhibited by parental HK1-ras or HK 1-TGFa line homozygosity (Dominey et al., 1993; Greenhalgh et al., 1993a). In addition, HKlras/TGFa papillomas arose with only a moderately reduced latency (again more typical of line homozygosity). To date, HK1-ras/TGFa papillomas have not exhibited significantly different numbers or growth rates. In addition, these papillomas are not autonomous and regress in a similar fashion to parental tumors. These observations suggest that, in being members of the EGFR/ MAP kinase signaling pathway and with the idea that TGFa overexpression possibly provides a similar genetic insult to that of Ha-ras activation, a redundancy is created at these early stages of epidermal carcinogenesis. If so, this data may also suggest that the mild synergism observed centers on the alternative ras independent TGFa signaling pathways such as p91, and are again associated with a promotion role. Moreover, these transgenic data imply that while simple deregulation in membrane signaling is sufficient for production of a benign tumor, further progression requires a downstream anomaly and, possibly, deregulation of specific transcription factors. This observation is in direct agreement with classical in vitro cooperation studies which demonstrated the requirement for two distinct complementary groups, often including a membrane signal transducer and a nuclear oncogene (Land et at., 1983; Ruley, 1983; Weinberg, 1989). Furthermore the HK1-ras/TGFa data predict that where MT-TGFa expression replaces the requirement for TPA promotion in DMBA-initiated MT-TGFa transgenic skin (Jhappan et at., 1994), as tumors also arose without Ha-ras activation it is not only Ha-ras but possibly an additional DMBA-mutated (nuclear?) gene(s) that is the cooperative initiation event in MT-TGFa tumorigenesis. These cooperation experiments between Ha-ras, fos, and TGFa demonstrate the power of transgenic mouse models to dissect otherwise elusive features of carcinogenesis, and not only highlight the necessity for multiple events but also show that particular mutagenic events have to impart the appropriate synergism for tumor progression, and that the synergisms observed may be different in nontransgenic models.
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B. EFFECTOF ~ 5 TUMOR 3 SUPPRESSOR GENELoss The design of molecular carcinogenesis models would be incomplete without investigating the rofes of tumor suppressor genes such as p53, since cancer etiology may be biased toward tumor suppressor gene failure rather than dominant oncogene activation (Knudson, 1986; Marshall, 1991). Mutations in the p53 gene are some of the most frequently observed genetic lesions in human cancer and are associated with the inherited Li-Fraumeni cancer susceptibility syndrome (Malkin et ul., 1990). T h e observed mutations have a propensity to occur in highly conserved regions of exons 5-8, with distinct hot spots for mutations occurring at amino acid residues 175, 248, and 273 (Hinds et al., 1990). These mutations are presumed to predispose affected individuals to cancer, particularly when the remaining normal allele becomes either somatically lost or mutated. ’I‘hese mutant forms of p53 are capable of transforming cells in ziitro and can cooperate with the H a m s oncogene (Eliyahu et al., 1984; Parada et al., 1984). Conversely, the wild-type p53 gene is able to reduce transfortned cell growth and tumorigenicity (Chen rt al., 1990). Its potency as a tumor suppressor is demonstrated by an ability to block mutated p53-ras cooperation (Finley et al., 1989). Researchers believe that wild-type p53 inhibits cyclin dependent kinases, for example, via transactivation of the WAF l/CIP 1 protein (El-Deiry et d.,, 1993; Harper et al., 1993), to block a cell in C, and give time to repair damaged DNA prior to replication, or divert it into apoptosis (programmed cell death) (Hartwell, 1992; Perry and Levine, 1993). This role as “molecular policeman” (Lane, 1992) makes p53 an obvious candidate for failure in tumorigenic processes, and a particular target for inactivation by viral oncoproteins such as the E6 gene of HPV16 or - 18 (Werness rt al., 1990). This idea was further strengthened with the discovery that MDM2, a protein involved in p53 inactivation, is amplified in many sarcomas (Oliner P t al., 1992). Thus, in normal cells when DNA is damaged, some as yet unknown mechanism triggers the accumulation of wild-type p53 and subsequent G, arrest, to allow for repairs or to trigger apoptosis (Hartwell, 1992; Perry and Levine, 1993). Tumor cells, in which p53 is inactivated via mutation or sequestered by viral (HPV16 E6) or cellular (MDM2) proteins, do not have this option and are thus less stable genetically, leading to accumulated mutations, chromosomal rearrangements, and carcinogenesis (Hartwell, 1992; Lane, 1992). This idea explains why p53 knock-out mice develop normally but then succumb to numerous lymphomas and sarcomas by 5-6 mo of age (Donehower et al., 1992), and why p53 is such an attractive target for inactivation by viral oncoproteins such as HPVl6 E6. Furthermore, the
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mutations that abrogate normal p53 function can fall into two classes. Since p53 transcriptional regulation is via a tetrameric complex (Hartwell, 1992; Perry and Levine, 1993), the potential exists for heterotetramer formation with p53 mutants from the mutated allele that block normal p53 function. Such dominant negative mutants can be considered equivalent to p53 loss or viral/MDM2 inactivation. A second class of p53-specific missense mutations may result in a “gain of function” (Dittmer et al., 1993); tumors with these missense mutations may be more aggressive than those lacking p53 by chromosomal rearrangements or dominant negative p53 mutants (Dittmer et al., 1993). In support of this idea, in vitro transfection identified several mutant forms with markedly different transforming potentials (Hinds et al., 1990). Several studies have already identified the spectrum of mutations in human skin cancer (Brash et al., 1991; Pierceall et al., 1991c)and indicate a distinct role for UV irradiation from sunlight in the generation of these mutations, since most are typical pyrimidine dimerizations, including a high frequency of C + T mutations (Brash et al., 1991; Pierceall et al., 1991~). A spectrum of p53 mutations was also obtained in chemical carcinogenesis studies on mouse skin in which the p5? becomes mutated and the normal allele subsequently appears to be lost in carcinomas (Burns et al., 1991; Ruggeri et al., 1991). Thus, in the chemical carcinogenesis mouse skin model, loss of $153appears to be a later event. Conversely, results in humans demonstrated that preneoplastic solar keratosis exhibited p5? mutations (Gusterson et al., 1991; Sim et al., 1992). Also, the observation that mutant p53 could induce epithelial proliferation in vitro (Wyllie et al., 1993),coupled to the finding that p53 cooperates with Ha-ras in induction of hyperplasia in uivo (Lu et al., 1992), suggests the idea that p53 may be involved at an earlier stage of carcinogenesis. Therefore, by performing a series of mating experiments between HKI mice expressing Ha-rm, fos, and TGFa mice null for p5? by virtue of a knock-out experiment (Donehoweret al., 1992),the number and nature (growth factor, transcription regulator) of events necessary before p53 anomalies become causal could be assessed. To date, the HK1-radnull, HK1-fos/null, and HK1-TGFdnull genotypes have been developed together with their hemizygous and wildtype p53 sibling controls (D. A. Greenhalgh, unpublished data). In these experiments, hemizygous animals were identical to wild-type sibling and parental HKl transgenic mice. However, in the null genotype, an unexpected and confusing result has been obtained. Instead of an expected accelerated progression of papillomas to malignancy, tumorigenesis is distinctly repressed in all single transgenic genotypes. For example, the spontaneous wound-associated (ear tag) papillomas in HK l-ras line
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1205, which can appear as early as 5-6 wk of age in 100% of animals (Fig. 2A), did not appear 1205/null animals until 18-20 wk in only 15% of animals, were much smaller, and grew slowly. Whether these papillomas would have become malignant could not be assessed since null mice succumbed to spontaneous internal sarcomas and lymphomas by .5-6 mo. Equally, only one HK1-fos/null p53 transgenic mouse to date exhibited the wound-associated HK 1-fos ear keratosis. In attempts to accelerate H K 1-ras/null papillomatogenesis by TPA promotion, to give more time to assess conversion, this phenomenon was graphically demonstrated. Normally, HK 1-ras 1205 animals are exquisitely sensitive to TPA promotion, producing large tumors following two applications of TPA, as were their hemizygous p53 progeny. HoMTever, 1205inull mice gave no skin tumors after multiple applications until their death at 5-6 mo. What can be concluded from these results? Clearly, p53 loss does not appear to have a cooperative effect with ray, fox, or TGFa in the early stages of papillomatogenesis. This is consistent with a late role for p53 loss in mouse skin carcinogenesis, and is in agreement with the massive degree of malignant conversion observed when p53 null mice were employed in classical two-stage chemical carcinogenesis experiments (Kemp et al., 1993b). Thus, HK1-fos/ras null and HK1-fos/TGFa null 1153 genotypes are under development to assess whether autonomous papillomas expressing two genetic hits can achieve the tumor stage at which p53 loss becomes causal, or whether a HK1-foslrasiTGFa null genotype, that is, a “4-hit mouse,” is necessary to achieve conversion. ’I‘his causal role for p53 at a late stage of murine skin carcinogenesis does not necessarily preclude a role for p53 mutations found at earlier stages of neoplasia in human skin, such as preneoplastic actinic keratosis. As outlined earlier, both H a m s and TGFa appear to have early and late stage functions; this may be the case for p53 also. An early role for a “gain-of-function” p53 mutant may give cells a selective advantage, resulting in a hyperplastic response such as actinic keratosis, consistent with the proliferative effect of mutant p53 in normal keratinocytes (Wyllie et al., 1993) or cooperation with Ha-ras in early stage epithelial hyperplasia (Lu et al., 1992). Alternatively, early inactivation of the p53 surveillance system (p53 loss, dominant negative mutants) may be necessary to acquire the genetic mutations responsible for hyperplasia, but, consistent with the above HK1 transgenic data, the main effect of p53 loss manifests later when the uncontrolled proliferation of late stage benign tumor cells allows an accumulation of mutations at an accelerated rate, rapidly leading to malignant conversion and subsequent metastasis. Although no obvious progressive synergism with the HK1 transgenes
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could be attributed to p53 loss, which possibly highlights the requirement for “gain-of-function mutants” at these early stages, the protective apparently tumor-suppressive effect of p53 loss was totally unexpected. Interestingly, a similar phenomenon was observed in the two-stage chemical carcinogenesis experiments on p53 null mouse skin, in which a decrease in the numbers of papillomas was recorded (Kemp et al., 1993b). In this study, Balmain and co-workers cleverly suggested that, in the absence of p53 cell cycle regulation surveillance, the initiated cells progress into S phase with a burden of unrepaired DMBA-induced mutations, which prevents successful completion of the cell cycle; subsequently, cell death is initiated, with a corresponding reduction in tumor numbers. In support of this idea, TPA promotion of DMBA initiated p53 null mouse skin displayed areas of necrotic tissue and epidermal loss (Kemp et al., 1993b). Although this attractive scenario cannot be ruled out, it does not appear to apply to HK1 transgenics, since only a single genetic hit is present and therefore putative DNA damage is minimized. Moreover, phenotypic HK1-rashull and HK1-TGFdnull hyperplastic newborn epidermis shows no areas of necrosis or epidermal loss. One intriguing observation is that this phenomenon appears to be restricted to inhibition of phenotypes that required a wound-associated or TPA-promotion stimulus, but not HK 1 transgene-induced newborn hyperplasia. This result raises several interesting possibilities and the speculation that p53 has alternative roles in epidermal cells. Primarily, the function of the epidermis is as a barrier; blocking epidermal proliferation or inducing apoptosis under conditions of wounding would be undesirable, yet it would be essential for the epidermal cell to retain the p53 role as a surveillance system for DNA damage from such agents as UV irradiation. Thus, perhaps p53 has additional functions in epidermal cells that are of necessity separate from G, arrest and induction of apoptosis. Could p53 be involved in a proliferative response and, under certain very specific conditions such as wounding or TPA promotion or in certain cell types, could p53 expression be necessary for proliferation? Therefore, absence of wild-type p53 expression somehow minimizes the effects of a promotion stimulus in the HKl transgenic mice. Clearly, p53 mutants induce proliferation of epidermal cells an vitro; thus “gainof-function” mutants may function in part by activating a putative pathway of p53-regulated proliferation. Alternatively, since cells somehow “sense” DNA damage and induce p53 expression, G, arrest, and growth block for repair, can this mechanism “sense” the loss of p53? Since the epidermis is one of the tissues most exposed to the effects of environmental carcinogens, a redundancy and provision for multiple back-up systems for DNA repair appears logical. If so, such surveillance proteins,
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or an activation of downstream p53 target proteins such as WAFlICPl independent of p53 expression, appear to compensate effectively for p53 loss in the early stages of murine skin papillomatogenesis. IV. Development of a Rapid Screening System for Tumor Promoters and Chemical Carcinogens
T h e successful establishment of transgenic mice expressing oncogenes in the epidermis provides an in uivo system genetically predisposed to the development of cancer, with a uniform genetic background and a known genetic insult. This latter point, coupled to the accessibility of the epidermis, creates the potential to develop highly sensitive or very specific models in which to assess and identify the potency of novel promoters and carcinogen. For instance, the cooperation between chemical treatment and transgene expression is likely to accelerate the neoplastic process and significantly reduce the time and, thus, the expense of in ‘LIZZIO bioassays in rodents (Tennant and Zeiger, 1993). Moreover, a rapid transgenic assay model that could reduce the dependence on longterm bioassays in rodents may be essential, given the fact that short-term tests for genotoxic chemicals, originally designed as fast inexpensive assays, failed to detect three of the most potent carcinogens identified in long-term rodent bioassays (Tennant et al., 1987). To date, several groups have explored the screening potential of transgenic mice, including the TG:AC transgenic line, which expresses v-Ha-rux from a y-globulin promoter (Leder et ul., 1990). Using these animals, Spalding et al. ( 1993) assessed the relative tumor-promoting activities of benzoyl peroxide, 2-butanol peroxide, and TPA. Papillomas were induced in mice treated with all three tumor promoters, with tumors observed in some treatment groups as early as 3 wk into treatment. The relative activity of the tumor promoters was TPA > 2-butanol peroxide > benzoyl peroxide. The short latency period for papilloma development and the high incidence of papilloma induction indicated that TG:AC mice possess high sensitivity to these tumor promoters. Unfortunately, these mice also develop spontaneous internal tumors (Leder et al., 1990),which may compromise their effectiveness in assessing weaker agents over longer time courses. This limitation would be overcome by using the HK 1 targeting vector, which limits expression of the oncogene to the epidermis. thereby limiting cooperative actions of chemical agents to this tissue. This specific targeting of oncogene expression to the epidermis also minimizes confounding actions of chemicals in other tissues that could affect the health, fecundity, or life-span of the mice. As in the previous report on TG:AC transgenic mice (Leder et al., 1990; Spalding
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et al., 1993), initial experiments have assessed the sensitivity of HK1-ras, HK1-TGFa, and HK1-fos mice to TPA promotion. Studies on HK1-ras animals utilized three lines: 1205, which was sensitive to a wound promotion stimulus; 1203, which developed sporadic, often non-wound-associated papillomas; and 1276 which, although a competent HK l-ras expresser, gave only rare papillomas. The results showed a remarkable sensitivity of 1205 mice to TPA promotion; the entire treatment area@)developed into a single, massive papilloma within 3 wk (two administrations of 2.5 kg TPA/100 k1 acetone; one application per week). The non-wound-associated lines 1203 and 1276 developed more sporadic tumors under our administration regime, after 8-10 wk of promotion (D. A. Greenhalgh et al., unpublished data). T h e initiated nature of our HK1-ras mouse skin, particularly the remarkable sensitivity to TPA promotion of 1205, is encouraging for the use of these animals as screening systems for even weak tumor promoters. Furthermore, on removal of the TPA, the tumors regressed over 34 wk. No spontaneous malignant conversion was observed, suggesting that the HK1-ras mice would be useful in an assay for identification of complete carcinogens (i.e., those with both initiating and promoting activity). In a similar fashion, HKl-TGFa mice were also sensitive to this TPA promotion regime, producing papillomas as early as 4-5 wk (three treatments) without any evidence of malignant conversion after 60 wk of promotion, but with immediate regression on removal of the TPA promotion stimulus (Wang et al., 1994). Thus, these data show that TGFa overexpression can be an initiating event for TPA promotion, presumably by substitution for Ha-ras activation. This result was in agreement with those of an earlier study by Vassar et al. (1992), in which TGFa was overexpressed in the epidermis by a keratin K14 promoter (K14-TGFa) and TPA promotion produced papillomas without an activated Ha-ras. T h e sensitivity of HKl-TGFa mice to lower doses of TPA than K14TGFa mice probably simply reflects the lower expression levels in K14TGFa mice. Thus, HK1-TGFa transgenic mice can potentially detect strong promoters and/or a specific spectrum of carcinogens. Equally, considering the result .of DMBA initiation on MT-TGFa mouse skin, in which constitutive TGFa expression rendered promotion unnecessary (Jhappan et al., 1994), and the association of TGFa with promotion (DiGiovanni, 1992), HK1-TGFa mice may be biased to the identification of novel initiators. Given the role of wound promotion in the etiology of HK1-fos phenotypes (Greenhalgh et al., 1993b), and the TPA induction of c-fos expression in chemical carcinogenesis (Rose-John et al., 1988), a surprising
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lack of sensitivity to TPA promotion was exhibited by HK1-fos mice (D. A. Creenhalgh et al., unpublished data). No effect was observed until 22 wk of TPA treatment, when mice began to develop 2-3 small papillomas per mouse. After 60 wk of promotion, HK1-fos TPA papillomas grew larger but, whereas HK1-ras o r HK1-TGFa TPA-induced papillomas regressed, removal of the TPA promotion stimulus resulted in persistence of HK1-fos TPA papillomas, and most converted to malignancy. This HK1-fos TPA papilloma etiology is consistent with the acquisition of an additional genetic insult, prior to overt tumor formation. ‘Therefore, HK l-fos-induced tumors will be assessed for both spontaneous Ha-ras activation and p53 mutations, as well as for the characteristic chromosomal changes associated with chemical carcinogenesis (Aldaz et al., 1989; Bianrhi et al., 1991; Kemp et al., 1993a). The requirements for an earlier genetic event in HK1-fos TPA promotion suggests that, whereas HK1-ras and HK1-TGFa may cooperate with tumor promoters and complete carcinogens, the HK 1-fos mice may have the potential to identify novel classes of tumor initiators. V. Summary and Future Prospects
In this chapter, by way of’ example, we have reviewed our data employing an epidermal targeting vector t o demonstrate the importance of developing transgenic models for carcinogenesis. In our attempt not only to understand the molecular requirements for skin cancer but hopefully to identify mechanisms of carcinogenesis applicable to epithelia in general, we have assessed interactions between relevant oncogenes, tumor suppressor genes, and growth factors, to eventually design clearly defined models that represent all the discrete stages of skin carcinogenesis. Viable phenotypic transgenic mice have been obtained for all oncogene constructs targeted, and we have begun to assess cooperation in 7~1710.T h e results to date show that several synergistic events are required to achieve each distinct tumor stage prior to malignancy Zn v i m This conclusion has been found by numerous other groups involved in targeting to the skin (Bailleul et al., 1990; Vassar and Fuchs, 1991; Tinsley P t al., 1992; Lambert et al., 1993) and has been found in the development of a targeted transgenic mouse model for colon cancer (Kim et al., 1993). Currently, the HK 1 transgenic data support at least a four-stage mechanism to achieve malignancy: ( 1) a genetic initiation event, for example, Ha-ra~or TGFa; (2) a lesion-eliciting event (TPA promotion, wounding, or genetic); (3) autonomous growth (genetic event, e.g.,fos); and (4)malignant conversion (genetic event, e.g., p53 losslmutation). Furthermore, it appears that we have developed a transgenic mouse
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model system in which preneoplastic and benign lesions do not undergo rapid progression to malignancy. Therefore, we cannot only assess the molecular interactions between genes at particular stages of carcinogenesis but also investigate the role of suspected environmental carcinogens such as UV light in the context of known genetic insults. This aspect may potentially be employed to significantly reduce the time and cost of screening suspected tumor promoters and carcinogens. T h e genetic predisposition of these animals to carcinogenesis may also represent an ideal opportunity to test the efficacy of various antitumor agents at different stages of neoplasia, including bryostatin, which inhibits TPA promotion (Hennings et al., 1987); staurosporine, which inhibits the growth of Ha-rus infected keratinocyte papillomas in nude mouse grafts (Strickland et al., 1993); and azatyrosine, which has antitumor activity in two-stage chemical carcinogenesis experiments (Izwawa et al., 1992). One of the most exciting therapeutic possibilities is the application of somatic gene therapy (Anderson, 1992). An obvious use of transgenic models will be in the assessment of gene therapy approaches prior to clinical trials in humans. Important advances have been achieved in the past few years in methods of in uiuo gene transfer (Morgan and Anderson, 1993; Mulligan, 1993) that may allow significant gene expression. Moreover, the ability to create the so-called “bystander” effect (Culver et al., 1992; Vile and Hart, 1993), in which death of a single tumor cell is thought to result in the death of surrounding tumor cells, may offset the requirement of transducing all neoplastic cells in a given tumor. One can also envision transducing a tumor cell in viuo to enhance the host immune system, and possibly coupling cytotoxicity with an immune response (Nabel, 1992; Rosenberg, 1992). Transgenic mice may represent the only system that can (1) assess delivery routes, side effects, expression characteristics, and in uiuo efficacy and (2) assess agents designed to specifically counter a known genetic defect. As molecular and cellular techniques progress, we envision the continuing discovery and identification of new oncogenes or tumor suppressor genes. These will be coupled to the design of new targeting vectors that result in the evolution of highly sophisticated transgenic models with which to assess molecular carcinogenesis and to provide avenues for novel therapeutic intervention. ACKNOWLEDGMENTS We thank Dr. Joseph Rothnagel for design of the vector, Dr. Xiao-Jing Wang and Dr. A. M. Dominey for their expertise on these projects, Joshua Eckhardt, Donnie Bundman, Mary Ann Longley, and Xin-ru Lu for continued excellent technical help, Dr. J. Tschan
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(St. Joseph's Hospital, Houston, ' l x a s ) for histological assessment, and N. J. Laniinack for preparation of. the manuscript. This work was supported in part by National Institutes of Health Grants HD25479, CA52607, 11130283; the Texas Advanced Technology Program (ATP 004949048); and a gift from Johnson and Johnson.
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INDEX
C
A Accessory chain, FcyRs, 218 Actin, as PKC substrate, 172-173 Antibody, FcyRII-regulated production, 230-232,237 Antigen presentation, role of FcyRs, 218 Apoptosis, programmed, 259 Astrocytoma, specific PKC isozyme expression, 187-188 Autophosphorylation as molecular switch, 88 and SH2 binding sites, 94
Calmidazolium, effect on RB phosphorylation, 47 Calphostin C, PKC-specific inhibitor, 167 Carcinogenesis cervical, model, 4 early stage, role of p53, 283 multistage cooperation between Ha-ras and fos, 258 and mouse models, 248 role of PKC, 191-192 skin Ha-ras role, 257 models representing discrete stages, 288-289 TGFa role, 267-268 Carcinogens chemical, development of rapid screening system, 286-288 environmental in multistep oncogenic process, 3 potential targets, 248 role in context of known genetic insults, 289 Cell activation, and FcyR expression, 224-225 Cell cycle progression, and RB phosphorylation, 74 regulated phosphorylation of RB, 3248 Cell growth arrest, preventive role of E6, 16 inhibition in culture, 31-32
B B cells antibody production, regulation by FcyRII, 230-233 FcyR expression, 221 FcyRII expression, 213, 224-225 PKC isozymes in, 179-180 Bcr-Abl, and Ras activation, 102103 BCR-ABL, role in leukemia, 90 Beta cells, FcyRII expression, 224 Bisindolylmaleimides, PKC-specific inhibitor, 167 Bone marrow, reconstitution, and PymT expression, 144-148 Bryostatin in neoplasia, 289 PKC activation, 164-165
297
INDEX
Cell growth ( c ( ~ d ~ ~ d ) regulation, 3-7 Cell line, .wr also specific cell lines tumor-derived, and elevated c-Src kinase, 117 Cervical cancer development, role of pKB activity loss, 7 E6 and E7 expression, 18 HPV-positive, and somatic p53 mutations, 14 r-elation to HPV, 1-3 Chelerythrine chloride, PKC-specific inhibitor, 167 Chimera, embryonic stem cell, 135- 140 Colon carcinoma elevated p ~ 6 0 ' - ~1 ~ I5~ , and PKC substrate changes, 195- 196 Cornification, role for fk,259, 263-264 Cross-linking, FcyRs, 2 18-220 Cyclic AMP, effect on RB phosphorylation, 47 Cyclin, D family association with RB, 62-63, 68 complex with pRB, 8, 10 Cyclin-dependent protein kinase GI -specific, role of P I C l , 17 KB as substrate, 33-34 in regulation of cell growth, 5-6 C y tokine effect on FcyR expression, 223-224, 238 release induced by FcyR cross-linking, 218 signaling, and STAT proteins, 96-97 Cytotoxicity, coupling with immune response, 289
D Dephosphorylation K B by phosphatase, 34-36 tyrosine residue in p ~ 6 0 " - ~114 r~, Diacylglycerol, in PKC activation, 162164 Differentiation nnonialous keratinocyte, 279 associated inactivation of RB phosphorylation, 39-43
epidermal fos role, 263 TGFa role, 265 keratinocyte, 182- 183 and malignant progression, 3-4 myeloid, 177- I79 neuroblastonia, role of PKC, 186-187 neuronal, and neurite outgrowth, 185186
and PKC isoforms, 176-187 and PKC isozynie expression, 170 terminal, 5
E E6 and E7 and HPV immortalization, 4-5 and targeting conimon pathway, 1618
HPV, targeting, 269-274 HPV 16, cooperation with Ha-rm, 273 interactions with p53, 11 consequences, 13-15 in oncogenesis, 14- 15 E7 HPV, targeting, 269-274 interactions with pRB family, 7 consequences, 9- 1 1 pRB-independent activities, 11 Early gene, induction, and PKC, 167-168 Endocytosis, immune complexes, FcyRmediated, 2 18 Endothelial cells, PymT-transformed, 149-153 Epidermal cells, p.53 alternative roles, 285 Epidermal growth factor receptor PKC phosphorylated, 173 TGFa binding, 268 Epithelial ceils, normal proliferation, perturbed by HPV types, 4
F FcyR, S P P Fcy receptor Fc receptor expression on tumor cells, 233-235
299
INDEX
soluble and membrane-bound in immune defense, 238 in malignancies, 235-237 Fcy receptor binding sites, and ligand binding, 215217 class I active binding site, 216 structural features, 2 13 class I1 conformational alteration, 233 role in antibody regulation, 230-232 structural features, 213-214 class I11 cross-linking, 220 specificity and signal-inducing capacity, 216 structural features, 2 14-2 15 expression, 221-225 mediated signal transduction, 2 19-22 1 membrane-bound, 217-219,227-230 regulatory role, 230-233 sFcyR production by proteolytic cleavage, 228-230 shedding, 228 soluble, 225-227 regulatory role, 230-233 Fibroblasts F6, specific PKC isozyme expression, 188 mitogen-activated RB phosphorylation, 36-38 Fos, complex with alternative jun members, 259 fos
activated, targeting, 258-264 cooperation with Ha-ras, 274-278 TGFa, 278-280
G Gene, see also Oncogene; Proto-oncogene early, induction, and PKC, 167-168 growth inhibitory, role of RB, 76 Gene targeting, in study of carcinogenesis, 249 Gene therapy, and transgenic models, 289
Genetics, in FcyR differential expression, 221-222 Genome, HPV, and replication, 4 Genotype multiple transgenic, development, 274286 single transgenic, development, 250274 G proteins, coupled receptors, PKC phosphorylated, 174 Grb2, multiple complexes in growth factor-stimulated cells, 103 Growth factor, see also specijic growth factors
components, as potential oncogenes, 6 signaling, role of PKC, 169-170 Growth factor receptor, activated, association with SH2, 92 Growth suppression, reversal, correlation with RB phosphorylation, 72-73
H HamT binding to Fyn, 131-132 expression in uivo, 148 Ha-ras activated, targeting, 25 1-258 cooperation with fos, 274-278 HPVlG E6,273 p53, 282-286 TGFa, 280-28 1 in mouse skin tumor, 252 in skin carcinogenesis, 257 HL-60 cells differentiation, role of PKC, 177-178 RB phosphorylation, inactivation during differentiation, 39-43 HPV, see Human papillomavirus Human papillomavirus and cervical cancer, 1-3 encoded oncoproteins, in viral life cycle, 15-16 high- and low-risk, interaction of E7 with p107, 16 high-risk association with malignancies, 2-5 encoded oncoproteins, 4-5
300
INDEX
Human papillomavirus ( c o n t t n u d ) high-risk E6 proteins, interaction with p.53. 1 1 low-risk, iii benign lesions. 2-5 oncoproteins, tools a i d targets, 18- 19 subset 18, viral oncogene targeting, 269-274 H yperplasia aswciated cooperation between p.53 a n d Ha-rus. 283 and Ha-t-a\ activation, 277 H K I -fos-induced, 263-264 v-Ha-rcu-induced, 257 I Irperproliferatioii. IiPV-induced, 2-4
K K562 cells differentiation, role of PKC, 183, 197 RB phosphorylation, inactivation during differentiation, 41-42 Keratinocyte differentiation anomalous, 279 role of PKC, 182- 183 immortalization, 273 normal, hyperplasia with, 268 Keratosis. in HKI-fos mice, 260-262 Killer cells, FcyRIII expression, 214; SPY also Natural killer cells Kinetics, turnorigenesis, 249
I I nirnortalizatiori L HI'\'. and E6 and E7. 4-5 keratinocyte, 273 Lesion. verrucous, 271-274 Ininiune response Leukemia, role of HCX-ABL oncogene, 90 coupling with c!totoxicity 289 Leukeniogenesis, role of Rb-I inactivation, nialignaricy-rriggeI-ed, 236-257 28-29 Inimunoglobulin effect on FcyR expression, 222-223 IgG, crass-linking with FcyK, 2 18 M IgG-BF. production mechanisms, 227230 Macrophage, FcyRIlI expression, 214 isoty pes, differential FcyR binding, Malignancy 215-217 and FcR. 235-237 Inimunoglobiilin-),itrding factor HPV-induced, 270-27 1 immunoglobulin-indiice(1 release. 23 1Malignant progression 232 HPV-infected cells, and cervical cancer, relation t o FcyKs. 226-227 1-3 soluble truncated, 237-238 and prerieoplastic and benign lesions, Insulin, signaling 289 and IRS-1, 95 MARCKS, as PKC substrate, 172-173 role of PKC, 168-169 and turnorigenesis, 189- 190 Insulin receptor, PKC phosphorylation M 1 cells, K B phosphorylation, inactivation sites. 153 during differentiation, 40-4 1 Interferon-a, effect o n RB phosphor!Slelanocytes, specific PKC isozyme expreslation, 45-46 sion, 187- 188 Interleukin-6, production, role of FcyR MELC cells cross-lin king, 2 1X differentiation, role of PKC. 183- 184 Interleukin-3, signaling, role of PKC, RB phosphorylation, inactivation durI 69 ing differentiation, 42-43 IKS- I , and insulin >ignaling, 95 Metastasis, and p53 loss, 284 1-(.5- Isoquinolinylsulf~~nyl)-2-1nettiylpiper- hlitogen, activated RB phosphorylat ion, azine, PKC- specific inhibitor, I67 36-39
30 1
INDEX
Models cervical carcinogenesis, 4 RB molecular function, 69 transgenic mouse, 117-120, 134-144, 247-289 Molecular matchmaker, role of RB, 6770 Monocytes FcyRII expression, 213 FcyRIII expression, 214 Mutagen, exposure, and oncogenic progression, 4 Mutation RB, naturally occurring, 51 Rb-l in tumor progression, 29-30 tumors, 26-30 ultraviolet light-generated, 283 Mutational analysis, E7, 7, 9-10
N Natural killer cells, FcyRIII expression, 214 Neoplasia bryostatin efficacy, 289 cervical intraepithelial, and HPV, 3-4 HPV-induced, 270-271 role of TGFa, 265 Neurite formation, role of PKC, 185 Neuroblastoma, differentiation, role of PKC, 186-187 Neurons differentiation, role of PKC, 185186 transplantation, and PymT expression, 144 Neutrophils FcyRII expression, 213 FcyRIII expression, 214 Nuclear tethering, RB, 65-66
0 Okadaic acid, effect on RB phosphorylation, 46 Oncogenes, see also specijic oncogenes activated tyrosine kinases, 89-90
potential, and growth factor components, 6 single-step, PymT as, 143 viral, transformed cells, role of PKC, 191-193 Oncogenesis and E6, 14-15 and E6-p53 interaction, 1 1 and E7-pRB interaction, 7 Oncoproteins high-risk HPV-encoded, 4-5 viral, RB-binding, 54, 77
P P53 cooperation with ras, 282-286 interactions with E6, 1 1 consequences, 13-15 normal functions, 12-13 as surveillance system for DNA damage, 285 Papilloma HKI-fos, 275 and secondary events, 255-258 squamous, 271, 274 TGFa in, 265-269, 278-279 PC 12 cells PKC in, 184-185 RB phosphorylation, inactivation during differentiation, 41 Phagocytes, mononuclear, FcyRI expression, 213 Phorbol ester, see also speczpc phorbol esters PKC activation, 163-164 role in tumor promotion, 193-194 Phospholipase C, FcyR-mediated activation, 219 Phospholipid, metabolism, role of SH2containing proteins, 92-94 Phosphorylation, see also Autophosphorylation; Dephosphorylation multiple sites in RB, 77-78 physiological targets by PKC isozymes, 171-175 protein tyrosine, target molecules, 143 RB, cell cycle regulated, 32-48 regulated RB function, 70-75
302
INDEX
Phosphorylation (continued) serine, FcyRII, 229 tyrosine, 2 19-220 regulation of SH2 binding, 91 Phosphotyrosine phosphatase, SH2containing, 104 PKC, sep Protein kinase C Platelets, FcyRII expression, 213 Polyomavirus early region, controlled PymT expression, 133-140 hamster, 130- 132 middle T antigen, see HamT middle T antigen, see PymT murine, induced transforming events, 126-127 pp601 association with receptor tyrosine kinase, I16 elevated activity in colon carcinoma, I I5 role in PymtT-mediated tumorigenesis, 1 I 7- 1 19 PP60”~s’1 with intrinsic tyrosine kinase activity, 112 transforming ability, 114 PKB independent activities of E7, 1 1 interactions with E i , 7 consequences, 9- I 1 normal functions, 7-8 Proliferation cell, and tumor suppressor genes, ti epithelial cell, perturbed by HPV types, 4 Promoter element, controlled PymT expression, 133-144 Protein kinase C: activation and translocation to membranes, 162-165 down-regulation, 165-167 isoforms in differentiation, 176-187 in tumorigenesis, 187- 196 isozynies differential expression and distribution, 170-171 overexpression, 194-195 structure, 160- 162 -(I‘
role in insulin signaling, 168-169 substrates, 171-175 Protein phosphatase type 1, in dephosphorylation of RB, 34-36 type I - u ~ , RB-binding, 64 Proteins, see nlso Oncoproteins cytoskeletal, PKC substrates, 173 G, sre G proteins intracellular signaling with SH2, 92-94 and tyrosine kinases, 87-89 PKC-associated, identification, 175- 176 STAT, and cytokine signaling, 96-97 Proto-oncogene, c-fos down-regulation, 263 role in epidermal carcinogenesis, 2582.59 PymT hiochemical properties, 127- 130 expression in vivo, 133-148 mediated mammary tumorigenesis, 1 l i - I 19 oncogenic action iu vzvo, 133 in transformation initiation, 127 transformed endothelial cells, 149- 153
R Ras activation, and SH2/SH3 adaptor proteins, 100-102 regulatory cycle, 100 rm
synergistic relationship with PKC, 197 transformed cells, role of PKC, 190-191
KB binding proteins, 54-65 dephosphorylation by phosphatase, 3436 growth-inhibitory activity, 30-32 mitogen-activated phosphorylation, 3639 as molecular matchmaker, 67-70 nuclear tethering, 65-66 phosphorylation effects of antimitogens, 43-48 inactivation during differentiation, 39-43
303
INDEX
phosphorylation-site-specificregulation, 73-75 protein-binding domains, A/B pocket, 48-54 protein-binding function, inactivation by phosphorylation, 70-71 role in transcription, 66-67 as substrate of cyclin-dependent protein kinases, 33-34 viral oncoprotein-mediated E2F release, 57-58 R6-1 inactivation and induction of pituitary tumor, 27 in RB, 26-27 role in leukemogenesis, 28-29 reconstituted, effect on cell growth, 30 RbAp48, RB-binding protein, 63-64 RBP60, interaction with RB, 64-65 Receptor tyrosine kinase activation of intracellular biochemical pathways, 88 association with pp60~-5"",116 SH2-containing signaling proteins, 93-94 Neu, complex with pp60c-src,119 Reconstitution, bone marrow, and PymT expression, 144-148 Replication, HPV, 3-4 Retinoblastoma tumor suppressor protein, see RB RNA, messenger, alternative splicing, 227-228
Signal transduction cascade, in regulation of cell growth, 5-6 downstream of tyrosine kinases, 93 FcyR-mediated, 2 19-221 genetics, 104 role for PKC, 166, 197 and SH3 and PH domains, 99 and tumorigenesis, 11 1 Sphingosine, effect on RB phosphorylation, 46-47 Src homology 2 domain containing phosphotyrosine phosphatase, 104 direct binding to phosphorylated tyrosine residues, 113 and IRS-1, 95 low affinity for Tyr 527 site, 114 mediated signaling in T cells, 95-96 multiple functions, 97-98 and SH3, characterized receptorbinding proteins, 88-89 signaling proteins with, 92-94 structure and binding properties, 9092 Src homology 3 domain and PH domains, 99 in STAT proteins, 96 structure and binding properties, 98-99 Staurosporine, PKC-specific inhibitor, 167 Stem cells embryonic, chimera, 135- 140 hematopoietic, RB-resistant, 75 uncontrolled proliferation, role of R6-1 loss, 28-29 Stimulation, prolonged, and PKC downregulation, 165-166
S Serine, phosphorylation of FcyRII, 229 73hsc, complex with RB, 64 SH2, see Src homology 2 domain SH3, see Src homology 3 domain Signaling cytokine, and STAT proteins, 96-97 insulin and IRS-1, 95 role of PKC, 168-169 SH2-mediated, in T cells, 95-96 Signal transducers, intracellular, and PKC activation, 174-175
T T cells expressing FcyRs, 226-227 mitogen-activated RB phosphorylation, 38-39 PKC isozymes in, 180-182 SH2-mediated signaling, 95-96 12-0-Tetradecanoyl phorbol- 13-acetate effect on PKC subcellular distribution, 169 RB phosphorylation, 47-48
304
INDEX
12-0-Tetradecanoyl phorbol- 13-acetate (ronhtled ) in papillomatogenesis, 284-288 PKC activation, 163 prolonged treatment, in PKC downregulation, 166, 196 TGFu, see Transforming growtfi factor a
321) cells, differentiation, role of PKC, 178-179 Tissue, specific patterns of PKC isozyme expression, l70-li1, 196 TPA. see 12-O-71etradecanoylphorbol- 13acetate Transcription, role of RB, 66-67 Transcript ion factor E2F family, interaction with pKB protein family, 7-1 1 RB-binding ATF-2. 60 c-% yc-, l ti 1 E2F family. .55-58 Elf- 1, 59-60 MyOD, 58-39 PLI. 1 . 60 Transduction. physiological stimuli on PKC activation, 168-170 Transtormat ion cultured cells, role of I'GFa, 265 endothelial cells I>?P!m'l, 134, 149153 malignant, and changes in PKC expression, 189 by oncogenes, role of PKC, 190I93 Transforming growth factor a cooperation with fos and Ha-rms, 27828 1 overexpression, 287 targeting, 264-269 Transforniing growth factor @, effect on RB phosphorylation, 43-43 Transgenes expression, cooperation w i t h chemical treatment, 266-288 HKl-ras, 2.53-258 synergism, 248 'I'ranslocation, and activation of protein kinase C, 162-165
Transplantation, neuronal, and PymT expression, 144 Tumor cells, FcR expression, 233-235 Tuniorigenesis and acquisition of oncogenic genetic lesions, 12 in HKI-ras mice, 255-257 kinetics, 249 mammary, PymT-mediated, 117-1 19 as multistep process, 153-154 role of E6 and E7, 15 PKC isoforms, 187- 196 and signal transduction pathway, 111 Tumor necrosis factor malignancy-associated continuous release, 236-237 secretion induced by FcyR cross-linking, 218 Tumor promoters, development of rapid screening system, 286-288 Tumors induction, and PKC isozyme overexpression, 194- 195 mammary, and elevated c-Src kinase, 117 mouse skin, role for Ha-ray, 252 pituitary, induction by Rb-I inactivation, 27 ~>ro~notiori, role of phorbol esters, 193194
Rb-I, mutation, 26-30 relationship with host, role of FcyR, 212 Tumor suppressor gene encoded negative control of cell proliferation, 6 identification, and transgenic models, 289 products, inactivation by E6 and E7, 18 'isrosine, phosphorylation, 2 19-220 Tyrosine kinase c-Abl. interaction with RB, 61-62, 76 Fyn, binding to HamT, 131-132 and intracellular signaling proteins, 8789 mitogenic signaling, 105 oncogenic activation, 89-90
305
INDEX
Src family, 112-1 17 association with transforming proteins of polyomaviruses, 132 protein activation, 115-1 17 regulation, 113-1 15 structure, 112-1 13
v Vector, epidermal targeting and chemical action, 286 development, 250-25 1
W U-937 cells, differentiation, role of PKC, 179 Ultraviolet light damage, induction of p53 activity, 12 generation of skin cancer mutations, 283
W-7, effect on RB phosphorylation, 47 Wart, genital, and HPV, 1-2 Wound associated papillomas, 255-258, 267 and p53, 285 role info5 mice, 263-264
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