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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
ACADEMIC PRESS
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San Diego New York Boston London Sydney Tokyo Toronto
This book is printed on acid-free paper.
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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.
Academic Press, Inc. A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495 (Jnired Kingdom Edition published b.v Academic Press Limited 24-28 Oval Road, London NWI 7DX
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 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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20
KAREN H. VOUSDEN
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,
(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
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 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.
-
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).
214
JANOS
GERGELY AND GABRIELLA SARMAY
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
FCY RECEPTORS IN MALIGNANCIES
215
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.
216
JANOS
GERGELY AND GABRIELLA
SARMAY
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).
FCY RECEPTORS IN MALIGNANCIES
217
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
218
JANOS GERGELY AND GABRIELLA SARMAY
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
FCY RECEPTORS I N MALIGNANCIES
219
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
220
JANOS CERGELY A N D CABRIELLA
SARMAY
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
FCY RECEPTORS IN MALIGNANCIES
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)
222
.jA~osGERGELY
AND GABRIELLA
SARMAY
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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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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