Advances in Cancer Research Volume 60

ADVANCES IN CANCER RESEARCH VOLUME 60

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ADVANCES IN CANCERRESEARCH Edited by

GEORGE F. V A N E WOUOE ABL-Basic Research Program NCI-Frederick Cancer Research and Development Center Frederick, Maryland

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

Volume 60

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

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0 1993 by ACADEMIC PRESS, INC.

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CONTENTS

CONTRIBUTORS TO VOLUME 60 ....................

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ix

Structural and Functional Diversity in the FGF Receptor Multigene Family DANIEL E . JOHNSON

AND

LEWIST. WILLIAMS

I . Introduction .................................................... I1. The FGF Family of Polypeptide Mitogens .......................... 111. Early Binding and Cross-Linking Studies of the FGF Receptor ...... IV. Nomenclature of the FGF Receptor Genes ......................... V. Purification. cDNA Cloning. and Characterization of FGFR 1 ....... VI . Characterization of EFGR 2. FGFR 3. and FGFR 4 ................. VII . Multiple Forms of FGFR 1 and FGFR 2 Are Generated by Alternative Splicing ........................................... VIII . Multiple Forms of FGFR 1 ....................................... IX . Multiple Forms of FGFR 2 .................................... X . FGFR 3 and FGFR 4 ...................... XI . Ligand Binding Specificities of the Cloned FGF Receptors . . . . . . . . . . XI1. Alternative Splicing in the Third Ig Domain Is Important for Determining Ligand Binding Specificities ...................... XI11. Analogous Splice Variants from Different FGF Receptor Genes Encode Receptor Forms with Different Ligand Binding Specificities ......... XIV. Regulation of FGF Receptor Expression ........................... xv. Cell- and Tissue-Specific Alternative Splicing of FGF Receptor mRNAs ........................................ XVI . Differential. Tissue-Specific Expression of the Different ' FGF Receptor Genes ......................................... XVII . The Drosophilu FGF Receptor ..................................... XVIII . FGF Receptor-Mediated Signal Transduction .......................

V

1 2 6

7 8 11

15 15 20 23 24 25 27 28 28 29 30 31

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CONTENTS

XIX. Concluding Remarks ............................................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

33 35

Protein Tyrosine Kinase Growth Factor Receptors and Their Ligands in Development, Differentiation, and Cancer

ANDREW F. WILKS ............... I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . 11. Structure of Receptor Protein Tyrosine Kinases . . . . . . . . . . . . . . . . . . . . 111. RTKs in Development . . . , . . . . . . . . . V. CHnetics and Can

43 45 49 60 65 67

The Molecular and Genetic Characterization of Human Soft Tissue Tumors COLINS. COOPER I.

........................ 11. Tumor Etiology . . . . 111. Genetic Susceptibility to Soft Tissue Tumors . . . . . . . . . . . . . , . . . . . , . . .

Detection of nu Gene Activation . . . . Chromosomal Abnormalities . . . . . . . Tumor Suppressor Cxnes . . . . . . . . . . Gene Amplification . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . , . . . . . . . . Predictors of Tumor Behavior . . . . . . . . . . . . , . 1X. Molecular Cloning of Translocation Breakpoints . . . . . . . . . . . . . . . . . . . X. Future Developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I v. V. VI. VII. VIII.

....................... .. . . . . . . .. . . . . . . . .. . . .

75 78 80 87 88 100 104

105 107 109 110 120

Genomic instability and Tumor Progression: Mechanistic Considerations

KEITH C. CHENGAND LAWRENCE A. LOEB I.

Introduction

....................................................

11. Genomic Stability and Instability: Background and Implications . . . . . 111. Mechanisms of Genomic Stability and Instability . . . . . . . . . . . . . . . . . . . IV. Quantitative Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

121 124 127 142

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CONTENTS

V. Summary and Perspectives ....................................... References ......................................................

145 148

Function and Regulation of the Human Multidrug Resistance Gene KHEW-VOON CHIN.IRAPASTAN.AND MICHAEL M . GOTTESMAN I . The Multidrug Transporter as a Cause of Multidrug Resistance ..... I1. Regulation of Expression of mdr Genes ............................ I11. Summary and Conclusions ....................................... References ......................................................

157 169 176 176

Relationship between MYC Oncogene Activation and MHC Class I Expression PETERI . SCHRIER AND LUCYT. C . PELTENBURG I . Introduction .................................................... I1 . MHC Class I Expression and Cancer .............................. I11. Modulation of MHC Class I Expression by Oncogenes ................................................... IV. Molecular Mechanism of MHC Class I Regulation by Oncogenes .... V. Biological Consequences of MHC Class I Downmodulation by Oncogenes ...................................................... VI . Concluding Remarks ............................................ References ......................................................

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Immunosuppressive Factors in Human Cancer

Dov SULITZEANU Introduction .................................................... Immunosuppression in Cancer-Recent Evidence .................. Immunosuppressive Factors Produced by Tumor Cells/Cell Lines . . . . Well-Characterized Immunosuppressive Molecules .................. Partially Characterized Immunosuppressive Factors ................. VI . Immunosuppressive Factors in Sera and Effusions .................. VII . Other Immunosuppressive Factors ................................ VIII . Concluding Remarks: Immunosuppressor or Growth-Regulatory Cytokines? .................................... References ...................................................... I. I1. 111. IV. V.

247 248 248 249 254 254 255 257 262

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CONTENTS

Lysosomes. Lysosomal Enzymes. and Cancer

MICHAELJ . BOYERAND IAN F. TANNOCK I . Introduction ............................ ........................ I1 . Methods for Studying Lysosomes . . . . . . . . . ........................ 111. Lysosomal Enzyme Activity in Tumors . . . . ........................ 1V. Lysosomes. Tumor Invasion. and Metastasis ....................... V. Lysosomes and Tumor Microenvironment . ........................ VI . Lysosomes and Anticancer Therapy . . . . . . . ........................ ........................ VII . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................ References ..............................

269 270 271 275 278 281 286 287

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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CONTRIBUTORS

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

MICHAEL J . BOYER,Departments of Medicine and Medical Biophysics, Ontario Cancer Institute, Toronto, Ontario M4X lK9, Canada (269) KEITHC. CHENC,'Department o f Pathology,Joseph Gottstein Memorial Cancer Research Laboratory, University of Washington, Seattle, Washington 981 95 (121) KHEW-VOON CHIN,* Laboratory o f Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 (157) COLINS. COOPER,Molecular Carcinogenesis Section, The Institute of Cancer Research, The Haddow Laboratories, Belmont, Sutton, Surrey S M 2 5NG, England (75) MICHAELM. GOTTESMAN, Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 ( 157) DANIEL E. JOHNSON, Howard Hughes Medical Institute, Program of Excellence in Molecular Biology, and Cardiovascular Research Institute, University of California, San Francisco, California 94143 (1) LAWRENCE A. LOEB,Department of Pathology,Joseph Gottstein Memorial Cancer Research Laboratory, University of Washington, Seattle, Washington 98195 (121) IRAPASTAN,Laboratory of Molecular Biology, National Cancer Institute, National Institutes o f Health, Bethesda, Maryland 20892 (157) LUCYT. C. PELTENBURG, Department of Clinical Oncology, University Hospital, 2300 RC Leiden, The Netherlands (181) 'Present address: Department of Pathology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania 17033. "resent address: Genetics Center, University of Texas Health Science Center, Houston, Texas 77225.

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CONTRIBUTORS

PETERI. SCHRIER, Department of Clinical Oncology, University Hospital, 2300 RC Leiden, The Netherlady (181) Dov SULITLEANU, The Lautenberg Center for General and Tumor Immunology, Hadassah Medical School, The Hebrew University, Jerusalem 91010, Israel (247) IAN F. TANNOCK, Departments of Medicine and Medical Biophysics, Ontario Cancer Institute, Toronto, Ontario M4X 1K9, Canada (269) ANDREWF. WiLus, Melbourne Tumor Biology Branch, Ludwig Institute for Cancer Research, Victoria 3050, Australia (43) LEWIST. WILLIAMS, Howard Hughes Medical Institute, Program of Excellence in Molecular Biology, and Cardiovascular Research Institute, University of California, San Franctsco, California 9414? ( I )

STRUCTURAL AND FUNCTIONAL DIVERSITY IN THE FGF RECEPTOR MULTIGENE FAMILY Daniel E. Johnson and Lewis T. Williams Howard Hughes Medical Institute, Program of Excellence in Molecular Biology, and Cardiovascular Research Institute, University of California, San Francisco, California 94143-0724

I. Introduction 11. The FGF Family of Polypeptide Mitogens

111. IV. V. VI. VII.

VIII.

IX.

X. XI. XII. XIII. XIV. XV.

XVI. XVII. XVIII. XIX.

Early Binding and Cross-Linking Studies of the FGF Receptor Nomenclature of the FGF Receptor Genes Purification, cDNA Cloning, and Characterization of FGFR 1 Characterization of FGFR 2, FGFR 3, and FGFR 4 Multiple Forms of FGFR 1 and FGFR 2 Are Generated by Alternative Splicing Multiple Forms of FGFR 1 A. Variations Involving Ig Domain I B. The Inclusion or Exclusion of Two Amino Acids in the Extracellular Domain C. Three Alternative Exons for the Second Half of Ig Domain 111 Multiple Forms of FGFR 2 A. Variations Involving Ig Domain I B. Variations Involving the Acid Box Domain C. Alternative Exons for the Second Half of Ig Domain 111 D. The Inclusion or Exclusion of Two Amino Acids in the Juxtamembrane Domain E. Three Alternative Exons for the C-Tail Domain FGFR 3 and FGFR 4 Ligand Binding Specificities of the Cloned FGF Receptors Alternative Splicing in the Third Ig Domain Is Important for Determining Ligand Binding Specificities Analogous Splice Variants from Different FGF Receptor Genes Encode Receptor Forms with Different Ligand Binding Specificities Regulation of FGF Receptor Expression Cell- and Tissue-Specific Alternative Splicing of FGF Receptor mRNAs A. Cell- and Tissue-Specific Alternative Splicing of the Third Ig Domain B. Tissue-Specific Alternative Splicing Involving Ig Domain I Differential, Tissue-Specific Expression of the Different FGF Receptor Genes The Drosophila FGF Receptor FGF Receptor-Mediated Signal Transduction Concluding Remarks References

1 ADVANCES IN CANCER RESEARCH, VOL. 60

Copyright 8 1993 by Academic Press. Inc. All rights of reproduction in any form reserved.

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1. introduction

The fibroblast growth factors (FGFs) constitute a family of closely related polypeptide mitogens (Burgess and Maciag, 1989; Folkman and Klagsbrun, 1987; Gospodarowicz et al., 1986b; Thomas, 1987). Currently, seven members of this family have been identified on the basis of amino acid sequence homologies. T h e FGF family has distinguished itself from other growth factor families by virtue of the pleiotropic actions of its members. In addition to their abilities to stimulate proliferation of a wide variety of cells, FGFs exhibit potent neurotrophic and angiogenic activities (R.S. Morrison et al., 1986; Walicke et al., 1986; Folkman and Klagsbrun, 1987; Anderson et al., 1988). FGFs also have the capacity to induce differentiation, inhibit differentiation, or maintain a differentiated phenotype of cells in culture (Linkhart et al., 1981; Serrero and Khoo, 1982; Broad and Ham, 1983; Lathrop et al., 1985; Togari et al., 1985; Wagner and DAmore, 1986; Anderson et al., 1988). Furthermore, a substantial body of evidence indicates that FGFs play important roles during development. The effects of FGFs are known to be mediated by high affinity receptor tyrosine kinases. T h e recent purification and cDNA cloning of FGF receptors have led to the discovery of a family of structurally related FGF receptor molecules. Within this family there is an enormous degree of complexity. Four distinct FGF receptor genes have been identified, and in the case of at least two of these genes, multiple mRNA transcripts are known to be generated by alternative splicing. A number of studies have now shown that the structurally diverse receptor molecules are also functionally different. Moreover, characterization of structural and functional diversity within the FGF receptor family is beginning to shed new light on differences in the mechanisms of action among members of the FGF familv.

II. The FGF Family of Polypeptide Mitogens The first members of the FGF family to be purified and characterized were acidic FGF (aFGF) and basic FGF (bFGF). Both factors were purified on the basis of their mitogenicity toward fibroblasts, using bovine pituitary (Armelin, 1973; Gospodarowicz et al., 1974; Gospodarowicz, 1975) and brain (Trowel1 et al., 1939; Hoffman, 1940; Gospodarowicz et al., 1978) as sources. In subsequent studies aFGF and bFGF were purified from a wide variety of sources including adrenal gland (Gospodarowicz et al., 1986a), bone (Hauschka et al., 1986), cartilage (Sullivan

FGF RECEPTOR MULTIGENE FAMILY

3

and Klagsbrun, 1985), corpus luteum (Gospodarowiczet al., 1985a), hypothalamus (Klagsbrun and Shing, 1985), kidney (Baird et al., 1985a), liver (Ueno et al., 1986), placenta (Gospodarowiczet al., 1985b), prostate (Nishi et al., 1985), retina (Baird et al., 1985b), testis (Ueno et al., 1987), and thymus (Gospodarowiczet al., 1986b).A significant improvement in the purification of aFGF and bFGF was made when it was discovered that both factors bind to heparin (Maciag et al., 1984; Shing et al., 1984). This led to the development of standardized purification protocols using heparin affinity chromatography (Shing et al., 1983; Lobb et al., 1986). The highly purified preparations of FGFs were subjected to amino acid sequencing, and this ultimately made possible the cloning of cDNAs for both aFGF (Gimenez-Gallegoet al., 1985; Thomas et al., 1985;Jaye et al., 1986) and bFGF (Esch et al., 1985; Abraham et al., 1986a,b; Kurokawa et al., 1987). The cloned cDNAs for aFGF and bFGF each encode proteins of 155 amino acids. Curiously, the predicted amino acid sequences of both factors do not contain signal peptide sequences. In this respect, aFGF and bFGF resemble the interleukin-1 (IL1) proteins, which also lack signal peptide sequences. Despite considerable attention to this issue, it remains unclear how growth factors without signal peptides are secreted from the cell. The predicted sequences for human aFGF and human bFGF are 55% identical at the amino acid level. Both proteins are also highly conserved across species (Thomas, 1987). Human and bovine aFGF differ by only 12 amino acids, and human and bovine bFGF differ by only 2 amino acids. The proteins also share limited homology to IL1-a and IL1-p (25-27% amino acid identity). The cDNAs encoding aFGF and bFGF are derived from distinct, single-copy genes. The human gene encoding aFGF is located on chromosome 5 Uaye et al., 1986),whereas the human bFGF gene is located on chromosome 4 (Mergia et al., 1986).Despite their different chromosomal loci, the FGF genes have a similar structural organization. Both genes are composed of three exons, and contain two large introns at similar locations (Mergia et al., 1986; Abraham et al., 198613). Biological studies using purified or recombinant aFGF and bFGF have shown that both factors are potent mitogens for a wide variety of cells of mesenchymal and neuroectodermal origin (Burgess and Maciag, 1989; Gospodarowicz et al., 1986b). Of particular interest has been the discovery that both factors act as mitogens and chemoattractants for endothelial cells in uitro (Burgess and Maciag, 1989), and exhibit potent angiogenic activity in uzuo (Folkman and Klagsbrun, 1987).Hence, FGFs may play an important role in the normal development of the vascular

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DANIEL E. JOHNSON A N D LEWIS T. WILLIAMS

system through their action on endothelial cells. At the same time, however, aberrant production of FGFs or other aberrations in FGF response pathways may contribute to pathological conditions that result from either too much or too little vascularization. Thus, it is important to consider the potential involvement of FGFs in the vascularization of tumors, in wound healing, and in vascular diseases such as diabetic retinopathy. FGFs also stimulate cellular production of collagenases and plasminogen activator (Gross et al., 1982, 1983; Presta et al., 1985; Moscatelli et al., 1986; Mignatti et al., 1989), and this could help potentiate neovascularization or tumor invasiveness (Folkman and Klagsbrun, 1987). FGFs may also lead to cellular transformation through autocrine or paracrine mechanisms. Several groups have demonstrated that constitutive expression of exogenous FGF in transfected cells promotes growth in serum-free media and soft agar, and is tumorgenic in mice. The results that are obtained in these experiments, however, vary with the host cell line that is used. With some cell lines, certain parameters of transformation are highly dependent on the secretion of FGF Uaye et al., 1988; Rogelj et al., 1988; Sasada et al., 1988),whereas with other cell lines transformation is independent of FGF secretion (Neufeld et al., 1988; Jaye et al., 1988). In addition to their ability to promote cellular proliferation, FGFs also influence the differentiation of a variety of cell types. FGFs induce the differentiation of preadipocyte fibroblasts into adipocytes (Serrero and Khoo, 1982; Broad and Ham, 1983) and stimulate neurite outgrowth from hippocampal neurons (Walicke et at., 1986), cerebral cortical neurons (R. S. Morrison et al., 1986), and rat PC12 cells (Togari et al., 1985; Wagner and D’Amore, 1986). While both factors can act to induce a differentiated phenotype, they can also act to inhibit differentiation. This is the case in skeletal muscle myoblasts, where addition of FGFs acts to inhibit differentiation to myotubes (Linkhart et al., 1981; Lathrop et al., 1985).FGFs also support the survival of lesioned cholinergic neurons in vivo (Anderson et al., 1988), indicating a role in the maintenance of differentiated cells. The ability of FGFs to influence the differentiation of a variety of cell types suggests that these factors may play important roles during development. This idea is supported by experiments showing that addition of‘ FGFs to Xen@ embryos leads to mesoderm induction (Kimelman and Kirschner, 1987; Slack et al., 1987). The presence of FGF in early Xen0fni.s embryos indicates that FGFs may serve in this capacity during normal embryogenesis (Kimelman et al., 1988). Indeed, recent experiments have shown that selective disruption of FGF receptor-mediated

FGF RECEPTOR MULTIGENE FAMILY

5

signaling pathways in developing Xenopus embryos leads to dramatic inhibition of mesoderm formation and developmental defects in gastrulation and posterior development (Amaya et al., 1991). Over the last several years, five additional members of the FGF family have been identified on the basis of amino acid sequence homologies, These proteins are approximately 35 to 45% identical with aFGF and bFGF and include the product of the int-2 oncogene (Moore et al., 1986), the product of the hst oncogene (Kaposi sarcoma FGF) (Taira et al., 1987; Bovi et al., 1987), FGF-5 (Zhan et al., 1988), FGF-6 (Marks et al., 1989), and keratinocyte growth factor (KGF; Finch et al., 1989; Rubin et al., 1989). In addition to sequence homologies, these proteins also share some physical and biological properties with aFGF and bFGF, such as the ability to bind heparin and the ability to stimulate proliferation of a variety of cells of mesenchymal and neuroectodermal origin (Burgess and Maciag, 1989). In contrast to aFGF and bFGF, however, int-2 hstlKFGF, FGF-5, FGF-6, and KGF contain signal peptide sequences encoded by their mRNA transcripts. The int-2 oncogene was originally identified as a preferred site of integration for mouse mammary tumor virus (Peters et al., 1983). Mice containing viral integration near the int-2 gene frequently display transcriptional activation of the int-2 oncogene and develop mammary carcinomas (Peters et al., 1983, Dickson et al., 1984; Moore et al., 1986).The hst oncogene was identified in a focus forming assay following transfection of 3T3 cells with genomic DNA from a human stomach cancer cell line (Taira et al., 1987). The same gene was identified when DNA from Kaposi sarcoma cells was used to transfect cells (Bovi et al., 1987).We will refer to this gene as the hstlKFGF gene. FGF-5 was identified as a human gene whose fortuitous rearrangement during DNA transfection led to focus formation in 3T3 cells (Zhan et al., 1988). cDNAs for FGF-6 were isolated on the basis of nucleic acid hybridization with a hstlKFGF probe (Marks et al., 1989). Keratinocyte growth factor was cloned using amino acid sequence data obtained from the purified protein (Finch et al., 1989; Rubin et al., 1989). The FGFs are known to exert their effects by binding to high affinity receptors on the surface of responsive cell types. The existence of multiple members of the FGF family has raised the question whether all members of this family bind to a common receptor molecule. Alternatively, there could be multiple FGF receptors, each possessing different ligand binding properties. Recent advances in the FGF receptor field have shown that both of these possibilities are partially correct.

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DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

Ill. Early Binding and Cross-Linking Studies of the FGF Receptor Early characterization of FGF receptors focused on binding and cross-linking studies using iodinated aFGF or bFGF. These studies revealed that a variety of cells express saturable high affinity receptors with Kds of 50-500 pM and 10-200 pM for aFGF (Schreiber et al., 1985; Baird et al., 1986; Libermann el al., 1987) and bFGF (Neufeld and Gospodarowicz, 1985; Moenner et al., 1986; Olwin and Hauschka, 1986; Moscatelli, 1987), respectively. Competition analyses demonstrated that either ligand was able to compete for high affinity binding of the other ligand (Neufeld and Gospodarowicz, 1986; Olwin and Hauschka, 1986). This provided the first indication that aFGF and bFGF might share a common receptor. A related study has also shown that hstlKFGF causes downregulation of high affinity binding sites for bFGF, indicating that hstIKFGF also binds to the same receptor (Moscatelli and Quarto, 1989). When either aFGF or bFGF was used in cross-linking experiments, receptor species in the range 125-165 kDa were detected on SDS-polyacrylamide gels (G. Neufeld and Gospodarowicz, 1985; G. Neufeld and Gospodarowicz, 1986; Friesel et al., 1986; Moenner et al., 1986; Olwin and Hauschka, 1986, 1989; Libermann et al., 1987; Courty et al., 1988). Frequently two prominent cross-linked bands of 125 and 145 kDa were seen. Initially it was thought that the 125-kDa protein represented a proteolytic cleavage product of the 145-kDa protein. Recent evidence, however, indicates that the two proteins are derived from different alternatively spliced forms of the FGF receptor mRNA (see discussion in Section VII1,A). As expected, either ligand was able to block the crosslinking of the same or different radiolabeled ligand to both the 125-kDa and the 145-kDa proteins (Neufeld and Gospodarowicz, 1986; Olwin and Hauschka, 1986). In summary, the results of binding and crosslinking studies strongly supported the hypothesis that different members of the FGF family shared a common receptor. The idea of a common receptor for all members of the FGF family did not fit, however, with data obtained for KGF. This growth factor, although potently mitogenic for epithelial cells, failed to stimulate the growth of endothelial cells and fibroblasts, cells that are responsive to both aFGF and bFGF (Rubin et al., 1989). Thus, KGF did not appear capable of binding to the receptors for aFGF and bFGF that are present on these cells (Bottaro et al., 1990). As will be described in further sections, the KGF receptor represents a unique splice variant of an FGF receptor gene. Before the discussion on the binding of FGFs to cell surface receptors

7

FGF RECEPTOR MULTIGENE FAMILY

is concluded, it should be noted that cells also express a large number of low affinity binding sites for FGFs (Neufeld and Gospodarowicz, 1985; Moenner et al., 1986; Olwin and Hauschka, 1986; Clegg et al., 1987; Moscatelli, 1987). Considerable evidence indicates that the low affinity sites represent heparan sulfate proteoglycan molecules located on the cell surface or in the extracellular matrix (Moscatelli, 1987, 1988; Bashkin et al., 1989). Binding to low affinity sites occurs with a Kd of 2 to 10 nM and can be removed by incubation with heparin, washing with 2.0 M NaC1, or treatment with heparinase (Moscatelli, 1987). Recent evidence indicates that the binding of FGFs to low affinity sites plays a role in potentiating binding of FGFs to high affinity receptors (Rapraeger et al., 1991; Yayon et al., 1991). This information has recently been reviewed elsewhere (Klagsbrun and Baird, 1991)and will not be discussed further. Instead, this review will focus on the high affinity cell surface FGF receptors. IV. Nomenclature of the FGF Receptor Genes

Since the isolation of the first complete FGF receptor cDNA in 1989 (Lee et al., 1989),our understanding of the complexity of the FGF receptor field has increased dramatically. To date, four distinct FGF receptor genes have been identified. Furthermore, in the case of at least two of these genes it is clear that alternative splicing gives rise to multiple forms of the receptor. The different FGF receptor genes and splice variants of these genes are described in the literature using many different names (see Table I and Fig. 5). In an effort to simplify our understanding of TABLE I NOMENCLATURE OF THE DIFFERENT FGF RECEPTORGENES FGFR 1

FGFR 2

FGFR 3

FGFR 4

"g bFGFR Cekl N-bFGFR h2, h3, h4, h5 FGFR 1

bek Cek3 K-Sam K-Sam K-Sam' TK14 TK25 KGFR FGFR 2

Cek2 FGFR 3

FGFR 4

Note. The table shows some of the names that have been used to describe the different FGF receptor genes and cDNAs derived from alternatively spliced FGF receptor mRNA transcripts. The publications that were used to compile this list are referenced in the text.

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DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

FGF receptor diversity, we will refer to the different FGFR genes as FGFR 1, FGFR 2, FGFR 3, and FGFR 4, in the chronological order in which they were first identified and characterized. V. Purification, cDNA Cloning, and Characterization of FGFR 1

The first FGF receptor (FGFR 1) to be characterized in detail was purified from chicken embryos. In the purification scheme that was used, extracts from chicken embryos were incubated with biotinylated FGF in the presence of heparin (Lee et al., 1989). Biotin-FGF/FGFR complexes were then purified on an avidin-agarose column. Different ligand-affinity purification schemes have been employed by other laboratories (Imamura et al., 1988; Burrus and Olwin, 1989). The inclusion of heparin in the original purification protocol appears to have been critically important for purifying the high affinity receptor, as other approaches that have not included heparin have resulted in the isolation of low affinity binding proteins (Kiefer et al., 1990). The amino acid sequence obtained from peptides of the purified chicken FGF receptor showed striking similarity with the predicted amino acid sequences of two previously published partial cDNA clones: humanflg (Ruta et al., 1988)and mouse bek (Kornbluth et al., 1988).The human flg (fm-like gene) cDNA had been isolated from an endothelial cell cDNA by virtue of it hybridization with a c-fm probe. The region of cross-hybridization of this probe is presumably in the coding sequence for the tyrosine kinase domain of flg; otherwise JEg and c$m are not homologous. The mouse bek (bacterially expressed kinase) cDNA had been isolated from a cDNA expression library by probing with antiphosphotyrosine antibodies. Although the function of the flg and bek proteins was unknown at the time of the discovery of their partial cDNAs, it has since come to light that the full-length fig and bek cDNA clones represent specific splice variants of the FGFR 1 and FGFR 2 genes, respectively (see Sections V I I I and IX, and Fig. 5). A cDNA encoding the chicken FGFR 1 was isolated using oligonucleotide probes based on the amino acid sequence from the purified protein (Lee et al., 1989). The cDNA encoded a protein with a deduced molecular mass of 92 kDa (not including carbohydrate side chains) that contained several features commonly found in growth factor receptors (see Fig. 1). The protein contained a single membranespanning region, an amino-terminal signal peptide, and three extracellular immunoglobulin-like (Ig-like) domains (Williams and Barclay, 1988). Between the first (I) and the second (11) Ig-like domain, the

FGF RECEPTOR MULTIGENE FAMILY

9

FIG. 1. Schematic diagram of the chicken FGFR 1 structure. The following structural features are identified: hydrophobic leader sequence (striped box), three extracellular Iglike domains (labeled I, 11, and 111), acid box domain (open box), transmembrane domain (solid box), kinase 1 and kinase 2 domains (stippled boxes).

receptor contained a unique domain that has not been seen in other growth factor receptors. This domain consists of eight consecutive acidic residues and is referred to as the “acid box.” The intracellular domain of the FGFR 1 protein contained consensus tyrosine kinase sequences. This confirmed earlier biochemical evidence which indicated that the receptor was a tyrosine-specific protein kinase (Huang and Huang, 1986; Coughlin et al., 1988).The tyrosine kinase sequence of FGFR 1 is split by an insertion of 14 amino acids. The length of this kinase insert region is considerably shorter than those of the PDGF-P (Yarden et al., 1986) and CSF-1 (Coussens et al., 1986) receptors (104 and 70 amino acids, respectively), but comparable to those of the insulin and insulin-like growth factor-1 receptors (Ullrich et al., 1985, 1986). Another interesting feature of the chicken FGFR 1 protein is the length of the juxtamembrane domain. This domain consists of the region between the transmembrane domain and the first kinase domain (kinase 1). The FGFR 1juxtamembrane domain is 79 amino acids long, compared with juxtamembrane domains of 49 to 5 1 amino acids for the PDGF-f3 (Yarden et al., 1986),CSF-1 (Coussens et al., 1986),EGF (Ullrich et al., 1984), HER1 (Coussens et al., 1985), and insulin (Ullrich et al., 1985) receptors. Complementary DNA clones encoding similar FGFR 1 forms have subsequently been isolated from a variety of species including human (Dionne et a,!., 1990; Johnson et al., 1990; Eisemann et al., 1991; Hou et al., 1991), mouse (Mansukhani et al., 1990; Reid et al., 1990; Werner et al., 1992a),chicken (Pasquale and Singer, 1989),and Xenopus (Musci et al., 1990). The degree of amino acid identity between FGFR 1 proteins from different species is striking (see Fig. 2). Overall, when compared to the human FGFR 1 protein, the mouse, chicken, and Xenopls FGFR 1 proteins are 98, 91, and 78% identical, respectively. The most highly conserved regions of the receptor molecule are the kinase 1 and kinase 2 domains (92 and 95% identity, respectively, between human and Xenopus). The least conserved regions are the signal peptide region (50%, human to Xenopw), Ig domain I (54%), the membrane-proximal

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DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

HUMAN

vs

MOUSE

HUMAN

vs

HUMAN

vs

CHICKEN XENOPUS

Signal peptide

93 %

70

Yo

50 Yo

lg domain I

93 Yo

80 Yo

54 Yo

97

Oh

83 %

73 Yo

100 Yo

95 %

79 Yo

97 Yo

98 Yo

79 Yo

Membraneproximal TM

100 % 90 %

75 Yo 86 %

44 Yo

JM

100 Yo

92 Yo

81 Yo

Kinase 1

100 Yo

99 Yo

92 Yo

93

Oh

71 Yo

50 Yo

99 %

99 Yo

95 Yo

95 Yo

80 %

75 Yo

98 %

91 Yo

78 Yo

(119)

Acid box

(149)

Ig domain I1 (248)

Ig domain 111

Kinase insert Kinase 2

C-tail

57 Oh

FIG.2. Comparison of FGFR 1 proteins from different species. The figure shows the degree of amino acid identity between different domains of the human FGFR 1 protein (Dionne et al., 1990) and the corresponding domains of FGFR 1 proteins from mouse (Reid ~t al., 1990), chicken (Lee et al., 1989). and Xenopvs (Musci et al., 1990). All of the proteins compared represent 3 Ig domain receptor forms containing IIIc-type sequences in the third Ig domain. The numbers in parentheses indicate the last amino acid of each domain

FGF RECEPTOR MULTIGENE FAMILY

11

domain (44%), the transmembrane domain (57%),and the kinase insert domain (50%).

VI. Characterization of FGFR 2, FGFR 3, and FGFR 4 Following the isolation of the chicken FGFR 1 cDNA, cDNAs derived from three additional FGFR genes (2,3,and 4) were isolated and characterized by several laboratories. The proteins encoded by these genes are structurally similar to the FGFR 1 protein and are highly conserved at the amino acid level. Comparison of the mouse FGFR 1 sequence and the partial sequence of mouse bek revealed several differences at the amino acid and nucleotide levels. The isolation of full-length bek clones led to the discovery that bek was in fact the product of a different gene (Dionne et al., 1990). We will refer to this gene as FGFR 2 for the remainder of this review. Additional FGFR 2 cDNAs have been isolated as cDNAs hybridizing with amplified DNA fragments from a human stomach cancer cell line (Hattori et al., 1990). Also, cDNA clones encoding the receptor for keratinocyte growth factor, a unique splice variant of FGFR 2 (see further sections), were isolated using a novel expression cloning strategy (Miki et al., 1991). Even more FGFR 2 cDNA clones have been isolated using PCR or library screening (Saiki et al., 1988; Pasquale, 1990; Houssaint et al., 1990; Champion-Arnaud et al., 1991; Crumley et al., 1991; Raz et al., 1991; Sat0 et al., 1991; Dell and Williams, 1992). Complementary DNA clones derived from a third FGFR gene (FGFR 3) were obtained by low stringency screening of a human K-562 (chronic myelogenous leukemia cell line) cDNA library with a v-sea oncogene probe (Keegan et al., 1991). Human cDNA clones derived from a fourth

in the human FGFR 1 protein (numbering based on Fig. 3). The signal peptide region contains signal peptide sequences as well as sequences between the signal peptide and the beginning of Ig domain I. The boundaries of Ig domain I, the acid box domain, and Ig domains I1 and 111 were defined by intron/exon junctions (see Fig. 6 and Johnson et al., 1991). The boundaries of the membrane-proximal domain were defined by the introdexon junction at the end of Ig domain 111 (N-terminus) and the beginning of the transmembrane domain (C-terminus). The boundaries of the juxtamembrane domain were defined by the end of the transmembrane domain (N-terminus) and the beginning of the kinase 1 domain (C-terminus). The boundaries of the kinase 1, kinase insert, and kinase 2 domains were defined by comparison with consensus kinase sequences as reported by Hanks et al. (1988). Sequences following the end of the kinase 2 domain constitute the C-tail domain.

FGFRl MWSWKCLLFWAVLVTAT--LCTARPSPTLPEQAQ------PWGAPVEVESFLVH-PGDLL FGFRZ .V..GRFICLV.VTM..--.SL....FS.V.DTTLEPEEP.TKYQISQPEVY.AA..ES. FGFR3 .GRPA.A.ALC.A.AIVAGASSESLGTEQRWGRAAEVPG.EPGQQ.Q---..FGS..AV FGFR4 .RLLLA..GVLLS.PGPPV.SLEASEEVEL.PCLA-----.SLEQQEQELT-VA-L.QPV

51 58 57 53

FGFRl QLRCRLRDDV--QSINWLRDGVQLAESNRTRITGEEVEVQDSVPADSGLYACVTSSPSGS FGFR2 EV . . L.K .AA--- V.S.TK ...H.GPN ...VLI ..YLQ1KGAT.R....... TA.RTVD. FGFR3 E.S.PPPGGGPMGP TV.VX.. TG .VP. E.VLVGPQRLP.LNASHE...A.S.RQRLTQRV FGFR4 R.-.CG.AERG---GH.YKE.SR..PAG.V.GWRGRL.IASFL.E.A.R.L.LARGSMIV

109 115 117 109

FGFRl FGFR2 FGFR3 FGFR4

DTTYFSVNVSDALPSSEDDDDDDDSSSEE-KETDNTKPNRMPVAPYWTSPEKMEKKLHAV E.W ..M ...T..I-..G..E..T.GAE.FVS.NS.NS.N.R-----.....NT.....R.... LC-H ...R.T ..-...G..E.GE.EAEDTGVD.G--------- .....R..R.D ...L.. LQ-NLTL1TG.S.T ..N ..E.PK-.HRDPSNRHSYPQQ----- .....H.QR

........

168 169 166 162

FGFRl PAAKTVKFKCPSSGTPNPTLRWLKNGKEFKPDHRIGGYKVRYATWSIIMDSWPSDKGNY 228 F G F R ~ ...N ....R..AG.N.M..M..........QE.........NQH..L..E.......... 229 FGFR3 . . .N ..R . R . . A A . N.T.SIS .....R..RGE.....I.L.HQQ..LV.E......R... 226 FGFR4 . .GN ....R . . A A . N.T ..I....D.QA.HGEN....IRL.HQH..LV.E......R. T. 222 FGFRl FGFRZ FGFR3 FGFR4

.....

288 289 206 282

FGFRl WLKHIEVNGSKIGPDNLPWQILKTAGVNTTDKEMEVLHLRSFEDAGEYTCLAGNSIG FGFRZ .I..V.K .... Y...G...LKV..A. ........ I...YI...T................ FGFR3 .... V . . . . . . V...GT...TV.....A ...... L...S.H..T................ FGFR4 .....VI ... SF.AVGF....V. ...DI.SS--.V...Y.....A.... ...........

348 349 346 340

FGFRl FGFR2 FGFR3 FGFR4

LSHHSAWLTVLEALEER-PAVMTSPLYLEIIIYCTGAFLISCMVGSVIVYKMKSGTKKSD

1.F ........ P.PGRE-KEITA..D ....A...I.V...A...VT..LCR..NT...P. F . . . . . . . V..P.E..LVE.DEAGSV.AG.LS.GV.F..FILV.AA.TLCRLR.PP.. GL . .YQ .......PEEDPTWT.AAPEAR.TD..L.AS.SLALAVLLLLAGL.RGQALHGRHP

407 408 406 400

FGFRl FGFW FGFR3 FGFR4

FHSQMAVHKLAKSIPLRRQIPPVSADSSASMNSGVLLVR-PSRLSS-SGTPMLAGVSEYEL .S . .P.....T.R...........E..S....NTP...ITT....TAD............ --GSPT IS-RF.. K...SLESNA.--.S.NTP...I-A....-GEG.T..N...L.. -RPPAT.Q..S-RF..A..FSLESG..G--K.SSS...-GV....-..PAL...LVSLD.

465 468 459 454

FGFRl FGFR2 FGFR3 FGFR4

PEDPRWELPRDRLVLGKPLGEGCFGOVVLAEAIGLDKDKPL

525 528 519 514

TCIVENEYGSINHTYQLDVVERSPHRPILQAGLPANKTVALGSNVEFMCKVYSDPQPHIQ . .V H....................ASTW.GD...V......A..... V ...KF RQ T. ..L................Q.AV...D...H......A..... L...AV...RYN.L. ..L...... T . A W D..LL...... A

............ .. ... .. ..

..........

..

...

....K ..F...K.T..............M...V.I.....KEAVT.......D.......

.A..K...S.A..T..............M.....I...RAAKPVT.......D...D... .L..L..F....................R...F.M.PAR.DQAST.......DN.SD...

FIG.3. The amino acid sequences of four human FGF receptor proteins derived from distinct receptor genes. The amino acid sequences of the human FCFR 2 protein (Dionne ~t al., 1990), the human FGFR 3 protein (Keegan el al., 1991), and the human FGFR 4 protein (Partanen et al., 1991) are shown in comparison to the sequence of the human FGFR 1 protein (Dionne el al., 1990). Each sequence represents a 3 Ig domain receptor form containing IIIc t y p e sequences in the third Ig domain. For FGFRs 2, 3, and 4, only sequences which differ from the FGFR 1 sequence are shown. Dashed lines indicate gaps that have been introduced into the sequence.

13

FGF RECEPTOR MULTIGENE FAMILY

FGFRl SDLISEMEMMKMIGKHKNIINLLGACTQDGPLWIVEYASKGNLREYLQAPGLEYCY FGFR2 V... R ......M.. S. FGFR3 ...V G.....L....A......F.R....... D.SF FGFR4 A..V....V ..L..R .........V ...E........C.A......F.R...... PDLSP

...

......................................... ........................

585 588 579 574

FGFRl FGFR2 FGFR3 FGFR4

NPSHNPEEQLSSKDLVSCAYQVARGUEYLASKKCIHRDLAAFUWLVTEDNVMKIADFGLA 645 648 DINRV ....MTF ......T ..L.........Q................N........... DTCKP TF.. Q. 639 DGPRSS.GP ..FPV Q E.R................ ............ 634

FGFRl FGFR2 FGFR3 FGFR4

RDIHHIDYYKKTTNGRLPVKWMAPEALFDRIYTHQSDVWSFGVLLWEIFTLGGSPYPGVP V M............. I. V.NL V................. ..........I. .GV S.................V...........I............... I.

.....

................. ........................... ............ ..

............................. .. ........................ .........

.............

705 708 699 694

FGFRl VEELFKLLKEGHRMDKPSNCTNELYMMMRDCWHAVPSQ~TFKQLVEDLDRIVALTSNQE 765 LT..T. E. 768 FGFR2 .................A HD. ..I..E. ...A................VLTV..T D. 759 FGFR3 .................A E. 753 FGFR4 .....S ..R......R.PH.PP...GL..E....A............A..KVL-.AVS

.................................. ...

FGFRl FGFR2 FGFR3 FGFR4

YLDLSMPLDQYSPSFPDTRSSTCSSGEDSVFSHEPLPEEPCLPRHPAQLANGGLKRR .....Q ..E.....Y....-.S....D.....PD.M.Y.....QY.HI--.. S-VKT .....A.FE ....GGQ..P-.SS...D....AHDL..----- .AP.SS---. .S-.T ....RLTFGP ....GG.AS-.....S- ......D...--LGSSSF.F----.SGVQT

822 821 806 802

FIG.3. (cont.)

FGFR gene (FGFR 4)were isolated using PCR with tyrosine kinase specific primers followed by library screening with the amplified fragments (Partanen et al., 1991). The amino acid sequences of 3 Ig domain forms of human FGFR 2, FGFR 3, and FGFR 4 proteins in comparison to the human FGFR 1 protein are shown in Fig. 3. Table I1 shows the overall level of amino TABLE I1 COMPARISON OF THE DIFFERENT HUMANFGF RECEPTORGENES FGFR 1 FGFR 2 FGFR 3

FGFR 2 72%

FGFR 3 62% 66%

FGFR 4 55% 57% 61%

Note. The table shows the overall degree of amino acid identity between the four amino acid sequences shown in Fig. 3. All of the proteins compared represent 3 Ig domain receptor forms containing lIIc type sequences in the third Ig domain. Percentage identity was calculated by dividing the number of identities by the number of amino acids in the larger of the two receptor proteins being compared.

14

DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

FGFR 1

FGFR 1

FGFR 1

FGFR 2

FGFR 3

FGFR 4

43 Yo

17 Yo

20 %

40

Yo

27 Yo

19 Yo

43

Yo

33 Yo

27

79 Yo

64 %

61 Yo

78 Oh

81 %

74

Yo

38 62

31 Yo 33 Yo

13 24

Yo Yo

76 Yo

46

39 Yo

88%

83 Yo

75 Yo

50 Yo

43 Yo

7 Yo

62 Yo

46 %

42 Yo

vs

Signal peptide

q-

(30)

Ig domain I

vs

vs

(119)

Acid box

(149)

Ig domain I1

Yo

(248)

Ig domain I11 Membrane-

-.=

lPiloxima' JM

Kinase 1 Kinase insert

C-tail

I

(360) (376) (397)

. _..: ...... ........ ...... .__._. ......... ;:.:. ..... ........ :.:...... ...... ........ .:::._

(476)

...... ..... .._._. ..... ..... _..: ......

(580)

.... ........

(594)

...... .:....... ....... ..... .,I._ .: ... ......

FIG.4. Comparison of human FGF receptor proteins derived from four different receptor genes. The figure shows the degree of amino acid identity between different domains of the human FGFR 1 protein (Dionne et nl., 1990) and the corresponding domains of human FGFR 2 (Dionne et al., 1990). human FGFR 3 (Keegan et al., 1991), and humall FGFR 4 (Partanen et al., 1991). All of the proteins compared represent 3 Ig domain receptor forms containing 1IIc-type sequences in the third Ig domain. The numbers in parentheses indicate the last amino acid of each domain in the human FGFR 1 protein (numbering based on Fig. 3). Domain boundaries were defined as described in the legend tor Fig. 2.

FCF RECEPTOR MULTIGENE FAMILY

15

acid identity between the different human FGFR gene products, and Fig. 4 shows a domain by domain comparison of all four human receptor proteins. Overall, the proteins encoded by the four different human genes are strikingly similar. The most closely related proteins are FGFR 1 and FGFR 2 (72% amino acid identity), whereas FGFR 1 and FGFR 4 are the least closely related (55% identity). It is interesting to note that these levels of identity are considerably higher than those observed among different members of the FGF family (35 to 55%). A comparison of the domains of the different human FGF receptors (Fig. 4) reveals a pattern of conservation similar to that observed when comparing FGFR 1 domains across species (Fig. 2). The most highly conserved regions of the different human proteins are the kinase 1 and kinase 2 domains (75 and 84% identity, respectively, between FGFR 1 and FGFR 4). The least conserved regions are the signal peptide region (20%), Ig domain I (19%), the membrane-proximal domain (13%), the transmembrane domain (24%),and the kinase insert domain (7%).

VII. Multiple Forms of FGFR 1 and FGFR 2 Are Generated by Alternative Splicing A striking discovery in the FGF receptor field has been the isolation by several groups of multiple, distinct cDNAs encoding variant forms of FGFR 1 and FGFR 2 (Dionne et al., 1990;Johnson et al., 1990; Reid et al., 1990; Champion-Arnaud et al., 1991; Eisemann et al., 1991; Hou et al., 1991; Miki et al., 1991). Evidence obtained from studies of the organization of the FGFR 1 and FGFR 2 genes indicates that alternative splicing of mRNA is responsible for generating the diverse receptor forms (Champion-Arnaud et al., 1991; Johnson et al., 1991). Although alternative splicing is not unprecedented in the growth factor receptor field, the sheer number of alternative FGF receptor forms far outweighs that seen for any other growth factor receptor. Figure 5 shows a schematic diagram of the different FGF receptor proteins encoded by FGF receptor cDNAs that have been isolated to date. As shown in the figure, alternative splicing results in either (a) the inclusionlexclusion of additional amino acids or (b) the use of alternate coding exons with no net gain or loss of amino acids. In either case, the resultant proteins are structurally different. In subsequent sections of this review, the different splice variants and the potential function of these variants are discussed. VIII. Multiple Forms of FGFR 1 This section discusses the various regions of diversity that have been observed for FGFR 1, beginning at the amino terminus and proceeding

FGFR 1 1

I

h4

*

FGFR 2 1

iQbFGFR

I K-sarn’

KGFR

K-sarn

~

BEWTKl4

FGFR 3 BEK

TK25

I

FGFR 4

nn

(Vlll)

(xtii)

FIG. 5. Schematic diagram of FGF receptor protein structures. The figure shows the structure of variant receptor forms predicted by published cDNAs. T h e names of some receptor variants as they appear in the literature are written directly above the structure. Although cDNAs encoding the receptor variant depicted by an asterisk (iii) have not been isolated, PCR and Northern blotting experiments have identified mRNA transcripts encoding this receptor form (Johnsonet al., 199 1 ; Werner et al., 1992a). Furthermore, both 3 Ig and 2 Ig domain forms of this receptor mRNA appear to exist. T h e following structural features are identified in the figure: the 32 unique amino acids at the C-terminus of the FGFR 1 Ig domain I secreted form (solid oval), acid box domains (open boxes), alternative sequences for the second half of Ig domain 111 labeled IIIa, IIIb, or IIIc (thick black line), transmembrane domains (solid boxes), kinase 1 and kinase 2 domains (stippled boxes), and the unique C-tail domains of 2 FGFR 2 proteins (checkered box and striped box). The following sources were used to compile this figure: (i, Eisemann et al., 1991),(ii,Johnson et al., 1990), (iv, Lee et al., 1989; Pasquale and Singer, 1989), (v, Johnson e l al., 1990; Mansukhani et al., 1990; Reid el al., 1990). (vi, Champion-Arnaud et al., 1991). (vii, Miki el al., 1991), (viii, Hattori et al., 1990), (ix, Dionne et al., 1990; Houssaint et al., 1990), (x, Champion-Arnaud et al., 1991), (xi, Champion-Arnaud et al., 1991), (xii, Keegan et al., 1991), and (xiii, Partanen et al., 1991). It is predicted that more structures will be added to this figure in the coming years.

FGF RECEPTOR MULTIGENE FAMILY

17

toward the carboxyl terminus. Also, reference will be made to published information regarding the organization of exons and introns in the human FGFR 1 gene (Johnson et al., 1991). A. VARIATIONS INVOLVING IG DOMAIN I The first region of FGFR 1 receptor diversity that was observed was in the first Ig-like domain (I). Although the first reported FGFR 1 cDNA encoded a protein with three Ig domains (Fig. 5, iv), several laboratories have subsequently reported the isolation of cDNAs encoding FGFR 1 proteins that are missing Ig domain I (Fig. 5, ii and v) (Johnson et al., 1990; Mansukhani et al., 1990; Reid et al., 1990).Analysis of the FGFR 1 gene revealed the presence of an intron separating Ig domain I from the remainder of the FGFR 1 coding sequence (Fig. 6) (Johnson et al., 1991). Thus it appears likely that the presence or absence of Ig domain I is mediated by alternative splicing. Binding studies have shown that the 3 Ig domain form of FGFR 1 has affinities of 20-80 pM and 50- 150 pM for aFGF and bFGF, respectively (Dionne et al., 1990; Johnson et al., 1990). Similarly, the 2 Ig domain form of FGFR 1 has affinities of 50 and 100 pM for aFGF and bFGF, respectively (Johnson et al., 1990). Thus, Ig domain I does not appear to be necessary for high affinity binding of aFGF and bFGF. Currently, the function of this domain remains unknown. The existence of FGF receptor forms containing 3 or 2 Ig-like domains helps to explain earlier results obtained from cross-linking studies. As discussed previously (Section 111), two prominent bands of 145

111.

FIG.6. The human FGFR 1 gene. The figure shows the arrangement of introns and exons in the human FGFR 1 gene as described by Johnson et al. (1991). The figure also contains one additional exon that was not included in the original description of the gene (solid oval). Arrows indicate the positions of introns and numbers above the arrows indicate the size of the intron in kilobases. The following structural features are identified: the exon encoding the 32 unique amino acids at the C-tail of the Ig domain I secreted form (solid oval), the acid box domain (open box), the three alternative exons for the second half of Ig domain I11 (thick black line; labeled IIIa, IIIb, or IIIc), stop codons at the end of the secreted and membrane-spanning forms (asterisks), the 3’ nontranslated region that is unique to secreted form mRNA transcripts (dashed line), the transmembrane domain (solid box), and the kinase 1 and kinase 2 domains (stippled boxes).

18

DANIEL E. JOHNSON AND LEWIS

T. WILLIAMS

and 125 kDa were frequently seen when aFGF or bFGF were crosslinked to cell surface receptors. More recent studies have utilized cell lines transfected with cDNAs encoding either 3 Ig or 2 Ig domain forms of the receptor (Dionne el al., 1990; Johnson et al., 1990; Mansukhani et al., 1990). The results of these experiments show that cross-linked receptors containing 3 Ig domains are about 145 kDa in size, while crosslinked receptors containing 2 Ig domains are about 125 kDa in size. Thus it appears likely that the 145- and 125-kDa bands originally detected in cross-linking experiments represent the 3 Ig and 2 Ig receptor forms, respectively. In support of this, coexpression of 3 Ig and 2 Ig mRNA transcripts has been detected in a variety of cell lines using PCR (Johnson et al., 1990; Eisemann et al., 1991), Northern blotting (Reid et al., 1990; Eisemann et al., 1991), and RNase protection analyses (Werner et a!., 1992b).

Another receptor variant involving the first Ig domain has been described by Eisemann et al. (1991). The cDNA isolated by this group encodes a complete signal peptide and a complete Ig domain I, followed by 32 unique amino acids and a stop codon (Fig. 5, i). The 32 unique amino acids are encoded by an separate exon located between the exon for Ig domain I and the exon encoding the acid box domain (Fig. 6). Presumably the protein encoded by this cDNA represents a secreted form of Ig domain I. Although the function of this protein is unknown, it is interesting that similar forms of N-CAM molecules are generated via alternative splicing (Cunningham et al., 1987).

B. THEINCLUSION OR EXCLUSION OF Two AMINO ACIDS IN THE EXTRACELLULAR DOMAIN Several groups have reported the isolation of cDNA clones encoding proteins containing (or missing) two additional amino acids just downstream from the acid box Uohnson et al., 1990; Eisemann et al., 1991). In the human FGFR 1 sequence (Fig. 3), these amino acids correspond to Arg (148) and Met (149). Clones encoding proteins containing these amino acids and clones encoding proteins missing these amino acids have been identified for both the 3 Ig and the 2 Ig receptor forms (Johnson et al., 1990; Eisemann et al., 1991). Analysis of genomic DNA indicates that this dipeptide variation does not reflect an allelic difference. Instead, these amino acids are encoded at the immediate 3' end of the acid box domain exon. Therefore, it seems likely that the inclusion or exclusion of these amino acids results from the use of slightly different splice donor sites at this exon/intron boundary (Eisemann et

FGF RECEPTOR MULTIGENE FAMILY

19

al., 1991).The functional significance of this subtle variation in receptor structure is not known. C. THREE ALTERNATIVE EXONS FOR 111 HALFOF Ig DOMAIN

THE

SECOND

Johnson et al. (1990) have reported the isolation of a human cDNA encoding a protein that is identical to the 2 Ig membrane-spanning form of FGFR 1 until a point approximately halfway through Ig domain 111 (Johnson et al., 1990). At this point, the novel protein (see Fig. 5 , ii) diverges completely and then terminates 79 amino acids downstream. The novel protein does not contain a hydrophobic membrane-spanning domain and represents an additional secreted form of the receptor protein. Further studies have shown that a 3 Ig domain form of this secreted protein also exists. Although the function of the secreted FGF receptor is currently unknown, Duan et al. (1992) have shown that this protein binds bFGF. It is possible that the secreted FGF receptor acts as an extracellular reservoir of FGF, regulating the availability of FGFs to cell surface receptors. In the human FGFR 1 gene, sequences encoding the second half of Ig domain I11 which are unique for the secreted receptor form (labeled exon IIIa in Fig. 6) follow immediately downstream from sequences encoding the first half of Ig domain 111 (Johnson et al., 1991). In contrast, sequences encoding the second half of Ig domain I11 which are associated with the membrane-spanning receptor form (labeled exon IIIc in Fig. 6) are located several kilobases downstream. Also, in the process of sequencing this region of the human gene an additional exon was discovered that is highly homologous to the IIIc exon (labeled exon IIIb in Fig. 6). At the amino acid level, the IIIb and IIIc sequences exhibit 45% amino acid identity. PCR and Northern blotting analyses indicate that this new exon is part of mRNA transcripts encoding a membrane-spanning receptor. Thus, there are three alternative exons for the second half of Ig domain 111. One of these exons (IIIa) is a part of mRNA transcripts encoding a secreted receptor form, while the other two (IIIb and IIIc) are a part of transcripts encoding membrane-spanning receptor forms. These three exons have been named in the order of their linear appearance in the FGFR 1 gene (Fig. 6; Johnson et al,, 1991). As will be discussed shortly, corresponding IIIb and IIIc exons are also seen in the FGFR 2 gene. Furthermore, the IIIb and IIIc sequences confer distinct ligand binding specificities to the membrane-spanning forms of both FGFR 1 and FGFR 2.

20

DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

IX. Multiple Forms of FGFR 2 A. VARIATIONSINVOLVING Ig DOMAIN As is the case for FGFR 1, cDNAs encoding 3 Ig domain and 2 Ig domain forms of FGFR 2 have also been identified (see Fig. 5 ) (Dionne et al., 1990; Hattori et al., 1990; Houssaint et al., 1990; Champion-Arnaud et al., 1991; Crumley et al., 1991; Miki et al., 1991). Binding studies have shown that both forms of FGFR 2 exhibit similar high affinities for aFGF and bFGF (Dionne et al., 1990; Crumley et at., 1991).Thus, Ig domain I of FGFR 2 does not appear to be necessary for high affinity binding of aFGF and bFGF. Crumley et al. (1991) have reported the isolation of an FGFR 2 cDNA that encodes a signal peptide, a single Ig-like domain, an acid box domain, and a stop codon. Comparison of this protein to the secreted Ig domain I form of FGFR 1 (Fig. 5, i) indicates that these two proteins are structurally related, but not identical.

B. VARIATIONSINVOLVING THE ACIDBox DOMAIN

To identify cDNA clones for the KGF receptor Miki et al. (1991) employed a novel expression cloning strategy. The approach that was taken involved transfecting 3T3 cells expressing KGF, but not the KGF receptor, with cDNAs derived from cells that express the receptor. Transformed foci that were obtained were then studied in further detail. Cells from one of the transformed foci were found to express high affinity binding sites for KGF. This led to the isolation of a KGF receptor (KGFR) cDNA. Interestingly, the cDNA encoding the KGF receptor represents a unique splice variant of the FGFR 2 gene (Fig. 5, vii). The protein predicted by this cDNA is missing both Ig domain I and the acid box domain, and contains sequences corresponding to the IIIb exon in the third Ig domain. This represented the first example of an FGF receptor protein that was missing the acid box domain. In the FGFR 1 gene, the acid box domain is encoded by a single distinct exon (Fig. 6). It remains to be seen, however, whether alternatively spliced mRNAs that code for FGFR 1 proteins without the acid box domain exist. The acid box represents a peculiar hallmark feature of the FGF receptors. At present, though, the function of this domain remains unclear. Since the KGF receptor also contained sequences in the third Ig domain different from sequences in other FGFR 2 proteins (Fig. 5 , ix and x) whose binding properties were known to be different, the differences in ligand binding specificities between these receptors could not be attributed solely to the presence or absence of the acid box domain.

FGF RECEPTOR MULTIGENE FAMILY

21

In the case of FGFR 1, however, deletion of this domain from 3 Ig or 2 Ig receptor forms does not affect the affinity of these receptors for either aFGF or bFGF (de Vries and Williams, 1992).

C. ALTERNATIVE EXONS FOR I11 HALFOF Ig DOMAIN

THE

SECOND

Several groups have reported the isolation of cDNAs encoding FGFR 2 proteins with different amino acid sequences in the second half of the Ig domain 111. Some of these cDNAs contain sequences corresponding to the IIIb exon of FGFR 1 [Hattori et al. (K-Sam), 1990; ChampionArnaud et al. (K-Sam’), 1991; Miki et al. (KGFR), 1991; Sat0 et al., 1991; Dell and Williams, 19921, whereas the others contain sequences corresponding to the IIIc exon of FGFR 1 [Dionne et al. (BEK), 1990; Houssaint et al. (TK14), 1990; Pasquale (CekS), 1990; Champion-Arnaud et al. (TK25), 1991; Raz et al., 19911. No FGFR 2 cDNAs that contain sequences corresponding to the IIIa (secreted form) exon of FGFR 1 have been reported. Thus, at a minimum there are at least two alternative exons for the second half of Ig domain I11 in FGFR 2. Analyses of the region of the human FGFR 2 gene encoding Ig domain I11 have revealed that the FGFR 2 gene is organized in a fashion nearly identical to that of the FGFR 1 gene (Champion-Arnaud et al., 1991;Johnson et al., 1991). The linear arrangement of the IIIb and IIIc exons in the FGFR 1 and FGFR 2 genes is identical, and the positions and sizes of intron sequences are also similar. In contrast, the putative IIIa exon of the FGFR 2 gene (based on corresponding location to that of the FGFR 1 IIIa exon) would contain an open reading frame that codes for only four amino acids (Johnson et al., 1991). Thus, a putative FGFR 2 (IIIa) secreted form would be considerably shorter than the FGFR 1 (IIIa) secreted form. More studies are needed to determine whether an authentic FGFR 2 IIIa exon is present in the FGFR 2 gene and whether this exon is expressed in vivo. Figure 7 shows a comparison of the IIIb and IIIc sequences of FGFR 1 and FGFR 2. It is interesting to note that the FGFR 1 IIIb sequence is more closely related to the FGFR 2 IIIb sequence (78% identity) than to the FGFR 1 IIIc sequence (45%). Also, the FGFR 1 IIIc sequence is more closely related to the FGFR 2 IIIc sequence (83%) than to the FGFR 1 IIIb sequence (45%). The FGFR 2 IIIb and IIIc sequences exhibit 51% identity. These numbers demonstrate that there is greater divergence between similar exons of the same gene than between corresponding exons of different genes. This suggests that the existence of the IIIb and IIIc exons is primordial to the existence of a multigene family.

22

DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

A

LL.U

LLLC

IUh ~~

I..I.,k

-HSGINSSDAE--VLTLFNVTEAQSGEYVCKVSNYIGEANQSAWLTVTRP I I I I II I II I l l 1 I l l IIIIII -TAGVNTTDKEMEVLHLRNVSFEDAGEYTCLAGNSIGLSHHSAWLTVLE

45

HSGINSSNAE--VLALFNVTEADAGEYICKVSNYIGQANQSAWLTVLPK00 __ I I I II Ill I I I I I I I l l IIIIIIII AAGVNTTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFHSAWLTVLP

51 %

%

C FGFR 1 Luh

U

HSGINSSDAEVLTLFNVTEAQSGEYVCKVSNYIGEANQSAWLTVTRP IIIIIII IIII IIIIIII I l l IIIIIIII IIIIIIII1 HSGINSSNAEVLALFNIPPEADAGEYICKVSNYIGQANQSAWLTVLPKQQ

78

%

D k X J 3 X m TAGVNTTDKEHEVLHLRNVSFEDAGEYTCLAGNSIGLSHHSAWLTVLE IIIIIIIII Ill Ill 111lI1IIIIlIIIII I OIIIIII FGFR 2 IIIC AAGVNTTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFHSAWLTVLP

83 %

FIG. 7. Comparison of the IIIb and IIIc amino acid sequences of human FGFR 1 and human FGFR 2. The figure shows (A) an alignment of the FGFR 1 IIIb and FGFR 1 IIIc sequences; (b) an alignment of the FGFR 2 IIIb and FGFR 2 IIIc sequences; (C) an alignment of the FGFR 1 IIIb and FGFR 2 IIIb sequences; and (D) an alignment of the FGFR 1 IIIc and FGFR 2 IIIc sequences. The numbers on the right indicate the degree of amino acid identity between the two sequences. The FGFR 1 IIIb sequence was derived from sequences in the human FGFR 1 gene Uohnson el al., 1991). The remaining sequences were derived from cDNA sequences discussed in the text.

D. THEINCLUSIONOR EXCLUSION OF Two AMINO ACIDSIN THE JUXTAMEMBRANE DOMAIN The isolation of FGFR 2 cDNAs encoding proteins containing (or missing) two additional amino acids in the juxtamembrane region has been reported by several groups (Hattori et al., 1990; Houssaint et al., 1990; Champion-Arnaud et al., 1991). In the human FGFR 2 sequence shown in Fig. 3 these amino acids correspond to T h r (429) and Val (430). The location of this dipeptide corresponds precisely to the location of an

FGF RECEPTOR MULTIGENE FAMILY

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exonhntron boundary in the FGFR 1 gene. It remains to be seen whether the presence or absence of this dipeptide results from the use of slightly different splice donor sites.

E. THREE ALTERNATIVE EXONSFOR THE C-TAILDOMAIN The FGFR 2 sequence shown in Fig. 3 (also Fig. 5, ix) contains a C-tail domain of 61 amino acids. Two additional cDNA clones have been isolated from human tumor cDNA libraries that encode two distinct C-tail domains [Fig. 5 , viii (K-Sam)and Fig. 5, xi (TK25); Hattori et al., 1990; Champion-Arnaud et al., 19911. The two distinct C-tail domains both diverge from the FGFR 2 (Fig. 3) sequence following amino acid 760 (Ile). The sequence of one of these C-tail domains [Fig. 5, viii (K-sam)]is only 12 amino acids long and is highly divergent from the C-tail sequences of other members of this family (Fig. 5). The sequence of the other C-tail domain [Fig. 5, xi (TK25)] is 27 amino acids long and bears considerable homology to a portion of the C-tail sequence found in other FGFR 2 proteins. It remains to be determined whether these shorter, variant C-tail domains are expressed in normal cells. This is an important point, since previous studies have shown that mutations in the C-tail domains of receptor tyrosine kinases can lead to increased transforming potential of these proteins (Ullrich and Schlessinger, 1990).

X. FGFR 3 and FGFR 4 Currently, only single cDNAs have been isolated for FGFR 3 and FGFR 4 (Fig. 3 and Fig. 5, xii and xiii). In each case, the cloned cDNAs encode 3 Ig domain receptor forms containing IIIc type sequences in the third Ig domain (Keegan et al., 1991; Partanen et al., 1991). The FGFR 4 protein is somewhat unique in that it contains a core of only four consecutive (or five of seven) acidic residues in the acid box domain (see Fig. 3). This is somewhat shorter than the core sequence found in the acid box domains of FGFR 1 (eight consecutive acidic residues), FGFR 2 (six of seven acidic residues), or FGFR 3 (seven of eight acidic residues). In general, though, the structures of FGFR 3 and FGFR 4 closely resemble those of FGFR 1 and FGFR 2. In the future it will be interesting to determine whether multiple forms of FGFR 3 and FGFR 4 mRNA transcripts also exist.

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DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

XI. Ligand Binding Specificities of the Cloned FGF Receptors

Prior to the cloning of FGF receptor cDNAs, it was unclear whether multiple members of the FGF family could bind to a common receptor, and whether multiple forms of the receptor, if they existed, would exhibit different ligand binding specificities. Binding studies on cells transfected with cloned FGF receptor cDNAs have now provided conclusive answers to these questions. Initial studies demonstrated that 3 Ig and 2 Ig FGFR 1 forms containing IIIc-type sequences (Fig. 5, iv and v) bind either aFGF (Kd = 20-80 pM) or bFGF (Kd = 50-150 pM) with high affinity (Dionne et al., 1990; Johnson et al., 1990). Both ligands also activate receptor tyrosine kinase activity and receptor-mediated signaling (Dionne et al., 1990; Johnson et al., 1990; Mansukhani et al., 1990). Additional experiments have shown that both receptor forms also bind hstlKFGF, albeit with reduced affinity compared to the binding of aFGF and bFGF (Dionne et al., 1990; Mansukhani et al., 1990). Taken together, these experiments make two important points: (a) Multiple members of the FGF family can bind to the same receptor species and ( 6 ) the first Ig domain (I) of FGFR 1 is not essential for high affinity binding of aFGF or bFGF. Similar conclusions can also be drawn from studies of FGFR 2. In this case, a 3 Ig FGFR 2 form containing IIIc-type sequences (we will use the following nomenclature to refer to this receptor: 3 Ig/IIIc/FGFR 2; see Fig. 5, ix) has been shown to bind and become activated by aFGF (Kd = 40-100 pM), bFGF (Kd = 80-150 pM), and hstlKFGF (Dionne et al., 1990). Acidic and basic FGF also stimulate FGFR 3 activation (Keegan et al., 1991). Thus, as with FGFR 1, multiple members of the FGF family bind and activate both FGFR 2 and FGFR 3 proteins. In view of the fact that multiple FGF receptor proteins can bind multiple FGFs, how then could cells o r tissues selectively respond to individual members of the FGF family? Selective responsiveness to individual FGFs would seem to make sense if it is important to maintain such a large family of closely related ligands. indeed, several examples of ligand-specific responsiveness have been observed. Basic FGF, but not aFGF, is reported to stimulate mitogenesis in human melanocytes (Halaban et al., 1987). Also, aFGF and bFGF stimulate the proliferation of fibroblast and endothelial cells, whereas KGF does not (Rubin e l al., 1989).Thus, there must be mechanisms for achieving selective responsiveness to different members of the FGF family. One possibility is that variant receptor forms derived from the same gene by alternative splicing could exhibit different Iigand binding characteristics. Tissue-specific

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responsiveness to different FGFs could then be achieved by tissue-specific alternative splicing. Another possibility is that analogous splice variants derived from different FGF receptor genes encode receptor proteins with different ligand binding properties. In this case selective responsiveness could be achieved by tissue-specific expression of the different genes. As it turns out, both of these possibilities appear to be true. XII. Alternative Splicing in the Third lg Domain Is Important for Determining Ligand Binding Specificities

The isolation of cDNAs encoding a receptor for KGF provided a starting point for answering questions regarding the role of alternative splicing in determining receptor-ligand interactions. The receptor encoded by the KGFR cDNA (Fig. 5, vii) binds KGF (Kd = 180-480 pM) and aFGF with equal and high affinity (Miki et al., 1991). However, bFGF is 15- to 20-fold less effective than KGF or aFGF at competing with the binding of iodinated KGF to this receptor (Bottaro et al., 1990; Miki et al., 1991). These results differ from those obtained with a 3 Ig form of FGFR 2 containing IIIc sequences (3 Ig/IIIc/FGFR 2; Fig. 5, ix) (Dionne et al., 1990). This receptor protein binds aFGF (Kd = 40-100 pM) and bFGF (Kd = 80-150 pM) with high affinity, but presumably does not bind KGF (since fibroblasts that express this receptor are nonresponsive to KGF). These two receptor species differ in three regions: (a) KGFR is missing Ig domain I; (b) KGFR is missing the acid box domain; and (c) KGFR contains IIIb-type sequences in the third Ig domain, whereas the 3 Ig/IIIc/FGFR 2 form contains IIIc-type sequences. Hence, the different binding specificities of these proteins must be due to one or more of these three domains. Experiments with FGFR 1 proteins have helped to clarify the importance of the aforementioned three domains for ligand binding. As previously discussed (Section XI), both the 3 Ig and the 2 Ig forms of FGFR 1 bind aFGF and bFGF with nearly equal affinities (Dionne et al., 1990; Johnson et al., 1990). Thus Ig domain I is not essential for high affinity binding of these two ligands. Likewise, removal, via mutagenesis, of the acid box domain from either receptor form does not affect binding affinities for aFGF and bFGF (de Vries and Williams, 1992). These results suggested that the reduced affinity of the KGF receptor for bFGF may be due the presence of IIIb (as opposed to IIIc) sequences in this receptor form. To examine the functional importance of the second half

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DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

of Ig domain 111, Werner et al. (1992a) replaced IIIc sequences in the 2 Ig/IIIc/FGFR 1 (Fig. 5 , v) protein with IIIb sequences. T h e resulting 2 Ig/IIIb/FGFR 1 protein bound aFGF with high affinity, but had a much weaker affinity for bFGF. Thus, binding specificity for bFGF can be determined on the basis of which exon (IIIb or IIIc) is used to code for the second half of Ig domain I. Further experiments are needed to determine whether the 2 Ig/IIIb/FGFR 1 protein will also bind KGF. A summary of binding data for FGFR 1 and FGFR 2 forms containing different sequences in the third Ig domain is shown in Table 111. In a further series of experiments, Duan et al. (1992) have shown that a secreted FGFR 1 form containing IIIa sequences in the third Ig domain binds bFGF with higher affinity than aFGF. This leads to the conclusion that alternative splicing in the third Ig domain also is important for determining binding specificity for aFGF. Additional convincing evidence regarding the importance of alternative splicing in the third Ig domain has been demonstrated for the FGFR 2 proteins. Crumley et al. (1991) have recently isolated a cDNA encoding an FGFR 2 protein that is identical to the KGF receptor (Fig. 5 , vii) except that it contains IIIc-type sequences instead of IIIb-type sequences in the third Ig domain. This protein binds both aFGF and bFGF with high affinity. Thus, from these experiments, it can be concluded that alternative splicing in the third Ig domain of FGFR 2 is important for determining binding specificity for bFGF. In a related study, Dell and Williams (1992) have isolated a cDNA encoding an FGFR 2 protein that

TABLE 111 BINDING OF FGFs TO FGFR 1 A N D FGFR 2 FORMS CONTAINING DIFFERENT SEQUENCES IN THE THIRD Ig DOMAIN (111) FGFR 1 IIIa IIIb IIIC

bFGF > aFGF aFGF > bFGF aFGF = bFGF

FGFR 2 IIIb IIIC

aFGF = KGF > bFGF aFGF = bFGF (KGF does not bind)

Nofolu. This fable s h o w the relative affinities of different FGF receptor forms for aFGF, bFGF, and KGF. The depicted receptors differ in the sequences of their third Ig domains, containing either IIla. Illb, or IIlc sequences. The following sources were used to compile this figure: Dionne el al. (1990); Johnson ef al. (1990): Mansukhani et 01. (1990);kliki d al. (1991);Crumley et al. (1991); Werner et af. (l992a); and Dell and Williams (1992).

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27

is identical to 3 Ig/IIIc/FGFR 2 (Fig. 5, ix) except that it contains IIIbtype sequences. This protein binds aFGF with high affinity but exhibits only low affinity binding for bFGF. The findings presented above can be summarized as follows (see Table 111): (a) Acidic FGF binds with high affinity to FGFR 1 and FGFR 2 forms containing either IIIb or IIIc sequences regardless of whether Ig domain I and the acid box domain are also present. In contrast, aFGF binds with only low affinity to a 2 Ig FGFR 1 form containing IIIa sequences. (b) Basic FGF binds with high affinity to FGFR 1 and FGFR 2 forms containing IIIc sequences regardless of the presence or absence of Ig domain I and the acid box domain. Also, bFGF binds to the 2 Ig FGFR 1 form containing IIIa sequences. In contrast, bFGF binds with only low affinity to FGFR 1 and FGFR 2 forms that contain IIIb-type sequences. (c) KGF binds with high affinity to an FGFR 2 form that is missing Ig domain I and the acid box domain, and contains IIIb type sequences. In contrast, KGF does not bind to FGFR 1 and FGFR 2 proteins containing IIIc-type sequences. It remains to be determined whether the presence or absence of Ig domain I and the acid box domain have any additional influence on KGF binding (although one might predict that they do not). Furthermore it remains to be determined whether KGF binds to FGFR 1 forms containing either IIIa or IIIb sequences. At any rate, these binding experiments clearly demonstrate that alternative splicing in the third Ig domain is important for determining receptor binding specificities for all three FGFs that have been comparatively studied. XIII. Analogous Splice Variants from Different FGF Receptor Genes Encode Receptor Forms with Different Ligand Binding Specificities Two observations can be cited as proof that analogous receptor forms from different genes exhibit different ligand binding specificities: (a)A 3 Ig FGFR 4 form containing IIIc sequences (3 Ig/IIIc/FGFR 4; Fig. 5, xiii) binds aFGF with high affinity but does not bind bFGF (Partanen et al., 1991). Thus, the FGFR 4 receptor differs from corresponding 3 Ig/IIIc forms of FGFR 1 (Fig. 5, iv) and FGFR 2 (Fig. 5 , ix) which are known to bind aFGF and bFGF with comparable affinities (Dionne et al., 1990;Johnson et al., 1990). Since these three proteins are identical with respect to domain structure, the difference in affinities for bFGF cannot be attributed to alternative splicing and must be the result of other differences between the genes encoding these proteins. (b) It has been reported that hst/KFGF binds with high affinity to the 3 Ig/IIIc/FGFR 2 form (Fig. 5 , ix), but with reduced affinity to 3 IglIIIclFGFR 1 and 2

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DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

Ig/IIIc/FGFR 1 forms (Fig. 5, iv and v) (Dionne et al., 1990; Mansukhani et al., 1990). This difference also cannot be attributed to alternative splicing and must be the result of additional differences between the FGFR 1 and FGFR 2 genes. XIV. Regulation of FGF Receptor Expression

The hypothesis that cells or tissues could potentially achieve selective responsiveness to different FGFs by mechanisms involving alternative splicing or preferential expression of different FGF receptor genes is well supported by the binding studies presented above. The prediction that differential expression of different FGF receptor forms and genes actually occurs in nivo has recently been verified by studies from several laboratories. These studies have revealed several examples of cell- and tissue-specific alternative splicing as well as differential expression of the different FGF receptor genes in a variety of tissues. XV. Cell- and Tissue-Specific Alternative Splicing of FGF Receptor mRNAs

A. CELL-AND TISSUE-SPECIFIC ALTERNATIVE SPLICING OF THE THIRD Ig DOMAIN In their original studies with KGF, Rubin et al. (1989) noted that keratinocytes express receptors for KGF, but fibroblasts and endothelial cells do not. In contrast, fibroblast and endothelial cells express receptors that bind bFGF. From the binding studies discussed above it is clear that KGF binds to FGFR 2 (and possibly FGFR 1) forms that contain IIIb sequences and not IIIc sequences, whereas bFGF binds to FGFR 1 and FGFR 2 forms that contain IIIc sequences and not IIIb sequences. Therefore the different binding properties of keratinocytes and fibrohlasts/endothelial cells most likely reflect differential expression of IIIb and IIIc exons in these cells. Johnson et al. (1991) have shown that several human cell lines simultaneously express transcripts containing the IIIa, IIIb, or IIIc exons of FGFR 1. In each of these cell lines, however, expression levels of the IIIc exon appear to be much higher than those of the IIIa or IIIb exons. One cell line, foreskin fibroblasts, was found to express only the IIIc exon. Werner et al. (1992a) have demonstrated differential expression of the IIIa, IIIb, and IIIc exons of FGFR 1 in mouse tissues. Whereas the IIIc exon was expressed in all tissues examined with the exception of liver, the IIIa and IIIb exons exhibit more restricted patterns of expression.

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The IIIa exon was expressed in brain, skeletal muscle, and skin. The IIIb exon was expressed predominantly in skin, and at lower levels in brain, kidney, muscle, and placenta. As was observed in studies of human cell lines, in all cases where there is simultaneous expression of more than one exon, expression levels of the IIIc exon appear to be much higher than those of the IIIa or IIIb exons.

B. TISSUE-SPECIFIC ALTERNATIVE SPLICING INVOLVING Ig DOMAIN I Although the first Ig domain of the FGFR 1 may prove unimportant for ligand binding, it is clear that removal of this domain via alternative splicing occurs in a tissue-specific fashion. Northern blotting and RNase protection assays show that 3 Ig domain forms of FGFR 1 are the predominant forms of receptor expressed during mouse embryogenesis (Reid et al., 1990; Werner et al., 1992b). In fact, 2 Ig domain forms are not detected by protection analyses until after birth. Following birth, 3 Ig and 2 Ig forms are simultaneously expressed at nearly equal levels in a number of different tissues, including heart, lung, and muscle (Werner et al., 1992b).In brain and kidney, however, 3 Ig domain forms continue to be the predominant, if not exclusive, form expressed.

XVI. Differential, Tissue-Specific Expression of the Different FGF Receptor Genes Several studies that help to document differential expression of the different FGF receptor genes in a variety of tissues have been completed (Kornbluth et al., 1988; Reid et al., 1990; Heuer et al., 1990; Wanaka et al., 1990, 1991; Sat0 et al., 1991; Stark et al., 1991; Peters et al., 1992a,b; Werner et al., 1992b). These studies have utilized the techniques of Northern blotting, RNase protection analyses, and in situ hybridization, and have used a variety of probes. It is important to note that although these studies clearly delineate expression patterns of the different FGF receptor genes, typically they do not provide information on expression of specific alternatively spliced mRNAs. In general, the FGFR 1 and FGFR 2 genes exhibit broad but distinct patterns of expression during development and in adult animals. On the other hand, the FGFR 3 and FGFR 4 genes appear to have more restricted patterns of expression. In the developing embryo, FGFR 1 transcripts are expressed predominantly in the brain and mesenchymal tissues (Heuer et al., 1990; Wanaka et al., 1990, 1991; Peters et al., 1992a),

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DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

whereas FGFR 2 transcripts are expressed predominantly in brain and epithelium (Peters el al., 1992a; Werner et al., 1992b). FGFR 3 transcripts are expressed predominantly in brain, spinal chord, and cartilage rudiments of developing bone (Peters et al., 1992c). FGFR 4 transcripts are expressed in developing endoderm and the myotomal component of the somite, as well as myotomally derived skeletal muscle (Stark et al., 1991). N o expression of FGFR 4 transcripts was seen in cardiac muscle, however (Stark et al., 1991). In adult animals, FGFR 1 transcripts are detected in brain, bone, kidney, skin, lung, heart, and muscle, but not in liver (Heuer et al., 1990; Wanaka et al., 1990, 1991; Peters et al., 1992a; Werner et al., 1992b). FGFR 2 transcripts in adult animals are detected in brain, kidney, skin, lung, and liver, but not in heart, spleen, or muscle (Kornbluth et al., 1988; Peters et al., 1992a; Werner et al., 1992b). The pattern of expression of FGFR 1 in the central nervous system is consistent with neuronal expression, whereas the pattern of FGFR 2 expression in the CNS is more consistent with glial cell expression (Heuer et al., 1990; Wanaka et al., 1990, 1991; Peters et al., 1992a,b). FGFR 3 transcripts are detected in adult animals in brain, kidney, skin, and lung (Peters et al., 1992c).

XVII. The Drosophila FGF Receptor Glazer and Shilo ( 1 99 1) have recently reported the isolation of genomic clones for a Drosophila FGF receptor gene by low stringency screening of a Drosophila genomic library with a mouse FGFR 1 cDNA probe. The genomic clones were then used to isolate a nearly full-length cDNA clone (missing 5' nontranslated and signal peptide sequences) from a Drosophzla cDNA library. The protein encoded by this cDNA is structurally similar to the other membrane-spanning FGF receptors w e have discussed. The extracellular region of the Drosophilu FGF receptor contains 3 Ig-like domains and an acid box domain (core sequence: EDNDDDVE). The intracellular regon contains a relatively long juxtamembrane domain of approximately 90 amino acids, a tyrosine kinase domain that is interrupted by a kinase insert region of 22 amino acids, and a C-tail domain of approximately 70 amino acids. Comparison of the genomic and cDNA clones revealed that they are colinear with the exception of a single 85-bp intron in the kinase domain. The location of this intron is conserved in the human FGFR 1 gene (Johnson et al., 1991). What is striking about the genomic sequence of the Drosophila FGF receptor is the lack of introns in the extracellular coding region. This finding allows two conclusions to be drawn regarding the complexity of

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FGF receptor expression in Drosophila. First, since the coding region for the first Ig domain (I) is not separated from the remainder of the receptor gene by intron sequences, the potential for removing this domain via alternative splicing appears to be restricted to higher eukaryotes. The fact that higher eukaryotes express predominantly 3 Ig receptor forms (as opposed to 2 Ig) during development suggests that Ig domain I may have a function that is critically important during development. Experiments using Drosophila embryos may help to identify the function of this domain. Second, because the extracellular domain of the Drosophila gene does not contain introns, there is only one coding sequence for the second half of Ig domain 111. The amino acid sequence of this domain exhibits nearly equal identity with the human FGFR 1 IIIb sequence (38%) and the human FGFR 1 IIIc sequence (40%). In higher eukaryotes, receptors containing different sequences ( M a , IIIb, or IIIc) in this region exhibit distinctive ligand binding specificities (see Section XII). The existence of only one alternative for Ig domain I11 in the Drosophila FGF receptor indicates that this receptor is considerably less flexible, and suggests that the Drosophila FGF family may be less complex than that of higher eukaryotes. Glazer and Shilo (1991) also conclude that there is only one FGF receptor homolog in Drosophila based on their inability to isolate more than one class of FGFR clones. This conclusion also draws support from studies of FGF receptors from higher eukaryotes. As discussed in Section IX,C, comparison of alternative exons for Ig domain 111, both within the same gene and also between different genes, suggests that the existence of alternative exons for this receptor region is primordial to the existence of a multigene family. Since the Drosophila FGF receptor does not contain alternative exons in this region, it makes sensqto argue that there is only one FGF receptor gene in Drosophila. It seems likely, therefore, that both the FGF family and the FGF receptor family are considerably less complex in Drosophila than they are in higher eukaryotes.

XVIII. FGF Receptor-Mediated Signal Transduction Treatment of cells with FGFs leads to increased intracellular pH and intracellular Ca2+ levels (Tsuda et al., 1985; Halperin and Lobb, 1987); increased hydrolysis of polyphosphoinositides (Brown et al., 1989); increased phosphorylation of cellular proteins (Huang and Huang, 1986; Pelech et al., 1986; Coughlin et al., 1988); and increased transcription of a subset of cellular genes, including c-myc and c-fos (Kruijer et al., 1984; Muller et al., 1984; Stumpo and Blackshear, 1986). Depending on the

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DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

cell type, exposure to FGFs ultimately leads to proliferation, differentiation, inhibition of differentiation, or maintenance of a differentiated phenotype. The signaling mechanisms that give rise to these ultimate manifestations, however, remain largely unknown. It is known that FGF-dependent signaling is initiated immediately following the binding of FGF to its receptor. The binding of aFGF or bFGF to its receptor induces receptor dimerization (Bellot et al., 1991; Ueno et al., 1992), similar to what has been observed for several other growth factor receptors (Williams, 1989; Ullrich and Schlessinger, 1990). Interestingly, both homodimeric and heterodimeric receptor species can be formed between the FGFR 1, the FGFR 2, and the FGFR 3 proteins (Bellot et at., 1991; Ueno et al., 1992). Binding also leads to activation of FGF receptor tyrosine kinase activity and receptor autophosphorylation (Huang and Huang, 1986; Coughlin et al., 1988; Mansukhani et al., 1990). Phosphorylation of dimerized receptors appears to occur via an intermolecular transphosphorylation mechanism (Bellot et al., 1991).Activation of the receptor tyrosine kinase also leads to increased tyrosine phosphorylation of a number of cellular proteins (Huang and Huang, 1986; Coughlin et al., 1988; Bottaro et al., 1990; Burgess et al., 1990; Mansukhani et al., 1990; Miki et al., 1991; Peters et al., 1992d). Phosphorylation of a 90-kDa protein (and possibly others) may be unique to FGF receptor signaling pathways (Coughlin et al., 1988). It remains unclear, however, how many (if any) of these proteins associate with and are directly phosphorylated by the FGF receptor. Proteins that are known to associate with other growth factor receptors include: Raf-1 (Morrison et al., 1989), GTPase-activating protein (GAP) (Molloy et al., 1989; Ellis et al., 1990; Kaplan et al., 1990; Kazlauskas et al., 1990), pp60c-src (Krypta et al., 1990), the p85 subunit of phosphatidylinositol 3-kinase (Coughlin et al., 1989; Bjorge et al., 1990; Kazlauskas and Cooper, 1990; Escobedo et al., 1991; Otsu et al., 1991),v-Crk (Matsuda et al., 1990; Mayer and Hanafusa, 1990), and phospholipase C-y (PLC-y) (Wahl et al., 1988, 1989; Kumijan et al., 1989; Margolis et al., 1989; Meisenhelder et al., 1989; Burgess el al., 1990; Morrison et al., 1990). Currently, only one protein, PLC-y has been identified as a candidate substrate of an FGF receptor. PLC-y is phosphorylated on tyrosine residues following FGF stimulation (Burgess et al., 1990) and direct association with the receptor has been demonstrated (Mohammadi et al., 1991; Peters et al., 1992d). Mohammadi et al. (1991) have shown that the SH2 (src homology region 2) domain of PLC-y mediates binding to the FGF receptor. In addition, a 28 amino acid peptide derived from the C-tail region of FGFR 1 has been shown to bind to the SH2 domain of PLC-y (Mohammadi et al., 1991). This peptide contained phosphorylated Tyr

FGF RECEPTOR MULTIGENE FAMILY

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(766) (see Fig. 3). This tyrosine residue is conserved across all four FGF receptor genes. Mutation of Tyr (766) to Phe generates a receptor protein that does not associate with or phosphorylate PLC-y and does not mediate FGF-dependent phosphotidylinositol turnover (Peters et al., 1992d). The mutant receptor does, however, autophosphorylate and mediate increased tyrosine phophorylation of other cellular proteins (Peters et al., 1992d). Additional experiments with this mutant receptor should help to determine the role of PLC-y in FGF receptor-mediated signaling. Although the experiments described above have shed new light on the interaction between PLC-y and FGF receptors, the precise role of PLC-y in FGF receptor-mediated signaling remains unclear. In CCL39 cells (Chinese hamster lung fibroblasts),for instance, FGF does not stimulate hydrolysis of polyphosphoinositides (Magnaldo et al., 1986), indicating that the FGF receptors on these cells do not couple to PLC-y signaling pathways. Also, in cells where FGF does stimulate polyphosphoinositide breakdown, the magnitude of this response is much less than that observed following stimulation with other growth factors, such as PDGF (Peters et al., 1992d). Furthermore, cells expressing the Tyr (766) to Phe (766) mutant FGF receptor proliferate in response to FGF (Peters et al., 1992d), indicating that PLC-y may not be important for pathways leading to FGF-induced mitogenesis. The potential involvement of other signaling molecules in FGF receptor-mediated signaling remains largely untested. It appears likely though, that GAP protein, in contrast to its interaction with the PDGF receptor, does not associate with the FGF receptor (Molloy et al., 1989). Stimulation of cells with FGF does, however, lead to hyperphosphorylation of c-Raf-1 (Morrison et al., 1988). Currently, it is not known whether c-Raf-1 is directly phosphorylated by the receptor, or whether the phosphorylation of c-Raf-1 affects its kinase activity. Finally, although several cellular proteins are known to become phosphorylated on tyrosine residues in response to FGF treatment, the identities of these proteins (with the exception of PLC-y) remain unknown. XIX. Concluding Remarks

The FGF receptor field has grown increasingly complex in recent years with the discovery of four distinct FGFR genes. Alternative splicing of mRNA transcripts from at least two of these genes has introduced even further complexity. Binding studies discussed in this review have shown that although the multiple FGF receptor forms exhibit some common binding properties, considerable differences in binding specificities

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DANIEL E. JOHNSON AND LEWIS T. WILLIAMS

also exist. Functional differences between the different receptor forms were observed at two levels. First, different FGF receptor forms derived from the same gene via alternative splicing have different ligand binding properties. Specifically, the binding properties of a receptor can be determined in part by which exon (IIIa, IIIb, or IIIc) is used to code for the second half of Ig domain 111. Second, analogous splice variants from different FGF receptor genes bind different members of the FGF family. For instance, FGFR 1 and FGFR 4 forms that are identical with respect to domain structure show differences in their ability to bind bFGF. Recently, studies of FGFR mRNA localization have demonstrated tissuespecific alternative splicing of FGFR mRNA transcripts as well as tissuespecific expression of different FGFR genes. Thus, it can be concluded that mechanisms involving alternative splicing or differential expression of genes can be used to achieve selective responsiveness of different tissues to different members of the FGF family. The discovery of multiple forms of the FGF receptor raises many important questions for future investigations. It is particularly important to elucidate the roles of each of the different FGF receptor forms, particularly during development. Based on preliminary studies of FGFR mRNA localization, it seems likely that the different receptor forms have distinct roles. To determine these roles, it will be necessary to selectively knock out, by genetic or other means, specific receptor forms. Amaya et al. (199 1) and Ueno et al. (1992) have already achieved some success in knocking out FGFR function using dominant negative technology. However, it is clear that new approaches will be needed to selectively knock out individual receptor forms. Other questions that need to be addressed concern the nature of FGF receptor-mediated signal transduction. What are the signaling pathways (and molecules) used by the FGF receptors? Do the products of different FGFR genes couple to different signaling molecules and pathways? Do receptor forms that differ only in the sequences of their third Ig domain or C-tail region couple to different pathways? Do different members of the FGF family induce distinct signaling mechanisms? These and other questions need to be addressed in order to understand how FGFs can exert such a wide variety of effects in responsive cells types. Answers to these questions will provide a basis for understanding which receptor forms and which signaling pathways are involved in FGF-dependent cellular responses such as mitogenesis, chemotaxis, neurite outgrowth, neuronal maintainance, and inhibition of differentiation. Another important avenue for future research is the rational design of agonists and antagonists of FGF actions. The design of such molecules will benefit greatly from studies of the ligand binding domains of the

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different FGF receptor forms. Initially these studies will be complicated by the fact that there are several members in the PGF family, and by the fact that there are multiple FGFR genes and multiple exons for the second half of the third Ig domain. The advantage of studying FGFFGFR interactions, however, is that ligand binding is defined by a relatively short region of amino acids. Specifically, it has been demonstrated that 2 Ig domain forms of the FGF receptor can bind aFGF, bFGF, hstlKFGF, or KGF. Thus, ligand binding requires a maximum of 2 Ig domains (227 amino acids). Furthermore, binding studies of receptor forms with different sequences in the second half of the third Ig domain show that specificity for different FGFs can be achieved by minor variation in the composition of this short region (47-48 amino acids). Mutational analyses coupled with X-ray crystallography of ligand/receptor complexes should provide meaningful information on the ligand binding domaints) of the FGF receptor. The recent publication of the threedimensional structures of aFGF and bFGF also should provide valuable information for these studies (Zhu et al., 1991). The long-range goal of designing effective agonist and antagonists of FGF action has considerable therapeutic value. Agonists of FGF might be useful for accelerating wound healing and neovascularization, and maintaining viability of certain neuronal populations. Antagonists of FGF, on the other hand, might be useful for blocking angiogenesis in pathological conditions such as diabetic retinopathy and tumor neovascularization. ACKNOWLEDGMENTS We are deeply indebted to many colleagues for helpful insights and suggestions, and for providing data prior to publication. Without their cooperation and generosity this review would not have been possible. We thank Kevin Peters, Dah-Shuhn R. Duan, Sabine Werner, Carlie de Vries, Pauline Lee, Hikaru Ueno, Khoi Le, Dan Mirda, and Michael Jaye. This work was supported by the National Institutes of Health Program of Excellence in Molecular Biology (HL43821). D.E.J. was supported by American Heart Association Fellowship 94-1219116.

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Le Bon, T., Kathuria, S., Chen, E., Jacobs, S., Francke, U., Ramachandran, J., and Fugita-Yamaguchi, F. (1986). EMBO J. 5,2503-2512. Wagner, J. A., and DAmore, P. A. (1986).J. Cell Biol. 103, 1363-1367. Wahl, M. I., Daniel, T. O., and Carpenter, G. (1988). Science 241, 968-970. Wahl, M. I., Olashaw, N. E., Nishibe, S., Rhee, S. G., Pledger, W. J., and Carpenter, G. (1989). Mol. Cell. Biol. 9, 2934-2943. Walicke, P., Cowan, W. M., Ueno, N., Baird, A., and Guillemin, R. (1986).Proc. Natl. Acad. S C ~U . . S. A. 83, 3012-3016. Wanaka, A., Johnson, E. M., Jr., and Milbrandt, J. (1990). Neuron 5,267-281. Wanaka, A., Milbrandt, J., and Johnson, E. M., J . (1991).Deuelmt(Cambridge, UK) 111, 455-468. Werner, S., Duan, D.3. R., de Vries, C., Peters, K. G., Johnson, D. E., and Williams, L. T. (1992a). Mol. Cell. Biol. 12, 82-88. Werner, S., Peters, K. G., and Williams, L. T. (1992b). In preparation. Williams, A. F., and Barclay, N. A. (1988). Annu. Rev. Immunol. 6 , 381-408. Williams, L. T. (1989). Science 243, 1564-1570. Yarden, Y., Escobedo, J. A., Kuang, W.-J., Yang-Feng, T. L., Daniel, T. O., Tremble, P. M., Chen, E. Y., Ando, M. E., Harkins, R. N., Francke, U., Friend, V. A., Ullrich, A,, and Williams, L. T. (1986). Nature (London) 323, 226-232. Yayon, A., Klagsbrun, M., Esko,J. D., Leder, P., and Ornitz, D. M. (1991). Cell (Cambridge, MOSS.)64,841-848. Zhan, X., Bates, B., Hu, X., and Goldfarb, M. (1988). Mot. Cell. Biol. 8, 3487-3495. Zhu, X., Komiya, H., Chirino, A., Faham, S., Fox, G. M., Arakawa, T., Hsu, B. T., and Rees, D. C. (1991). Science 251, 90-93.

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PROTEIN TYROSINE KINASE GROWTH FACTOR RECEPTORS AND THEIR LIGANDS IN DEVELOPMENT, DIFFERENTIATION, AND CANCER Andrew F. Wilks Melbourne Tumor Biology Branch, Ludwig Institute for Cancer Research, Victoria 3050, Australia

I. Introduction 11. Structure of Receptor Protein Tyrosine Kinases 111. RTKs in Development

A. Developmental Mutants of the Mouse B. Developmental Mutants of Drosophikz melanagaster 1V. RTKs and Tumorigenesis A. RTKs as Retroviral Oncogenes B. RTKs as Cellular Oncogenes V. Genetics and Cancer References

I. Introduction The protein tyrosine kinases (PTKs) are a thematic protein family (comprising at least seven structural variants), built around a highly conserved domain capable of phosphorylating protein substrates on tyrosine residues. The physiologically powerful catalytic activity of this domain has been harnessed to a wide range of metabolic demands, from transduction of extracellular growth stimuli (Yarden and Ullrich, 1988; Ullrich and Schlessinger, 1990) to responding to changes in intracellular REDOX potential (Bauskin et al., 1991).Members of this family are thus made up as a mosaic of sensory, regulatory, and effector domains, a composition best understood in one structural variant of the family: the growth factor receptor PTKs (receptor tyrosine kinases or RTKs). Members of this class of protein each share a common transmembrane topology, with an often highly glycosylated N-terminal ligand binding domain presented to the extracellular milieu, and an intracellular protein tyrosine kinase domain toward the C-terminus of the protein. A single hydrophobic “transmembrane” region lies between these two domains (Fig. 1). The structure of signal transduction molecules such as these facilitates a directional flow of information from the exterior of the cell to the interior of the cell. This process is specific for the correct recep43 ADVANCES IN CANCER RESEARCH, VOL. 60

Copyright 8 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Class f Class III

LEU

EGF R MU ErbB3 Xmrk DER let23

#

PDGFa-R PDGFfi-R CSFl-R c-kt FLT

Class 11

Class IV Class V

u

-I

INSULIN-R IGH-R IRR3

FGF R BEK CEK2 FGFR4

FLT3 NEwFLkl

EPH ELK ERK EEK ECK HEK

= Fibronectin type III repeat

= Trans-membrane Domain

= Cysteine Rich Domain I

= Qrosine Kinase Domain

= Cysteine Rich Domain 11 = Cysteine Rich Domain In

= Ig-like Domain

= Insert Domain 4

= Liganddependent Autophosphorylation Sites

FIG. 1. RTK families. Five of the six classes of RTK are shown, displaying structural features which define each class. T h e nature of each of these structural features is shown at the right. Classes I, 11, and I l l are as defined by Yarden and Ullrich (1988); the remaining classifications are discussed in the text. References describing each of the RTKs listed below their prototypical structure are found in the text.

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torlligand interaction and results in significant amplification (or “gain”) as the signal (phosphorylation of substrates upon tyrosine residues) is transmitted within the cell. Under the strictest control, this combination of specificity and thematic signal transduction has been frequently used in building the repertoire of possible differentiation processes that are presumably available to the totipotent cells of a preimplantation embryo. Unconstrained, it is also, by virtue of its pivotal position in most widely accepted schemes of signal transduction processes, a likely mechanism by which cancer cells acquire some of the features of their tumorigenic phenotype, such as their apparent independence of extracellular growth stimulation (Aaronson, 1991; Bishop, 1991; Hunter, 1991; Cross and Dexter, 1991). Both of these aspects of the biochemistry of RTKs will be addressed in this review.

II. Structure of Receptor Protein Tyrosine Kinases Members of the RTK family hold a number of structural features in common. These are an extracellular ligand-binding domain; a single transmembrane domain [in contradistinction to the seven transmembrane receptor family (e.g., Noda et al., 1984)]; a single protein tyrosine kinase domain; and at least one regulatory domain, which serves to reign in the catalytic activity of the PTK domain. Within the rather loose constraints of the foregoing description there are at least seven distinct morphotypes of RTK, some of which have become evolutionarily successful combinations of structural elements, and upon which structures family clusters of RTKs have been elaborated (Hanks et al., 1988; Hanks and Quinn, 1991). These structures are shown in Fig. 1. Yarden and Ullrich (1988) described three classes of RTK based on structural features. Class I included the EGF receptor (Ullrich et al., 1984)and its siblingNEUlerbB2 (Bargmann et al., 1986a),erbB3 (Kraus et al., 1989),and Xmrk (see Section IV,A and B) (Wittbrodt et al., 1989).The Drosophilu EGF receptor-related protein (Livneh et al., 1985) and the let23 gene of Caenmhabditis elegans (Aroian et al., 1990) were discovered subsequently, and each RTK clearly exhibited an overall structural similarity to the prototypical members of this class. Class I11 RTKs included the platelet-derived growth factor p (PDGFP) receptor (Yarden et al., 1986), c-kit (subsequently discovered to be allelic to the W locus, see Section II1,A) (Yarden et al., 1987), and the colony-stimulating factor 1 (CSFI) receptor (Coussens et al., 1986; Rothwell and Rohrschneider, 1987). More recently RTKs with a related structure have been uncovered, such as the PDGFa receptor (Claesson-Welsh et al., 1989),jlt

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(Shibuya et al., 1990),flt3/flk-2(Matthewset al., 1991a; Rosnet et al., 1991) and fEk-1 (Matthews et al., 1991b). Class I1 RTKs included the homodimer receptors for insulin (Ebina et al., 1985),and IGFl (Ullrich et al., 1986), to which has recently been added a third family member, IRR (insulin-related receptor) (Shier and Watt, 1989). The recent explosion of reports of the discovery of P T K related sequences wrought by the advent of screening protocols based around the high degree of sequence similarity within the catalytic domain of members of this family of proteins [including low-stringency cDNA library screening (e.g., Kraus and Aaronson, 1991),or the polymerase chain reaction (PCR) (Wilks, 1989, 1991), or upon the catalytic activity of the PTK domain itself (Letwin et al., 1988; Lindberg et al., 1988; Lindberg and Pasquale, 1991)] has rendered the original classification of the RTKs obsolete. Among the newly discovered RTKs novel structural formats have been discovered. Highly populated familial clusters have been described; for example, the fibroblast growth factor (FGF) receptor class of RTKs accommodates four mammalian-derived RTKs (Reid et al., 1990; Kornbluth et al., 1988; Keegan et al., 1991; Partanen et al., 1991; Stark et al., 1991) and additional representatives from Xmopus htwis (Musci et al., 1990) Drosophila melanogaster (Glazer and Shilo, 1991), and chicken (Lee et al., 1989; Pasquale, 1990). The cluster of related RTKs based around the structure of EPH (Hirai et al., 1987) at present includes six members [EPH (Hirai et al., 1987);ECK (Lindberg and Hunter, 1990);ELK (Letwin et al., 1988; Lhotak et al., 1991); ERK (Chan and Watt, 1991);EEK (Chan and Watt, 1991);HEK (Boyd et al., 1992)]. A third cluster of related sequences is centred around the trk protooncogene (Martin-Zancaet al., 1986,1990a) and, to date, includes three members [trk (Martin-Zanca et al., 1986); t d B (Klein et al., 1989); trkC (Lamballe et al., 1991)l. In order to extend the original nomenclature devised by Yarden and Ullrich (1988), it is proposed that these three families of RTK be denominated the Class IV, Class V, and Class VI RTKs, respectively, in acknowledgment of the frequency with which their structure reoccurs. Finally, a host of “orphan” receptors have also been uncovered, whose structures are, at present, unique (see Fig. 2). Presumably other family members for each of these receptors will be discovered in time. At present, the number of putative RTKs far exceeds the number of ligands available for assignment. The process of marrying ligand to receptor is thus still at an early stage. However, some degree of success has been achieved. For example, putative ligands for the MET receptor (Bottaro et al., 1991; Naldini et al., 1991), c-kit (Williams et al., 1990; Copeland et al., 1990; Zsebo et al., 1990a,b; Martin et al., 1990; Huang et al., 1990; Flanagan and Leder, 1990; Anderson et al., 1990), and NEU

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ORPHAN RECEPTORS

!I!

LTK

MET

SEA

= Fibronectin type

a

SEVENESSIROS

III repeat

= Trans-membrane Domain

DI

= Tyrosine b a s e Domain

= Cysteine Rich Domain = Ig-like Domain

34 4

= Ligand-dependent Autophosphorylation Sites

FJG.2. Orphan receptors. RTKs unrelated to other known classes of RTK (see Fig. 1) are shown. The structural motifs encountered in these proteins are defined at the right. The references describing these receptors are cited in the manuscript, with the exception of RYK (Hovens, S. Stacker, and A. F. Wilks, unpublished results).

(Lupu et al., 1990; Tarakhovsky et al., 1991) have been described. Recently, each member of the trk family of RTKs (see Fig. 3) has been paired with its respective ligand. Members of this family of RTKs are expressed predominantly in tissues of neural origin. trk was originally discovered as an oncogenic fusion protein combining the catalytic domain of trk and a portion of the nonmuscle tropomyosin gene (MartinZanca et al., 1986) (see Section IV). More recently other members of this family of RTKs have been uncovered using low-stringency cDNA library screening techniques. These include trkB (Klein et d.,1989) and trkC (Lamballe et al., 1991). A major advance was made when it was discovered that trk was the receptor for nerve growth factor (NGF)

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ANDREW F. WILKS

NGF

NT-3

BDNF

m

= Cysteine Rich Domain I = Cysteine Rich Domain 11 = Ig-like DO= Trans-membraneDomain = Leucine Rich Domain = Tyrosine Kinase Domain

trk

trkC

trk B

gp140

gp145

gp145

FIG.3. The trk family of RTKs and their ligands. The three members of the trk family (Class VI) of RTKs, trk, trkB, and trkC, are shown displaying the structural motifs that have been encountered in their sequence. The extracellular domains of trk and trkC are 54% identical, and the extracellular domains of trkB and trkC are 53% identical. The PTK domains of trk and trkC are 76% identical, and the PTK domains of trkB and trkC are 83% identical. The ligands for each of these RTKs are shown above their respective receptor. The trkC ligand, NT3, appears to be able to bind to the other two members of the trk family, albeit with lower affinity.

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(Cordon-Cardo et al., 1991; Kaplan et al., 1991a,b; Klein et al., 1991a), and that trkB and trkC were the cellular receptors for two related growth factors, namely brain-derived neurotrophic factor (BNDF) (Klein et al., 1991b; Soppet et al., 1991; Squint0 et al., 1991) and neurotrophin-3 (Lamballe et al., 1991),respectively. Each of these growth factors is capable of binding with low affinity to other members of the trk family (Lamballe et al., 1991)and the complex interrelationship between these receptors and their ligands remains to be elucidated. Although it is not within the purview of this article to consider the nature of the signal transmitted within the cell by the PTK domain of members of the RTK family (for a review of this aspect of the biochemistry of PTKs, see Ullrich and Schlessinger, 1990),it is appropriate at this point to reflect on the multiplicity of cellular responses that appear to be mediated by members of this family. Thus, the specific inductive events mediated by the D. melanoguster sevenless gene product (detailed elsewhere in this review) may well involve the production of intracellular signals distinct from those induced by, for example, the murine RTK, c-kit. While acknowledging this fact, for the purposes of this review, the function of each of these RTKs is considered to be broadly equivalent: in both cases intracellular processes that affect the developmental fate of cells bearing these RTKs are triggered. Thus RTKs will be considered tools with which various developmental programs are established, and the question of mechanism will not be approached. With this caveat in mind, it is nonetheless evident that RTKs feature in developmental processes in several distinct modes. Examples of a number of diverse developmental roles are presented below. Ill. RTKs in Development

The expression patterns of many human, murine, and Drosophila RTKs have been determined by Northern analysis of mRNAs from adult tissues. For some murine and Drosophila RTKs in situ hybridization analysis has also been performed. Using this latter approach, the expression patterns of these mRNAs appear to range from widespread [e.g., the D. melanoguster EGF receptor homolog DER (Katzen et al., 1991)] to exquisitely highly localized [e.g., members of the trk family of RTKs (Klein et al., 1989; Martin-Zanca et al., 1990b; Lamballe et al., 1991)JMoreover, analyses of expression of RTKs during embryogenesis have provided insight into the possible developmental role played by certain members of this family. Thus, in situ hybridization analyses of CSFl and its receptor (Regenstreif and Rossant, 1989) firmly place both ligand and receptor in the region of the developing trophoblast during the development

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of the placenta. Similarly, angiogenesis during human extraembryonic development has been demonstrated by the same technique to involve the spaciotemporal expression of PDGFP and its receptor (Holgren et al., 1991). Studies such as these strongly favor the interpretation that these receptor/ligand pairs have important roles to play in the induction events that mediate the developmental processes occurring at these locations. More definitive demonstration that members of these classes of molecules are involved has come from the investigation of a number of developmental mutants of both the mouse and the fruit fly D. melanogmter. These studies are outlined below.

A. DEVELOPMENTAL MUTANTSOF THE MOUSE A large number (> 1000) of mutant mouse strains have been described (Lyon and Searle, 1989), each exhibiting an unusual developmental phenotype that is the result of mutation at a single genetic locus. Three such mutants, dominant white (W) spotting (Little, 1915; Gruenberg, 1942; Russell, 1970), patch (Gruenberg and Truslove, 1960), and steel (Bennett, 1956), are of particular interest in the context of the role of RTKs in development. Each of these loci maps to a gene encoding a growth factor or an RTK growth factor receptor, and the profound developmental phenotypes associated with the loss of each of these genes are a testimony to the importance of molecules of each of these classes to the progress of normal murine development. The W, steel, and patch mutants are particularly informative of the types of processes in which these molecules are involved. In view of their importance, each of these mutants will be discussed separately.

I . The Dominant White Spotting (W) and Steel Loci The W locus has been the focus of genetic analysis for over 75 years (Little, 1915) and at least 25 distinct alleles of this locus have been described in the mouse (see references in Lyon and Searle, 1988) and one in the rat (Niwa et al., 1991; Tsujimura et al., 1991) The phenotypes of these alleles range from severe [e.g., W37, where heterozygotes are mostly white and homozygote embryos become inviable soon after implantation (Geissler et al., 19811 to mild [for example, W57 (Reith et al., 1990), where heterozygotes have a single white spot and homozygotes have white patches and are mildly anemic]. Overall, the W mutants appear to have common defects of differing severity in melanogenesis, hematopoeisis, and primordial germ cell production. Analysis of embryonic chimeras has shown that the W gene product itself is required to be produced by the afflicted cells themselves (Russell, 1979). In contrast,

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the steel ( S I ) mutation (located on chromosome 10 in the mouse and represented by at least 10 alleles), whose phenotype shares deficiencies in pigment cells, blood cells, and germ cells with that of the W mutant, appeared to perturb some aspect of the microenvironment of these cells (Russell, 1979). This led to the suggestion that these two loci were intimately involved with the transmission of a common developmental signal to these cells and were most likely a growth factor receptor and its cognate ligand. Recently, the S I gene has been shown to encode stem cell factor (SCF) (Zsebo et al., 1990a,b)[also known as mast cell growth factor (MCGF) (Williams et al., 1990; Anderson et al., 1990; Copeland et al., 1990)], the ligand for the growth factor receptor encoded by the W locus. SCF is a membrane-bound glycoprotein of 36 kDa, with a 31- to 33-kDa processed form that is secreted (Anderson et al., 1990; Zsebo et al., 1990a; Nocka et al., 1990; Flanagan et al., 1991). The W locus was mapped to chromosome 5 in the mouse (Yarden et al., 1987) and has subsequently been demonstrated to be allelic to the gene for the RTK c-kit (Chabot et al., 1988; Geissler et al., 1988) and the protooncogene borne by the feline retrovirus HZ4-FeSV (Besmer et al., 1986a).The same RTK gene was demonstrated to be mutated in the Ws allele of the rat (Niwa et al., 1991; Tsujimura et al., 1991). Intriguingly, this same locus has emerged as the mutant allele in the human autosoma1 genetic disorder piebaldism (Morgan, 1786; Rizzoli, 1877, 1878), which is characterized by congenital patches of skin and hair that are completely devoid of melanocytes. In this mutant human allele, a single point mutation in the PTK domain of the c-kit gene appears to be the genetic basis of this disorder (Giebel and Spritz, 1991; Fleischman et al., 1991).Thus mutation in the c-kit gene in mouse (Reith et al., 1990; Tan et al., 1990), rat (Niwa et al., 1991; Tsujimura et al., 1991), and human (Giebel and Spritz, 1991; Fleischman et al., 1991) appears to be the basis for similar developmental defects in all three species. A particularly elegant study (Kishet et al., 1991) examining, by in situ hybridization, the expression pattern of c-kit and its cognate ligand SCF, during the development of the postimplantation mouse embryo, has shed light on the mechanism of action of these two molecules, and laid a biochemical basis for the genetic studies performed on these loci. A particular focus of this study was the migrating stem cell populations known to be affected in W and S I mutant mice. In these two mutant phenotypes three populations of migratory cells are affected. The migration of the primordial germ cells from their site of origin, in the allantois, through the mesentery and hindgut, to the forming genital ridges occurs between Embryonic Days 9.5 and 11.5 (Donovan et al., 1986; Bennett, 1956; Mintz and Russell, 1957). At the time of onset of

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the primordial germ cell migration, SCF mRNA appears to be expressed at its highest level in the developing genital ridge, whereas, the c-kit mRNA is found associated with cells in the mesentery in the vicinity of the dorsal aorta, the location of the migrating primordial stem cells. Similarly, melanoblasts derived from neural crest cells begin to migrate around at Day 8.5 of embryonic life and are fully dispersed in the trunk endoderm by Day 11.5. Again, SCF appears to be expressed in the precursor of the skin (dermamyotome), whereas c-kit expression appears to be expressed only in migrating melanoblasts. At Embryonic Day 15.5, the patterns of SCF and c-kit established earlier in the proceedings remain, even though essentially all of the migration of the melanoblasts is complete. During this phase, however, there is a considerable expansion of the numbers of melanocytes at this location. Each of these patterns of expression fit well with the phenotype of the W and S l mutants described above. Nonetheless, c-kit and SCF expression was also detected at sites in the neural tube and developing brain, tissues which are not affected by the loss of these genes in the W and S 1 mutant strains. Accordingly, there remain elements of uncertainty in combining the data emerging from the analysis of the developmental mutants with that derived from the studies of the expression patterns of these proteins during embryogenesis. Several plausible interpretations are possible. First, the embryonic lethality characteristic of homozygotes with the more severe W and SZ alleles has been attributed to the consequences of the severe anemia that results from these mutations. It may well be that the actual cause of the lethality resulting from these mutant alleles lies with the neurological defects caused by the absence of their gene products from normal sites of expression in the neural tube and developing brain. A possible alternative explanation of these data may lie in a builtin redundancy of the molecules required for the induction of the neurological development of the embryo. The steel-Dickie ( s l d ) mutant is a semidominant mutant that arose spontaneously in the DBA/2J inbred strain (Bernstein, 1960). s l d l s l d homozygotes are viable, although they are severely anemic and sterile. The s l d mutation lies within the SCF (MCGF) gene and consists of a 4-kb intragenic deletion that removes sequences encoding the normal C-terminus of the SCF protein (Flanagan et al., 1991; Brannan et al., 1991). The new C-terminus of the mutant protein is taken from adjacent intron sequences, and the sld-allele-derived SCF no longer bears a transmembrane domain. The truncated SCF appeared to be fully biologically active, when measured as a mitogen on a murine mast cell line. Further, injection of sld/sldmice with pharmacological doses of a soluble form of SCF was able to partially rescue the hematopoietic defects in these mice

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(Zsebo et al., 1990b).Thus, if there is biologically active SCF produced in the SId mutant mouse, why is the phenotype so severe? Although the answer to this question remains unknown, it is probable that the membrane-bound form of SCF is required during embryonic development, when the three classes of migratory cells are “in transit.” In this way, the location of the primordial germ cells and melanoblasts could be accurately defined so that their subsequent development proceeds properly. This sort of function could not be carried out by the soluble sId-derived SCF. After birth, however, the alleviation of the usually fatal macrocytic anemia that is characteristic of this mutant phenotype can be achieved by the soluble SCF, and the sId mutant therefore remains viable. Thus SCF may have multiple functions, both as a soluble growth factor and as a membrane-anchored growth factor. What has the detailed analysis of the S I and W loci and their incumbent growth factor and growth factor receptor genes taught us about the roles played by these classes of molecules in embryonic development? First, the impact of these mutations on developmental fate appears to differ with the cell type under consideration. Thus, W alleles can demonstrate severe defects in melanoblast migration and exhibit the macrocytic anemia characteristic of this mutant, but nonetheless remain fertile (Lyon and Searle, 1989). Whether this is a product of a combination of the reduced capacity of the mutant c-kit gene to transduce the appropriate signal coupled with a lack of availability of sufficient ligand in particular locations in the developing embryo, or is the result of a differential sensitivity of each of these classes of cells to the type of signal transmitted by the c-kit RTK, is at present unresolved. A third possibility would be the presence of an overlapping induction system (for example, in the migrating germ cell population) that could compensate, at least in part, for the reduced signaling capacity of the mutant c-kit protein. These issues remain to be resolved. Second, although the nature of the inductive event remains enigmatic, it appears likely that there exists a “gradient” of SCF ligand along which the migratory melanoblasts, germ cells, and hemopoeietic stem cells move. These cells appear to stop their migratory phase at locations of highest SCF levels, and begin to divide in these sites. Thus the c-kitlSCF interaction appears to have the effect of permitting the movement of these migratory cells, and ultimately fixing their correct position for an expansionary phase later in embryonic development. Although clearly present at the time of this expansionary phase, it is at present unknown whether c-kit and its ligand have a role to play in this process. Further, whether the apparent dependence of these cell migrations upon c-kit signaling is wrought as a consequence of a mitogenic effect of

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this RTK or flows from a chemotactic response to the presence of SCF is equally mysterious. Third, the lack of an obvious neurological defect in W mice, despite strong evidence that both this RTK and its cognate ligand are expressed in the developing neural tube, appears to be at odds with the otherwise good correlation of the W phenotype with the expression pattern of the c-kit gene (Kishet et al., 1991). Although it is possible that such a defect would only manifest itself in the Wl W homozygote, and on this account may in fact be the cause of the lethality of such a genotype, it is equally plausible that the development of this particular tissue has a built-in level of redundancy, which secures its development even in the absence of the developmental cues usually transmitted by the c-kit gene product. Clearly, much work remains to be done to resolve these issues. 2. Patch (Ph) Patch (Ph) is a semidominant mutant of the mouse that arose spontaneously in the C57/BL strain. Mice heterozygous for the mutant patch allele have sharply defined white spots in the belt region, due to an absence of functional melanocytes in the torso and a somewhat deformed face, due to a wider prefrontal bone (Gruenberg and Truslove, 1960). Homozygous PhlPh mice do not survive to birth and usually die in utero sometime after Day 10 of embryonic life. Embryos that survive beyond Day 10 have profound deformities in the region of their snout and palate. T h e P h locus is on mouse chromosome 5 and is allelic to the receptor for platelet derived growth factor a (PDGFa-R) (Stevenson et al., 1991; Smith et al., 1991), which lies very close to the c-kit locus in both mouse and humans (Yarden et al., 1987; Lalley et al., 1989). Although the precise boundaries of the regions lost have not yet been mapped (D. Bowen-Pope, personal communication), the PDGFa-R is clearly absent, and the nearby c-kit gene remains unaffected. It nonetheless remains a formal possibility that there are other genes in addition to the PDGFa-R gene that are lost at the mutant locus, and whose loss may play a part in the establishment of the phenotype. PDGFa-R expression in the embryo follows a distinctive course, which to a greater or lesser extent is predictive of the phenotype of these mutants. For example, on the basis of in situ hybridization studies performed on the developing mouse embryo (D. Bowen-Pope, personal communication), most mesoderm-derived tissues appear to express the PDGFa-R. Rarely, tissues that are derived from the ectoderm, such as the lens of the eye and the choroid plexus, also express significant levels of PDGFa-R. Thus, PhlPh homozygotes reach several critical developmental stages beyond which only a proportion of them are able to proceed. By Embryonic Day 9, many of the

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tissues derived from the mesoderm that express PDGFa-R in normal embryos have failed to develop appropriately. Approximately two-thirds of these PhlPh embryos die around this time. Surviving embryos manifest defects in many tissues derived from the mesoderm, including the heart, dermis of the skin, and tissues derived from the nonneurogenic cells of the neural crest. Defects in the ectodermally derived tissues normally expressing PDGFa-R, such as the lens and the choroid plexus, are also evident (D. Bowen-Pope, personal communication). The foregoing discussion of the patch phenotype, and its purported provenance as a null mutation in the PDGFa receptor gene, raises the question of functional redundancy of molecules such as these in development. The mere fact that one-third of PhlPh survive beyond Day 9 of embryogenesis in the complete absence of the PDGFol receptor suggests that the absence of a functional PDGFa receptor can be compensated for, to some extent, by other mechanisms. The further observation that only rarely do surviving embryos reach term, and that there is a progressive and continued loss of viability between Day 9 and term, suggests that there is no single crisis point reached by these embryos, but rather a gradual reduction in viability of their tissues. Thus, although functional redundancy is a strong candidate for the basis of the survival of these embryos, this process is highly inefficient and ultimately ineffective in reestablishing a normal developmental program in the PhlPh embryo. The semidominant nature of the W and patch mutants is particularly revealing of the biochemistry of RTK activity. Three alleles of W, namely W, Wx, and WlgH, are essentially null mutations of the c-kit locus (Geissler et al., 1988). Their dominant phenotype extends only to their effects on melanocyte migration and is due to haploinsufficiency of the remaining normal c-kit allele. Other alleles of W 1e.g. W4*, W3’, Wv, and W41 (Nocka et al., 1990)] have a more severe phenotype than the presumed null alleles mentioned above. The implication here is that the presence of a mutant version of the c-kit receptor suppresses even the reduced signal emanating from the normal gene product. In a functional sense this indicates that the normal and mutant proteins must interact with each other, either as dimers or as larger oligomers. This type of suppressive mutant gene product was termed an “antimorph” by Muller (1932), although in more current parlance the term “dominant negative mutant” (Herskovitz, 1987) seems to have replaced this original term. Recent work has demonstrated that many of the dominant W mutants are the product of point mutations in the coding sequence of the c-kit gene (Reith et al., 1990; Nocka et al., 1990; Tan et al., 1990).The molecular basis of their dominant effect appears to lie in the fact that each of these point mutations lies in an important segment of the PTK

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catalytic domain of c-kit and results in the (usually) complete abrogation of the intrinsic kinase activity of the mutant proteins (Reith et al., 1990; Nocka et al., 1990). Upon mutual contact of the receptor and its ligand, a process of dimerization occurs, whereupon a process of cross-phosphorylation is initiated (Ullrich and Schlessinger, 1990). Failure to enact this cross-phosphorylation event results in failure to transmit an appropriate intracellular signal. Thus, equimolar quantities of mutant and normal c-kit receptor on the surface of a cell will produce only 25% functional dimers upon ligand activation, the remaining 75% being nonfunctional heterodimers o r mutant homodimers. Thus a greater severity of the W phenotype is observed in heterozygotes bearing antimorph alleles of c-kit than with null mutant heterozygotes. These data have been used to strengthen the case for ligand-induced receptor dimerization developed from purely biochemical studies in other systems (Ullrich and Schlessinger, 1990). MUTANTSOF B. DEVELOPMENTAL Drosophila Melanogaster

Our understanding of the genetics of D . melanogaster is more detailed than that of any other multicellular organism. T h e utility of D . wlanogaster as a system for the analysis of developmental programs stems from this fact coupled with the availability of sophisticated protocols capable of screening and propagating homeotic mutants and maternal effect zygotic mutants (Nusslein-Volhard and Wieschaus, 1980) and enhancers o r suppressors of these mutant loci (Strecker et al., 1991; Simon et al., 1991). In the light of the power of D . melanogaster as a tool for seeking genetic events that perturb development, it is not surprising that a proportion of these mutants are allelic to genes encoding RTKs. Drosophila RTK mutants include loss-of-function mutants such as jib (Nusslein-Volhard et al., 1984), torpedo (Schuepbach, 1987), and sevenless (Harris et al., 1976; Campos-Ortega et al., 1979) and gainof-function mutants, such as torso-D4021 (Sprenger et al., 1989) and ellipse (Baker and Rubin, 1989). Each mutant has been informative in framing our current notions of how members of the RTK family contribute to the process of embryogenesis, and are worth examination in some depth.

I . jlb, ellipse, and torpedo The D. melanogaster EGF receptor homolog (DER) (Livneh et al., 1985) is allelic to genetic loci of three Drosophila developmental mutants. These are faint little ball cflb) (Schejter and Shilo, 1989), torpedo (Price et al.,

PROTEIN TYROSINE KINASE GFR

57

1989),and ellipse (Baker and Rubin, 1989).jb is a loss-of-functionzygotic mutant whose complex phenotype affects all germ layers of the embryo. In severe alleles ofjb, anterior head structures fail to develop properly, most probably as a result of cell death rather than a defect in cell determination (Schejter and Shilo, 1989). Further, the germ band does not retract and the embryonic central nervous system does not develop appropriately. torpedo is a recessive female-sterile mutation (Schuepbach, 1987) that affects the pattern of the eggshells of homozygous females. Although demonstrably allelic to each other (Price et al., 1989), torpedo alleles are weaker than jlb alleles, and homozygous mutant flies survive to reproductive age. Female homozygous torpedo flies are nonetheless sterile due to their failure to establish the dorsoventral pattern of the eggshell and embryo. Thus DER appears to be involved in the establishment of this positional patterning, the loss of which is lethal to the developing embryo. These mutational analyses have been correlated with the expression pattern of DER (Katzen et al., 1991), wherein the differential regulation of the DER transcript during embryogenesis appears to explain the susceptibility of certain tissues to the pleiotropic effects of the mutant alleles. Similar studies on the expression pattern of the DER protein (Zak et al., 1990) underscore these findings. Thus, not surprisingly, widespread and pleiotropic effects appear to be the consequence of mutation at a widely expressed RTK locus.

2. sevenless In the developing compound eye of D. melanogaster, multipotent precursor cells undergo a timed series of inductive events, during which the coordinate production of 8 neuronal photoreceptor cells (six outer cells, Rl-R6, and two inner cells, R7 and R8) and 12 to 14 accessory cells, each held in a precise array, are produced in each ommatidium (Tomlinson, 1988; Ready et al., 1976) (see Fig. 4). Each eye bears around 800 such units. The retinal primordium is derived from the eye imaginal disc (Garcia-Bellidoand Merriam, 1969) and during the third-instar of larval development a wave of differentiation (called the morphogenic furrow) sweeps across the disc epithelium from posterior to anterior. Ahead of this furrow, the cells are undifferentiated and behind it the photoreceptor neurons Rl-R8 have already formed into the highly organized structure of the adult compound eye. The sequence of inductive events at the morphogenic furrow is as follows: The central R8 cell differentiates first, followed by the pairwise differentiation of the R2 and R5 cells, the R3 and R4 cells, and the R1 and R6 cells. The R7 cell, which lies touching the R8, R6, and R1 cells at the apex of the cluster of neuronal cells, is the last to undergo differentiation. As a developmental system,

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time

1 I

I

I

I

I

3hr

6hr

9hr

12hr

15hr

I

I

*

FIG.4. Ommatidial development in Drosophzla melanogaster. A single ommatidium is shown. The initiation of each array begins with the determination of the R8 photoreceptor cell. This is followed after 5-6 hr by the R5 and R2 cells, and between 8 and 10 hr by the R4 and RS cells. The R1 and R 6 cells are determined 12 hr after the R8 cell, and finally the R7 cell after 15 hr. The sevenless phenotype occurs after failure of the R8 cell to induce photoreceptor differentiation in the R7 cell.

the fate of the R7 cell has been the focus of various elegant studies which have identified several genes that have a role to play in the induction of the appropriate developmental program in this cell. Two proteins in particular are central to the theme of this review; they are the product of the homeotic gene sevenless, an RTK, and its ligand, bride ofsevenless (also known as boss). T h e elegant studies performed in this system have shed light on some of the processes of embryonic induction, and in particular the role that the RTKs play in them. In the homeotic mutant sevenless the R7 cell fails to respond to its developmental cue and instead becomes a cone cell (Tomlinson and Ready, 1986). T h e sevenless protein bears all of the hallmarks of an RTK (Hafen et al., 1987; Bowtell et ul., 1988), albeit with an uncommon structure (Fig. 1). T h e sevenless gene encodes a protein of 2559 amino acids (290 kDa), with two putative transmembrane domains, one at the Nterminus of the protein, and a second located approximately 400 amino acids from the C-terminus of the protein (Bowtell et al., 1988). The PTK domain is situated within the intracellular C-terminal portion of the protein. T h e extracellular domain is more than 2100 amino acids (290 kDa) in length and is post-translationaly cleaved to two chains of 220 and 58 kDa after synthesis (Simon et ul., 1989).T h e mammalian RTK, ROS (Birchmeier et al., 1990; Matsushime and Shibuya, 1990), is structurally related to sevenless, although it appears to be functionally quite distinct and does not play an equivalent role in mammalian eye development.

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Genetic mosaic analyses of the boss gene provided the first evidence that the R8 cell delivered an inductive cue for the R7 cell (Reinke and Zipursky, 1988). An immediate inference made from this observation was that the boss gene product was the ligand for the sevenless RTK. The molecular cloning of the boss gene (Hart et al., 1990) drew the surprising conclusion that the boss gene encoded a protein with seven putative membrane spanning domains and a large (498 amino acids) extracellular domain. Further work by the same group (Kraemer et al., 1991) elegantly demonstrated that the 120-kDa boss glycoprotein, when expressed in vitro on the surface of S2 cells, was able to mediate the homotypic aggregation of other S2 cells bearing the sevenless gene product. Further, the physical proximity of the two proteins on the adjacent surfaces of the R8 (boss-bearing) and the R7 (sevenless-bearing) cells (Kraemer et al., 1991) provided compelling evidence that these two proteins have the opportunity to interact with each other during the induction of R7 cells by their R8 neighbors. Thus, superficially,the induction of the R7 cell by the R8 cell appears to be a simple process requiring the expression of the sevenless receptor on the R7 cell and its inductive ligand, boss, on the R8 cell. However, several additional layers of complexity have been added to the picture, and some anomalies remain to be resdved. For example, at one time or another, all of the cells R1 through R6 express the sevenless gene, but these cells are unable to respond to the same inductive stimulus provided by the R8 cell in their midst. Second, expression of a ligandindependent activated sevenless protein in flies under the control of the sevenless promoter (Basler et al., 1991) results in the recruitment of many of the surrounding cone cells to an R7 fate; meanwhile, the R3 and R4 cells, which clearly express the activated sevenless protein, do not alter their fate. Further, the expression in flies of boss under a heat shock promoter is capable of recruiting cone cells, which surround the neuronal Rl-R8 cells, to adopt an R7 neuronal phenotype, although again, the Rl-R6 cells, which express the sevenless receptor, fail to change their own developmental fate (Van Vactor et al., 1991). It has therefore been postulated that although the sevenless receptor is sufficient to determine the fate of the R7 cell, this program is overridden in the Rl-R6 cells by a mechanism, or mechanisms, as yet unknown. These observations serve to underline the complexity of the inductive process, and demonstrate the importance of the hierarchical influences that impinge upon the ultimate fates of cells during organogenesis. Whether the bosslsevenless interaction is typical of a wider range of embryonic inductions is not known at present. Indeed whether this process can be said to be representative of growth factorlgrowth factor

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ANDREW F. WILKS

receptor interactions in general is moot. Neither protein is typical of either of these two classes of molecule, nor is their interaction at the interface of the R8 and R7 cells mechanistically similar to, for example, the EGF/EGF receptor interaction, upon which biochemical basis many of our notions of this type of system have been developed. Moreover, whether the inductive event is a promotion of cellular growth at all (and therefore the act of a “growth factor”) is unlikely, since the presumptive R7 cell does not become extinct in the absence of the inductive event, but simply alters its developmental fate and becomes a cone cell. Thus, the activation of the sevenless PTK domain has a measurable impact on the fate of the R7 cell, an observation in apparent counterpoint to the role that the c-kit P T K domain presumably plays in the migration of melanoblasts in midgestation mouse embryos (see Section 111, A). Thus a wide range of apparently distinct cellular processes appear to be responsive to the catalytic activity wrought by the P T K domain of RTK family members.

IV. RTKs and Tumorigenesis Tumorigenesis is a complex multistep process, resulting from the accumulation of gain-of-function (or loss-of-function) mutations in genes whose gene products regulate cell growth and/or differentiation. Among the many protooncogenes described to date, a significant number of them encode either growth factors or growth factor receptors (Hunter, 1991). This is, perhaps, not surprising considering the pivotal role members of these two families play in signal transduction processes. The oncogenic capabilities of RTKs have generally been uncovered through one of two mechanisms: capture by an acutely transforming retrovirus (often coupled with subsequent mutation) or as a transforming gene in a 3T3-based transformation assay. In addition, RTKs have been detected at elevated levels in particular types of tumor [e.g., NEU (Slamon et al., 1987, 1989; Hung, 1988; Zhang et al., 1989). Table I lists the RTKs implicated in the etiology of various tumors and the presumed molecular basis for their activation. In summary, in order to transform cells, RTKs may be overexpressed (Slamon et al., 1987, 1989; Hung, 1988; Zhang et al., 1989) and/or inappropriately expressed (Janssen et al., 1991; O’Bryan et al., 1991) or be expressed in a ligand-independent activated form (Martin-Zanca et al., 1986). In this review, several aspects of the oncogenicity of members of the RTK family will be considered. Discussion of the signal transduction pathways downstream of the activated receptor are expressly absent

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PROTEIN TYROSINE KINASE GFR

TABLE I IMPLICATED IN TUMORICENESIS RTK ONCOGENES Type of tumor

Oncogene EGF receptor NeU

trk

i. Squamous cell carcinoma

Amplification (1)

ii. Astrocytoma

Amplification (2)

i. Neuroblastoma (carcinogen induced) ii. Breast carcinoma i. Colon carcinoma ii. Breast carcinoma

RET ROS K-sam arllUF0

Mode of activation

iii. Breast carcinoma iv. Papillary thyroid carcinoma Papillary thyroid carcinoma Astrocytoma Carcinoma of the stomach Chronic myeloid leukemia

Point mutation in

T M domain (3) Amplification (4) Rearrangement (5) Rearrangement (6)* Rearrangement (7)* Rearrangement (8) Rearrangement (8,9) Rearrangement ( 10) Amplification ( 1 1) Overexpression (12)

Note. The involvement of certain RTKs, and their apparent mode of activation, for a number of different tumors is shown. References for each of these relationships are as follows: 1. Ishitoya et al. (1989); 2. Burgart el al. (1991); 3. Bargmann elal. (1986); 4. Slamon et al. (1989); 5. Martin-Zanca et al. (1986); 6. Kozma et al. (1988); 7. Ziemiecki el al. (1990); 8. Bongarzone et al. (1989); 9. Grieco et al. (1990); 10. Fasanoetal. (1984); 11. Nakatanietal. (1990); 12. OBryanetal. (1991);Janssenetal. (1991). The asterisks denote RTKs rearranged as part of the transformation process.

from the material presented here. These important aspects of the subject have been well reviewed in other articles (e.g., Cantley et al., 1991). Instead, this section will focus on the structural alterations associated with the acquisition of oncogenicity by members of this class of proteins. A. RTKs

AS RETROVIRAL ONCOGENES

The capture of RTK sequences into the genomes of acutely transforming retroviruses has been one of the predominant mechanisms by which these molecules become overexpressed or inappropriately expressed. For example, the RTKs erbB (chicken) (Ullrich et al., 1984),sea (chicken) (Smith et al., 1989), kit (feline) (Besmer et al., 1986a), and fm (feline) (Hampe et al., 1982) were first uncovered in this fashion. Both overexpression and mutation appear to be features of the RTK sequences captured by retroviruses. N-terminal truncations, which usually remove a significant portion of the extracellular ligand-binding domain

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ANDREW F. WILKS

are found in v-kit (Besmer et al., 1986a), v-erbB (Ullrich et al., 1984), and v-sea (Smith et al., 1989). This is presumably part of the mechanism whereby the RTK is rendered free of the requirement for its ligand to activate it. T h e absence of a ligand-binding domain is often accompanied by its replacement with viral sequences, such as the gag protein [e.g., ROS (Neckamayer and Wang, 19851 and the e m protein [e.g., sea (Smith et al., 1989)l. There is some limited evidence that mere truncation may be insufficient to render certain RTKs tumorigenic. For example, deletion of the extracellular domain of the insulin receptor was unable to ftransform chicken embryo fibroblasts (Poon et a!., 1991). In contrast, the same deletion with an added N-terminal gag sequence was able to render these cells fully transformed, suggesting that the viral gag component had a role other than the mere provision of a translation start codon (Poon et al., 1991). The C-terminus of an RTK often bears regulatory elements, such as tyrosine autophosphorylation sites (Ellis et al., 1986; Margolis et al., 1989; reviewed in Ullrich and Schlessinger, 1990). Truncation of the Cterminus is thus, not surprisingly, a feature of several retroviral transduction events. For example, the v f m proteins borne by the feline acutely transforming retroviruses SM-FeSV (McDonough et al., 197 1; Donner et al., 1982) and HZ-5 (Besmer et al., 1986b) each bear deletions at their C-termini, compared with the cfm protein. Although these deletions remove negative regulatory elements (Browning et al., 1986; Roussel et al., 1987), they were unable to unleash the transformation potential of c-fms (Roussel et al., 1990), suggesting that the trigger of the ligand-independent signal transmitted by the oncogenic forms of this protein lay elsewhere in the protein. Intriguingly, a single point mutation, in the region of the v-fms gene that codes for the extracellular domain of this protein, was able to confer transforming ability to an otherwise wild-type cfm protein (Roussel et al., 1990). T h e conversion of the wild-type leucine residue, located at position 301 in the extracellular domain of this protein, to a serine, threonine, glutamic acid, or proline rendered the human CSF 1 receptor capable of transforming murine 3T3 cells. Thus, the oncogenic potential of the cfm gene was unmasked by the combination of an N-terminal point mutation, probably coupled with a C-terminal deletion. T h e rigors of capture by a retroviral genome are highly likely to lead to point mutation and deletion in any gene so transduced, and the selection procedure for oncogenic transformations is very sensitive. In the light of such highly mutagenic mechanisms, whether there is a role for RTKs in nonviral tumorigenesis is a legitimate question.

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B. RTKs AS CELLULAR ONCOGENES 1 . Rearrangement of RTK Genes The development of DNA transfection techniques (Graham and Van der Eb, 1973; Shih et al., 1979; Krontiris and Cooper, 1981; Murray et al., 1981; Perucho et al., 1981)provided a significant leap forward in the isolation of many of the genes capable of transforming cells. Several loci encoding proteins with all of features of growth factor receptors were isolated from the DNA of tumors in this way. These include MET (Cooper et al., 1984), NEU (Schechter et al., 1984, 1985; Hung et al., 1986; Bargmann et al., 1986a), trk (Martin-Zanca et al., 1986; Kozma et al., 1988), RET (Takahashi et al., 1985; Takahashi and Cooper, 1987), ROS (Fasano et al., 1984; Birchmeier et al., 1986)and ajcllUF0 (O’Bryan et al., 1991; Janssen et al., 1991) (see Table I). Some RTK loci seem to be, in some degree, intrinsically unstable. Both trk (Oskam et al., 1988; Kozma st al., 1988) and RET (Ishizaka et al., 1989; Koda, 1988) have been demonstrated to undergo an oncogenic rearrangement during the process of transfection into the recipient 3T3 cells. The original trk oncogene (trk-lh)was generated by rearrangement of the trk locus with the gene for nonmuscle tropomyosin (Martin-Zanca et al., 1986). Subsequently, fusion proteins of trk with a portion of the ribosomal protein L7a (trk-2h) (Kozma et al., 1988) and a second unknown protein (Kozma et al., 1988) were also isolated after transfection of NIH/3T3 cells. In the case of the activation of RET, there also appears to be some degree of instability intrinsic to the locus encoding this RTK gene. Initial studies demonstrated that the gene became truncated and activated after, and possibly as a consequence of, the transfection process. In one case, in a human T-cell lymphoma, the RET gene fused with a putative zinc finger protein, fp, to form a fusion protein with PTK activity (Takahashi et al., 1988). Subsequently, the RET gene was found to be rearranged following, but not preceding, transfection of DNAs from a human gastric carcinoma (Ishizaka et al., 1989) and a human colon carcinoma (Koda, 1988). In papillary thyroid carcinoma, however, the bona fide oncogenic potential of the human RET locus was uncovered after transfection into 3T3 cells (Grieco et al., 1990). In this case, whether a truncated RET gene product is formed, or a fusion protein with an as yet uncharacterized 5’ partner, is not known. It is intriguing to note that the location of the most extreme portion of the RET protein that remains in the oncogenic version of this protein is only 13 amino acids N-terminal of the ATP binding site of the catalytic domain and corresponds to the location of a splicejunction (Ishizaka et al.,

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ANDREW F. WILKS

1989). No suitable AUG start codon appears to be present in the portion of the RET protein remaining at the mutated locus, suggesting that this may come from the upstream fusion partner and be spliced into place after transcription. In all cases where a fusion protein is formed from the junction of a RTK and another cellular sequence, the P T K domain is always located at the C-terminus of the protein and never at the Nterminus. 2 . Amplification of RTK Genes Amplification of RTK genes appears to be another mechanism whereby these genes can be rendered oncogenic. T h e oncogene K-sum, for example, was discovered as a component of a highly amplified region of the genome of a stomach-cancer-derived cell line KATO-I11 (Hattori et al., 1990). This gene bears considerable similarity to members of the FGF receptor family (see Section 11). Although highly tentative, the murine gene bek (Kornbluth et al., 1988) appears to be the most similar to the K-sum sequence. Amplification of the K-sum gene appears to be restricted to poorly differentiated types of stomach cancer (Nakatani et al., 1990). T h e normal K-sum transcripts are 4.5-kb mRNA with a minor component at 3.5 kb. Amplification of the K-sum gene results in the highly abundant expression of the 3.5-kb transcript. There does not appear to be any mutation present in the K-sum mRNA that could explain its oncogenicity, and it is presumed that overexpression of this gene is all that is required. Reports of amplification of other members of the FGF receptor family in breast carcinoma (Adnane et al., 1991) confirm the possible importance of this type of event in the etiology of a significant proportion of tumors. In this study, bek andflg were found to be amplified in 11.5 and 12.5% of breast tumors, respectively. In this case, correlation with prognosis had not been accomplished. T h e NEU (c-erbB2 or HER-2) protooncogene was first identified (Schechter et al., 1984, 1985; Hung et al., 1986; Bargmann et al., 1986a) by transfection of DNA from chemically induced neuroglioblastomas in a 3T3 transformation assay similar to that described above. The NEU protein is a member of the EGF receptor family (Class I RTK), and several candidate ligands have been detected for it (Lupu et al., 1990; Tarakhovsky et al., 1991). Remarkably, the mutated, and oncogenic, version of the rat NEU gene differs by only a single point mutation (a valine to glutamic acid alteration in the transmembrane domain) from the normal allele (Bargmann et al., 1986b). In one study, four independent cell lines containing activated rat NEU alleles bore exactly the same activating point mutation (Bargmann et al., 1986b), suggesting that there

PROTEIN TYROSINE KINASE GFR

65

are only a limited number of mutagenic events capable of oncogenically activating this RTK. The human NEU gene also has oncogenic potential. In one particular study, amplification of the NEU gene was detected in 28% of 189 primary breast cancers (Slamon et al., 1987) and a correlation between NEU amplification and poor prognosis was observed. An extended study by the same authors examined the status of the NEU gene in patients with breast cancer or ovarian cancer (Slamon et al., 1989). The amplification of NEU was demonstrated to be a strong indicator of both future relapse and/or survival in these patients. The absence of any alteration in the coding sequence of NEU transcripts isolated from tumors, most notably the activating point mutation detected in the rat system, suggested that the oncogenic potential of the NEU gene product was a function of its overexpression. Moreover, the raised levels of NEU protein detected in these tumors were often not accompanied by amplification of the NE U gene. The demonstration that overexpression of the normal NEU gene could transform 3T3 cells to tumorigenicity provides an in vitro correlate of the in vivo situation (Bargmann et al., 1986b). Overexpression of an otherwise normal receptor thus appears to be the mode of activation of several RTKs. The protooncogene axllUF0 (Janssen et al., 1991; OBryan et al., 1991) is a growth factor receptor uncovered as the transforming gene of two (of eight) (O'Bryan et al., 1991) or a single (Janssen et al., 1991) primary human chronic myeloid leukemias. Overexpression of this gene in murine 3T3 cells appears to be sufficient to transform these cells to a tumorigenic phenotype. In other studies, overexpression of other RTKs results in neoplastic transformation of recipient cells [e.g., EPH (Maru et al., 19901. Although the cognate ligand of the axl or the EPH receptors are unknown it may be that an autocrine loop is established, indeed in the case of the ax1 gene, the overexpression of this gene appears only to be a feature of CML and no other tumors (OBryan et al., 1991), suggesting that a downstream (i.e., intracellular signal transduction event) or an upstream (i.e., ligand binding) event is a feature of this type of cell alone. It may be that coexpression of the ligand for this receptor is thus required before the transforming capacity of the ax1 protooncogene reveals itself. V. Genetics and Cancer

One fascinating example of the transformation potential of RTKs is demonstrated by the Tu locus in Xiphophow hybrids (Wittbrodt et al., 1989). The platyfish X . maculatus and the swordtail X . helleri are related

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species, each of which are normally uniformly gray in coloration. Certain varieties of X . muculatus, however, bear spot patterns (macromeianophores) arising from large accumulations of the melanocytic pigment cells from which they derive their normal grey body colouration. In cross-breeding experiments between grey X . hellerz and spotted X . maculatus, these macromelanophores develop into malignant melanoma. The genetic locus encoding the dominant oncogene Tu, whose activity becomes derepressed in these hybrids, has been demonstrated to be an RTK similar in structure to the EGF receptor. This protooncogene has been designated Xmrk, for X i p h o p h o m melanoma receptor tyrosine kinase (Wittbrodt et al., 1989). The precise genetics of the melanoma produced as a result of the unmasked expression of the Xmrk remain to be elucidated; however, genetic analyses suggest that the oncogenic potential of the Xmrk gene is suppressed in the X . muculatus background by a repressor, “R,” which is absent in the X . helleri background. The Xmrk locus thus encodes an activated RTK whose tumorigenic potential is suppressed in wild-type platyfish. The fine genetics of the Xmrk locus are complex and still remain to be fully resolved. The Xmrk genes are borne on the X and Y chromosome of the platyfish. A third copy is present on one of the autosomes. The oncogenic activation of Xmrk appears to be a consequence of the overexpression of the Xmrk gene, due to transcriptional activation (Adam et al., 1991). In tumor-prone animals, a 5.8-kb transcript from one of the Xmrk genes is accompanied by an apparently tumor-specific 4.7-kb transcript (Adam et al., 1991). The smaller mRNA may be derived from a tumor-specific promotor, or may be the product of premature termination/poIyadenylation of the Xmrk transcript. Thus, since the tumorigenic Xmrk protein is essentially identical to the protein produced in the non-tumor-prone genetic background, the transformation process appears to be dependent solely upon the unregulated expression of this RTK. In the Xzphophorus system, the genetic predisposition of certain strains to melanoma appears to be due to the absence of a protein that controls the expression of the oncogenic form of the Xmrk gene. However, overexpression of RTKs is a feature of several mammalian tumors. Thus, the overexpression of otherwise structurally normal RTKs, such as ax1 (Janssen et al., 1991; O’Bryan et al., 1991),NEU (Slamon et al., 1987, 1989), and EPH (Hirai et al., 1987), may be a product of the mutation of an erstwhile transcriptional regulator. These “indirect” events have yet to be described, but are likely to be a feature of turnongenesis involving RTKs and other protooncogenes.

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ACKNOWLEDGMENTS I thank Dr. Helen Cooper and Dr. Steven Stacker for reading early drafts of this review, and for suggesting improvements.

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THE MOLECULAR AND GENETIC CHARACTERIZATION OF HUMAN SOFT TISSUE TUMORS Colin S. Cooper Molecular Carcinogenesis Section, The Institute of Cancer Research, The Haddow Laboratories, 15 Cotswold Road, Belmont, Sutton. Surrey, SM2 5NG, United Kingdom

I. Introduction 11. Tumor Etiology 111. Genetic Susceptibility to Soft Tissue Tumors

IV. V.

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VII. VIII.

IX. X.

A. Fibromatosis B. Li-Fraumeni Syndrome C. Von Recklinghausen’s Neurofibromatosis D. Sarcoma in Retinoblastoma Families E, Beckwith-Wiedemann Syndrome F. Benign Tumors Detection of YUJ Gene Activation Chromosomal Abnormalities A. Lipomas B. Leiomyomas C. Lipsarcomas D. Synovial Sarcomas E. Rhabdomyosarcomas F. Malignant Fibrous Histiocytomas G. Other Tumor Types Tumor Suppressor Genes A. The p53 Gene B. The RBI Gene C. Studies on Chromosome Loss Gene Amplification Predictors of Tumor Behavior A. Tumor Cell Ploidy B. Multidrug Resistance Molecular Cloning of Translocation Breakpoints Future Developments References Note Added in Proof

I. Introduction Soft tissue is defined as “nonepithelial extraskeletal tissue of the body exclusive of the reticuloendothelial system, glia, and supporting tissue of various parenchymal organs” (Enzinger and Weiss, 1988).This includes 75 ADVANCES IN CANCER RESEARCH, VOL. 60

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smooth and striated muscle, fat, fibrous tissue, and the vessels that serve these tissues. By convention it also encompasses the peripheral nervous system. Soft tissue tumors are a heterogeneous group of tumors that arise as soft tissue masses and usually exhibit the differentiated features of adult soft tissue although in some cases there is no clearly defined normal tissue homolog. The major histogenic categories of malignant soft tissue tumor (Table I) include leiomyosarcoma (smooth muscle), rhabdomyosarcoma (striated muscle), liposarcomas (fat), and malignant peripheral nerve sheath tumors (Schwann cells and perineurial cells). Examples of sarcomas that apparently have no obvious normal counterpart are malignant fibrous histiocytoma and synovial sarcoma. The term malignant fibrous histiocytoma was originally used to describe a group of soft tissue tumors that were thought to be derived from histiocytes (Ozello et al., 1963; OBrien and Stout, 1964; Newland et al., 1975). However, the majority of evidence now indicates that cells from these tumors are more closely related to fibroblasts (Roholl et al., 1985a,b; Wood et al., 1986). The term synovial sarcoma was originally used to describe a group of tumors that commonly occurred around the large joints and that had a microscopic resemblance to normal synovium (Smith, 1927; Knox, 1936). Its origin from synovid tissue has, however, never been proven. The label soft tissue sarcoma can also be extended to include mesotheliomas, extraskeletal Ewings sarcoma, and extraskeletal TABLE I MAJORHISTOLOGICAL CLASSES OF S o n TISSUE TUMORS ~

~

_

_

_

Tumor type

Related normal tissue

Malignant Liposarcoma Leiomyosarcoma Rhabdomyosarcoma Malignant fibrous histiocytoma Fibrosarcoma Deep fibromatosis Synovial sarcoma Malignant peripheral nerve sheath tumor Mesothelioma Extraskeletal chondrosarcoma Angiosarcoma

Fat Smooth muscle Striated muscle Unknown Fibroblasts Fibroblasts Unknown Schwann cells and perineurial cells Mesothelial cells Chondroblasts Endothelial cells

Benign Lipoma Leiomyosarcoma

Fat Smooth muscle

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osteosarcoma but these particular tumors will not be considered in detail in this review. Malignant soft tissue tumors occur at frequencies of around 1-2 per 100,000 population accounting for about 1% of all cancers and 2% of cancer deaths. There have been reports of increases in the incidence of sarcomas but it is not clear whether this genuinely represents an upward trend or whether it is a reflection of improvements in diagnostic procedures and/or greater interest in this tumor type. Soft tissue sarcomas can occur anywhere in the body and at any age although the age distribution varies according to the histological type. For example, rhabdomyosarcomas occur most frequently in children, accounting for 6-7 % of childhood cancer, whereas malignant fibrous histiocytoma occurs predominantly in old age. Benign tumors are at least 100 times more common than their malignant counterparts and represent the most common group of human neoplasms. Benign tumors closely resemble normal tissue, are noninvasive, and are usually not life-threatening. Nonetheless in particular instances (e.g., for leiomyoma of the uterus) they may require surgical intervention. Many of the major conceptual advances in our understanding of the process of malignant transformation have been provided through studies on the transformation of mesenchymal cells (usually fibroblasts) in culture and 6n the induction of soft tissue sarcomas in animal models. Such studies have provided evidence that malignant conversion is a multistep process and in particular have given rise to the concept that immortalization and transformation can represent different steps along this pathway. Thus Newbold and Overall (1983) demonstrated that although primary hamster dermal fibroblasts could be immortalized following treatment with chemical carcinogens an additional transformation step is required to obtain the fully malignant phenotype. Similarly, the experiments of Land et al. (1983) demonstrated that malignant transformation of mesenchymal cells (e.g., primary rat embryonic fibroblasts) can be achieved through cooperation between different classes of activated oncogenes (e.g., the ras and m y genes). Studies on virally and chemically induced sarcomas have, for example, led to the identification and isolation of retroviral oncogenes and provided the original identification of the p53 suppressor protein as a protein overexpressed in chemically induced sarcomas cells (DeLeo et at., 1979). Mesenchymal cells have also been used in developing assays, such as the NIH3T3 mouse fibroblast transfection-transformation assay, that can be used to detect activated oncogenes in human tumors. Although studies on human soft tissue tumors have in general lagged

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behind those on the more common malignancies, such as cancer of the breast, lung, and colon, in the last 5 years a number of key advances have been made in understanding the molecular mechanisms underlying sarcoma development and in the identification of new diagnostic and prognostic indicators for this tumor group. It is well established that certain families show an inherited predisposition to soft tissue tumors and many of the genes involved in these disorders have now been localized and cloned. There have been a number of reports describing the involvement of tumor suppressor genes and dominant transforming genes in sarcoma induction and exciting advances have been provided by cytogenetic analysis demonstrating the presence of specific chromosomal translocations in certain classes of soft tissue tumor. The discovery of these tumor-specific translocations and the identification of genes that control differentiation cloning soft tissue lineages have, in addition, provided new molecular markers that can be used in tumor diagnosis. Finally, studies in which attempts have been made to correlate the presence of specific classes of genetic alteration or abnormalities in gene expression with tumor behavior have identified a number of new prognostic indicators. It is these recent developments that are the subject of this review.

II. Tumor Etiology

Although the factors responsible for the development of the human soft tissue tumors are in most cases still unknown, a variety of causes have been suggested, including exposure to chemical carcinogens and radiation, physical trauma, immunological factors, and constitutional genetic abnormalities. Identification of the precise cause is, however, often extremely difficultbecause of the relative rarity of many individual types of soft tissue tumor and the possibility of long latency periods between exposure and sarcoma development. A small proportion (< 1%) of patients undergoing intensive radiotherapy for breast carcinoma or other malignant tumors develop soft tissue sarcomas within the field of radiation (Laskin et al., 1988). The mean latency period is around 10 years and although any type of soft tissue sarcoma may arise the most commonly observed tumors are malignant fibrous histiocytomas, extraskeletal osteosarcomas, and fibrosarcomas (Laskin et al., 1988). A variety of chemical agents have been suggested as possible causes of soft tissue sarcoma. This is an attractive hypotheses because it is well established that many structurally distinct classes of chemical carcinogens can induce soft tissue sarcomas in animals. There have been somewhat conflicting results about the possible

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association of soft tissue sarcomas and occupational exposure to herbicides and pesticides in, for example, forestry and agricultural workers. An excess of soft tissue sarcoma was found in workers in Sweden (Hardell and Sandstrom, 1979; Eriksson et al., 1981; Wingren et al., 1990), Denmark (Lynge, 1985), Italy (Veneis et al., 1987), and New Zealand (Reif et al., 1989) exposed to phenoxyherbicides and chlorophenols. In contrast, other studies have failed to confirm this finding (Balarajan and Acheson, 1984; Wiklund and Holm, 1986; Hoar et al., 1986; Wiklund et al., 1988). Dioxin exposure has also been suggested as a possible etiological factor because an increased incidence of sarcomas has been reported in some forestry and agricultural workers exposed to dioxincontaining herbicides (Hardell and Sandrom, 1979) and in workers exposed to dioxin in industrial accidents. However, this idea was not supported by case-control studies that failed to demonstrate an elevated incidence of sarcoma in soldiers exposed to the dioxin-containing defoliant Agent Orange (Smith et al., 1984; Kang et al., 1986; Wiklund and Holm, 1986). Furthermore, angiosarcomas of the liver have been attributed to exposure to thorotrast (da Motta et al., 1979; Falk et al., 1979), vinyl chloride (Popper et al., 1978; Falk et al., 1979), inorganic arsenic (Popper et al., 1978), and androgenic steroids (Falk et al., 1979). Preliminary epidemiological studies have indicated that exposure to a variety of other agents including chloramphenicol and chewing tobacco or snuff may also be risk factors in sarcoma induction (Zahm et al,, 1989). Exposure to asbestos, mainly in the form of crocidolite and chrysolite, has been implicated in the induction of mesothelioma in miners and industrial workers and in those who come in contact with asbestos used in insulation, building, etc. (Wagner et al., 1960; Selikoff et al., 1964). There is also some epidemiological evidence suggesting higher incidence of other types of soft tissue sarcoma in workers exposed to asbestos (Wingren et al., 1990). Isolated reports of sarcomas arising at the site of an injury in scar tissue following surgical procedures, at fracture sites, and adjacent to plastic or metal implants have led to the suggestion that trauma or injury may be a factor in determining sarcoma development. More thorough epidemiological studies are, however, required before a firm link between injury and sarcoma induction can be established. A small fraction of cases may result from genetically determined immunodeficiency diseases (Spector et al., 1978) or from immunosuppression following the administration of immunosuppressive drugs during organ transplants (Hoover and Fraumeni, 1973; Kinlen et al., 1979). In addition immunosuppression resulting from HIV infection has been implicated in the development of Kaprosi's sarcoma (Ross et al., 1985). Apart from this

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there is no clear evidence for the involvement of viral agents in the etiology of human soft tissue sarcoma. 111. Genetic Susceptibility to Soft Tissue Tumors There is now a considerable body of evidence demonstrating that genetic factors can predispose toward the development of most types of human cancer including both benign and malignant soft tissue tumors. A genetic susceptibility to cancer development may be recognized in several different ways. In the most dramatic cases families can be identified in which there is a clear excess of cancer. In certain families the predisposition is inherited as a well-defined Mendelian trait associated with a greatly elevated risk of developing cancer. This includes autosoma1dominant disorders such as familial adenomatous polyposis where individuals may develop colon cancer as well as autosomal recessive cancer-prone diseases such as xeroderma pigmentosum where affected individuals have an elevated incidence of skin cancer. Other families may have an unquestionably elevated risk of cancer development but it may be difficult to identify a clear pattern of inheritance. In many cases these families adhere to strictly defined criteria and are unlikely to arise by chance. For example, in the Li-Fraumeni syndrome (see Section II1,B) families exhibit clustering of breast cancer, soft tissue sarcoma, and a variety of other cancers. Finally, even where family clustering is not apparent there may nonetheless be associations of a particular trait with an increased risk of cancer development. T h e association of nasopharyngeal carcinoma with certain HLA types (Simons et al., 1974) and the correlation between ability to metabolize 4-hydroxydebrisoquine and lung cancer risk (Ayesh et al., 1984) are good examples of this. The development of cancer is a multistep process requiring the mutation of several genes for the expression of the fully malignant phenotype. A genetic abnormality associated with an increased risk of cancer development may substitute for one of these mutations, thus reducing the number of genetic events that need to occur during tumor induction. This type of effect is observed in, for example, familial retinoblastoma and familial polyposis coli. Alternatively the abnormality may predispose to the accumulation of mutations, for example, by affecting the metabolism of carcinogenic chemicals or by interfering with DNA repair.

A. FIBROMATOSIS The term fibromatosis refers to fibrous neoplasia that are intermediate in character between benign fibrous lesions and fibrosarcomas.

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These tumors are locally infiltrating with a high rate of recurrence but are never metastatic. Two groups of fibromatosis can be distinguished (Enzinger and Weiss, 1988). Superficial fibromatoses, which are slow growing and usually quite small, arise in the palm, sole of the foot, penis, and knuckle pad. By comparison deep fibromatoses (also referred to as desmoids), which most commonly involve deep structures, are more aggressive than the superficial fibromatosis and have the potential to grow to large size. For both superficial and deep fibromatoses there is evidence for heritable predisposition. Desmoids are found asa complication in around 10%of patients with familial adenomatous polyposis (FAP). Familial adenomatous polyposis is an autosomal dominant disease that affects around 1 in 10,000 individuals. Affected patients develop multiple adenomatous polyps in the colon and rectum that, if not surgically treated, can progress to malignant tumors. The association of FAP with desmoids, osteomata, cutaneous cysts, and other soft tissue tumors such as lipomas and leiomyomas is known as Gardner syndrome (GS). Gardner syndrome is believed to be a variant form of FAP rather than a separate genetic disorder. This hypothesis was initially supported by the observation that these two disorders can occur in different individuals within the same family (McAdam and Goligher, 1970;Jones et al., 1986;Jagelman, 1987) and by the finding that they appear to be linked to the same genetic locus at chromosome 5q21-22 (Hererra et al., 1986; Bodmer et al., 1987; Leppert et al., 1987; Hockey et al., 1989). Several candidate genes that are closely linked to the FAP locus at 5q21-22 have been cloned and sequenced (Kinzler et al., 1991a,b; Nishisho et al., 1991; Groden et al., 1991). Two of these genes, called MCC and APC, were mutated in tumors from sporadic colon cancer patients but only APC was mutated in the germ line of FAP and GS patients. These observations demonstrated that both APC and MCC may be involved in the genesis of colonic cancer and that mutations of the APC gene were responsible for both FAP and GS. Nishisho et al. (199 1) extended these findings by demonstrating that individuals with a G + T transition of codon 302 of the APC gene can either have the range of symptoms associated with GS or have FAP with no evidence of extracolonic manifestations. These observations indicate that the conditions specifically associated with GS may be controlled by other genetic or environmental factors. Clearly it will be important in future studies to identify those factors and to establish the role of the MCC and APC genes in the development of fibromatoses. There have been reports of kindreds containing multiple desmoids as the only manifestation of a genetic abnormality (Zayid and Dihmis, 1969). The existence of these

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families provides further support for the idea that a second genetic locus distinct from APC may predispose toward the development of desmoids. Alternatively, it is conceivable that particular types of mutations of the APC gene predispose toward the development of desmoids without resulting in other symptoms associated with GS. Molecular analysis of the APC gene in these rare desmoid families should resolve this issue. There is also evidence that superficial fibromatoses may, in some cases, arise through a genetic predisposition. Support for this idea is provided by the observation superficial fibromatoses are often bilateral and occur in many generations of the same family (Ling, 1963). However, the genetic abnormalities responsible for such kindreds have not been characterized at the molecular level.

B. LI-FRAUMENI SYNDROME In 1969 Li and Fraumeni carried out a review of archival documents for 648 children who had developed rhabdomyosarcoma, and identified four families in which there was a higher than expected incidence of childhood soft tissue tumors, breast cancer, and other neoplasms (Li and Fraumeni, 1969a,b). Confirmation of the existence of this syndrome (the Li-Fraumeni syndrome) has been provided through prospective observations of the original families, segregation analysis of a consecutive series of children with soft tissue sarcoma, studies on breast cancer among mothers of unselected children with sarcoma, and other descriptive reports (Li and Fraumeni, 1975, 1982; Strong et al., 1987; Li, 1988; Li et al., 1988; Birch, 1990). T h e spectrum of cancers associated with the Li-Frdumeni syndrome is now known to include soft tissue sarcoma, breast cancer, brain tumors, osteosarcomas, leukemia, and adrenocortical carcinoma and possibly also melanoma, germ cell tumors, lung cancer, prostate cancer, and pancreatic cancer. Frequently multiple tumors are observed in individual family members and tumors may have an unusually early onset. Difficulties encountered in carrying out linkage analysis on LiFraumeni families due to the relative rarity of the families and high mortality prompted Malkin et al. (1990) to examine the p53 suppressor gene as a possible candidate. T h e p53 gene was selected because previous studies had shown that it was inactivated in sporadic forms of many of the tumors associated with the Li-Fraumeni syndrome. Remarkably mutations in the p53 gene were found in all five families examined in this study and in a single Li-Fraumeni family examined by Srivastava P t al. ( 1990), demonstrating that inherited mutations on the p53 gene are

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probably responsible for the Li-Fraumeni syndrome. An example of one of the families examined in these studies in shown in Fig. 1. Mutations of the p53 gene in sporadic tumors occur at a variety of sites but are most commonly found in exons 5-9. By comparison the mutations initially reported for the Li-Fraumeni syndrome appear to have a more restricted distribution since all were located within exon 7 of the p53 gene at positions 245,248,252, and 258. These mutations are found together with a normal copy of the p53 gene in noncancerous cells and presumably do not interfere with growth and development of the individuals carrying them. Furthermore the levels of p53 found in fibroblasts from members of Li-Fraumeni families were low compared to those usually found in tumor cell lines containing p53 mutations. The presence of the altered p53 gene appears to have a predisposing effect in carcinogenesis.Consistent with this view Bischoff et at. (1991) found that fibroblasts from Li-Fraumeni patients, in contrast to those from normal controls, become immortal spontaneously when grown in culture.

C. VON RECKLINGHAUSEN’S NEUROFIBROMATOSIS Von Recklinghausen’s (Type I) neurofibromatosis (NF1) is a disease that affects an estimated 1 in 3500 individuals. This autosomal dominant

Li-Fraumeni family

BR.30

BR.28

STS.l Brain.5 OS.8

FIG. 1. Li-Fraumeni pedigree. Symbols represent females with cancer (O),females males with cancer males without cancer (0),and the presence of a without cancer, (0), constitutional mutation the p53 gene (*). The abbreviations used are BR, breast; STS, soft tissue sarcoma; and OS, ostosarcoma. Ages at which the tumors arose are indicated. Adapted from Malkin et al. (1990).

(m),

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disorder is characterized by the presence of cafe-au-lait spots, skin neurofibromas, and pigmented iris hamartomas called Lisch nodules. In addition, a variety of other abnormalities may also be observed in a proportion of cases (Ponder, 1990). Around 5 % of individuals with NF1 develop Schwannomas of the peripheral nerves and 2% of cases develop malignant peripheral nerve sheath tumors (MPNST). The locus (called NFl) that is responsible for this disease was originally mapped to the long arm of chromosome 17 (17q11.2) (Barker et al., 1987; Seizinger et al., 1987) and a candidate gene has now been cloned and characterized (Cawthon et al., 1990; Viskochil et al., 1990; Wallace et al., 1990). Sequence comparisons have shown that the NF1 gene encodes a protein with substantial homology to mammalian GAP that is responsible for activating the GTPase activity of ras proteins. Germ line mutations of the NFI gene have been detected in NFl patients and it will be interesting to assess the role of this gene in the development of neurofibromosis and MPNST in von Recklinghausen’s patients as well as in tumors occurring spontaneously. IN RETINOBLASTOMA FAMILIES D. SARCOMA

Retinoblastoma is an intraocular eye tumor that is usually found in children under 5 years of age. Like many cancers both sporadic and hereditary forms can be distinguished. The sporadic disease accounting for around 70% of cases is characterized by solitary tumors with an average age of onset of 18 months. By comparison the hereditary form is almost always bilateral and multifocal (four to five tumors per eye) with a mean age of onset of 10 months. Around 10-20% of patients with the hereditary disease develop secondary tumors later in life (Draper et al., 1986; Derkinderen et al., 1988). Osteosarcoma is the most common secondary tumor in the decade following the presentation of retinoblastoma, whereas soft tissue sarcomas are more frequent later in life. Knudson (1971) noted that the number of tumors that developed in hereditary cases followed a Poisson distribution, suggesting that a single random event was required for transformation of a retinal cell in those individuals. On the basis of this observation he proposed that two events were required for tumor development in both the hereditary and the nonhereditary forms. In hereditary retinoblastoma one hit or mutation is carried in the germ line; only a single additional hit is therefore required for tumor development. By comparison, in the sporadic retinoblastoma both hits must be acquired by somatic mutation. The location of the gene mutated in the germ line in hereditary retinoblastomas was deduced from the presence of constitutional deletions of chromo-

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some 13q14 in a small proportion of patients (Knudson et al., 1976; Cowell et al., 1986) and from genetic linkage studies (Sparkes et al., 1983).Examination of chromosome abnormalities in retinoblastoma further suggested that the second hit may involve the same region of chromosome 13 (Balaban-Malenbaum et al., 1981). The gene located at 13q14 that is responsible for the inherited predisposition to the development of retinoblastoma in children and osteosarcoma and soft tissue sarcoma in adults has now been cloned and characterized (Friend et al., 1986; Fung et al., 1987). This gene (called RB1) appears to play a key role in controlling transition through the cell cycle and has the functional attributes of a tumor suppressor gene. Thus its introduction into human cell line lacking functional RB 1 protein can result in suppression of the malignant phenotype (Huang et al., 1988). E. BECKWITH-WIEDEMANN SYNDROME

Beckwith-Wiedemann syndrome (BWS) is a condition characterized by an enlarged tongue and abdominal organs, neonatal hypoglycemia, and atypical cranio-facial features. The 1lp15 region of human chromosome 11 seems to contain a gene or genes involved in determining this condition and in the genesis of several types of childhood tumors, including Wilms’ tumor, rhabdomyosarcoma, and hepatoblastoma, that are associated with BWS (Sotelo-Avila et al., 1980). Support for the importance of this chromosomal region has been provided by reports of cytogenetically identifiable germ line deletions of the short arm of chromosome 11 in BWS patients (Pettenati et al., 1986) and by studies demonstrating linkage to genetic markers from 1lp15 in BWS families (Koufos et al., 1989; Ping et al., 1989).Further support is provided by the observation that uniparental paternal disomy (both alleles at a particular locus derived from a single parent) frequently occurs in the 1lp15 region in BWS patients (Henry et al., 1991). The potential importance of a gene located at the end of the short arm of chromosome 11 in the development of soft tissue tumors has also deduced from analysis of allele loss in rhabdomyosarcoma (see Section V1,C). F. BENIGNTUMORS 1 . Lipomas

There is also good evidence that genetic factors can predispose toward the development of benign soft tissue tumors such as lipomas and leiomyomas. Lipomas are benign tumors that differ little in microscopic

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appearance from surrounding fat. T h e cutaneous or superficial lipomas are most common, occurring predominantly in the regions of the back, shoulders, upper neck, and abdomen. Approximately 1 in 20 individuals with lipomas have multiple tumors and in a small proportion of patients with this condition, there is evidence of a hereditary trait that has features of autosomal dominant transmission (Humphrey and Kingsley, 1938; Kurzweg and Spencer, 1951; Shanks et al., 1957; Stephens and Isaacson, 1959; Enzinger and Weiss, 1988). An example of a multiple lipoma family where three successive generations are affected is shown in Fig. 2. In patients that have multiple lipomas or the related angiolipomas, apparently without a family history, it is possible that nonhereditary factors account for the multiple tumors. Alternatively it is possible that they represent a less penetrant form of genetic predisposition. There is no evidence indicating that multiple lipoma families have a excess of liposarcoma o r of other types of cancer. 2. L e i o m y o m

Leiomyomas are benign tumors that show the differentiated features of smooth muscle. Leiomyomas are found most commonly in the uterus where they pose a major clinical problem and are known as fibroids. It has been proposed that there is an inherited predisposition to developing uterine leiomyomas but it is extremely difficult to prove this for a tumor that may be present in up to one in four women. More rarely leiomyomas, either solitary or multiple, may be found in the skin of young adults where they frequently arise from the pilar erector muscles. Multiple liporna family

I

m

FIG. 2. Multiple lipoma family pedigree. Symbols represent affected males (B)and females (0).and unaffected males (0)and females (0).

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For a proportion of patients with multiple leiomyomas there is evidence of an inheritance as an autosomal dominant tract (Kloepfer et al., 1958; Fisher and Helwig, 1963; Verma et al., 1973). Multiple leiomyomas have however been associated with a variety of other conditions including HLA-B8, dermatitis herpetiformis, and Type I multiple endocrine adenomatosis. VI. Detection of ras Gene Activation

DNA transfection into NIH3T3 mouse fibroblasts has been used extensively to screen human tumors for the presence of dominantly acting oncogenes. In this technique tumor DNA is introduced into NIH3T3 cells as a coprecipitate with calcium phosphate and cultures are monitored for the appearance of foci or morphologically transformed cells that are formed when the mouse cells stably incorporate an activated oncogene. The majority of the genes detected using this assay are members of the ras gene family (H-, K-, and N-) although non-ras gene, such as met, raf, trk, and ret are occasionally observed (Hall, 1990). A selection of 46 soft tissue tumors and tumor cell lines that includes all the major classes of adult tumors has been examined using this assay (Krontiris and Cooper, 1981; Perucho et al., 1981; Pulciani et al., 1982; Marshall et al., 1982; Fukui et al., 1985; Stanton and Cooper, 1987; Gill et al., 1991). Activated K-ras was detected in a primary embryonal rhabdomyosarcoma (Pulciani et al., 1982) and in a primary leiomyosarcoma (Gill et al., 1991), whereas activated N-ras was detected in the RD rhabdomyosarcoma cell line (Hall et al., 1983; Chardin et al., 1985) and in the HT1080 fibrosarcoma cell line (Hall et al., 1983). In one study an activated gene apparently unrelated to ras was transferred from a leiomyosarcoma but the detailed characterization of this gene has not been reported (Fukui et al., 1985). In a more recent study, a non-ras gene was transferred from a liposarcoma (Gill et al., 1991). Cloned regions of this gene were localized to human chromosome 19 and were found to detect a 3-kb transcript in most human cell lines. Stratton et al. (1989a) used a different approach to screen a series of childhood rhabdomyosarcoma for the presence of activated ras genes. In this study DNA was prepared from formalin-preseved tumors from histopathological archives and the regions of the ras genes where activating mutations occurred (codons 12, 13, and 61) were amplified using the polymerase chain reaction (PCR). Mutant ras genes were detected by hybridization of amplified DNA to specific oligonucleotide probes. Using this approach mutated versions of N-ras and K-ras were detected in 35% (5/14) of embryonal rhabdomyosarcomas. It was also noted that

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a high proportion of tumors located around the urogenital tract (5/7) contained ras genes mutations, whereas mutations were not observed in tumors from other locations (0/7). When taken together these studies demonstrate that ras activation is implicated in the development of childhood rhabdomyosarcorna but is a relative rare event in major classes of adult tumors. V. Chromosomal Abnormalities

Because of problems such as low mitotic index and difficulties in obtaining satisfactory quality of mitotic figures, cytogenetic analyses of human solid tumors have lagged behind analogous studies on leukemia. However, recent important rnethological improvements have resulted in the identification of several specific chromosomal abnormalities in solid tumors. The methodological advances include (a) procedures for tissue dissagregation by prolonged incubation with collagenase that allow tumor cells to be grown in culture for a few days to increase the mitotic index; (b) improved methods of preparing mitotic figures; and (c) improvements in banding procedures (Gibas et al., 1984; Limon et al., 1986d; Trent et al., 1986). One of the most exciting consequences of these developments has been the discovery of specific chromosomal translocations that are unique to certain classes of soft tissue sarcoma. Moreover, these translocations commonly occur in a high proportion of tumors and are frequently found as the only karyoptypic abnormality. Cytogenetic analysis of leukemia is widely used in diagnosis and in distinguishing between subgroups of this disease that have different prognoses. Similarly the discovery of specific translocations in soft tissue tumors offers the prospect of their use in tumor diagnosis and prognosis. Where consistent chromosomal translocations have been documented in other classes of human neoplasm they usually correspond at the molecular level to rearrangements involving particular genes. For example, the translocation involving chromosomes 8 and 14 found in Burkitt’s lymphoma involves the c-myc protooncogene on chromosome 8 and the immunoglobin heavy chain locus on chromosome 14 (Dalla Favera et al., 1982; Croce et al., 1985). Similarly the t(9:22) translocation detected in almost all chronic myelogenous leukemia arises through the joining of c-abl locus on chromosome 9 to the bcr locus on chromosome 22 (Witte, 1986). By analogy it is assumed that the translocations found in soft tissue tumors correspond at the molecular level to alterations of particular genes and a major objective of work in this area is to clone and characterize the genes involved in these translocations. This work would not only provide important insights into the molecular mechanisms of

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induction of soft tissue sarcoma but would also yield molecular probes that could be used to detect the translocations and hence aid in diagnosis. A. LIPOMAS From karyotype analyses carried out on over 50 lipomas it has become apparent that four cytogenetically distinct groups can be distinguished. (a) Around 30-35% of tumors have normal karyotype. (b) Another 3035% contain a reciprocal translocation involving the q13-14 region of chromosome 12 (Heim et al., 1986, 2988a; Turc-Care1 et al., 1986c, 1988c; Mandahl et al., 1987, 1988a; Sreekantaiah et al., 1990a,b,c, 1991b).Although chromosome 12 can have many different translocation partners, a reciprocal translocation involving chromosomes 3 and 12 [t(3;12)(q27-28;q13-14)]has been observed repeatedly (Fig. 3). Genes located around the q13-14 region of chromosome 12, which are therefore considered as candidates for involvement in these translocations, include the int-1 and gli protooncogenes, and the gene encoding the receptor for apolipoprotein E. Southern anaylses of DNA have, however, failed to reveal rearrangements of these genes in lipomas containing abnormalities of 12q13-14 (Arheden et al., 1989b; Myklebost et al., 1989; Paulien et al., 1990). ( c ) Fifteen percent of lipomas contain ring chromosomes (Heim et al., 1987, 1988a; Mandahl et al., 1987, 1988a; Turc-Care1et al., 1987a).Although the origin of these ring chromosomes has not been determined unequivocably, it has been proposed that they arise through the end-to-end joining of the 3q+ chromosome that is formed as a consequence of the 3;12 translocation (Heim et al., 1986). ( d ) Finally, a small proportion of lipomas exhibit other classes of abnormality (Sandberg et al., 1986; Mandahl et al., 1987, 1988a; Heim et al., 1988a;), including possible recurrent abnormalities of 6p (Sait et al., 1989b; Sreekantaiah et al., 1990a) and 13q12-13 (Sreekantaiah et al., 1989). Although the origin of the variant translocations involving 12q13-14but not 3q27-28 has not been determined, it is conceivable that they correspond at the molecular level to rearrangements between a specific gene on chromosome 12 and selection of other genes located at different sites on the human chromosome. Alternatively it is possible that the variant translocations involve rearrangements of chromosomes 3 and 12 that are masked at the molecular level. In this regard it is worth while to note that a parallel situation exists in chronic myelogenous leukemia (CML) where at the molecular level translocations always involve the c-abl protooncogene on chromosome 9 and the bcr gene on

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23

22 21 14

13 12 11 11

12 13

c

21

22 23 24

15 21

25

22

8

26 23

c

27

28 29

3

24

M

U

12

der(l2)

E

der(3) FIG. 3. Schematic presentation of the specific chromosomal translocation between chromosomes 3 and 12, t(3;12)(q27-28; q13-14). found in lipomas. The translocation occurs at the positions indicated by the arrows and gives rise to the der(3) and der(l2) chromosomes.

chromosome 22. In contrast the involvement of chromosome 9 in CML is not always apparent at the cytogenetic level (Dube et al., 1989). Studies on multiple lipoma have been undertaken to determine the origin of the individual tumors. In one study three lipomas from the same region of a single patient were examined (Dal Cin et al., 1988~). Two contained the t(3;12) translocation shown in Fig. 3 and the third contained a 1:12 translocation that also involved the q13-14 region of chromosome 12. These observations were thought to provide support

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for the idea that each lipomas arises independently rather than by spread from a single tumor. Heterogeneity has been observed between different regions of a single lipoma. Thus Sreekantaiah et al. (1991a) found that a biopsy from a lipoma in a 64-year-old man contained three abnormal clones. One clone contained a t( 12;21) translocation, the second contained t(2; 12) and t( 19;20)translocations, and the third contained a ring chromosome. In a subsequent specimen the predominant abnormal clone contained the same t(2; 12) translocation but, in addition contained a t(3; 11) translocation and a ring chromosome. B. LEIOMYOMAS Cytogenetic studies of uterine leiomyomas reveal that the majority of tumors (30-80%) appear to be karyotypically normal, whereas those tumors exhibiting cytogenetic abnormalities can be divided into a number of distinct groups. Twenty-five to 30% of tumors with clonal alterations contain reciprocal translocations. Most commonly these involve 12q14-15 usually as t(12; 14)(q14-15;q23)(Fig. 4) but occasionally

i1

;; @

a -

c

22 21 15

der( 12)

23

22

24 31 32

24

12

14

der( 14) FIG. 4. Schematic presentation of the specific chromosomal translocation between chromosomes 12 and 14, t(12;14)(q14-15;q23), found in leiomyonas. The translocation occurs at the positions indicated by the arrows and gives rise to the der(l2) and der(l4) chromosomes.

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COLIN S. COOPER

as translocations with other chromosomes (Heim et al., 1988b; Nilbert et al., 1988b; Turc-Care1 et al., 1988b; Vanni and Lecca, 1988; Mark et al., 1989a; Sait et al., 1989a; Vanni et al., 1989, 1991; Nilbert and Heim, 1990; Kiechle-Schwarz et al., 1991; Rein et al., 1991). High-resolution mapping of this translocation has recently defined the breakpoints in chromosomes 12 and 14 to 12q15 and 14q24.1 (Pandis et al., 1990). Translocations involving 12914-15 are frequently observed as the only karyotypic abnormality, indicating that they may represent a primary event in tumor formation. Southern analysis of tumor DNA has been used to assess the involvement of the int-1 and gli protooncogenes and the COL2Al collagen gene, which all map around 12q13-14, but no alterations of these genes were detected (Arheden et al., 1989a). It is also worth while to note that a translocation involving chromosomes 1 and 2, [t(1 ;2)(p36:p24)], has recently been reported in a small proportion of tumors (Mark et ai., 1989a). Other classes of primary cytogenetic abnormality that are also observed, often as the only karyotypic alteration, include alterations of 6p, trisomy of 12, and interstitial deletions of the long arm of chromosome 7 (Boghosian et al., 1988; Nilbert et al., 1988c, 1989b, 1990b; Mark et al., 1989a; Sait et al., 1989a; Nilbert and Heim, 1990; Kiechle-Schwarz et al., 1991; Rein et al., 1991; Vanni at al., 1991). Together these account for a further 50% of leiomyomas containing clonal abnormalities. Deletions of the long arm of chromosome 13 have also been reported as the only cytogenetic abnormality in two leiomyomas, suggesting that in some cases this alteration may represent an important event (Meloni et al., 1991). A variety of nonrecurrent translocations account for the remaining 20-25% of abnormalities (Gibas et al., 1988; Mugneret et al., 1988; Nilbert et al., 1988c, 1989a; Mark el al., 1989a). Ring chromosomes and alterations of chromosome 1 are also found but these usually occur together with other cytogenetic abnormalities and are therefore considered to represent secondary changes (Nilbert et al., 1988b). Other recurrent chromosomal abnormalities include monosomy 22 (KiechleSchwartz rf ai., 1991) and changes of chromosomes 2 and 19 (Mark et al., 1990; Vanni et al., 1991). Karyotypes of tumors from individuals with multiple leiomyomas in a single uterus have been obtained (Mark et al., 1989b, 1991; Nilbert et al., 1989b). In some individuals different abnormalities were observed in two or more leiom yomas suggesting that individual tumors are entirely independent. In other patients similar abnormalities were observed in separate leiomyomas from a single patient, apparently indicating that independent leiomyomas may in fact arise from the same initial neoplastic clone. An alternative explanation suggested by Mark et al. (1991)

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HUMAN SOFT TISSUE TUMORS

is that similar patterns of cytogenetic abnormalities could arise from the involvement unidentified etiological factors that favor particular chromosomal alterations. In support of this idea he pointed out that abnormality involving deletion of the region of chromosome 7 that they observed in two leiomyomas from a single patient was also detected in tumors from other individuals. These cytogenetic studies are of considerable interest because together with the studies on lipomas described above they show that clonal chromosomal abnormalities are not restricted to malignant tumors and that the presence of cytogenetic alterations cannot, by itself, be used as an indication of malignancy.

C. LIPOSARCOMAS Cytogenetic analysis of these malignant proliferations has identified a specific translocation involving chromosomes 12 and 16, t( 12;16) (q13:pl l), that is frequently found as the only cytogenetic abnormality (Limon et al., 1986a; Turc-Care1et al., 1986d; Mertens et al., 1987; Smith et al., 198713; Bridges et al., 1988; Walters et al., 1988). Liposarcomas, which occur primarily in adult life between ages 40 and 60 years, are classified into four categories based on histological appearance: myxoid, round cell, well differentiated, and pleomorphic (Enzinger and Weiss, 1988). The t(12;16) translocation (Fig. 5 ) is found in around 50% of 13 12

11 11 12 13

c

13 14

12

15

1-1

21

11 12 13 21

22 23

22 23 24

24 ~

12

der(l2)

16

der(l6)

FIG.5. Schematic presentation of the specific chromosomal translocation between chromosomes 12' and 16, t(12;16)(q13;pl l), found in liposarcomas. The translocation occurs at the positions indicated by the arrow and gives rise to the der(l2) and der(l6) chromosomes.

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COLIN S. COOPER

myxoid tumors but is apparently not present either in the other histological categories of liposarcoma or in other types of myxoid tumor (e.g., myxoid chondrosarcomas). Recently the chromosomal breakpoints of the t(12;16) have been localized to subbands 12q13.3 and 16pl1.2 (Ener0th et al., 1990). Furthermore it has been suggested that trisomy of chromosome 8 may represent a nonrandom secondary change in myxoid liposarcoma (Sreekantaiah et al., 1991~). T h e region of chromosome 12 involved in this translocation is similar, perhaps identical, to that commonly found in lipomas. This observation raises the possibility that the same gene located on chromosome 12 might be implicated in the development of both benign and malignant adipose tissue tumors. Furthermore the genes mentioned above that are located on chromosome 12 and are candidates for involvement in the lipoma and leiomyoma translocations are also candidates for involvement in the t( 12; 16) translocation. Southern analysis of DNA from liposarcomas containing the t( 12;16) translocation has, however, failed to reveal rearrangement in the int-1 protooncogenes (Turc-Carel et al., 1987~).In addition Paulien et nl. (1990) used pulsed-field gel electrophoresis to construct a 600-kb restriction map spanning the gli protooncogene locus at 12q13-14. Their analysis demonstrated that both the liposarcoma 12:16 breakpoint and the lipoma 12q13-14 breakpoint (see Section V,A) lie outside this region. However, they also found that myxoid liposarcoma DNA contained altered restriction fragments detectable with gli probes that were highly specific and reproducible from case to case. These altered fragments resulted from highly specific and reproducible methylation differences that were unique to myxoid liposarcoma DNA. It was also suggested that those methylation differences might provide a useful diagnostic tool for distinguishing different subtypes of liposarcoma. I t is generally believed that liposarcomas are derived from primitive niesenchymal cells rather than mature fat cells and only very rarely arise by progression from existing lipomas. Support for this hypothesis is provided, for example, by the observation that liposarcomas occur only very infrequently in subcutaneous fat, which is a common site of lipomas (Enzinger and Weiss, 1988). Cytogenetic studies also provide support for the idea that liposarcomas are not derived from lipomas because the types of karyotypic abnormalities found in liposarcomas are not the same as those found in lipomas. In an attempt to assess the role of chromosomal abnormalities in the progression of myxoid liposarcomas Orndal et al. (1990) determined the karotype of different regions of a single tumor that exhibited histological heterogeneity. In one of three myxoid nodules and in the surrounding “lipoma-like” tissue the translocation t( 12; 16)(q13;pl1) was

HUMAN SOFT TISSUE TUMORS

95

found as the only chromosomal abnormality. In the other two nodules additional alterations involving chromosomes 1, 12, and 16, which were probably secondary to the t( 12;16) translocation, were also found. The presence of these more complex alterations in regions of the tumor that had a more malignant histology led the authors to suggest that acquisition of secondary chromosomal aberrations may arise during tumor progression.

D. SYNOVIAL SARCOMAS Synovial sarcomas occur most commonly in young adults between ages 15 and 40 and are usually found at the extremities in the vicinity of the large joints (Enzinger and Weiss, 1988). Although estimates of its incidence vary, it is generally considered to account for 5-10% of soft tissue tumors. Two histologically distinct pattern of synovial sarcoma can be distinguished: the biphasic pattern, containing both epithelial-like cells and spindle cells, and the monophasic pattern, containing only spindle cells. Cytogenetic studies have demonstrated that a specific translocation involving chromosome X and 18 is found in a high proportion of both biphasic and monophasic synovial sarcoma (Limon et al., 1986d, 1989; Turc-Care1et ah, 1986b, 1987b; Smith et al., 1987a; Griffin and Emanuel, 1987; Noguera et al., 1988; Ueda et al., 1988; Nojima et al., 1990b). This translocation (Fig. 6) can be found as the only karyotipic abnormality. Usually chromosomes X and 18 are involved in a simple reciprocal translocation [t(X;18)(pl1.2;ql1.2)] giving rise to the der(X) and der( 18) chromosomes shown in Fig. 6. However, complex translocation involving a third chromosome in addition to chromosomes X and 18 are occasionally observed; for example, the (X;18;21)(pl1.2;ql1.2;p13) translocation detected by Turc-Care1et al. (1987b).There have also been reports of the X;18 translocation in a fibrosarcoma (Mandahl et al., 1988b) and in a malignant fibrous histocytoma (Turc-Care1et al., 1987b) but the significance of these reports is unclear because it is often difficult to distinguish these tumors from the monophasic variant of synovial sarcoma. Genes located around the position of the translocation on the X chromosome that are considered candidates for involvement in the X; 18 translocation include the c-elk and hA-raf protooncogenes, a gene called A1S9 that is involved in cell cycle control, the gene encoding a protein designated TIMP that has a dual role of inhibitor of metalloproteinase and as an erythroid potentiating factor, the gene encoding on the TFE3 helix-loop-helix transcription factor, and an anonymous gene designated SB1.8 (Huebner et al., 1986; Rao et al., 1989; Beckmann et al.,

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COLIN S . COOPER

22.3 22.2 1

n. 21 11.4 11.3 11.2 11.1 11 12 11.32 11.31

13

11.2 11 1 11.1 11.2 12.1 12.2 12.3 21.1 21.2 21.3

21

22

23 24 25

26 27

22

28

23

X

der(X)

18

der(l8)

FIG.6. Schematic presentation of the tumor-specific translocation t(X, 18)(p11.2; q l l . 2 ) , found in a high proportion of synovial sarcomas. The arrows indicate the position of the translocation.

1990; Zacksenhaus et al., 1990). Other genes located in these regions are those encoding, synapsin I and monoamino oxidase (Human Gene Mapping 10, 1989). There is, however, currently no evidence that any of these genes are altered in synovial sarcomas. Conflicting reports have been obtained in attempts to map the position of the breakpoint relative to representative genes and markers on the X chromosome. Both Reeves et at. (1989a,b) and Gilgenkrantz et al., (1990) prepared hybrids containing the der(X) chromosome in the absence of other material from human chromosomes X and 18 by fusing synovial sarcoma cells to rodent cells. In the der(X) examined by Reeves et d. (1989a) the breakpoint was mapped close to the centromere between two markers designated DXS146 and DXS14. By comparison the der(X) examined by Gilgenkrantz et al. (1990) appears to contain the breakpoint further away from the centromere between markers DXS146 and TIMP. These differences might reflect heterogeneity of the breakpoint on the X chromosome or could result from a secondary rearrangement or deletion in one of the der(X) chromosomes. To distinguish between the possibilities additional tumors must be examined.

HUMAN SOFT TISSUE TUMORS

97

E. RHABDOMYOSARCOMAS Rhabdomyosarcoma is the most common soft tissue sarcoma in children and adolescents. In the majority of cases the tumors can be assigned to one of three histological groups (Enzinger and Weiss, 1988). However, it is difficult to draw sharp divisions between the different groups, and for some individual tumors it is not clear in which group they should be placed. Embryonal rhabdomyosarcoma, which mainly affects children under 15 years of age, accounts for around 70% of all rhabdomyosarcomas. Alveolar rhabdomyosarcoma, which occurs predominantly in subjects between 10 and 25 years of age, are less frequent, accounting for 20% of tumors. Finally, pleomorphic rhabdomyosarcomas, the rarest group, occur predominantly in mature adults. Cytogenetic analyses of rhabdomyosarcomas have revealed that one of the most common karyotypic abnormalities is a reciprocal translocation involving chromosomes 2 and 13, t(2; 13)(q37;q14) (Seidal et al., 1982; Garvin et al., 1986; Turc-Care1 et al., 1986a; Lai et al., 1987; LizardNacol et al., 1987; Rowe et al., 1987; Douglass et al., 1987; Engel et al., 1988; Wang-Wuu et al., 1988; Nojima et al., 1990a). This translocation (Fig. 7) is frequently observed as the only cytogenetic abnormality, suggesting that it may be a primary pathological event, but in one study it was observed in late but not in early passages of a cultured rhabdomyosarcoma, indicating that it could also play a role in tumor progression. The t(2; 13) translocation is observed in around 50% of alveolar rhabdomyosarcomas, but there are isolated reports of its occurrence in embryonal tumors and in tumors designated undifferentiated (Douglass et al., 1987). Furthermore the derivative chromosome 13 is absent in many tumors, indicating that it is the formation of der(2) that is important in tumor development. Variant translocations have been detected in an alveolar tumor, t(2; 1l)(q37; 13), and in an embryonal tumor, t(2;8)(q37;q13) (Whang-Peng et al., 1986; Hayashi et al., 1988), whereas constitutional translocation involving 2q37, t(2;5)(q37;q3l), has been observed in a patient with embryonal rhabdomyosarcoma (Morijama et al., 1986). Although embryonal rhabdomyosarcomas have no common structural abnormality Wang-Wuu et al. (1988) found trisomy 2 in all eight tumors examined in their study. In addition, in these eight tumors seven had +20, six had + 13, six had +8, five had + 11, and five had + 19. Two different approaches have been used to map the breakpoint relative to markers from chromosome 13. Mitchell et al. (1991) created somatic cell hybrids containing the derivative chromosome 13. This derivative chromosome hybridized to the loci p7F12 and pHUlO but failed

98

COLIN S. COOPER 25 24 23 22 21 16

15 14 13

;; %?

12 11 11 12 13 14

!I

der( 13)

21 22

22

23

31

24

32 33 34

31

13

32 33 34 35 36

37

2

der(2) FIG. 7. Schematic presentation of the specific chromosomal translocation t(2;13) (q37;q14)foundin rhabdomyosarcoma.The arrows indicate the position of the translocation.

to hybridize to the esterase D and RB1 gene probes and to pG14E1.9 and pG24E6.8 probes. When considered together with order of these loci deduced from genetic linkage studies, these studies indicated that the breakpoint occurs between pG14E1.9 (DS1322) and either p7F12 (D1351) or pHUlO (D1356). These conclusions are in agreement with the results obtained by Valentine et al. (1989), who has used in situ hy-

HUMAN SOFT TISSUE TUMORS

99

bridization to place the breakpoint distal to pHUlO and p7F12 but proximal to esterase D and RB 1. Barr et al. ( 1991) examined the involvement of five groups of candidate genes mapped to either chromosome 2 or 13. These included the genes for signal transduction proteins (RB1, inhibin a, FLT1, and Hox4B), muscle-specific proteins (myosin light chain, desmin, nictonic cholinergic receptor subunits y and 6), extracellular matrix proteins (collagen type V1 a 3 chain, elastin, and fibronectin), transformation-associated products (intestinal alkaline phosphatase and Lplastin), and esterase D. Southern blot analysis of DNA separated on conventional gels or by pulsed-field gel electrophoresis failed to detect rearrangements of these genes. HISTIOCYTOMAS F. MALIGNANTFIBROUS There have been a number of studies reporting karyotypic abnormalities in MFHs (Bridges et al., 1987, 1990; Genberg et al., 1989; Mandahl et al., 1985, 1989; Rydholm et al., 1990; Kanzaki et al., 1991) but perhaps the most interesting observation was the identification of a rearrangement involving 1 9 ~ 1 in 3 primary tumors. Other abnormalities include ring chromosomes, translocations and deletions of l l p and 3p, and the presence of the cytogenetic signs of gene amplification (homogeneously staining regions and double minute chromosomes). Furthermore, there appeared to be a correlation between the presence of the 19p marker chromosome and tumor behavior, since distant metastases and/or local recurrence occurred in 8 of the 9 patients with the 19p+ marker but in only 4 of the 13 without (Mandahl et al., 1989; Rydholm et al., 1990). The only other cytogenetic parameter that appeared to affect tumor relapse was the presence of ring chromosomes; relapse was found in 2 of 8 patients with ring markers compared to 10 of 14 patients without. +

G. OTHERTUMOR TYPES Cytogenetic studies have also been carried out on other tumor types, including fibrosarcomas (Adam et al., 199l), chondrosarcomas (Hinrichs et al., 1985; Turc-Care1et al., 1988a; Fletcher et al., 1989; Bridges et al., 1989; Mandahl et al., 1990), and leiomyosarcomas (Limon et al., 1986b; Dal Cin et al., 1988a,b,d; Nilbert et al., 1988a, 1990a,c; Sait et al., 1988; Boghosian et al., 1989; Mark et al., 198913; Sreekantaiah and Sandberg, 1991). It has been suggested that leiomyosarcomas can be divided into three karyotypically distinct groups: a pseudodiploid group that frequently contains simple reciprocal translocations that are unique to indi-

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COLIN S. COOPER

vidual tumors; a hypodiploid subgroup that often exhibits monosomy of chromosomes 18 and 22; and a third group with more complex karyotypes (Boghosian et al., 1989). Other observations of particular note include the preliminary indication that myxoid chondrosarcomas may contain translocations involving 9p3 1 and 2 2 ~ 1 3(Hinrichs et al., 1985; Turc-Carel et al., 1988a) and the report of a congenital fibrosarcoma containing a deletion of the short arm of chromosome 17, the location of the p53 gene, as the sole chromosomal abnormality (Adam et al., 1991). VI. Tumor Suppressor Genes

In contrast to ras and other oncogenes where the inappropriate function of specific genes can cause cancer in the case of tumor suppressor genes (also called recessive oncogenes or anti-oncogenes) loss or inactivation of both copies of the gene is usually required for tumor development. Several suppressor genes and putative suppressor genes have now been cloned and characterized. These include the RB1 gene located at 13q14, the p53 gene located at 1 7 ~ 1 3the , Wilms’ gene at llp13, the DCC gene at 18q2 1, the APC gene at 5q2 1-22, and the NF1 gene located at 17q 1 1.2. A role for the RB 1 and p53 genes in sarcoma development has now been established and the evidence for this is reviewed. In addition, as described in Sections Il1,A and III,C the APC and NF1 genes have been proposed as candidates for involvement in the genesis of, respectively, fibromatoses and MPNSTs. A. THEp53 GENE

It is now emerging that mutations in the p53 gene are one of the commonest abnormalities in human cancer (Hollstein et al., 1991). The p53 nuclear phophoprotein was originally detected in a protein that bound to the large T antigen of the simian virus SV40 (Lane and Crawford, 1979; Linzer and Levine, 1979) and also as a transplantation antigen expressed in 3-methylcholanthrene-induced mouse fibrosarcomas (DeLeo et al., 1979). Genomic and cDNA clones of p53 were isolated and it was demonstrated that the cloned p53 gene could immortalize cells in culture and cooperate with other oncogenes such as as to fully transformed primary rodent embryo fibroblasts (Eliyahu et al., 1984; Parada et al., 1984; Jenkins ct al., 1984). These observations suggested that the p53 gene had the properties of a dominant transforming gene. However, the interpretation of these experiments came under scrutiny when it was discovered that the constructs used in immortaliza-

HUMAN SOFT TISSUE TUMORS

101

tion and transformation studies contained mutated versions of p53 (Hinds et al., 1989). Recent studies suggest that p53 in fact has many of the properties of a tumor suppressor gene. For example, the wild-type p53 gene can eliminate the tumorigenic potential of cells in culture (Chen et al., 1990), cause arrest of cells at the G1 phase of the cell cycle (Baker et al., 1990; Diller et al., 1990), and inhibit the transformation of cells in culture by other oncogenes (Finlay et al., 1989; Eliyahu et al., 1989). However, it is also apparent that versions of the p53 allele containing point mutations can, in some cases, negate the normal suppressor function of p53. This might occur if mutated p53 acquires a new activity that overcomes the effect of wild-type p53. Support for this idea is provided by the observation that introduction of mutated p53 into cells that do not express p53 can enhance their tumorigenicity (Wolf et al., 1984).Since oligmerization of mutated p53 protein has been reported, as an alternative hypothesis, it has also been proposed that an excess of mutated p53 protein may sequester smaller amounts of normal p53 protein through the formation of hybrid oligomers (Green, 1989). The p53 gene is located on a region of the short arm of chromosome 17 that is frequently lost in human cancer including cancer of the lung, breast, and colon. When one allele of the p53 gene is lost the remaining allele usually contains point mutations, indicating that loss or inactivation of both alleles of the p53 gene is required for tumor development (Nigro et al., 1989; Baker et al., 1989; Hollstein et al., 1991). Loss of the short arm of chromosome 17 and point mutation of p53 has also been reported in several types of soft tissue sarcoma including rhabdomyosarcoma, MFH, and leiomyosarcoma (Mulligan et al., 1990; Stratton et al., 1990).Homozygous loss (loss of both alleles) and gross rearrangement of the p53 gene have also been detected in soft tissue sarcomas (Mulligan et al., 1990; Stratton et al., 1990). These types of structural alterations were uncommon in many of the types of cancer previously examined (Baker et al., 1989; Nigro et al., 1989) but have been detected in osteosarcoma and blast crisis CML (Masuda et al., 1987; Ahuja et al., 1989). It is possible that the distinct patterns of inactivating mutations observed in different tumor types may reflect the nature of the carcinogen involved in tumor induction. This hypothesis is supported by studies on point mutations of the p53 gene in hepatocellular carcinoma, which in certain areas of the world is believed to arise through exposure to aflatoxin. In a study of hepatocellular carcinoma from the high-incidence region of Qidang, China, all eight p53 mutations occurred at codon 249 and seven were G to T transversions, precisely the mutations expected to arise following

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interaction of activated metabolites of aflatoxin with DNA (Hsu et al.,

1991). B. THERB1 GENE The discovery that soft tissue sarcomas frequently arise as secondary tumors in survivors of familial retinoblastomas (Draper et al., 1986; Derkinderen et al., 1988) prompted an examination of soft tissue sarcomas for loss of chromosome 13 and for abnormalities of the RBI gene. Loss of a single copy of the RBI gene was found in around 30-40% of tumors (Stratton et al., 1989b).Using RB1 cDNA probes several groups have detected homozygous alterations of the RBI gene and abnormalities of RB1 transcriptions (Friend et al., 1987;Mendoza et al., 1988; Stratton et al., 198913;Wunder et al., 1991).In some cases these alterations involve rearrangements within the RB 1 gene itself or deletions of specific regions of the RB 1 gene. Abnormalities have been detected most frequently in MFHs and leiomyosarcomas, but also in liposarcomas and synovial sarcomas. When considered together the data provided strong evidence that alteration of the RB 1 gene is involved in sarcoma development. In a combined study of 36 bone and soft tissue sarcomas Wunder et al. ( 1991)examined the association of deletions of the RB 1 gene with tumor grade. The RBI gene was altered in 10125 of high-grade tumors but in only 1 / 1 1 low-grade tumors. Accordingly it was proposed that RB 1 alterations may function as a marker for high-grade tumors and it was suggested that RBI gene status should be assessed as an independent prognostic indicator. Stratton et al. (1990)found that 4 of 1 1 tumors containing alterations of the p53 gene also had abnormalities of the RBI gene. Futhermore similar patterns of coincident inactivation of the p53 and RB1 genes have also been found in small cell lung cancer and in osteosarcoma. When considered together these observations suggest that for some tumor types inactivation of both the p53 and the RB1 suppressor genes may be required for tumor development. These observations are of particular interest in relation to recent studies demonstrating that proteins encoded by two viruses (SV40 and adenovirus) capable of inducing sarcomas in rodents interact with the products of the RBl and p53 genes. Thus the adenovirus Ela and Elb proteins bind respectively to the RBI (Whyte et al., 1988)and p53 (Sarnow et al., 1982)proteins, whereas the SV40 large T antigen binds to the products of both the p53 (Lane and Crawford, 1979;Linzer and Levine, 1979)and the RBI (DeCaprio et al, 1988)genes. It has been proposed that a consequence of these

HUMAN SOFT TISSUE TUMORS

103

interactions is to remove the tumor suppressing properties of the p53 and RBI proteins (Green, 1989). Thus alterations occurring at the genetic level in some human tumors appear to reproduce the pattern of effects caused by SV40 and adenovirous proteins at the level of protein interaction.

C. STUDIESON CHROMOSOME Loss Indirect evidence for the involvement of a suppressor gene can be obtained by demonstrating the consistent loss of particular chromosome segments using probes or PCR primers that allow individual alleles to be distinguished. Using this type of analysis frequent loss of the end of the short arm of chromosome 11 (1lp15) has been observed in embryonal rhabdomyosarcoma (Koufos et al., 1985; Scrable et al., 1987, 1989).Notably the loss of chromosome 11 was specific to the embryonal class of rhabdomyosarcomas and was not observed in alveolar tumors. It was therefore proposed that loss of chromosome 11 may act as a diagnostic marker that could be used to distinguish the alveolar and embryonal histological groups. Analysis of the mechanism of loss of chromosome 11 has been undertaken (Scrable et al., 1987). In around 50% of cases the loss of one entire homolog of chromosome 11 was accompanied by duplication of the remaining mutant allele. The other 50% of cases had lost heterozygosity of a variable number polymorphic loci on the short arm of chromosome 11 but retained constitutional heterozygosity on the long arm. Nonetheless cytogenetic and densitometric analyses revealed that each tumor had two complete copies of chromosome 11. It was therefore proposed that the loss of heterozygosity arose following mitotic recombination between the two progenitor copies of chromosome 11 (Fig. 8). Consistent loss of the end of the short arm of chromosome 11 has also been observed in Wilms’ tumor. Furthermore as mentioned above (Section II1,E) the Beckwith-Wiedemann syndrome, which is associated with a high risk of developing rhabdomyosarcoma, Wilms’ tumor, and hepatoblastoma, has been genetically linked to markers at 1lp15. When considered together these observations strongly support the idea that loss of a suppressor gene located at 1lp15 is required for the development of rhabdomyosarcoma and Wilms’ tumor. Although the end of the 1lp15 chromosomal region is currently the subject of considerable attention, the suppressor gene located in this region has not yet been cloned. This locus is, however, distinct from the Wilms gene located at llp13 (Call et al., 1990) and the muscle-specific MyoDI gene, which is also located on the short arm of chromosome 11 (Scrable et al., 1990).

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Chromosome 1 1

[m............................

Constitutional

.a.................-*.

..............................@..................

TlJmor genotype

*-

*.

...................

FIG. 8. Schematic model showing mitotic recombination of chromosome 1 1 in rhabdomyoscromas. "+" indicates wild-type allele and "*" indicates the presence of a mutated allele at 1 lp15. The figure shows the presumed genotype in a cell predisposed to rhabdomyosarcama by either an inherited or a somatic mutation at the 1 lp15 locus (constitutional) and the proposed structure of chromosome 1 1 that results from mitotic recombination between the two progenitor copies of chromosome 1 1 (tumor genotype). Adapted from Scrable et al. (1987).

VII. Gene Amplification Amplification and overexpression of protooncogenes have been observed in several classes of human cancer. For example, amplification of the N-myc gene is frequently observed in neuroblastoma (Hall, 1990), whereas c-erbB-2 is commonly amplified in breast cancer. The cytogenetic hallmarks of gene amplification (double minute chromosomes and homogeneously staining regions) have been found in malignant fibrous histiocytomes, liposarcomas, and in both alveolar and embryonal rhabdomyosarcoma (Douglass et al., 1987; Mertens et al., 1987; Wang-Wuu et al., 1988; Coccia et al., 1989; Mandahl et al., 1989). In agreement with these observations there have been isolated reports of N-my, c-myc, and gli protooncogene amplification in embryonal rhabdomyosarcomas (Garson et al., 1986; Mitani et al., 1986; Tsuda et al., 1988; Roberts et al., 1989; Hayashi et al., 1990) and in one study N-myc amplification was found in a tumor recurrence but not in the original embryonal tumors, suggesting that N-myc amplification may be involved in tumor regrowth (Carson et al., 1986). By comparison Dias et al. (1990) found a 5- to 20fold amplification of N - m y in 4 of 6 alveolar tumors but in none of 7 embryonal rhabdomyosarcomas. It has been reported that the c-myc gene is overexpressed in rhabdomyosarcomas and that increased c-myc expression may be linked to the loss of ability to differentiate in myogenic cell lines. However, Dias et al. (1990) failed to find abnormalities of the c-myc gene in their series of 13 tumors. Kopald et al. (1990) used DNA fingerprint analysis as a first step in identifying abnormalities in soft tissue sarcomas. In this procedure Southern blots of HaeIII- and HinfI-digested DNA was hybridized to

HUMAN SOFT TISSUE TUMORS

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minisatellite probes. For all 14 soft tissue sarcomas the fingerprint of the tumor DNA exhibited changes when compared with the corresponding control DNA. In one liposarcoma a 1.5-kb DNA fragment was highly amplified. This sequence was cloned and mapped to human chromosome 7. Coccia et al. (1990) have used an in-gel renaturation technique to identify and isolate sequences amplified in soft tissue sarcomas. This technique allows the deletion and cloning of any sequences within the genome that are amplified greater than 15-fold. Using this procedure DNA amplification was detected in two of four malignant fibrous histocytomas with documented cytogenetic evidence of gene amplification and it was established that the amplification unit did not contain any of 15 known oncogenes including N-myc and c-my. Clones from the amplified region were isolated and screening of a panel of sarcomas identified amplification of these sequences in 218 MFH, 1/4 liposarcomas, and 1/1 leiomyosarcomas. Vlli. Predictors of Tumor Behavior

The identification of factors that can be used to predict the response of tumor to treatment and survival of individual patients is a major goal of clinical research. In addition to standard indicators such as grade, tumor size, and stage it is now recognized that, in certain tumors, the presence of particular genetic abnormalities correlates with clinical behavior. For example, in breast cancer amplification and overexpression of the c-erbB-2 gene are significant predictors of both overall survival and time to relapse (Slamon et al., 1987). Furthermore in neuroblastomas amplification and overexpression of the N-myc gene are frequently found in stage I11 and IV tumors, but rarely found in tumors at stages I and I1 (Brodeur et al., 1984).Similar correlations between the presence of specific genetic abnormalities or the expression of particular gene and clinical behavior have been observed for some types of soft tissue sarcoma. Correlations between the presence of a specific translocation involving chromosome 19 and tumor behavior and between the presence of alterations in the RBI gene and tumor grade were considered, respectively, in Sections V,F and V1,B. In this section studies on tumour ploidy and expression of multidrug resistance genes are reviewed. A. TUMOR CELLPLOIDY Abnormalities in the cells DNA content (ploidy) have been linked to prognosis in a variety of malignancies including breast cancer, neu-

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roblastoma, and acute lymphoblastic leukemia. In a study on unresectable rhabdomyosarcoma Shapiro et al. (1991) examined the relationship of tumor cell ploidy to histoglogical subtype and to patient response to treatment. They found the embryonal tumors had either a diploid DNA content or a hyperdiploid DNA content (1.1- 1.8 times the DNA content of diploid cells). By comparison alveolar tumors usually had either a diploid DNA content or a near tetraploid DNA content (1.8-2.0 times the DNA content of diploid cells). They also found that cell ploidy had a significant impact on treatment outcome. Patients with tumors with hyperdiploid DNA content had significantly longer survival times than did patients with diploid tumors. Patients with near tetraploid tumors exhibited a response intermediate between those observed for near diploid and diploid tumors. A similar association between hyperdiploid tumors and a favorable response has been observed for neuroblastoma and acute lymphoblastic leukemia. B. MULTIDRUCRESISTANCE The resistance of malignant cells to chemotherapeutic agents poses a major problem in the treatment of many cancers. Resistance may be intrinsic or may develop during the chemotherapy program and usually extends to several structurally and functionally unrelated agents. This phenomenon of multidrug resistance is associated with the expression of a 170-kDa membrane glycoprotein called P-glycoprotein, which is an energy-dependent pump that removes drugs from the cells. P-glycoprotein is encoded by a gene called mdr-1 that is frequently amplified and overexpressed in drug-resistant tumor cell lines. Chan et al. (1990) examined the relationship between expression of Pglycoprotein and prognosis in children with soft tissue sarcoma (mainly rhabdomyosarcoma) that were treated with chemotherapeutic agents (Fig. 9). In this study all nine patients that expressed P-glycoprotein relapsed after an initial response. By comparison, of 20 tumors that were consistently P-glycoprotein negative only one relapsed. This astonishing correlation between the absence of P-glycoprotein expression and longterm survival indicates that the expression of P-glycoprotein is a major factor in determining the long-term response of childhood sacomas to chemotherapy. These observations also raise hope that in the future novel treatment regimes involving the use of monoclonal antibodies against P-glycoprotein or inhibitors of P-glycoprotein such as cyclosporin, verapamil, and quinidine in combination with chemotherapy may improve long-term survival for patients with P-glycoproteinpositive tumors.

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1.0m P-glycoprotein-negativesarcoma

3 0.8> .-

s

0 0

) . ) .

0.61 0.6-

w. c

a 0.4 Q

n 0.2-

I

L

2

4

6

8

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Survival (years) FIG.9. Kaplan-Meier plot of the duration of survival in children with P-glycoproteinpositive and P-glycoprotein-negativesoft tissue sarcoma.

IX. Molecular Cloning of Translocation Breakpoints One of the most important goals of future research must be the cloning of genes involved in the set of specific chromosomal translocations found in soft tissue tumors. In this regard it is probable that there are lessons to be learned from the wealth of studies on translocation found in leukemias and lymphomas. Indeed, the application of molecular biological procedures to the analysis of specific translocations found in leukemias and lymphomas has resulted in profound insights into the molecular basis of development of these neoplasms. The translocations usually, but not exclusively, involve the immunoglobulin or Tcell receptor genes and are believed to result from errors in the gene rearrangement process (Hall, 1990). As a consequence of these errors the immunoglobin or T-cell receptor genes are juxaposed to a variety of genetic loci including genes that have been previously characterized (e.g., c-myc) together with an impressive array of novel candidate oncogenes (e.g., Lyl- 1, TallScl, Rhom- 1, and Rhom-2). These somatically acquired defects usually result in transcriptional deregulation of the transposed cellular gene. Many other translocations found in leukemias and lymphomas do not involve T-cell receptor or immunoglobulin loci. In some cases candidate genes previously localized to the general region of the chromosome involved in the translocation have been implicated. For example, it was discovered that the c-abl protein tyrosine kinase gene located on chromosome 9 is joined to the bcr gene in the t(9;22) translocation found in

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chronic myelogenous leukemia (Witte, 1986), whereas the E2A transcriptional factor gene located on chromosome 19 becomes fused to a homeobox gene as a result of the t( 1 ;19) translocation observed in childhood pre-B-cell acute leukemia (Nourse et al., 1990; Kamps et al., 1990). Southern and Northern analyses have been used to assess the involvement of candidate protooncogenes that are located at the appropriate chromosomal regions in the translocations found in soft tissue sarcomas but so far without success. Nonetheless it is notable that a surprisingly high proportion of genes activated by translocations in leukemia and lymphomas encode proteins that are members of the helix-loop-helix (my,E2A, Lyl- 1, and Tal/Scl) homeobox (Hox 1 1) and cysteine-rich/zinc finger (Rhom 1 and Rhom2) families of transcriptional regulatory tnolecules. New members of these gene families must therefore be considered as particularly strong candidates for involvement in the translocations found in soft tissue tumors. T h e DNA transfection/transformation assay might also provide a method for detecting genes that are activated by translocations. Indeed the NIH3T3 mouse fibroblasts would seem to be an ideal target cell for detecting genes activated in mesenchymal tumors. Unfortunately these types of studies have failed to identify activated oncogenes in synovial sarcomas containing the t(X; 18) translocation and in rhabdomyosarcomas containing the t(2; 13) translocation. A non-ras gene was apparently detected by transfection of DNA from a liposarcoma but this gene mapped to chromosome 19, not to chromosome 12 or 16 (Gill et al., 1991). Genes involved in chromosomal translocations may also be involved using a more indirect procedure generally referred to as reverse genetics. In this approach a combination of techniques is used to clone and create long-range restriction maps of the chromosomal region spanning the breakpoint. Pulsed-field gel electrophoresis, a technique used to separate large fragments of genomic DNA, can be used to map chromosomal regions and localize translocations using probes that may be several hundred kilobases from the position of the translocation. Probes flanking the translocation can also be used in genomic DNA walking and jumping experiments to isolate sets of cosmid and yeast artificial chromosome (YAC) clones that span the breakpoint. Candidate genes may then be identified by locating CpG-rich islands, by looking for regions of DNA that are highly conserved among different species, and by hybridizing the cloned DNA to Northern blots or cDNA libraries. This type of approach has been used to isolate a new oncogene called can that is involved in a specific translocation between chromosomes 6 and 9 [t(6;9)(p23;q34)]found in a subtype of acute nonlymphocytic leukemia

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(von Lindern et al., 1990)and to isolate the genes involved in the t( 15;17) translocation found in acute promyelocytic leukemia (Borrow et al., 1990).

X. Future Developments Tumor development is generally considered to be a multistep process. Evidence for this is provided by analysis of the pathology of cancer development, by studying the transformation of mesenchymal cells in tissue culture, and by studies on the kinetics of appearance of cancer (Nordling, 1953; Foulds, 1969). Furthermore transformation of primary cells in tissue culture can, in some cases, be achieved through cooperation between activated oncogenes. For example transformation of primary embryonic fibroblasts from rat can be achieved by cooperation between activated forms of myc and rus oncogenes, whereas each oncogene on its own is insufficient to induce transformation (Land et al., 1983).These observations suggest that several genetic alterations may be required to achieve full transformation. In agreement with this idea studies on the development colon cancer suggest that the stepwise activation of one oncogene (rus) and inactivation of three suppressor genes (APC, DCC, and p53) may be required for development of colon carcinoma (Nigro et al., 1989; Fearon et d., 1990; Kinzler et d., 1991a; Nishisho et al., 1991). For many classes of soft tissue sarcomas only a single consistent genetic abnormality has been detected (i.e., the presence of a specific chromosomal translocation, activation of a rus gene, or inactivation of a suppressor gene). The identification of other genetic changes and the elucidation of the ways in which the different types of genetic change cooperate during the development of sarcomas are clearly important areas for future studies. In this regard the assessment of the role of new suppressor genes such as NF1, DCC, and APC, mentioned above, in the development of sarcomas may be very important. As key genetic alterations are identified it will also of course be necessary to determine whether their presence has any prognostic significance. During the past decade there have been major changes in the classification of soft tissue sarcomas and improvements in diagnosis through the use of new immunohistochemical reagents and refinements in ultrastructural analysis. Despite these advances difficulties are still encountered in the diagnosis of certain tumor groups (Enzinger and Weiss, 1988). For example, it has recently been suggested that many tumors diagnosed as malignant fibrous histiocytomas may in fact belong to other tumor groups. Moreover it is often difficult to distinguish between

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poorly differentiated rhabdomyosarcoma and poorly differentiated examples of other types of childhood tumors (Carter et al., 1990). The recent identification of tumor-specific genetic alterations (see Section 111) and of genes that are expressed in a lineage-specific fashion offers a completely fresh approach that has the potential to further improve methods of diagnosis and to improve consistency of diagnosis among different centers. Examples of genes with lineage-specific expression that may be of use in the diagnosis of soft tissue tumors are provided by the MyoD 1 (or mrf) gene family. T h e four members of this family (myf3, myf4, m y p , and myfi) encode transcription factors that are expressed exclusively in striated muscule and that when introduced into fibroblasts cause their conversion into cells with myogenic characteristics (Davies et al., 1987; Braun et al., 1989a,b, 1990; Wright et al., 1989). Since rhabdomyosarcomas are believed to be derived from cells that are differentiated toward striated muscle (Enzinger and Weiss, 1988), all rhabdomyosarcomas would be expected to express one o r more members of the myf gene family. i n agreement with this prediction, expression of myf3 was found in 85-100% of tumors and myf4 was expressed in 75% of tumors (Hiti et al., 1989; Scrable et al., 1989; Clark et al., 1991). The myf genes were not expressed in other classes of soft tissue and pediatric tumors, indicating that expression of myf3 and myf4 many provide an extremely useful marker in the diagnosis of rhabdomyosarcoma. Although studies have so far been restricted to the use of myf genes, it is probable that families of tissue-specific transcription factors will also be involved in controlling differentiation along other soft tissue lineages. The cloning of the genes encoding such factors should in time yield probes that can be used in the diagnosis of other classes of soft tissue tumor. ACKNOWLEDGMENTS C.S.C. is funded by the Cancer Research Campaign, UK. Thanks are due to Dr. M. C. Poirer for providing the information shown in Fig. 2. REFERENCES

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GENOMIC INSTABILITY AND TUMOR PROGRESSION: MECHANISTIC CONSIDERAT1ONS Keith C. Cheng’ and Lawrence A. Loeb Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology SM-30, University of Washington, Seattle, Washington 98195

I. Introduction 11. Genomic Stability and Instability: Background and Implications A. Definitions B. The Importance of Genomic Instability in Tumor Progression C. Genomic Instability and Tumor Progression: Limitations of Data 111. Mechanisms of Genomic Stability and Instability A. Fidelity of DNA Replication B. Postreplicative Mismatch Correction C. Prevention of Oxidative DNA Damage D. Repair of DNA Damage E. Immunoglobulin Gene Hypermutation F. Checkpoints G. Maintenance of Chromosomal Ploidy H. Inherited Human Chromosomal Instability Syndromes I. Recombination J. Epigenetic DNA Modification K. Other Mechanisms IV. Quantitative Considerations A. Mechanistic Considerations B. Factors Affecting the Probability and Impact of Genomic Instability Mutations C. Effects of Cancer Physiology on Probabilities V. Summary and Perspectives References

1. Introduction Each cell in a multicellular organism lives under cooperative restraint not required of its counterpart in the unicellular world. Zoologist Theodor Boveri observed at the turn of the century (Boveri, 1902) that abnormal development occurs in sea urchin and ascaris embryos when chromosome number is disturbed. This suggested to him that the intercellular cooperation between cell types required during embryogenesis 1 Present address: Department of Pathology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, PA 17033.

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may be conferred by particular chromosomes. He surmised that the disrupted growth patterns characteristic of human cancers may represent analogous defects in intercellular cooperation, and may also result from chromosomal aneuploidy (i.e., changes in chromosomal number; Boveri, 1914).In today’s vocabulary, the antisocial behavior of the cancer cell is associated with a variety of karyotypic (chromosomal) and submicroscopic genomic modifications. During tumor progression in spontaneous and experimentally induced cancers, malignant characteristics appear to be acquired in stepwise fashion (Foulds, 1954). T h e relative genomic instability of cancer cells compared to normal cells, most visibly in the form of karyotypic instability (Sandberg and Hossfeld, 1970; Weiner et al., 1974),led Nowell (1976) to suggest that genomic instability may itself contribute to that stepwise progression. T h e essence of the argument is that if multiple genomic changes are required for the full development of the cancer phenotype, then an increased rate of causing those changes would cause an increased rate of cancer (Fig. 1). In contrast to certain pediatric tumors that appear to require as few as two genomic changes to produce a cancer phenotype (Knudson, 1971, 1986; Haber and Housman, 1991), the common adult cancers appear to require more (e.g., Fearon and Vogelstein, 1990).

Spontaneous Genomic Chanae

k1

Cancer Genes

.+

Cancer

+

Cancer

Genom ic Instability

Cancer Genes

FIG. 1. Increased genomic instability increases the rate of tumor progression. Important steps in tumor progression may occur by genomic modification of cancer-related genes (see Table I). If mutation in genomic stability pathways causes the rate of genomic alterations to increase from the normal background rate kl to a greater value k2, genomic instability occurs. This results in an increased rate of cancer gene alteration, resulting in earlier onset of cancer. Genomic-instability mutations may be present in the germline, as in genomic instability syndromes such as xeroderma pigmentosum or Bloom’s syndrome, or arise by somatic mutation.

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The genomic alterations directly affecting tumor progression include activating mutations in oncogenes (Buckley, 1988; Weinberg, 1989), inactivating mutations in tumor suppressor genes (Klein, 1987; Stanbridge, 1990) or metastasis suppressor genes (Steeg et al., 1988; Bevilacqua et al., 1989),and the often uncharacterized consequences of abnormal karyotypes (e.g., Mitelman, 1984, 1991; Yunis, 1983). For example, point mutations in the p53 tumor suppressor gene have been detected in more than half of all human cancers tested (reviewed by Hollstein et al., 1991). Most of these suppressor gene mutations, however, are recessive, i.e., are phenotypically silent when present in the mutant/wild-type heterzygous state. These recessive mutations may then be unmasked by homozygosis to mutant alleles. Homozygosis to mutant alleles, also referred to as loss of heterozygosity, can occur via a variety of mechanisms, including mutation, recombination, or chromosome loss with or without duplication. Loss of heterozygosity at proven or hypothesized tumor suppressor gene loci occurs at frequencies as high as 75% in human cancers (reviewed by Ponder, 1988; Scrable et al., 1990; Haber and Housman, 1931). The progression of human glial tumors provides a striking correlation between loss of heterozygosity and tumor progression. In advanced astocytomas, also known as glioblastoma multiforme, uniform loss of heterozygosity has been reported for chromosome 10, probably through chromosomal nondisjunction; there is no corresponding loss of heterozygosity in lesser grade glial tumors (Bigner et al., 1988). Multiple chromosomal abnormalities (cataloged recently by Mitelman, 1991) have been reported in a variety of human cancers, including small cell lung carcinoma (Bierrer and Minna, 1989), colonic adenocarcinoma (Vogelstein et al., 1988), breast carcinoma (Ali et al., 1987; Sat0 et al., 1990), hepatocellular carcinoma (Buetow et al., 1989; Walker et al., 1991), malignant melanoma (Balaban et al., 1986; Dracopoli et al., 1989), renal cell carcinoma (Kovacs and Frisch, 1989; Ogawa et al., 1991),gliomas (Jameset al., 1988; El-Azouzi et al., 1989; Fults et al., 1989), esophageal adenocarcinoma (Rabinovitch et al., 1988; Meltzer et al., 1991),and endocrine neoplasms (Bale et al., 1991).The association of karyotypic abnormality with cancer suggests the possibility that karyotypic instability as a phenotype may underlie some fraction of those changes. The importance of genomic alterations in tumor progression and their association with cancer make it important to study the mechanisms by which they arise. In this review, we will define genomic instability and discuss how the phenotype of genomic instability may itself predispose to cancer and accelerate tumor progression. We will then discuss how

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genomic alterations identified in human cancers can be viewed in the context of cellular pathways normally dedicated to the maintenance of genomic stability. During our selective survey of cellular mechanisms involved in maintaining genomic stability, w e consider some examples of disruption of these pathways leading to genomic instability. We propose that the large number of components involved in those mechanisms may increase the likelihood that disruption of normal genomic stability mechanisms will occur during a normal lifespan and during the growth of benign neoplasms. We then consider various factors that can affect the frequency of genomic instability mutations. Finally, we discuss how an understanding of these mechanisms may suggest ways to delay or, in effect, prevent certain types of cancer.

II. Genomic Stability and Instability: Background and Implications A. DEFINITIONS

Multiple metabolic pathways govern the accurate duplication and distribution of DNA to progeny cells; other pathways control regulatory modifications of DNA during normal development (Table I). Together, these pathways may be regarded as genomic stability functions. For each of these functions, there is a normal baseline frequency with which errors occur, leading to spontaneous mutations and other genomic alterations. We postulate that increased genomic instability (resulting in an increased frequency of mutations, karyotypic alterations, o r recombination) may arise by mutational o r exogenous interference with these pathways. Mutations in genes that cause an increased frequency of single base substitutions, insertions, o r deletions are commonly referred to as mutator mutations. Other mutations may lead to an increased frequency of aneuploidy o r structural chromosomal aberrations; these may be called chromosomal instability mutations. There exist tightly controlled cellular mechanisms that may cause genomic disruption if inappropriately expressed; these include general or locus-specific recombination that occurs in certain DNA repair processes and in B- and T-cell differentiation, as well as immunoglobulin gene somatic hypermutation, which is central to the stepwise generation of high avidity antibodies. In addition, there are mechanisms that govern gene expression according to developmental o r environmental cues. Mutations in regulatory DNA sequences that yield inappropriate gene expression may also constitute steps in tumorigenesis. Mutations

GENOMIC INSTABILITY AND TUMOR PROGRESSION

TABLE I MECHANISMS OF GENOMIC STABILITY AND INSTABILITY, AND CANCER-RELATED GENE GENOMIC EFFECTS, TARGETS~

Mechanisms of genomic stability and instability Maintenance of primary DNA sequence Replication Postreplicative proofreading Prevention of DNA damage DNA repair Checkpoint proteins Recombination Maintenance of chromosomal ploidy and structure Genome organization Topological management of DNA Chromosome segregation Proper epigenetic programming Differentiation Physiological responses to environment Genomic effects Gene amplification Single base substitutions Insertions Deletions Recombination Chromosomal abnormalities Numerical loss or gain Structural Translocations Inversions Deletions Other Epigenetic change Inappropriate expression Loss of function Cancer-related gene targets Oncogenes Tumor suppresssor genes Migration (invasion) genes Metastasis genes Metastasis suppressor genes Senescence genes Genomic stability genes Immune tolerance genes Epigenetic regulation genes a

Adapted from Cheng and Diaz (1991)

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that impair any of these mechanisms and result in increased genomic instability may together be classified as genomic instability mutations. B. THEIMPORTANCEOF GENOMIC INSTABILITY IN TUMOR PROGRESSION

The idea that genomic instability mutations may play an important role in human cancer comes from several lines of reasoning. First, there is increasing evidence for multiple genetic alterations in common human tumors; individual colon cancers, for example, have been reported to contain multiple genomic alterations (Fearon and Vogelstein, 1990). Second, the incidence of most adult tumors exhibits an exponential rate of increase with age, suggesting a series of events each rendered more likely by the occurrence of a previous event (Armitage and Doll, 1954). Third, cancer incidence is increased among people afflicted with the genetic chromosome instability syndromes such as Bloom’s syndrome (German, 1983). We have argued on the basis of the probability of spontaneous mutations that a mutator phenotype is required for multistep carcinogenesis. The probability of the multiple genomic changes associated with cancer has been estimated from spontaneous mutation rates of normal human cells for a variety of genetic targets. That rate, estimated to be about 1.4 x mutations/base pair/cell division, is inadequate to account for more than t w o or three mutations in a given cancer cell (Loeb, 1991). C. GENOMIC INSTABILITY A N D TUMOR PROGRESSION: LIMITATIONS OF DATA

A causal relationship between genomic instability and tumor progression remains to be established. Some experimental systems demonstrate an association between higher mutation rates and malignant progression (Cifone and Fidler, 1981; Seshadri et al., 1987). Cifone and Fidler (1981) showed that the frequency of 6-thioguanine-resistant and ouabain-resistant cells was about 7-fold higher in cells of high metastatic potential compared to cells of low metastatic potential. Seshadri et al., (1987) found that human leukemic cells had about a 100-fold higher frequency of 6-thioguanine-resistant cells compared to phytohemagglutinin-stimulated normal human lymphocytes. Tlsty et al. (1989) rein ported that the frequency of gene amplification was as great as tumorigenic cells, but undetectable in normal cells (Tlsty, 1990). In contrast, the frequencies of 6-thioguanine- and ouabain-resistant cells did not differ between normal diploid fibroblasts and a chemically trans-

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formed cell line (Barrett and Ts'o, 1978). A lack of correlation between malignancy and high mutation rates has also been reported in other cell culture systems (Elmore et al., 1983; Yamashina and Heppner, 1985; Damen et al., 1989). These apparent contradictions may be explained in several ways. First, single-step in nitro transformation systems would not be expected to require genomic instability to occur. Second, chemical mutagens by themselves generate multiple mutations and thus substitute for a mutator phenotype. Third, the frequency of some types of genomic alterations that may be critical to tumor progression could not be distinguished in the experimental systems used; for example, even though loss of heterozygosity has been shown in some tumors to result from recombinational or nondisjunctional mechanisms, such mechanisms are not detected by the examination of X-linked 6-thioguanine-resistant variants, where recombination and chromosome loss are not feasible mechanisms for drug resistance. Fourth, the contribution of different genomic instability mechanisms may vary from tumor to tumor. Lastly, in cancers requiring few steps, such as hereditary retinoblastoma, there is no need to invoke a mutator phenotype. Assays that detect multiple (or at least other) types of genetic instabilities in human tumors are needed to better define the association of genomic instability and cancer. 111. Mechanisms of Genomic Stability and Instability

A. FIDELITY OF DNA REPLICATION The 3 X lo9 bases that constitute the human genome are replicated with fantastic accuracy: replicative errors persist at a frequency of only one in lo9 to 10" bases (Drake, 1969; Chu et al., 1988; Loeb, 1991).This high level of accuracy of cellular DNA replication is maintained by sequential mechanisms (Loeb and Kunkel, 1982), each of which has the potential to enhance accuracy as much as 3 orders of magnitude. These mechanisms include the maintenance of balanced nucleotide concentrations (de Serres, 1985), differences in the free energy of hydrogenbonding between complementary and noncomplementary bases (Loeb et al., 1974; Mildvan, 1974), protection of the chemical integrity of nucleotide precursors (Topal and Baker, 1982), base selection by DNA polymerases during the insertion step (Loeb and Reyland, 1987), lack of extension of mismatched 3' termini by DNA polymerases (Perrino and Loeb, 1989), exonucleolytic proofreading (Kunkel, 1988; Kornberg, 1980), and postsynthetic mismatch correction (Modrich, 1987, 1989).

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1. Base Selection, Nucleotide Precursors,

and Nucleotide Alteration

A significant contribution to the accuracy of DNA replication is determined by the ability of DNA polymerases to select and connect the proper base for insertion opposite each template base (Loeb and Kunkel, 1982; Boosalis et al., 1987). T h e inherent differences in chemical stability of base pairs alone (Topal and Fresco, 1976) is insufficient to account for the accuracy of base selection. Differences in free energy between correct and incorrect Watson-Crick base pairs yield the estimate that one error would occur for every 100 bases inserted (Mildvan, 1974); studies utilizing nonenzymatic polymerization in fact confirm such high error rates (Lohrmann and Orgel, 1980). T h e hydrophilic environment at the catalytic site of DNA polymerases may increase the free energy differences between correct and incorrect base pairs and thus provide a mechanism for base selection (Petruska et al., 1986). DNA polymerase catalytic subunit mutations that decrease the ability of the polymerase to choose the proper base would be predicted to increase the mutation rate. Such decreased base selectivity could account for the up to 100-fold increase in mutation rate caused by mutations in DNA polymerase 111 of Escherichzu coli (Hall and Brammar, 1973; Konrad, 1978; Sevastopoulos and Glaser, 1977) and of Bacillus subtilis (Bazill and Gross, 1973). The balance of nucleotide precursor concentrations in cells may also affect the frequency of misinsertion by DNA polymerases (Weymouth and Loeb, 1978; Fersht, 1979). Higher mutation rates have been observed in mammalian cells that contain imbalanced nucleotide precursor concentrations as a result of mutations in genes involved in nucleotide metabolism (Meuth et al., 1979; Ullman et al., 1980; Weinberg et al., 1981; Kaufman, 1988; Mattano et al., 1990). T h e production of unequal nucleotide pools by the viral ribonucleotide reductase of herpes simplex virus 2 has been postulated to play a role in transformation (Huszar and Bacchetti, 1983). Nutrient starvation of cells may also alter nucleotide pools and thus be a potential source of mutations (Duan and Sadke, 1987). The disruption of normal nucleotide pool concentrations is an often unconsidered effect of mutagens (Das et al., 1983). The extent to which the modification of nucleotide precursors might induce mutations in human or other cancers remains to be determined. The mutagenic potential of chemically modified nucleotides was first analyzed by Topal and Baker (1982). More recently, a prominent form of oxidative base damage, 8-hydroxyguanine (also known as 8-0x0guanine), was shown to cause A + C transversions when incorporated as

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a nucleotide substrate (Cheng et al., 1992), in contrast to the G + T transversions it causes when present in the DNA template (Wood et al., 1990; Shibutani et al., 1991; Cheng et al., 1992). T h e concept of mutagenic nucleotide precursors is supported by studies in E. coli suggesting the existence of cellular enzymes that degrade modified guanine deoxynucleoside triphosphate such as 8-hydroxydeoxyguanosine triphosphate which exist predominantly in a mispair-prone syn conformation (Bhatnagar et al., 1991; Maki and Sekiguchi, 1992).

2. Mispair Extension and Exonucleolytic Proofreading DNA synthetic errors can be corrected in two ways. First, enhanced fidelity of DNA replication can be achieved by the exonucleolytic removal of bases misinserted at 3’ termini. This activity is present after purification of all prokaryotic DNA polymerases but not all eukaryotic polymerases (Fry and Loeb, 1986). The 3’ + 5’ exonuclease activity that constitutes that proofreading mechanism may be built into the DNA polymerase, as in the case of E. coli DNA pol1 (Derbyshire et al., 1988); alternatively, proofreading may be accomplished by separate polypeptides such as the E subunit of E. coli DNA pol111 (Scheuermann and Echols, 1984). T h e contribution of proofreading to the fidelity of DNA synthesis is illustrated by increases in genomic mutation rates associated with mutations in the exonuclease activity of DNA polymerases. For example, mutator DNA polymerase mutants of bacteriophage T 4 (Speyer, 1965; Drake et al., 1969; Reha-Krantz et al., 1986) have decreased 3‘ + 5’ exonuclease activity (Muzyczka et al., 1972). Furthermore, mutation rates are 10,000-fold higher in mutants in the gene for the proofreading E subunit of E. coli DNA polIII, dnaQ (also called mutD; Fowler et al., 1974). As expected, the increase in mutation rate in mutD E. coli is accounted for by single base substitutions (Fowler et al., 1986). Second, in the case of eukaryotic DNA polymerase-a, additional accuracy is determined by the lack of extension of mismatched 3’ termini, effectively pausing for repair to occur before proceeding (Perrino and Loeb, 1989). Mammalian DNA polymerase-a, believed to function in DNA replication and DNA repair, and DNA polymerase-P, thought of primary as a DNA repair enzyme, lack proofreading activity (Fry and Loeb, 1986). It is likely that there are separate proofreading exonucleases that work in association with these polymerases to enhance the accuracy of DNA replication and DNA repair, respectively. Such activities may reside on other polypeptides or on other polymerases; for example, the proofreading activity of DNA polymerase-?? is able to excise bases misincorporated by DNA polymerase-a at 3’-termini (Perrino and Loeb, 1990).

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B. POSTREPLICATIVE MISMATCHCORRECTION DNA base mispairs that persist after DNA replication are subject to repair by separate mismatch repair mechanisms (reviewed by Modrich, 1987; Grilley et al., 1990). This mode of error correction has been most extensively studied in E. coli, where postsynthetic mismatch repair is accomplished primarily by the MutHLS mismatch repair system. I n this system, repair is directed from the methylated parental strand by the the lack of methylation of the newly synthesized DNA strand (reviewed by Modrich, 1989). Mutations in the methylation system or in any of the three mismatch repair genes, mutH, mutL, or mutS, cripple this repair, yielding mutation rates as much as 100-fold over background; these genes were among the first mutator genes to be studied (reviewed by Cox in 1976). The proteins involved in recognition, cleavage, and excision have been purified, and shown to function in vitro (Lu et al., 1983, Lahue et al., 1989). At least one other mismatch repair system exists in E. coli, called very short patch (VSP) repair (Lieb, 1983; Lieb et al., 1986). Mismatch repair systems have been identified and are being investigated in yeast (Bishop et al., 1987; Kramer et al., 1989) and in human cells (Glazer et al., 1987; Brown and Jiricny, 1989).

C. PREVENTION OF OXIDATIVE DNA DAMAGE A major potential source of DNA damage and mutations is oxygen free radicals. T h e cell must cope with highly reactive oxygen species generated both during normal oxidative metabolism (Fridovich, 1975) and as a result of a variety of exogenous agents including irradiation, metals, and tumor promoters (Slaga et al., 1981; Kasai et al., 1986; Fridovich, 1986; Floyd et al., 1986; Klebanoff, 1988; Gajewski et al., 1990; Wei and Frenkel, 1991). Reactive oxygen species are also released by phagocytes in inflamed tissues (Weitberg et al., 1983, Weitzman et al., 1985) and by anoxic tissues (Granger et al., 1986; Oliver et al., 1990); the latter may explain why tumor cells, which often outgrow their blood supply, appear to release more oxidative species (Szatrowski and Nathan, 1991). Even estrogens have been associated with the generation of oxygen radicals in DNA (Roy et al., 1991). Reactive oxygen species have been estimated to introduce on the order of 20,000 lesions in DNA per mammalian cell per day (Fraga et al., 1990). Oxygen free radicalinduced DNA damage has been shown to be mutagenic in both prokaryotes (Fenn et al., 1957) and eukaryotes (Hsie et al., 1986). Accordingly, reactive oxygen species have been suggested to play a role both in the

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initiation of cancers and in tumor progression (Totter, 1980; Weitzman and Gordon, 1990; Cerutti and Trump, 1991). In order to blunt the harmful effects of these oxidants, cells have evolved a variety of antioxidants, including vitamin E, p-carotene, glutathione, ascorbic acid, and uric acid, each of which may reduce the amount of oxidative damage to DNA (Ames, 1983).The transformation of superioxide radicals to hydrogen perioxide, for example, is catalyzed by cellular superoxide dismutases (SOD); the hydrogen peroxide is in turn converted to water by catalase. Escherichia coli (Farr et al., 1986) and yeast mutants (Gralla and Valentine, 1991) defective in superoxide dismutase express a mutator phenotype. These results imply that the accumulation of the superoxide radical is mutagenic. Fe2+ is known to produce hydroxyl radicals, particularly in the presence of H,02, and has been shown to be mutagenic in vitro (McBride et al., 1991). Mutagenesis by Fe2 could explain the epidemiologic correlations between increased iron stores with a number of human malignancies (Stevens et al., 1986). In view of these associations, the use of antioxidants has been suggested for the chemoprevention of cancer (Ames, 1983; Ito and Hirose, 1989), and provides the basis of a number of current clinical trials (Boone et al., 1990). +

D. REPAIROF DNA DAMAGE DNA is subject to continuous endogenous and environmental insults during the life of the cell. To compensate, a multitude of enzymes have evolved to repair DNA damage (Walker et al., 1985; Friedberg, 1984); mutation in any of these repair pathways may increase genomic instability. Some of the most carefully studied sources of damage to human DNA are produced by radiation and chemotherapies for cancer (e.g., van Duuren, 1988).DNA damage is associated with mutations and chromosome rearrangements (Friedberg, 1984), as well as the activation of normally repressed genes (Barr et al., 1986).There are two major classes of DNA repair pathways; one involves the excision of segments of DNA containing structural alterations, and the other involves the removal of individual altered DNA bases by specific enzymes such as DNA glycosylases (Lindahl, 1982). Ultraviolet irradiation and oxidative DNA damage can be considered to illustrate the role of different types of repair genes in maintaining genomic integrity. Ultraviolet (UV) light causes the formation of both pyrimidine dimers and 6-4pyrimidine photoproducts in DNA, both of which block DNA synthesis. These lesions are frequently removed to facilitate the prog-

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ression of fork movement during DNA replication (reviewed by Friedberg, 1984). A number of genes are involved in the repair of UV damage; mutants in those genes in E. coli are hypersensitive to UV light (van Houten, 1990). In man, individuals affected by the autosomal recessive disease xeroderma pigmentosum are defective in excisional repair (Cleaver, 1968), resulting in severe skin damage and skin cancers after exposure to solar UV light. T h e disease has at least seven complementation groups, and the defective genes for many of the groups are being cloned to determine the basis for this form of genomic instability (for group A, see Tanaka et al., 1989). These patients provide some of the strongest evidence linking defective DNA repair with the development of human cancers. Dozens of potentially mutagenic DNA lesions result from oxidative damage (Hutchinson, 1985). T h e most abundant of the DNA base modifications (Gajewski et al., 1990) is 8-hydroxyguanine (oh8Gua, also known as 8-oxoguanine). T h e free base and nucleoside are excreted in urine of rodents and man in amounts that suggest that each cell suffers more than 100 such modifications per cell per day (Shigenaga et al., 1989). Because of a more oxidative environment in mitochondria, this base modification is more abundant in mitochondria1 DNA than in nuclear DNA (Richter et al., 1988). Animals with higher basal metabolic rates excrete more of the free base and nucleoside (Shigenaga et al., 1989). In template DNA, oh8Gua causes misincorporation of adenine during in vztro DNA synthesis (Shibutani et al., 1991). In E . coli, oh8Gua causes G -+ T transversions (Wood et al., 1990; Moriya et al., 1991; Cheng et al., 1992). T h e repair of oh8Gua in DNA is believed to account for the urinary excretion of the base and nucleoside (Shigenaga et al., 1989),as well for the decrease of oh8Gua in liver DNA over time after yirradiation of rats (Kasai et al., 1986). Repair of oh8Gua in E. coli is catalyzed by a glycosylase/endonuclease (Boiteux et al., 1987; Michaels et al., 1991; Tchou et al., 1991). Mutants in the corresponding mutM gene (also known asfpg-1) exhibit an elevated frequency of G-C-+ T.A transversions (Cabrera et al., 1988). A compilation of damage-sensitive mutants such as the RAD mutants of yeast (Haynes and Kunz, 1981) foreshadowed the identification of DNA repair genes in higher eukaryotes. T h e complexity of DNA repair is also illustrated by the preferential repair of genes on the transcribed strand (Bohr et al., 1987), which points to the possibility of a DNA repair-RNA polymerase supercomplex. Mutations in any of the genes important to those functions may be expected to be the basis of mutator phenotypes as well as increased chromosome instability (e.g., Mortimer et al., 1981).

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E. IMMUNOGLOBULIN GENEHYPERMUTATION Normal development may be associated with mutator mechanisms (Huebner et al., 1989). The best documented example is the rapid generation of mutants in the variable region of immunoglobulin genes that allows the B cell to generate antibodies with progressively higher avidity (Rajewsky et al., 1987).The mutation rate in immunologically competent cells has been estimated to be as high as per base pair per cell division (Wabl et al., 1987, Reynaud et al., 1991), which is a million-fold greater than that estimated for spontaneous mutations (Chu et al., 1988; Loeb, 1991). An understanding of the molecular basis and control of immunoglobulin diversity may tell us whether disruptions in the mechanism for the targeting of these mutations may cause increased rates of mutations elsewhere in the genome or along other pathways of cellular differentiation. If gene conversion is operative in immunoglobulin hypermutation, as shown in birds (Reynaud et al., 1987; Thompson and Neiman, 1987), it might be expected that mutational loss of control could cause tandemly repeated genes to mutate at a much higher rate. If immunoglobulin diversity resulted from repetitive errors during reiterative DNA repair (Hackett et al., 1990), mutations affecting stringent localization of this process to immunoglobulin genes could cause mutations throughout the genome. Thus, loss of control over the localization of mutagenic mechanisms such as those involved in immunoglobulin diversity could result in a less restrictive mutator phenotype.

F. CHECKPOINTS Mechanisms that control the cell cycle help to preserve genomic stability. Checkpoints, originally defined by Hartwell and Weinert ( 1989), are control mechanisms that maintain the dependence of steps of the cell cycle upon earlier steps (see also Enoch and Nurse, 1991). One checkpoint important to the maintenance of genomic stability determines that DNA synthesis must be completed before mitosis can occur. This checkpoint thus prevents the segregation of either incomplete or damaged genomes to daughter cells. This dependence is governed by particular genes that, if altered, could produce genetic instability mutants. Mutants that can divide without completion of DNA synthesis have been extremely informative about mechanisms of cell cycle regulation and defective chromosome transmission. Such mutants have been observed in budding yeast (rad9 gene, Weinert and Hartwell, 1988), fission yeast [small (wee) mutant alleles of cdc2; Norbury and Nurse, 1989; Enoch and Nurse, 19901, Aspergallus nidulans (in minA and bamE genes;

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Osmani et al., 1988),and the mammalian BHK cells (in the hamster rccl gene; Nishimoto et al., 1978).Checkpoints are bypassed normally during development, as illustrated by the continuation of mitosis in early embryos despite inhibition of S phase (Hara et al., 1980; Kimmelman et al., 1987). Bypass can also be induced by toxins, as illustrated by the treatment of mammalian cells with caffeine (Schlegel and Pardee, 1986) or okadaic acid (Yamashita et al., 1990).Abnormal activation of checkpoint bypass or inactivation of checkpoints during mitosis thus can serve as mechanisms of increasing genomic instability. The predicted genomic instability phenotype of checkpoint mutants has been demonstrated in two Saccharomyces cerevisiae checkpoint mutants. First, rud9 deletion mutants (studied as checkpoint mutants by Weinert and Hartwell, 1990) exhibit a 7- to 21-fold elevation of chromosome loss. Normally, another checkpoint delays completion of M if microtubules are disrupted; mad2 mutants, isolated based upon sensitivity to the anti-microtubule drug benomyl, fail to arrest after exposure to benomyl, and have an increased frequency of chromosome loss (Li and Murray, 1991). G. MAINTENANCEOF CHROMOSOME PLOIDY

The maintenance of exact chromosome complements during cell division is a precisely regulated process that is beginning to be understood at the molecular level. Accurate chromosome transmission requires proper chromosome packaging and movement; proper coordination with the cell cycle; and proper interaction of DNA with topoisomerases, histone and nonhistone proteins, microtubules, centrioles, centromeres, and telomeres. As might be expected from the central role of these structures and functions, the fidelity of chromosome segregation is affected by mutations in genes associated with these functions (Pardue, 1991). Here, we briefly list such mutations that cause a decrease in the fidelity of chromosome transmission. The replication, recombination, and packaging of enormous lengths of DNA in small spaces results in entanglement of sister chromatids (Sundin and Vdrshavsky, 1980; DiNardo et al., 1984). Disentanglement requires cutting and rejoining of the DNA, which is catalyzed by the topoisomerases (reviewed by Wang, 1985).Accordingly, yeast mutants in topoisomerase I I have difficulty in separation of chromosomes during mitotic cell division, leading to the loss and breakage of chromosomes, and the segregation of nonviable cells at a high frequency (Holm et al., 1985; Uemura and Yanigida, 1986; Uemura et al., 1987). The nucleosomal structure of chromosomes is determined by interac-

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tions between the histones and DNA. Disturbing the normal ratios of histone types in yeast causes a loss in fidelity of mitotic chromosome transmission (Meeks-Wagneret al., 1986).Other chromosome-associated proteins may also be important in the proper segregation of chromosomes. Chromosome movement is mediated by the mitotic spindle apparatus, which is composed of spindle microtubules, the kinetochore (the chromosomal spindle attachment site), and the spindle pole body (the yeast equivalent of the centriole of higher eukaryotes). Disruption of the spindle pole body SPA1 gene is associated with a marked increase in the frequency of mitotic chromosome missegregation (Snyder and Davis, 1988). In a transgenic mouse model of tumor progression in which SV40 large T antigen expression in the pancreas is driven by the elastase promoter, tumor cell aneuploidy is preceded by the formation of tetraploid cells with more than four centrioles each (Levine et al., 1991). This sequence of tetraploidy leading to aneuploidy is strikingly reminiscent of a model proposed by Boveri in 1914 for the generation of aneuploidy in which he considers how the distribution of chromosome in a tetraploid cell can vary due to competition between centrioles for chromosomes, and is also consistent with the association in yeast between polyploidy and chromosome instability (Mayer and Aguilera, 1990). Other microtubular proteins also play an important role in proper chromosome segregation. The KARl gene of yeast is required for function of intranuclear and extranuclear microtubules. Mutations in or overexpression of this gene result in defective chromosome disjunction, causing polyploidy (Rose and Fink, 1987). Searches for mutants in yeast that cause an increase in the frequency of chromosome loss have led to the identification of tubulin and nontubulin genes that are required for microtubule stability (Hoyt et al., 1990). The kinesins are a family of microtubule motor proteins (reviewed in McDonald and Goldstein, 1990).Drosophih A homolog is kinesin the non-claret disjunctional (ncd) gene (McDonald and Goldstein, 1990); mutants in this gene exhibit an increase in meiotic and mitotic nondisjunction (reviewed in Sequeira et al., 1989). The importance of the kinesins is underscored by the existence of kinesin homologs in lower eukaryotes; an example is the nod gene of D. mehnogaster (Zhang et al., 1990), in which mutations result in nondisjunction (Carpenter, 1973; Zhang and Hawley, 1990). A proteinaceous complex is formed around the centromere during cell division, called the kinetochore. This complex represents the attachment site for the spindle microtubules to the chromosome (reviewed by Mitchison, 1988; Mazia, 1987).Among the many proteins purified from the yeast kinetochore is the CBFl (centromere binding factor) protein

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(Cai and Davis, 1990). Disruption of the cbfl gene causes an increased rate of chromosome loss/nondisjunction. Telomeres are repetitive sequences found at the ends of chromosomes that are required for chromosome stability and perhaps the completion of DNA replication of chromosome termini (reviewed by Blackburn and Szostak, 1984). A mutant with a defect in telomere elongation, estl (ever shorter telomeres), exhibits an increased frequency of chromosome loss (Lunblad and Szostak, 1989). Mutants in the rap1 yeast telomere binding protein gene also exhibit decreased chromosome stability (Conrad et al., 1990). The distribution of a full genome by chromosome segregation is only possible after DNA synthesis is completed. It is therefore not surprising that nearly all budding yeast cell cycle mutants have a high rate of chromosome loss (Hartwell and Smith, 1985). Direct searches for mutants or overproducing clones that exhibit an increased frequency of chromosomal loss in yeast have uncovered a large number of complementation groups (Meeks-Wagner et al., 1986; Kouprina et al., 1988; Spencer et al., 1990). Further insight into the mechanisms of chromosomal aneuploidy should be provided by molecular characterization of the corresponding genes and gene products. T h e number of chromosome instability mutants in lower eukaryotes suggests the existence of human homologs and the production of chromosomal instability by mutations in those genes. Given the large number of components dedicated to accurate chromosome transmission it is not surprising that there are a large number of chemicals that may also interact with these components and diminish the fidelity of chromosome transmission (Dellarco et al., 1986; Waters et al., 1986; Bond and Chandley, 1983). H. INHERITED HUMAN CHROMOSOMAL INSTABILITY SYNDROMES

Four human inheritable diseases, Bloom’s syndrome, ataxia telangiectasia, Fanconi’s anemia, and Werner syndrome, are associated with both chromosome instability and cancer (Knudson, 1977; Salk, 1982). T h e molecular basis of ataxia telangiectasia, Fanconi’s anemia, and Werner syndrome are not known, but Bloom’s syndrome is associated with alterations in DNA synthesis and chromosome instability. In particular, there is evidence for decreased production of DNA ligase I (Willis and Lindahl, 1987; Chan et al., 1987) despite the presence of wild-type ligase I DNA sequence (Petrini et al., 1991); there is also evidence in Bloom’s syndrome for an alteration in uracyl glycosylase (Seal et al., 1988).

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I.

RECOMBINATION

Recombination (reviewed by Low, 1988a; Smith, 1988a) plays an important role in different aspects of maintaining the genetic integrity of a cell, including DNA repair (Resnick et al., 1989),the proper segregation of chromosomes (Baker et al., 1976),and B- and T-cell differentiation (Hunkapiller and Hood, 1989; Huebner et al., 1989). However, recombination can also play a role in oncogene activation (Bishop, 1987) and in the loss of tumor suppressor gene function (Fig. 2; e.g., James et

1

DNA Replication

A @ no

B @ single

*

C double, @ e reciprocal

* * Homozygous diploids

D

c m gene

*

FIG.2. Loss of heterozygosity by homologous recombination. The recessive mutation indicated by the “x” occurred during an earlier somatic or germline event, resulting in a phenotypically normal cell heterozygous for the mutation. After DNA replication, prior to cytokinesis, homologous recombination can occur, resulting in any of a number of recombinants (“exchange” refers to a cross-over event, or a splice site where information from one chromosome is joined to information from another). No recombination (A) yields only heterozygotes. A single exchange between the homologous chromosomes can yield a cell homozygous for the mutation (B). Double, reciprocal exchange can result in the transfer of a patch of information from one chromosome to the other and, with appropriate assortment, can also yield a homozygous mutant (C). In gene conversion, information from one parent chromosome is transferred to the homolog without the loss of donor information (D). Other important mechanisms of loss of heterozygosity, including mutation and chromosome loss, are illustrated elsewhere (Scrable et al., 1990).

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af., 1989). T h e types of recombination (reviewed by Low, 1988b) are homologous recombination, site- or region-specific recombination (including immunoglobulin and T-cell receptor gene rearrangements), transposition, and illegitimate (nonhomologous) recombination. The recombination of DNA requires the breakage and rejoining of DNA duplexes. Enzymatic breakage by definition requires endonuclease activity. Homologous alignment of single-stranded DNA with double-stranded DNA is mediated by DNA pairing enzymes, which have been studied in E. cofi (Radding, 1981), yeast (Heyer et al., 1988; Sugino et al., 1988), cow (Kawasaki et af., 1989), and humans (Moore and Fishel, 1990). The importance of DNA ligases may be supported by the finding of deficient DNA ligase activity in human Bloom’s syndrome cells, which exhibit both elevated chromosomal instability and cancer susceptibility (see Section 111,H). Homologous recombination provides a mechanism for the loss of heterozygosity at tumor suppressor gene loci (Fig. 2). Beginning with a cell heterozygous for a mutation (indicated by the X on the light chromosome), DNA replication followed by cell division without recombination yields two heterozygous daughter cells. After DNA replication, the two chromatids of each newly duplicate chromatid pair (connected by a common centromere) segregate to a separate daughter cell (Fig. 2A). In contrast, reduction to homozygosity at the mutant locus can occur by one of three homologous recombination pathways. An odd number of crossover events between gene and centromer leads to the intermediate shown in B; the chromosomes can be segregated in either of two ways: one, shown under B, is the segregation leading to a cell homozygous for the mutation and the other (not shown) yields two heterozygous daughter cells. Double reciprocal exchanges can lead to exchanges of patches of information between the homologs (C). Again, one of two possible segregations of the daughter chromosomes leads to a cell homozygous for the mutation. In contrast, in gene conversion, the information on one strand is lost, with retention of information on the donor chromosome. In this case, either possible distribution of chromosomes yields one daughter that is homozygous for the mutation (D). Homologous recombination between repeated sequences can also cause loss of heterozygosity by looping out and deletion of the wild-type locus (Klein, 1988; Nelson et al., 1989; Yen et al., 1990; Vnencak-Jones and Phillips, 1990). The study of mutants that recombine at an abnormally low o r high frequency has led to the identification of enzymes required for homologous recombination in E. coli (e.g., Clark and Margulies, 1965; Konrad, 1977). Recombination by the RecBCD pathway, the major pathway of

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recombination of E. coli, is regulated at least in part by the presence of a certain sequence, Chi, and related sequences. The sequence 5’-GCTGGTGG-3’, comprising Chi, increases the frequency of recombination in its vicinity (Smith, 1988b). Some sequences similar to Chi, differing by one base pair, can also stimulate recombination, although to a lesser degree (Schultz et al., 1981; Cheng and Smith, 1984). An interesting implication of this work is that special sites and related sequences may drive homologous recombination, and account for discrepancies between genetic and physical maps of chromosomes (Cheng and Smith, 1984). The potential importance of homologous recombination sites is underscored by their existence in fungi (Gutz, 1971; Keil and Roeder, 1984)and humans (Wahls et al., 1990).There is an example of introduction of a recombination hotspot by a retrotransposon (Edelmann et al., 1989).Mutations that may either create (Sprague et al., 1978; Ponticelli et al., 1988) or destroy (Schultz et al., 1981; Schuchert et al., 1991) hotspots of recombination have been identified. Region-specific recombination, such as that occurring in B- and T-cell development for the generation of immunoglobulin genes and T-cell receptors, respectively (Blackwell and Alt, 1989; Perry, 1988; Hunkapiller and Hood, 1989), results in the deletion of segments of DNA based upon appropriate splice donor and receptor sites, leave a distinct pattern of nucleotides at the splice junction (Tonegawa, 1983; Akira et al., 1987), and depend upon expression of the RAG-1 and RAG-2 recombinase genes (Schatz et al., 1989; Oettinger et al., 1990). Loss of the regional specificity of that recombination would be expected to cause deletions or translocations with similar characteristics. This sort of mutation is illustrated in spontaneous h p t mutants selected from fetal T-lymphocytes. In contrast to adult T-lymphocytes, most fetal T-lymphocyte hprt mutations represent deletions characteristic of V(D)J recombinase activity (Fuscoe et al., 1991). Other relevant examples include the frequent association of the T-cell receptor locus with chromosomal translocations in T-lymphoblastic neoplasms (Reynolds et al., 1987), and chromosome 14 inversion in a human T-cell lymphoma mediated by illegitimate VH-Ja rearrangements. Translocations are frequent chromosomal aberrations associated with human cancers (reviewed by Heim and Mitelman, 1989; Cleary, 1991). It has become apparent of late that a large number of lymphomas associated with infection with HIV-1 are associated with translocations (reviewed in Karp and Broder, 1991). Unfortunately, the molecular mechanisms of translocations are not known. Even the basis of the first consistent tumor-associated chromosomal translocation, that causing the appearance of the Philadelphia chromosome (Ph’) in chronic my-

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elogenous leukemia (Nowell and Hungerford, 1960), remains unclear despite extensive study (Bernards et al., 1987). J. EPIGENETIC DNA MODIFICATION

During development, the differentiation of precursor cells into specialized tissues is the result of the coordinate expression of subsets of genes characteristic for that tissue, and the repression of genes not normally expressed in that tissue. Outside the recombination that occurs during immune cell differentiation, it is generally thought that differential gene expression occurs despite the presence of identical genomes in different cell types. Whatever the mechanisms are for that regulation, those same mechanisms for tissue-specific gene activation or inactivation may also be operative in the activation o r inactivation of oncogenes and tumor suppressor genes. We consider here the methylation of cytosine residues as an epigenetic developmental regulator of gene expression (Razin and Riggs, 1980; Conklin and Groudine, 1984; Cedar, 1988). Cytosine methylation of mammalian DNA occurs in the dinucleotide CpG to create 5-methylcytosine. T h e cell preferentially methylates cytosine in specific regions (such as highly repetitive DNA and centromeres), maintains methylation during cell division via methylases, and is able to demethylate sequences as appropriate. T h e extent of DNA methylation of genes is inversely proportional to transcriptional activity. The site-specific control of DNA methylation is currently unclear. However, methylated DNA binding proteins (Boyes and Bird, 1991) have been hypothesized to play an important role in the regulation of DNA methylation. Programmed DNA methylation has been studied extensively in two unusual situations in man: the fragile X mental retardation syndrome and X chromosome inactivation. T h e fragile X syndrome (Sutherland, 1985) is a common X-linked dominant form of mental retardation in humans with reduced penetrance, named after an isochromatid gap inducible in cell culture in the long arm o f t h e X chromosome (Xq27.3; Krawczun et al., 1985). T h e data appear consistent with an attractive hypothesis proposed by Laird, in which a DNA mutation blocks the reactivation of an inactivated X chromosome before oogenesis (Laird, 1987; Laird et al., 1987).Consistent with this hypothesis is the finding of aberrant hypermethylation at a CpG island in the fragile X region that correlates with absence of expression of the fragile X mental retardation (FMR-1) gene in affected individuals (e.g., Verkerk et al., 1991). Polymorphism in the length of CGG repeats within an FMR- 1 exon in fragile X chromosomes may influence the CpG island methylation (Verkerk et

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al., 1991). In X-chromosome inactivation (Gartler and Riggs, 1983), a major portion of one of two X chromosomes is not transcribed. Although the regulation of X-chromosome inactivation is not clear, inactivation is associated with DNA methylation (Gartler and Riggs, 1983) and the transcription of a certain region, XZST (X-inactivation-specific transcripts), exclusively on the inactive X chromosome (Brown et al., 1991). The components of these and other methylation systems that cause programmed gene inactivation could be imagined to cause transcriptional inactivation of tumor suppressor genes, causing epigenetic functional homozygosity despite the presence of genetic heterozygosity. An interesting set of experiments related -to unstable thymidine kinase transformants suggests changes in methylation as a plausible basis for instability of gene expression in cancer cells. Transfection of genetically TK-deficient cells with either unmethylated (Wigler et al., 1979)or methylated (Pollack et al., 1980) herpes virus tk DNA yields T k + transfectants containing demethylated DNA. However, these transfectants are unstable, frequently reverting to a T K - phenotype; the revertants have a highly methylated tk gene (Pellicer et al., 1980).

K. OTHERMECHANISMS Other mechanisms of genomic instability include gene amplification and transposition. Gene amplification,which was initially investigated in cultured cells (Schimke, 1988), represents one of the most common accompaniments of human cancer (Otto et al., 1989; Tlsty et al., 1989; Tlsty, 1990) and has prognostic significance in certain tumors (Brodeur et al., 1984; Ranzani et al., 1990). It is interesting that amplification appears to be associated with chromosomal deletion and integration; their integration (resembling transposition) may be associated with other forms of chromosome instability (Ruiz and Wahl, 1990). The expansion in the number of GC-rich triplet DNA repeat sequences has been associated with mutant phenotypes in Fragile X syndrome (as mentioned above) and in two other human genetic diseases. In the Fragile X syndrome, the repeated sequence is CGG (Verkerk et al., 1991; Fu et al., 1991). Increased numbers of repeats correlate with deficient transcription of the associated fmr-1 gene (Pieretti et al., 1991). Spinal and bulbar muscular atrophy (Kennedy disease) is associated with expansion in the number of CAG repeats (Edwards et al., 1991; La Spada et al., 1991). In myotonic dystrophy, the repeat is GCT (Fu et al., 1992; Mahadevan et al., 1992). The mechanism of the amplifications, as well as the relationships between methylation, the repeat sequence amplication, and other possible mutations, is currently unclear. For exam-

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ple, it is not known whether the mechanisms involved in amplifying the three different trinucleotide repeats differ fundamentally, or to what extent those mechanisms might involve mutation, recombination, and/or replication. It would be very important to know whether this form of genomic instability can also play a role in cancer. Transposition involves the insertion of DNA sequences either from other sites in the same genome, as in the case of transposons and retrotransposons (Weinstein, 1988; Brosius, 1991). o r from exogenous DNAs such as retroviruses (Temin, 1974). These inserted sequences can interrupt tumor suppressor genes, activate oncogenes by insertion of transcriptional activators, o r bring in activated oncogenes (Bishop, 1987). Although transpositions have been seldom observed in eukaryotic cells (Meuth, 1990), it is possible that they may play a role in a subset of human cancer. IV. Quantitative Considerations

A. MECHANISTICCONSIDERATIONS Because there are multiple genes that affect genomic stability and because there are multiple sites on each gene for inactivation, there is a high probability for the formation of genetically unstable clones during normal human development. In order to illustrate the potential importance of genomic instability mutations in humans, let us attempt to estimate the probability of genetically unstable clones arising per human lifetime. From the above lists of candidate genomic instability genes, a reasonable estimate of the number of genomic instability genes per genome is 100. If we estimate that on average one dominant mutation is possible per gene, the target size for dominant genomic instability mutations per genome is about 100 nucleotides. (By dominant, we mean that the mutant gene causes a phenotype despite the presence of a wild-type gene.) Of the 10I6 cell divisions estimated to occur during a human lifetime (Duesberg, 1987), perhaps one in a million (or 10lo cell divisions) yields cells that are capable of further division (in contrast to terminally differentiated neurons) and that are also retained within body tissues (in contrast to shed epithelial cells); this speculation derives from our estimates of cell turnover in different tissues. Given the probability of one mutation per 1 O ' O nucleotides per mammalian cell division, 100 dominant genomic instability mutations may occur per lifetime. Recessive mutations are those that cause no phenotype in the presence of a wild-type allele, but cause a phenotype when homozygous for

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the mutant allele. The recessive mutations important to this discussion include inactivating mutations in nonessential genomic instability genes and deleterious but noninactivating mutations in any genomic instability gene. Since inactivating mutations are included among the recessive mutations, their frequency is much higher than that of dominant mutations. If we use the h c Z a gene as a model, where mutation of about 40% of the nucleotides may cause gene inactivation (T. Kunkel, personal communication), an average gene with a 1000 nucleotide coding sequence would contain 400 nucleotides that, if mutated, may result in gene inactivation. If there are 25 nonessential genomic instability genes, then the genetic target for recessive mutations in nonessential genes causing genomic instability is about 10,000 nucleotides (25 genes x 400 nucleotides per gene). Based upon the calculations used at the end of the last paragraph, about 10,000 recessive genomic instability mutations in nonessential genes may occur per lifetime. By definition, cells containing recessive genomic instability mutations have no phenotype. However, cells homozygous for the mutations (i.e., containing two mutant and no wild-type alleles) would be expected to be genetically unstable. The process of generating a homozygous mutant from a heterozygote can be referred to as loss of heterozygosity or reduction to homozygosity. If the frequency of loss of heterozygosity in as estimated in uitro from human human somatic tissues is lov5 to cells (Hakoda et al., 1990)and in yeast (Hartwell and Smith, 1985),there is a real probability of any person developing a clone of cells exhibiting a genomic instability phenotype from the 10,000 cells hypothesized to develop recessive genomic instability mutations. Such clones would be subject to accelerated tumor progression. The large number of genomic instability genes also suggests that the probability of some people in the human population carrying a recessive mutation in a genomic instability gene is high. During 1O'O cell divisions, a probability of loss of heterozygosity of would be expected to yield in these individuals 100,000 cells per lifetime exhibiting genomic instability. As reviewed earlier (Fig. l), cells exhibiting increased genomic instability are expected to have an increased probability of progressing to a malignant phenotype. Moreover, a 1-cm benign internal tumor mass in one of these people, which approximates the lower limit of clinical detection and represents about lo8 cells,* together with a frequency of loss of heterozygosity of would yield 1000 individual clones per 1cm benign tumor with a genomic instability phenotype; these clones 2 This value represents a low estimate for the number of cells per 1-cc volume mass, given that tumor cells diameters range between about 12 and 20 I*..

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LOEB

would then be on a fast track to cancer. To put it another way, cancer may represent a delayed phenotype of genomic instability. It follows that genomic instability is expected to be more common among cancer cells than normal cells. We also expect that many uncharacterized genetic predispositions to cancer represent recessive genomic instability mutations. B. FACTORS AFFECTING THE PROBABILITY A N D IMPACT OF GENOMIC INSTABILITY MUTATIONS

Cell size and cell death are expected to affect the number of genomic instability mutations that might be expected in a tumor. For tumors containing small cells, such as small cell carcinoma of the lung, there are u p to 10 times more cells per unit volume, leading to a proportional rise in the number of genomic instability mutations. This increased tumor cell number at the time of clinical detection would therefore be predicted to allow further progression of the tumor by mutation compared to tumors composed of larger cells; this factor may contribute to the poor prognosis of small cell tumors in general and small cell carcinomas of the lung in particular. T h e number of generations necessary to generate a tumor mass would also be increased by cell death caused by either inadequate blood supply (also frequently the case for small cell carcinomas of the lung) or the occurrence of lethal genomic alterations; either would increase the probability of genomic instability mutations per unit volume of tumor even further. The timing of genomic instability mutations in the lifetime of a tumor is also expected to affect their potential contribution to tumor progression. T h e late occurrence of a genomic instability event would be expected to d o little; however, the early occurrence of a genomic instability mutation might be expected to cause a significantly larger number of genomic alterations during the growth of progeny cells, increasing the chance of late steps in tumor progression, and even increasing the chance of additional genomic instability mutations. T h e impact of individual genomic instability mutations on tumor progression is also dependent upon active cell division. Mitotically inactive cells would not be expected to be affected by mutations in genes important in the fidelity of DNA replication o r chromosome transmission. Similarly, mutations affecting the frequency or accuracy of recombination are more likely to be expressed if recombined chromosomes are being segregated. Genomic instability events causing their effect via loss of heterozygosity are irrelevant unless one of two alleles is mutated previously.

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The frequency of each genomic alteration is expected to differ according to the mechanism involved. Whereas the spontaneous rate of mutations, due to an aggregate of mechanisms, has been estimated to be on the order of lo-’ per gene (Oller et al., 1989; Monnat, 1989; Seshadri et al., 1987; Chu et al., 1988), current estimates for the frequency of spontaneous loss of heterozygosity are several orders of magnitude higher (Hakoda et al., 1990).The much lower frequency of spontaneous mutations compared to loss of heterozygosity suggests the possibility that the former is the rate-limiting step for two-step inactivation (mutation + loss of heterozygosity) of tumor suppressor genes; the strength of mutator phenotypes would be predicted to proportionally increase the probability of tumor progression.

C. EFFECTSOF CANCERPHYSIOLOGY ON PROBABILITIES Other factors that may affect the frequency of mutations or chromosome loss include different tendencies to generate endogenous mutagens such as free radical oxygen; anatomic or metabolic susceptibility to environmental mutagens (e.g., UV light does not usually affect the colon); and toxic metabolic effects such as anoxia, caused by poor oxygenation commonly seen in the centers of tumors. Sutherland (1988) showed that different regions in a tumor vary in oxygen tension, pH, nutrients, lactate, growth factors, and concentration of therapeutic agents. Stasis would be expected to result in the accumulation of potentially genotoxic waste products. Inflammatory cell infiltrates may also differ in different regions of a tumor; since inflammatory cells are associated with the production of DNA-damaging free radicals (Weitzman and Stossel, 1982),mutation rates may be increased locally. Finally, therapeutic interventions such as radiation and chemotherapy are also mutagenic.

V. Summary and Perspectives

A large number of genes are important in DNA metabolism; we have argued that this aggregate target makes it likely that mutations or chemical inhibition of these gene products could occur at a frequency high enough to contribute to the initiation and the acceleration of malignant progression in human cancer. It is important to note that these genes do not code for gene products that directly change the phenotype of a normal cell to a cancer cell. Instead, mutations in these genes cause an increased frequency of genomic changes throughout the genome; it is these secondary mutations (such as those affecting tumor suppressor

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genes) that can contribute more directly to tumor progression. A major focus of current cancer research has been on oncogenes and tumor suppressor genes. It is now important to study the mechanisms behind the genomic modifications affecting those genes. Work aimed at identifying the location of tumor suppressor genes does not adequately address the mechanisms of genomic instability. Some early work on retinoblastoma showed loss of heterozygosity by nondisjunction and recombination (reviewed by Scrable et al., 1990). However, other tumor systems have not been pursued with such attention on mechanism. More work is also required to address the question of frequency of those mechanisms in tumors, as well as genes that may be involved in causing the hypothesized increases in frequency of loss of heterozygosity. Experimental systems involving the analysis of mutants with genomic instability phenotypes are likely to yield further insight into the mechanisms of genomic instability. It is important to note that the mutational, recombinational, and nondisjunctional mechanisms of loss of heterozygosity described for tumor suppressor genes apply equally to genomic stability genes. T h e size of the aggregate genomic target for genomic instability mutations makes it likely that a good many people may inherit recessive mutations in genomic stability genes. Those people would be susceptible to somatic events resulting in homozygous mutant genotypes at those loci; those clones would then be expected to undergo accelerated tumor progression. A direct prediction of this hypothesis is that many uncharacterized cancer families have recessive mutations in genomic stability genes. Measures that might be found to lower the frequency of loss of heterozygosity may help lower the rate of cancer in such individuals. Implicit in the hypothesis of genomic instability accelerating tumor progression is the possibility that interrupting o r inhibiting this mutation cascade may delay the onset of clinically apparent cancer. There are at least two human cancers that are particularly pertinent to analysis in this context: hepatocellular carcinoma and prostatic cancer. Hepatocellular carcinoma is the major cause of cancer mortality in Asia and Africa and is associated with chronic hepatitis B virus infection. The time between initial infection with hepatitis virus and clinically detected hepatocellular carcinoma can be as great as 40 years (Beasley et al., 1981). infection with this virus is associated with a striking infiltration of hepatic parenchyma with inflammatory cells (Cotran et al., 1989a),which in turn generate potentially mutagenic oxygen free radicals (Cerutti and Trump, 1991). Although stimulation of cell replication by hepatitis virus and mutagenesis by environmental exposure to aflatoxin must be con-

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sidered in the etiology of hepatocellular carcinoma, let us consider for a moment the possibility that the generation of mutations by oxygen free radicals constitutes a rate-limiting step in the progression of this cancer. If mutations caused by oxygen free radical damage to DNA is a ratelimiting step in liver cancer, a 50% decrease in damage by oxygen free radicals by inhibition of inflammation or by treatment with free radical scavengers might significantly increase the latent period from cancer initiation to the appearance of tumor. Prostate cancer represents the third leading cause of cancer deaths in men. Its late onset suggests multistep progression. An alarming feature of this cancer is its high and increasing incidence. In one study, 30% of all prostates removed at autopsy contained foci of adenocarcinoma (Cotran et al., 1989b), although only 4% of American men over age 45 develop clinically diagnosed prostate cancer (Strahan, 1963). The late onset of this cancer offers an attractive opportunity for cancer chemoprevention efforts. If just one of the steps in this cancer can be delayed even 10 years, the death rate from prostatic cancer can be significantly reduced. Our knowledge of genomic instability is based upon important contributions from a wide variety of experimental systems. Critical information is gleaned from approaches ranging from phage and bacterial genetics to the histopathology of cancer cells, from the enzymology of DNA polymerases to the karyotypic analysis of chromosomes, from the study of yeast and Drosophila mutants to human cancer syndromes. Despite the wide variety of associations, predictions, examples, and calculations, the general prevalence and importance of increased genomic instability in human cancer remain to be established. The study of basic mechanisms of genomic instability in unicellular and multicellular organisms, in combination with molecular epidemiologic studies of cancer, should yield a deeper understanding of the importance of genetic alterations associated with cancer and suggest more precise therapeutic and preventative interventions.

ACKNOWLEDGMENTS We thank our colleagues Leland Hartwell, John Kreider, Raymond Monnat, Richmond Prehn, Dan Feig, and Joann Sweasy for their suggestions and Joan Hiltner for help with editing the manuscript. This work was supported by Fellowship DRG-049 from the Damon Runyon-Walter Winchell Cancer Research Fund to K.C.C. and NIH O.I.G. R35-CA-39903 to L.A.L.

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FUNCTION AND REGULATION OF THE HUMAN MULTIDRUG RESISTANCE GENE Khew-Voon Chin,' Ira Pastan," and Michael M. Gottesman Laboratory of Cell Biology and 'Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

I. The Multidrug Transporter as a Cause of Multidrug Resistance A. in Vitro Model Systems for Studying Drug Resistance B. Multidrug Resistance Results from Expression of a Drug Efflux Pump C. Cloning of the MDRl Gene D. Mechanism of Action of the Multidrug Transporter E. Expression of the MDRI Gene in Normal Tissues F. Expression of the M D f f 1 Gene in Cancer G. in Viva Model Systems H. Clinical Trials of Agents that Inhibit the Multidrug Transporter 11. Regulation of Expression of mdr Genes A. Increases in mdr RNA Levels after Toxic Injury B. Regulation by Heat Shock and Levels of CAMP-Dependent Protein Kinase C. Developmental Regulation of mdr Gene Expression D. Studies on mdr Promoters E. Regulation of MDR 1 Gene Expression during Tumor Progression F. Post-translational Regulation of P-Glycoprotein 111. Summary and Conclusions References

I. The Multidrug Transporter as a Cause of Multidrug Resistance Of the approximately 1,000,000 new cases of cancer in the United States each year only half are curable with surgery and radiation. T h e remainder, due to local spread or distant metastases, must be treated systemically using chemotherapy or biological response modifiers, including various forms of immunotherapy. Of these latter approaches, only chemotherapy reliably produces cures, but only in a minority of systemic cancers such as leukemias, lymphomas, sarcomas, neuroblastoma, testicular cancer, and choriocarcinoma. The majority of human 1 Present address: Genetics Center, University of Texas Health Science Center, Houston. TX 77225.

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Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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metastatic cancers are either intrinsically resistant to chemotherapy or acquire resistance to multiple drugs after an initial promising response to treatment. Thus, the circumvention of resistance to chemotherapy has become one of the primary goals of modern approaches to cancer treatment. There have been many reviews on the phenomenon of drug resistance as it has been studied in the laboratory. T h e reader is referred to these, including comprehensive reviews by Gottesman et al. (1991), Kane et al. (1990),Endicott and Ling (1989), Moscow and Cowan (1990), and Schinkel and Borst (1991) for detailed information about the biology, genetics, and biochemistry of simultaneous resistance to anticancer drugs. T h e purpose of the present review is to summarize the current state of understanding of a major form of multidrug resistance to natural product drugs, with a specific focus on the ways in which this form of multidrug resistance is regulated in normal cells and in cancer cells. A. INVITRO MODELSYSTEMS FOR STUDYING DRUGRESISTANCE The primary approach to analyzing the causes of chemotherapy resistance in human cancer has been to develop human cancer cell lines in tissue culture that are resistant to anticancer drugs. Because both intrinsic and acquired resistance affects many different drugs, models in which simultaneous resistance develops to multiple drugs (known as multidrug resistance, o r MDR) have been emphasized. Our laboratory has used human KB carcinoma cells (a subclone of HeLa obtained from the American Type Culture Collection) to study the phenomenon of multidrug resistance. Multidrug-resistant cell lines are readily developed in tissue culture after selection in such anticancer drugs as doxorubicin or vinblastine, or the antimicrotubule agent colchicine. These cell lines are also cross-resistant to many other natural product anticancer drugs (enumerated in Table I) as well as many other cytotoxic compounds (also shown in Table I). Close examination of the chemical structures of these compounds reveals no common chemical features, but all of the drugs have hydrophobic regions, and most, but not all, are positively charged at physiological pH. These in zdro model systems of drug resistance generally reflect the broad resistance patterns seen in many human cancers. However, they do not precisely mimic in vivo patterns of drug resistance, since many of the more water-soluble anticancer drugs, such as nucleoside analogs and alkylating agents, are not included in this pattern of cross-resistance (see also Table I).

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CYTOTOXIC DRUGSTO

WHICH

TABLE I MDR CELLSARE RESISTANTOR SENSITIVE

Resistant

Sensitive

Anticancer drugs

Other agents

Anticancer drugs

Vznca alkaloids Vinblastine, vincristine Anthracyclines Daunorubicin, doxorubicin, mitoxantrone Epipodophyllotoxins Etoposide, teniposide Mitomycin C Actinomycin D Taxol Topotecan Mithramycin

Colchicine Puromycin Podophyllotoxin Ethidium bromide Emetine Gramicidin D Valinomy cin

5-Fluorouracil Cytosine arabinoside Methotrexate Chlorambucil Melphalan Cyclophosphamide Carmustine Bleomycin Camptothecin

B. MULTIDRUGRESISTANCE RESULTS FROM EXPRESSION OF A DRUGEFFLUX PUMP Studies on MDR cell lines of the type described above indicated that intracellular accumulation of drugs to which the cell lines were resistant was reduced (Dano, 1973; Fojo et al., 1985a; Willingham et al., 1986). This reduced drug accumulation has been shown to be due to expression of an energy-dependent drug efflux pump (Dano, 1973; Willingham et al., 1986),associated with appearance in the plasma membrane of a 170,000-Daglycoprotein, called P-glycoprotein (Kartner et al., 1985). More recent studies have indicated that the mechanism by which drugs are extruded is complicated (see below) and involves a component of blocked influx into the cytoplasm, as well as active efflux. Although it has not yet been possible to purify P-glycoprotein to homogeneity and demonstrate its activity in a reconstituted liposome, vesicles prepared from MDR cells show ATP-dependent transport activity (Horio et al., 1988). OF THE MDR GENE C. CLONING

To gain a more detailed understanding of the mechanism of action of the multidrug efflux pump, it was necessary to clone the gene (or genes) responsible for multidrug resistance. To do this, advantage was taken of the fact that highly multidrug-resistant cell lines overexpress the gene

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responsible for multidrug resistance. Our MDR KB lines have amplified this gene (Fojo et al., 1985b). We were able to isolate a fragment of the gene responsible for MDR based on its amplification in MDR KB cell lines (Roninson et al., 1986).Gros et al. (1986) used a similar approach to isolate a probe for the mouse mdr genes. Other groups used either the overexpression of the RNA for the mdr gene (Scotto et al., 1986) or antibodies to P-glycoprotein to clone a cDNA fragment (Gerlach et al., 1986). It was quickly demonstrated that the mdr genes cloned on the basis of gene amplification or overexpression of mRNA encoded the 170,000-Da P-glycoprotein that had been previously associated with the MDR phenotype (Ueda et al., 1986). A full-length cDNA for the mdr gene from either human (Ueda et al., 1987a) or mouse (Gros et al., 1986) was shown to confer the complete phenotype of MDR on drug-sensitive cells, therefore proving that the correct gene had been cloned. The sequence encoded by the full-length MDR1 cDNA encodes a protein of 1280 amino acids (Chen et al., 1986).Computer-assisted analysis of this sequence reveals 12 hydrophobic amino acid stretches that are likely to be transmembrane regions, arranged so that there are 6 such regions in the amino half of the protein, and 6 in the carboxyl half. Two cytoplasmic regions contain sequences with considerable identity to other sequences in the protein database. These regions are believed to define ATP-binding and/or utilization sites that are involved in the transduction of the energy of ATP into the transport function of P-glycoprotein. In addition, the primary sequence contains three putative glycosylation sites in a region that appears to lie in the first extracellular loop of the protein. These data have been used to create a model of the way in which P-glycoprotein is inserted in the plasma membrane (Fig. 1). It should be noted that although some of the details of this model have been verified by biochemical data or immunological studies (Yoshimura et al., 1989), definitive studies that confirm which loops are intracellular or extracellular have not been done. Based on studies from a coupled in vitro translation translocation system (Zhang and Ling, 1991), a new topological model was proposed for the mouse mdrl (or mdr1b)-encoded P-glycoprotein, in which P-glycoprotein is predicted to have 10 transmembrane (TM) domains as opposed to 12. The eighth transmembrane domain (TM8) and the intervening residues between TM8 and T M 9 are found to be extracellular based on glycosylation of a site between TM8 and TM9 predicted to be cytoplasmic in the original 12 T M model. Although this form of P-glycoprotein may occur in a coupled in uitro translation-translocation system, the predicted glycosylation in the carboxyl half of the molecule has not been shown to occur in living human

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cells, providing support for the original 12 T M model (Bruggemann et al., 1989, 1992). Genetic and biochemical analyses of the human multidrug transporter have been used to define functional domains of P-glycoprotein. Such studies include genetic manipulations such as deletions, substitutions, and point mutations (Azzaria et al., 1989; Buschman and Gros, 1991; Choi et al., 1988; Currier et al., 1989; Gros et al., 1991; reviewed in Pastan et al., 1991) and biochemical studies in which labeling sites of photoaffinity analogs of drugs are identified (Bruggemann et al., 1989; Yoshimura et al., 1989; Greenberger et al., 1991).The results of some of these studies for human P-glycoprotein have been summarized in Fig. 1. Although these results are not based on an exhaustive analysis of structure-function relationships for P-glycoprotein, they indicate a role for the first intracytoplasmic loop, and T M l l in the choice of substrates for the transporter, since mutations in this loop and in T M l 1 affect substrate specificity (Choi et al., 1988; Kioka et al., 1989; Gros

1-1

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FIG. 1. Model of human P-glycoprotein showing important functional domains. A schematic representation of the 1280 amino acids of P-glycoprotein deduced from its cDNA sequence is shown, including putative glycosylation sites (wiggly lines), ATP binding sites (circled), and the position of two mutations which affect transport specificity (filled in balls at residues 183 and 185). The heavy bars define functional domains, including ATP sites (Chen et al., 1986), photoaffinity labeling sites (Bruggemann et al., 1989, 1992), and transport specificity sites (Choi et al., 1988; Currier et al., 1992). Adapted from Gottesman and Pastan (1988).

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1991; Currier et al., 1992). Photoaffinity labeling occurs in both halves of the molecule within the regions that include transmembrane segments and their associated intracellular and extracellular loops. This result has led to the suggestion that these regions come together in the three-dimensional structure of the transporter to form a single site through which drugs pass on their journey through the transporter. More than one mdr gene has been found in mammals. Humans have two such genes, called MDR 1 and MDR2. The MDR2 gene is adjacent to the MDR 1 gene on the long arm of chromosome 7 and encodes a cDNA with more than 80% sequence identity with MDR1. The MDR1 gene encodes a protein that confers drug resistance, but MDRZ does not. The function of MDRS, which is expressed as a mRNA primarily in liver, is not known. Rodents have three mdr genes, of which two (mdrl and mdr3, also known as mdrlb and mdrla, respectively) are closely related functional transporters with slightly different drug specificity, and one (mdr2) is not a functional multidrug transporter. The mdr genes have been found throughout nature, even in simple eukaryotes. They are thought to contribute to drug resistance in malaria and leishmaniasis (reviewed in Schinkel and Borst, 1991). As already noted, extensive sequence identity exists with other proteins in the two domains that are thought to bind ATP. This sequence identity defines a superfamily of proteins that harness the energy of ATP for a variety of functions, most associated with membrane transport. This superfamily has been called the ATP-binding cassette (ABC) superfamily based on this sequence identity and the belief that all of these proteins share functional homology (Higgins et al., 1990). Figure 2 summarizes the many different kinds of transport exhibited by this growing superfamily of proteins, including nutrient transport into bacterial cells, transport of pigments, export of polysaccharides, polypeptide toxins and hydrophobic peptides, transport across the peroxisomal membrane, and intracellular transport of peptides for class I antigen presentation across the membranes of the endoplasmic reticulum (reviewed in Gottesman et al., 1991; Kane et al., 1990). For some of these transport systems, such as the MDRI system and its close relative in yeast, STE6, which transports the hydrophobic sex peptide “a,” all of the components needed for transport are included in one protein; for other members of the family, the ATP-binding subunit is distinct, or linked to some, but not all, of the essential transmembrane regions. The precise functions of many members of the ABC superfamily are not currently known. et at.,

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polysaccharides polypeptide toxins

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EUKARYOTIC TRANSPORTERS

THER TRANSPORTERS CFTR MDR2

FIG. 2. The MDR or ABC superfamily of energy-dependenttransporters.This figure summarizes the various cellular sites in which ABC family members are thought to act. In some cases, such as the peroxisomal membrane pump and the class I antigen-presenting endoplasmic reticulum transporter, the function (and/or direction) of the pump is hypothetical.

D. MECHANISM OF ACTION OF THE MULTIDRUG TRANSPORTER T h e way in which the energy of ATP is harnessed to reduce drug accumulation in cells expressing mdr genes is not known. However, physiological, biochemical, and genetic studies suggest a novel mechanism of action of this transporter, illustrated schematically in Fig. 3. T h e essential features of this mechanism include the following. (a) Most drugs that are substrates for the transporter are hydrophobic, amphipathic molecules that can only cross the plasma membrane in their uncharged forms. Cells expressing P-glycoprotein show both decreased uptake and increased efflux of such drugs. One simple explanation for this result is that the drugs are intercepted by the transporter while they are still in the plasma membrane. Direct evidence that P-glycoprotein can remove

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MECHANISMOF MULTlDRUG TRANSPORT

B 1

extracellular medium

endogenous substrates

FIG.3. Model of how P-glycoprotein reduces drug accumulation in cells. P-glycoprotein is represented as a “hydrophobic vacuum cleaner” that removes drugs directly from the plasma membrane, thereby preventing their accumulation in the cytoplasm where they can affect cytotoxic targets. Either (A) uncharged drug diffusing through the membrane or (B) positively charged drug embedded in the inner leaflet of the plasma membrane m i l d be substrates for the transporter. Adapted from Gottesman ef al. (1991).

and expel drugs directly from the plasma membrane has been obtained (Raviv et al., 1990). (b) The transporter is not highly specific for drugs. Hence, the drug-labeling sites that are detected by photoaffinity studies are likely to represent points of contact with the drugs in the transporter, rather than tight binding sites. These contact sites are represented in the model of the cross-hatched areas, which come together to form a compartment or pore within the transporter. The specificity for substrates that does occur can be explained by either a filtering function of the transporter or an energy-dependent choice of which drugs are expelled from the transporter once they have entered. (c) Several studies have suggested that hydrogen ions or protonated compounds are extruded by cells that have active P-glycoprotein on their cell surfaces (Thiebaut et nl., 1990; Keizer and Joenje, 1989; Boscoboinik et al., 1990). Hence, a role for P-glycoprotein as a pump, in which the transport of protons is somehow linked to drug transport, has been suggested (Stein et al., 1992). Alternate hypotheses are that P-glycoprotein is essentially a “flippase” that can directly flip protonated drug from the inner leaflet of the lipid bilayer to the outer leaflet (Higgins and Gottesman, 1992) or a constantly moving waterwheel or sump pump that sweeps impurities out of the membrane. The role of the two ATP sites in P-glycoprotein is also somewhat

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mysterious. Both sites are essential for complete function (Azzaria et al., 1989), but they are not equivalent since they do not appear to be fully interchangeable (S. J. Currier, M. M. Gottesman, and I. Pastan, unpublished data). Several hypothesis have been suggested, including the idea that one site is required to get drug into the transporter, whereas the second site gets it out (I. Roninson, personal communication); that the sites work cooperatively to hydrolyze ATP; or that one site is a regulatory site for nucleotide triphosphates, whereas the other is catalytic. Further genetic and biochemical analyses should clarify whether any of these hypotheses is correct.

E. EXPRESSION OF THE MDR1 GENE IN NORMAL TISSUES To understand what the normal function of P-glycoprotein might be, it was necessary to determine where this protein was normally located. Using monoclonal antibody MRK- 16, P-glycoprotein was found in epithelial cells in the liver, kidney, pancreas, and small and large intestine (Thiebaut et al., 1987); in cells of the adrenal cortex (Thiebaut et al., 1987); in endothelial cells in the brain and testis (Thiebaut et al., 1989; Cordon-Cardo et al., 1989); in the placenta (Sugawara et al., 1988; Cordon-Carlo et al., 1989); and in secretory glands of the pregnant endometrium in the mouse (Arceciet al., 1988).Many of these sites of localization have been confirmed by studies in which levels of MDR 1 RNA were measured (Fojo et al., 1987). Since localization in epithelial cells is polarized along the lumenal surface of these cells, it seems likely that P-glycoprotein is involved in excretory transport of metabolites or xenobiotics in liver, kidney, and intestine. This idea has been supported by experimental evidence indicating that epithelial monolayers into which P-glycoprotein has been introduced express this protein apically, and efficiently transport MDR drugs from their basal to their apical surfaces (Horio et al., 1989). The localizations in the adrenal cortex and in secretory glands of the pregnant endometrium argue for a role for P-glycoprotein in steroid secretion. Some evidence suggests that some steroids, such as progesterone, may interact directly with P-glycoprotein (Yang et al., 1989, 1990; Qian and Beck, 1990). The specific localization of P-glycoprotein in endothelial cells in the brain and testis, as well as its localization in placenta, raises the possibility of a barrier function for this transporter. Thus, it may protect the brain, testes, and fetus from circulating xenobiotics or toxic endogenous metabolites.

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F. EXPRESSION OF THE M D R l GENEIN CANCER A large number of recent publications have confirmed that the MDR 1 gene is expressed in human cancers. Our initial studies, based on the analysis of M D R l RNA levels in over 600 human cancers (Goldstein et al., 1989; Fojo et al., 1987), demonstrated the following patterns of expression: 1. Cancers derived from tissues that normally express the M D R l gene, such as cancer of the colon, kidney, pancreas, adrenal, and liver, usually express high levels of MDR 1 RNA. Using a semiquantitative slot blot assay, M D R l RNA levels in these tumors are comparable to levels seen in selected tissue culture lines with at least four- to six-fold drug resistance (Akiyama et al., 1985; Shen et al., 1986). Thus, it seems likely that a component of the intrinsic resistance of these tumors is due to expression of P-glycoprotein, and, in some cases, direct evidence suggests that this is the case (Kakehi et al., 1988). 2 . High expression is sometimes found in untreated cancers, such as leukemias, lymphomas, non-small cell lung cancers with neuroendocrine properties, and chronic myelogenous leukemia in blast crisis in which the cell of origin of the tumor does not show high M D R l RNA levels. Why the MDRl gene is turned on in these cases, in the absence of selective factors, is unclear, but possible explanations will be considered in the section below on regulation of the M D R l gene. In the case of acute nonlymphocytic leukemia of adults, high MDR 1 gene expression at the time of tumor presentation predicts poor response to chemotherapy (Sato et al., 1990).T w o recent studies by Chan and co-workers (1990, 199 1 ) suggest that any appearance of P-glycoprotein, as detected histochemically, predicts poor response to treatment in childhood sarcoma and neuroblastoma. 3. Elevated M D R l RNA levels are seen in many tumors that have acquired resistance to chemotherapy and have recurred after successful initial treatment with anticancer drugs. Such cancers include breast and ovarian cancer, neuroblastoma, leukemias, and lymphomas. This is the expected result if P-glycoprotein protects cancers from chemotherapy with natural product drugs, and the use of these drugs selects resistant cells that have more P-glycoprotein on their surfaces. T h e same phenomenon, however, could occur in MDR 1 RNA expression increases in response to tumor progression, independent of the nature of the anticancer drugs to which the cancers are exposed. 4.There are many cancers, including many drug-sensitive tumors, in which M D R l RNA levels are low or nondetectable by standard techniques. Examples include untreated ovarian and breast cancer, Wilms ~

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tumor, and small cell and non-small cell lung cancer (Lai et al., 1989). This last disease is quite resistant to chemotherapy, but apparently this resistance does not relate to expression of the MDRl gene. While establishing that high-level MDR 1 gene expression is common in human cancer, these studies also raise some important questions which need to be addressed. One such question is how much P-glycoprotein does a cell need to become drug resistant? Although some studies suggest that any amount of expression is significant, this seems unlikely, unless P-glycoprotein expression is also a marker for malignant progression. This idea will be discussed below in the section on regulation. One way to approach the issue of how much expression is significant in a human cancer is to develop in vivo animal models with varying levels of P-glycoprotein (see next section). An even more critical approach is to develop clinical trials in which agents that inhibit the multidrug transporter are tested for their ability to reverse drug resistance in cancer with known levels of MDR 1 RNA or P-glycoprotein expression (see below). G. IN VIVOMODELSYSTEMS

Two general approaches to in vivo models include the use of MDR tumor xenografts or homografts, or the use of transgenic animals expressing the human MDRl gene. In the former approach, drug-resistant cell lines may come directly from human or animal cancers, or may be selected in nitro or constructed by introduction of a human MDRl gene. The latter case produces a genotype that is best defined, since it can be shown that drug resistance in vivo results directly from expression of the human MDR 1 gene. We have described such a system using xenografted HT-29 colon cancer cells infected with an MDRl retrovirus that becomes resistant in vivo to chemotherapy (Pearson et al., 1991). Probably the most studied system is the P388 mouse leukemia selected for doxorubicin resistance in vitro. Although this resistant tumor is known to express P-glycoprotein, the relative contribution of the multidrug transporter to the resistance of this cell line is not known. In this case, however, agents that inhibit the transporter can be shown to partially reverse resistance of P388 DoxR cells in vivo (Tsuruo, 1989). KB-8-5 cells, which are approximately four- to six-fold drug resistant and express levels of MDRl RNA comparable to those found in many human cancers, form drug-resistant cancers when injected subcutaneously into nude mice. The drug resistance of these tumors is reversible with the use of appropriate inhibitors of P-glycoprotein (unpublished

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data of the authors; S.-I. Akiyama, unpublished data). These data suggest an upper limit for the amount of MDRl RNA that is needed to produce drug resistance in vivo. An alternative in vivo model is the MDRl transgenic mouse in which expression of the MDRl gene in bone marrow renders white blood counts insensitive to normally toxic doses of natural product drugs such as daunorubicin, doxorubicin, actinomycin D, taxol, vinblastine, and vincristine (Galski et al., 1989; Mickisch et al., 1991a; reviewed in Mickisch et al., 1991b). In this model, MDRl RNA expression is also comparable to levels found in KB-8-5 cells, and the animals show from 50% to severalfold increased tolerance for cytotoxic MDR drugs. The MDRl RNA levels in the bone marrow cells in these MDR 1 transgenic mice appear to be close to the lower limit needed to produce detectable drug resistance. It therefore seems unlikely that the very low levels of MDRl RNA detectable by PCR, but not detectable by RNase protection o r slot blot analysis, would have physiological significance. Another use of the MDRl transgenic mouse is to test agents that reverse drug resistance. Many such compounds have been described, including verapamil and its analogs, dihydropyridines, cyclosporine analogs, nontoxic vinca and anthracycline analogs, phenothiazines, cephalosporins, quinidine, and reserpine (reviewed in Tsuruo, 1989). Most of these compounds seem to be substrates for P-glycoprotein and probably work by swamping out the ability of the transporter to handle cytotoxic drugs, which are present at lower concentrations. Efficacy, toxicity, and dosage schedules of these reversing agents can be worked out in the transgenic mouse system, with the hope that these data will accelerate the introduction of such agents into clinical trials (Mickisch et al., 1991~). H. CLINICAL TRIALS OF AGENTS THAT INHIBIT THE MULTIDRC'GTRANSPORTER

Although it is beyond the scope of this review to consider all of the clinical trials in which efforts have been made to interfere with Pglycoprotein-mediated drug resistance, preliminary results suggest that such a strategy is feasible. T h e most convincing published studies to date are those of Dalton and co-workers (1989), who studied multiple myeloma selected for resistance to vincristine, adriamycin, dexamethasone (VAD) therapy in patients in whom P-glycoprotein levels were increased following therapy. Addition of modest amounts of racemic verapamil to a VAD regimen resulted in clinical responses in about 50% of these patients with VAD-resistant cancers. Similar results have been obtained in a trial of combination chemotherapy in drug-resistant lymphomas at

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the National Cancer Institute using R-verapamil as the reversing agent (R. Wittes, W. Wilson, A. Fojo, and S. Bates, unpublished data). These results, in patients with massive burdens of drug-resistant tumors, suggest that addition of MDR-reversing agents to adjuvant or primary chemotherapy regimens, where tumor burden is much less and the frequency of MDR 1-expressing cancer cells is even rarer, might have significant impact on the chemotherapy of many human cancers. Leukemias, lymphomas, and breast and ovarian cancers seem like reasonable cancers in which to initiate these prospective clinical studies.

II. Regulation of Expression of mdr Genes Studies on the regulation of the mdr gene expression in normal tissues and cancers are few in number, and the mechanisms of regulation are thus not well understood at present. Understanding the manner in which mdr gene expression occurs may provide crucial information for the design of more rational chemotherapy for human cancers. A. INCREASES IN rndr RNA LEVELS AFTER TOXIC INJURY

Studies in rat liver show that expression of rndr RNA is modulated in response to toxic insults such as carcinogens, and mdr RNA is increased in regenerating rat liver following partial hepatectomy (Thorgeirsson et al, 1987; Fairchild et al., 1987). The increased mdr RNA levels in regenerating rat liver appear to be post-transcriptionally regulated since nuclear run-on analysis shows no major effect of hepatectomy on transcription (Marino et al., 1990).The presence of a destabilizing consensus motif in the 3’ noncoding region of the human MDR 1 gene suggests that a similar mode of post-transcriptional regulation may occur in the human (Marino et al., 1990). For post-transcriptional regulation to be effective, the half-life of rndr RNA should normally be short and should increase following toxic insult. The rndr RNA levels drop quickly several days after hepatectomy (Thorgeirrson et al., 1987), or after recovery from heat shock (Chin et al., 1990a),suggesting that, at least under some conditions, the half-life of mdr RNA is short (30-60 min). However, direct measurements of mdr RNA half-life under steady-state conditions have proved difficult, and a direct demonstration of increased half-life of rndr RNA as a result of toxic injury has been elusive. We have demonstrated that rndr RNA levels are also dramatically elevated after exposure of rodent cells to a variety of chemotherapeutic agents that are known to be substrates for the multidrug transporter as

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well as inhibitors of DNA topoisomerase I1 (Chin et al., 1990b). Again, direct effects of these agents on transcription as detected by nuclear runon experiments were not observed (Chin et al., 1990b). Increases in MDRl RNA levels were also not observed in human cell lines after treatment with these agents. In contrast to these results, Kuwano and colleagues, using a 2-kbp fragment of the human MDR 1 gene promoter fused with the chloramphenicol acetyltransferase (CAT) reporter gene, showed that human MDRl promoter activity could be upregulated in the presence of vincristine, daunomycin, or colchicine, by two- to fivefold, when transfected into human KB cells (Kohno et al., 1989). Changes in the level of expression of the endogenous MDR 1 gene, however, were not reported in this study (Kohno et al., 1989). The modest changes in CAT activity observed in these experiments, plus the lack of internal and reference controls, suggest the need for further experiments to determine whether transcription of the human MDRl gene may be regulated by chemotherapeutic agents.

B. REGULATION BY HEATSHOCK AND LEVELS OF CAMP-DEPENDENT PROTEIN KINASE We have noted the presence of heat shock consensus elements in the promoter region of the MDR 1 gene and subsequently discovered that its expression is regulated by heat shock, arsenite, and cadmium in a human kidney cell line (Chin el al., 1990a). The increase in P-glycoprotein level after heat shock or arsenite treatment correlates with a transient increase in drug resistance. Recent work from Ueda and colleagues indicates that the tandem repeats of heat-shock-responsive elements present in the MDR 1 promoter are required for transcriptional activation by arsenite (K. Ueda, personal communication), We have also shown that a CAMP-dependent protein kinase (PKA) mutant and a transfectant Chinese hamster ovary (CHO) cell line expressing the defective regulatory subunit (RI) for PKA from this mutant were sensitive to multiple drugs that are known to be substrates for the multidrug transporter (Abraham et al., 1987). The sensitivity of the kinase mutant and the transfectant cell lines to multiple drugs was a result of decreased P-glycoprotein mRNA, which led to decreased protein (Abraham et d.,1990; Chin et al., 1992a). A similar reduction in mdr mRNA was seen in PKA mutants in a mouse adrenal Y1 cell system (Chin et al., 1992a). Preliminary nuclear run-on results indicate that the reduced mdr RNA levels in the PKA mutant cells may be post-transcriptionally regulated, but the precise regulatory input of PKA in the basal expression of mdr genes in these cells remains undefined.

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C. DEVELOPMENTAL REGULATION OF mdr GENEEXPRESSION Studies described above in Section I, E, indicate that there are dramatic differences in levels of MDRl RNA levels in different human tissues, and similar results have been observed in rodents. These differences presumably reflect tissue specific regulation of mdr gene transcription and/or mRNA stability, but mechanisms responsible for most of these differences have yet to be determined. A striking example of the developmentally regulated expression of the mdr gene is illustrated by studies on the gravid mouse uterus. One of the mouse mdrl genes is expressed at extremely high levels in the secretory epithelium of the endometrium during pregnancy, in contrast to the relatively low levels of expression in the nongravid uterus (Arceci et al., 1988). In related studies, the mouse mdrlb RNA levels were found to be increased by estrogen and progesterone in secretory glands of the mouse uterus (Arceci et al., 1990). Similar results were obtained in studies of uterine expression of hamster pgp genes using monoclonal antibodies and immunohistochemistry (Bradley et al., 1990). The precise function of the uterine secretory glands is not known, but secretion of steroids or peptide hormones is likely, and P-glycoprotein is thought to play a role in this secretion. The mechanism of progesterone stimulation of the mdr gene appears to be transcriptional (S. B. Horowitz, personal communication). Induction of MDRl gene expression has also been observed with differentiating agents such as retinoic acid, sodium butyrate, dimethyl sulfoxide, and dimethylformamide, in human neuroblastoma and colon carcinoma cells (Bates et al., 1989; Mickley et al., 1989). Despite an increase in cell surface P-glycoprotein in the experiments, no detectable increase in drug resistance was observed in most of these cells. ON mdr PROMOTERS D. STUDIES

Progress in understanding transcriptional regulation of human and rodent mdr genes will require detailed knowledge of the structure and activity of the mdr promoter region. Genomic sequences encompassing 5' upstream regions and the complete structural genes for the human MDRl gene and the mouse mdrlb gene have been isolated (Chen et al., 1990; Raymond and Gros, 1989).Figure 4 shows a schematic diagram of the structure of the human MDRl gene, including its 5' regulatory region. Isolation of the promoter of the human MDR 1 gene and characterization of its transcription initiation site revealed that at least two

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different promoters are utilized in different multidrug-resistant cell lines (Ueda et al., 1987b,c). T h e downstream promoter is the major promoter used in normal tissues that express the MDR 1 gene, but transcription from a start site further upstream is seen in some multidrugresistant cell lines. T h e downstream promoter is TATA-less and contains a consensus CAAT box and two GC box-like sequences (Ueda et al., 1987~).Normal human adrenal gland, colon, and liver cells, the human hepatoma cell line HepG2, and vinblastine-selected human KB multidrug-resistant cells initiate transcription from the downstream promoter at two major sites corresponding to nucleotides 136 and 140 upstream from the ATG translational start codon. Analysis with a series of colchicine-selected multidrug-resistant KB cell lines showed that transcription starts from an upstream promoter as well as from the downstream T h e upstream promoter resides at a large promoter (Ueda et al., 1987~). distance from the downstream promoter and may represent an aberrant control region actively recruited only under specific selective conditions, since, as noted, normal human tissues and various other cell lines initiate transcription from the downstream promoter only. In nitro transcription results indicate that the region from +5 to + 127 around the transcriptional initiation site influences the transcriptional efficiency from the MDR 1 promoter (Cornwell, 1990). Consensus sequences in this region similar to downstream regulatory elements of the adenovirus major late promoter and other eukaryotic promoters were also noted. I n addition, an initiator-like consensus (Smale and Baltimore, 1989) is also present, flanking the initiation start site of the promoter. T h e presence of these sequence elements suggests that transcriptional

10 kb

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FIG.4. Structure of the human M D R l gene. The positions of the exons (numbered vertical bars) and the putative initiation sites for transcription are shown (Chen et al., 1990; Ueda ef al., 1987b). The ATG that initiates translation is in exon 2.

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regulation through the promoter of the MDRl gene resembles a large variety of cellular and viral TATA-less promoter genes (Smale and Baltimore, 1989; Pugh and Tjian, 1991). Kuwano and colleagues have also reported the isolation of the human MDR 1 gene promoter and, in addition, have located a tissue-specific enhancer within a 700-bp fragment claimed to be 10 kb upstream from the initiation site (Kohno et al., 1990). The activity of this enhancer has been demonstrated in a few cell lines of diverse species origin (monkey kidney CV- 1 cells, mouse adrenal cortical Y 1 cells, and human adrenal SW13 carcinoma cells). A promoter of the human MDRl gene that includes about 1 kb of DNA sequence 5' to the downstream initiation site is also capable of conferring cell-type-specific expression in a variety of cells (K.-V. Chin, I. Pastan, and M. M. Gottesman, unpublished data). The ability of this promoter fragment to direct cell-type-specific expression requires further analysis and comparison with the results reported by Kohno et al. (1990). Analyses of the mouse mdrla (Hsu et al., 1990; Ikeguchi et al., 1991) and mdrlb (Raymond and Gros, 1990; Cohen et al., 1991) promoters have also been described. The mdrlb promoter has been shown to contain consensus TATA, CCAAT, and GC sequence elements, and celltype-specific activity of the promoter was also observed, which correlated with endogenous mdrlb mRNA levels (Raymond and Gros, 1990). Horwitz and colleagues have identified putative AP-I and AP-2 sites, glucocorticoid responsive elements (GRE), and potential positive and negative ®ulatory elements in the mdrlb promoter (Cohen et al., 1991). T h e presence of such putative elements in the promoter should be interpreted with caution, as functional correlations have not yet been established for most of these regulatory elements. Sequence analysis of the mdrla promoter also reveals the presence of multiple putative cisacting consensus elements, including TATA and CAAT boxes, and Sp-1, AP-1, and AP-2 sites (Hsu et al., 1990; Ikeguchi et al., 1991). Differential initiation from two putative promoters and the alternative usage of different poly(A) signals have been noted to give rise to multiple forms of mdrla transcripts in various multidrug-resistant cell lines (Hsu et al., 1990). Kuo and colleagues (Ikeguchi et al., 1991) have demonstrated that the putative AP-1 element present in the promoter negatively regulates promoter activity, since deletion or mutagenesis of the consensus sequence enhances the basal promoter activity. In contrast, a putative AP-1 element in the hamster pgpl (mdrla) gene was shown to be essential for full promoter activity since mutagenesis of this element resulted in a substantial decrease in basal pgpl promoter activity (Teeter et al., 1991).

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Increased CAT activity was also observed in cotransfection experiments of both mouse and hamster mdrla promoters with expression vectors encoding the c j u n and c-Fos proteins, which interact with AP-1 sites. In contrast, cotransfection with the retinoic acid receptor results in suppression. T h e hamster pgpl promoter, like its human counterpart, is TATA-less and contains an Sp-1 site in addition to an AP-1 site. Some of the differences among the human MDR 1 promoter and the rodent mdr promoters may not be functionally significant, but other differences probably reflect the different ways in which these genes are regulated. These differences include response to chemotherapy (stimulation in rodent, but not human), heat shock (stimulation in human, but not rodent), and the differential developmental regulation of the two functional rodent mdr genes.

E. REGULATION OF MDRl GENEEXPRESSION DURING TUMOR PROGRESSION As described in Section I,F, expression of the MDRl gene occurs commonly in human cancers derived from normal tissues that express the multidrug transporter, as well as tumors that have acquired resistance to chemotherapy during relapse (Goldstein et al., 1989). Also, as previously noted, MDR 1 gene expression may also occur in some cancers not derived from MDR 1-expressing normal tissues, and not previously exposed to drugs, such as acute nonlymphocytic leukemias and chronic myelogenous leukemia in blast crisis (Goldstein et al., 1989; Pirker et al., 1989). These results suggest that expression of the MDR 1 gene is stimulated during tumor progression, o r regulated by genes involved in tumorigenesis. There is now strong evidence that implicates the RAS oncogene and the p53 tumor suppressor gene in the malignant progression of cancers, such as carcinoma of the colon, neuroblastoma, and chronic myelogenous leukemia in blast crisis (Bishop, 1991; Feinstein et al., 1991; Fearon and Vogelstein, 1990). Since RAS oncoproteins could affect gene expression (Barbacid, 1987), and recent data suggest that p53 protein might directly affect transcription (Fields and Jang, 1990; Raycroft et al., 1990), w e speculated that the emergence of multidrug resistance might be closely associated with tumor progression and that the MDRl gene could be turned on as a result of activation of RAS and inactivation of the p53 tumor suppressor gene which occurs through genetic lesions during tumorigenesis. We examined this hypothesis directly by cotransfecting an MDR 1 promoter CAT construction with expression vec-

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tors for c-Ha-RAS-1, wild-type p53, and mutant p53. Our results showed that the human MDRl gene promoter is a target for the c-HaRAS-I oncogene and the p53 tumor suppressor gene (Chin et al., 1992b). The stimulatory effect of c-Ha-RAS-1 was not specific for the MDRl promoter alone, since CAT activities driven by the chicken pactin, c-Ha-RAS-1, Rous sarcoma virus (RSV), and SV40 promoters are also strongly enhanced. On the other hand, a mutant human p53 harboring a substitution from arginine to histidine at amino acid 175 specifically stimulated the MDR 1 promoter and wild-type p53 exerted specific repression (Chin et al., 1922b). In addition, c-fos, c-jun, and c-myc oncogenes seemed to have no direct effect on the MDRl promoter. These results taken together imply that the MDR 1 gene can be activated during tumor progression associated with mutations in certain oncogenes and tumor suppressor genes. F. POST-TRANSLATIONAL REGULATION OF P-GLYCOPROTEIN P-glycoprotein is post-translationally modified by glycosylation, as already discussed, and by phosphorylation (Carlsen et al., 1977; Hamada et al., 1987; Mellado and Horwitz, 1987; Richert et al., 1988). Nonglycosylated P-glycoprotein appears to be active, as indicated by multidrug resistance of tunicamycin-treated cells (Beck and Cirtain, 1982) and the fact that multidrug-resistant cells can be selected from lectin-resistant mutants that do not carry out normal glycosylation (Ling et al., 1983). Thus glycosylation does not appear to be a major mechanism of posttranslational regulation of activity of P-glycoprotein. P-glycoprotein isolated from intact cells is phosphorylated on several sites. One such site is increased after TPA treatment of cells (Hamada et al., 1987), which may result in a small increase in multidrug resistance resulting from decreased drug accumulation (Fine et al., 1988). In vitro, P-glycoprotein is a substrate for protein kinase C (PKC), which is stimulated by TPA (Chambers et al., 1990) and is also a substrate for PKA, which is stimulated by CAMP(Mellado and Horwitz, 1987). More recently, it was demonstrated that multidrug-resistant MCF-7 cells transfected with the cDNA for the MDR 1 gene, when transfected in addition with the cDNA for PKCa, showed increased drug resistance and increased phosphorylation of P-glycoprotein (Yu et al., 1991). Drug resistance and levels of P-glycoprotein phosphorylation of PKCa-transfected cells could be further increased in the presence of phorbol ester. These studies, if confirmed, suggest that phosphorylation of P-glycopro-

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tein by PKCa leads to increased drug resistance in multidrug-resistant cells. The nature of the kinases responsible for the other phosphorylation sites in uiuo is unknown, but in vitro another membrane-associated kinase has been found to phosphorylate P-glycoprotein (Staats et al., 1990).The importance and significance of other putative protein kinases responsible for P-glycoprotein phosphorylation and function remain to be determined.

Ill. Summary and Conclusions The discovery of an energy-dependent pump system for natural product anticancer drugs has important implications for the biology of related energy-dependent transport systems as well as for the treatment of human cancer. To fully realize the therapeutic potential associated with manipulation of the multidrug transporter, it will be necessary to understand the mechanisms of action of the transporter and its mode of regulation. This review has summarized recent developments in these areas which suggest that both the activity of the pump and its genetic regulation are potential targets for new anticancer therapies. ACKNOWLEDGMENTS We thank our many co-workers for ideas and discussion throughout the course of this work, Joyce Sharrar for help in designing the figures, and Dwayne Eutsey for secretarial help and figure construction.

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RELATIONSHIP BETWEEN myc ONCOGENE ACTIVATION AND MHC CLASS I EXPRESSION Peter I. Schrier and Lucy T. C. Peltenburg Departmentof Clinical Oncology, University Hospital, P.O. Box 9600,2300 RC Leiden, The Netherlands

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A. Immune Defense against Cancer Cells B. Role of MHC Class I in Immune Defense C. MHC Class I Expression in Tumors Modulation of MHC Class I Expression by Oncogenes A, Adenovirus E l A B. Other Viral Oncogenes C. fos, raJ and ras D. myc Molecular Mechanism of MHC Class I Regulation by Oncogenes A. General Mechanisms of Regulation B. Regulation in Tumor Cells Biological Consequences of MHC Class I Downmodulation by Oncogenes A. MHC and Progression B. T Cells C. Other Factors Involved in the Immune Reaction against Tumor Cells D. HLA Class I Mediates Effect of c-ntyc on NK Sensitivity E. Other Factors Determining N K Susceptibility Concluding Remarks References

1. Introduction Over the past decade it has become manifest that cancer cells develop by multiple genetic alterations (Weinberg, 1989; Bishop, 1991). These alterations include activation of protooncogenes as well as inactivation of tumor suppressor genes. Protooncogenes, more commonly called oncogenes, exert important functions in cell proliferation and differentiation and their activity is usually tightly controlled to ensure a minimal risk of inappropriate activity. Oncogenes can be activated in animal and human tumors by several mechanisms including amplification, elevated expression, and point mutations. These genetic alterations usually result in an altered activity of the oncogene-encoded protein and this contributes to uncontrolled proliferation. In contrast, tumor suppressor genes 181 ADVANCES IN CANCER RESEARCH, VOL. 60

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are involved in cancer through their inactivation. T h e function of these genes is not precisely known, but it is thought that they encode proteins involved in the control of proliferation and differentiation and that their inactivation relaxes the constraint these proteins normally exert on cell division. Inactivation of tumor suppressor genes often occurs through deletions of the gene, but also point mutations have been found (see Marshall, 1991, for a recent review). T h e number of oncogenes and tumor suppressor genes thus far discovered is almost dazzling. More than a hundred oncogenes and a rapidly increasing number of tumor suppressor loci have been described (Stanbridge and Cavenee, 1989; Bishop, 1991; Marshall, 1991). Due to tight regulation of oncogenes in each cell, the individual seems rather weil protected against neoplasia. Nevertheless, the chance for cancer to develop during the lifetime of an individual is considerable, apparently suggesting that the control mechanisms are not always capable of preventing a cell from degenerating into a cancer cell. T h e increase of the risk to develop cancer with age indicates that multiple hits are involved in disturbing the regulatory mechanisms that confine the boundary between normal proliferation and cancerous growth. At least some of these hits consist of structural alterations in genes, leading to the appearance of altered proteins. An important question is whether the immune defense of the individual has the potential to recognize these alterations and eliminate (pre)neoplastic cells in which immunogenic hits have occurred. T h e answer should be positive, because the immune system has the manifest potential to detect altered self-antigens and therefore, altered oncogene o r tumor suppressor proteins are obvious candidates to be tracked down. Processed peptides of these antigens are presented to the immune system by proteins encoded by the major histocompatibility complex (MHC) Class I. The combination of MHC Class I and peptide is expressed at the cell surface and the complex can be recognized by potential immune effector cells, usually T lymphocytes (see Napolitano et al., 1989, for review). T h e elimination of tumor cells by natural killer (NK) cells, however, is also influenced by MHC Class I proteins (Hoglund et al., 1990). Considering the involvement of MHC Class I in the killing of tumor cells by T cells and N K cells, it is evident that the level of expression of MHC Class I proteins is decisive for a proper interaction of the immune system with malignant cells. In this context, it is highly relevant that in animal as well as in human tumor cells activation of certain oncogenes does largely influence the expression of MHC Class I. In this contribution w e will review this phenomenon, paying special attention to the myc oncogenes. Members of the m y oncogene family are expressed in all

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vertebrate cells, where they play a key function in the regulation of growth and differentiation (Collum and Alt, 1990; Liischer and Eisenman, 1990). Their expression increases at the onset of cell proliferation. Recently it has been shown that the c-my protein is capable of forming a heterodimer with another cellular protein resulting in a DNA-binding activity (Ariga et al., 1989; Blackwell et al., 1990; Blackwood and Eisenman, 1991). This suggests that the protein is involved in transcriptional regulation. In many forms of human cancer elevated expression of m y genes has been found. This activation may coincide with altered MHC Class I expression, which indicates that oncogenic properties induced by activation of the c-myc gene are directly coupled to an alteration of the sensitivity of the tumor cells to the cellular immune defense of the host. In this review, we will first discuss regulation of MHC Class I expression in tumors and ask which oncogenes might be involved. Then, we will look more closely at the mechanism of MHC Class I regulation and discuss possible mechanisms for the action of the myc oncogenes in down-modulation of MHC Class I expression. Finally, we will address the question what is the implication of the suppression of MHC Class I by myc genes for the immune sensitivity of tumor cells and what are the possible mechanisms underlying it.

11. MHC Class I Expression and Cancer A. IMMUNE DEFENSE AGAINST CELLCELLS Recent developments in mobilizing the immune system of the patient for the defense against neoplastic cells have drawn much attention. Remarkable results were obtained with therapies involving interleukin-2 (IL2) (Rosenberg et al., 1987, 1988; Rosenberg, 1988). In these treatment modalities, lymphocytes cultured in vitro with IL-2 are administered to the patients. Either lymphokine-activated killer (LAK) cells originating from peripheral blood mononuclear cells or expanded tumor-infiltrating lymphocytes (TIL cells) were used. A LAK cell population mainly consists of activated N K cells (Hersey and Bolhuis, 1987; Ballas and Rasmussen, 1990; Faure et al., 1990).These cells have a broad specificity and play an important role in the defense against viral infections (Herberman, 1989; Robertson and Ritz, 1990). In addition, they are capable of discriminating between tumor cells and normal cells thereby preferentially reacting with tumor cells (Hiserodt and Herberman, 1989). In the case of TIL cells, the active effector cells are predominantly activated cytotoxic T cells (CTLs) with specific antitumor activity (Muul et al., 1987; Topalian et al., 1989). These T cells interact with

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altered peptides presented by proteins encoded by the MHC gene complex (termed HLA in humans), a phenomenon commonly called MHC restriction. It is therefore likely that specific T cells in the T I L population react with peptides present in the tumor cells but not in normal cells. HLA Class I-restricted T cells specifically reacting with the tumor have been found in patients for only a limited number of human cancers (Degiovanni et al., 1988, 1990; Anichini et al., 1989; Belldegrun et al., 1989; Knuth et al., 1989; Topalian et al., 1989; Wolfel et al., 1989; Crowley et al., 1990, 1991; Gervois et al., 1990; Ioannides et al., 1991b). In animals, especially in those bearing virus-induced tumors, tumorspecific MHC Class I-restricted CTLs are a common phenomenon and have been well characterized (reviewed in Melief and Kast, 1990). For human melanoma, T cells with unique specificity for the autologous tumor have been described (Topalian et al., 1989; Wolfel et al., 1989; Gervois et al., 1990). CTL clones recognizing different antigens on one single tumor have been described (Degiovanni et al., 1988, 1990; Van den Eynde et al., 1989; Wolfel et al., 1989). However, in other reported cases, the CTLs also reacted with allogeneic tumors but only when identical HLA Class I molecules were present on the tumors (Crowley et al., 1990). T h e latter data suggest the occurrence of a common tumor-specific antigen present in at least several allogeneic melanomas. These findings stress a number of important points. First, the immune system seems capable of recognizing and eliminating tumor cells. Second, MHC Class I antigens do play an important role in the interaction between tumor cells and the immune system: potential tumor antigens of any kind (nuclear, cytoplasmic, o r cell membrane-bound) are processed in the tumor cell and the resulting peptides bind to MHC Class I molecules followed by expression of the complex at the cell surface. Third, tumor cells must express antigens that are more or less specific for the tumor or a particular tumor type to be recognized and killed by specific T cells. In several cases of virus-induced animal tumors, these antigens are well defined and the precise structure of the peptides presented by the MHC Class I molecules has even been elucidated (Kast and Melief, 1991). For human tumors, which are usually not virus-induced, such tumor-specific antigens have not been clearly defined thus far. Whatever their identity may be, a good expression of HLA Class I in the tumor cell will be required for their presentation at the cell surface and proper recognition by T cells. In contrast, tumor cells with low expression of MHC Class I proteins may evade T-cell immunity (Tanaka et al., 1988; Melief et al., 1989). Further evidence for a pivotal role of HLA Class I proteins in determining the sensitivity of tumors cells to the host’s immune defense came

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from another line of research. Tumor cells with low expression of MHC Class I antigens are more refractile to natural killer cells than counterparts expressing high amounts of MHC Class I protein (Ljunggren and Karre, 1990). The mechanism of this phenomenon has not been resolved, but will be discussed in more detail in Section v. Nevertheless, this finding puts the role of MHC Class I proteins in the immune defense against cancer cells in a wider perspective: low expression of these proteins does not necessarily result in an escape of the tumor cell from the immune system of the host because these cells, insensitive as they are to T cells, still can be eradicated by NK cells. B. ROLEOF MHC CLASSI IN IMMUNE DEFENSE 1. Structure and Function of MHC Class I

The elucidation of the structure of several human MHC Class I proteins by X-ray crystallography (Bjorkman et d., 1987a,b; Garrett et al., 1989; Madden et al., 1991) has led to new exciting data on the biology and function of HLA Class I antigens. We will briefly summarize the most important findings relevant for an understanding of the involvement of HLA Class I antigens in the interaction of immune effector cells with cancer cells. The MHC gene complex is localized on chromosome 6p in humans and on chromosome 17 in mice. It extends over more than 3000 kb, and the numerous loci can be subdivided in four classes, two of which, Class I and Class 11, are involved in antigen presentation. Both encode integral membrane proteins. The MHC Class I proteins represent the classical transplantation antigens, H-2 in mice (encoded by 3-6 loci dependent on the haplotype) and HLA in humans (encoded by 3 loci, HLA-A, -B, and -C). These proteins are heterodimeric glycoproteins consisting of a 45-kDa heavy chain noncovalently associated with a 12-kDa protein, p2microglobulin. The Class I1 complex includes the classical immune response genes (la in the mouse, HLA-D in humans) encoding heterodimeric proteins, which are expressed at high density specifically on antigen-presenting cells. Telomeric of the MHC Class I genes resides a group of genes with strong homology to Class I genes, which are often referred to as the nonclassical MHC Class I genes. These genes are present in the human as well as in the mouse genome; in the mouse they are defined as the Qa-Tla genes (Robinson, 1987; Heinrichs and Orr, 1990). A striking feature of the MHC gene complex is its high polymorphism. This holds true in particular for the Class I and Class I1

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genes: by now, at least 80 Class I HLA-A, -B and -C molecules and 35 Class I1 HLA-D, -P, and -Q molecules have been identified (Lawlor et al., 1990; Yunis and Yunis, 1991). T h e Qa-Tlu region in the mouse is far less polymorphic and in humans no polymorphism in the nonclassical MHC Class I genes has been described (Mellor et al., 1984). T h e polymorphism of the classical HLA genes is indicative for the biological function of the molecules: selective pressure has engendered a large variety of HLA-A and HLA-B alleles as a consequence of their capability to present antigens to the immune system. In contrast, HLA-C proteins do not present antigens due to a failure to be expressed at the cell surface and consequently are less polymorphic (Lawlor et al., 1990). The elucidation of the complete tertiary structure by X-ray crystaliography of three HLA Class I alleles, A2, Aw68, and B27 (Bjorkman et al., 1987b; Garrett et al., 1989; Bjorkman and Parham, 1990; Jardetzky et al., 1991; Madden et al., 1991), has provided a prototype MHC Class I structure. This has largely contributed to the understanding of the precise function of MHC Class I proteins in the presentation of peptides derived from altered self-antigens or foreign antigens to the immune system of the host. The polymorphic extracellular a, and a2 domains of the protein are situated on top of an Ig-like 6-sandwich structure formed by the agdomain and p2microglobulin and form a platform of an eight-stranded P-pleated sheet topped by two a-helices (Bjorkman and Parham, 1990). This results in a binding groove shaped in such a way that peptides can fit into it. Dependent on the particular MHC Class I allele, the size of the peptide as well as its sequence is critical for appropriate binding (Schumacher et al., 1991). Endogenous peptides are probably generated by proteasome complexes (Brown et al., 1991; Glynne et al., 1991) and are made accessible to MHC Class I molecules in the rough endoplasmatic reticulum or cis-Golgi (Schumacher et al., 1990; Townsend et al., 1990). Recently discovered transporter proteins with homology to the multidrug resistance family of transporters are involved in the transport of peptides into this compartment (Deverson et al., 1990; Monaco et al., 1990; Trowsdale et al., 1990; Spies and DeMars, 1991). T h e conformation of the HLA Class I heavy chain is largely determined by the association with peptide (Elliott et al., 1991) and it seems that only the peptide-bound molecule is able to assemble with 6,-microglobulin and form a stable heterodimer that is expressed on the cell surface (Townsend et al., 1990; Kozlowski et al., 1991; Silver Pt al., 1991). This implies that the availability of appropriate peptide in the cell is essential for surface expression of MHC Class 1. Recently, peptides eluted from immunoprecipitated HLA-A2, several murine MHC Class I molecules (Falk et al., 1991), and HLA-B27 (Jardetzky et al., 199 1) have been sequenced. Dominant motifs in the amino

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acid sequence of eluted peptides were found. Several viral peptides known to be presented by MHC Class I contained these motifs. In addition, some of them matched to earlier identified proteins including histones, ribosomal proteins, and heat shock proteins (Jardetzky et al., 1991). Also, dominant peptides of unknown origin were found. These could be self-peptides playing a role in the discrimination between selfdeterminants and non-self-determinants by immune effector cells. A possible role for such peptides in determining the sensitivity of tumor cells to natural killer cells will be discussed in Section V. In tumor cells, MHC Class I-bound peptides might represent (tumor-specific) altered self-epitopes that in principle can be recognized by surveilling T cells.

2. Immunogenicity of Tumor CelLs In the light of the crucial role that HLA Class I antigens play in the interaction of altered self-antigens or viral antigens with cytotoxic T cells, one would expect that antigens specifically present in tumors are presented by HLA Class I molecules. Such antigens might be viral antigens in the case of virally induced tumors or altered self-proteins in the case of tumors induced by xenobiotics such as carcinogens or radiation. Also, “spontaneously arisen” tumors are probably caused by xenobiotic agents, although a naturally occurring incorporation of mismatched base pairs in the process of DNA replication by inadequate repair cannot be excluded. In numerous animal and human tumors, mutations in oncogenes have been shown to be responsible for the tumorigenic properties of the tumor cell. These mutations are potential targets for recognition by the immune system of the host. A recent demonstration of an immune response against an oncogene protein is the presentation of mutated ras oncogene peptides by MHC Class I1 molecules to T helper cells resulting in proliferation of MHC Class II-restricted T cells recognizing the mutated peptide (Jung and Schluesener, 1991; Peace et al., 1991). It was shown that intact cells are capable of presenting exogenous native mutated rm protein, indicating that effective processing to the correct mutated peptide and binding to the HLA Class I1 molecule had occurred (Peace et al., 1991). Although so far no HLA Class I-restricted responses were seen, these results are encouraging with respect to T-cell responses against mutated oncogene proteins. Another candidate oncogene protein product that can be activated by various mutations is the p53 protein, involved in many forms of human cancer (see Harris, 1991, and Vogelstein, 1991, for comprehensive reviews). Specific MHC Class Irestricted T-cell responses directed against tumors have been seen in animals bearing carcinogen- or UV-induced tumors (Prain and Main, 1957; Kripke, 1988). New immunogenic antigens on tumor cells can

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easily be induced as shown by the isolation of highly immunogenic variants of tumor cells by treatment with carcinogens or UV radiation. This suggests that altered self-proteins can function as tumor antigens (Whitwell et al., 1984; Hostetler and Kripke, 1988). Strikingly, for a particular murine tumor and its variants, several genes encoding tumor-specific proteins have been cloned, and these turned out to be completely unrelated and probably not involved in the origin of the tumor (Lurquin et al., 1989; Sibille et al., 1990; Szikora et al., 1990; Van den Eynde et al., 1991). These genes were isolated using MHC Class I-restricted, in vivo elicited, CTL clones with strict specificity for the tumor. For human melanomas similar observations point to the existence of different tumor-specific antigens that can be discerned by HLA Class I-restricted cytotoxic T cells (Knuth et al., 1989; Van den Eynde et al., 1989; Degiovanni et al., 1990). I n two independent studies (Wolfel et al., 1989; Crowley et al., 1991), HLA-A2 could be assigned as the allele presenting a tentative tumor-specific peptide. However, the data are contradictory in that one group of investigators found unique antigens not present in allogeneic melanomas (Wolfel et al., 1989), whereas the other group found that the T-cell cultures cross-react with allogeneic HLA-A2positive melanomas from a number of different patients (Crowley et al., 1991). In the latter case, there should be a common tumor antigen present on different unrelated melanomas. This antigen might be a mutated oncogene activated in human melanoma, although it should be conceded that no consistent activation of any of the known oncogenes has been found in human melanomas so far. Alternate candidates for common tumor antigens in melanoma might be self-antigens expressed at elevated levels in the tumor cells. These include, for example, gp75 (Vijayasaradhi et al., 1990), p3.58 (ICAM-1) (Johnson et al., 1988), and p97, a member of the transferrin family (Rose et al., 1986). In the case of the latter protein, MHC Class 11-restricted T-cell clones specifically raised against the human p97 protein were found to be capable of promoting elimination of p97-positive murine tumor cells in an animal model (Kahn et al., 1991). Although these proteins are not supposed to induce an autoimmune reaction, one can imagine that tolerance to some of these self-antigens has failed, resulting in an autoimmune reactivity that becomes only manifest when the antigens are expressed at an elevated level such as in the tumor cells. A striking example of such a phenomenon has recently been documented for an immunogenic tumor antigen in the murine mastocytoma P815: the antigen is identical to a self-protein found in a mast cell line, but is not expressed in normal mast cells and other cell types (Van den Eynde et al., 1991). T h e combined findings so far indicate that T-cell responses against

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self-antigens on tumor cells can occur and that MHC antigens play a crucial role in the presentation of these antigens to T cells. From this point of view, it is evident that loss of MHC Class I expression will have severe consequences for the interaction of immune effector cells with tumor cells.

IN TUMORS C. MHC CLASSI EXPRESSION

1 . MHC Class I Downmodulation in Experimental Tumors

Downmodulation of MHC Class I expression has been shown in many animal and human tumors. Since this subject has been reviewed extensively over the past few years (Goodenow et al., 1985; Bernards, 1987; Festenstein, 1987; HZmmerling et al., 1987; Tanaka et al., 1988; Elliott et al., 1989; Napolitano et al., 1989), we will discuss only a few highlights with particular reference to the role of oncogenes in the regulation of expression of MHC Class I. Murine tumors often are devoid of MHC Class I antigens and losses do not seem restricted to particular tumor types. Well-known examples are the B16 melanoma cell line (Nanni et al., 1983; Taniguchi et al., 1985),the methylcholanthrene-induced T 10 fibrosarcoma (Katzav et al., 1983), and the spontaneous Lewis lung carcinoma (Isakov et al., 1983). Also, tumors of hemopoietic origin, e.g., the AKR lymphoma (Hui et al., 1984), can be H-2-negative. More recently, other experimental tumors lacking MHC Class I expression have been described (Garrido et al., 1986; Bahler et al., 1987; Nishimura et al., 1988). In all these cases the precise genetic alterations leading to transformation into tumor cells are not known and therefore, no link between a particular genetic defect and the modulation of MHC Class I expression can be established. A different situation exists in the case of cells transformed by oncogenic adenoviruses. Here, the transformation of murine cells by adenovirus 12 (Ad12) E l genes causes downmodulation of MHC Class I expression and it has been shown that the E I A oncogene is responsible for this (Schrier et al., 1983). The Ad5 E I A gene is not capable of exerting this effect. This may explain why Ad5-transformed cells are not oncogenic, in contrast to Ad 12-transformed cells, which are highly oncogenic: due to low MHC Class I expression the latter may evade T-cell immunity (Bernards et al., 1983). The regulation of MHC Class I by Ad12 E I A occurs at the transcriptional level (Ackrill and Blair, 1988; Friedman and Ricciardi, 1988; Lassam and Jay, 1989; Meijer et al., 1989). The precise

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sequences in the H-2 promoter region involved in the regulation by ElA are not yet known (see Section IV). In some cases the H-2 abrogation is specific for a certain locus. For instance, the 3LL Lewis lung carcinoma (H-2" origin) expresses the H-2D" locus gene product but almost completely lacks the H-2K" product (Plaksin e l al., 1988). T h e T10 sarcoma (of H-2" x H-2k F, origin) lacks the K locus products, but expresses either one o r both H-2D alleles (Wallich et al., 1985). As another example, various sublines of the murine lymphoma S49 exhibit locus-specific shut-off of K , D, or L loci (Keeney et al., 1989). In contrast to these murine tumors, no locus-specific downmodulation has been found in adenovirus-transformed rodent cells (Eager et al., 1985). 2. Allele-Specijk HLA Class I Downmodulation in Human Tumors Over the past years a great effort has been put in the analysis of HLA Class I expression in human malignancies (reviewed in Hammerling et al. 1987; Elliott et al., 1989; Napolitano et .al., 1989). In many tumor types, low expression was found in a considerable percentage of the specimens investigated (Table I). In older studies, antibodies against monomorphic determinants of the HLA Class I heavy chain or against P,-microglobulin were used to analyze HLA Qass I expression in tissue sections. In addition, in some studies, mRNA analysis was performed using a probe detecting monomorphic sequences. More recently, the expression of specific alleles was investigated with locus-specific tools, using several techniques. First, antibodies with locus specificity are available and with these, tissue sections can be stained for histochemical analysis or cells can be labeled for cytofluorimetric analysis. In this way selective loss of either HLA-A o r HLA-B proteins or both were revealed in colorectal, gastric, laryngeal, cervical, and lung carcinomas and in melanoma and acute lymphoblastic leukemia. However, caution should be taken in the interpretation of the data obtained with these antibodies since none of them is completely locus- o r allele-specific. Second, a more detailed analysis of expression of HLA Class I alleles can be performed by analysis of the proteins on isoelectric focusing gels (Neefjes et al., 1986). This technique permits a fair resolution of the individual HLA alleles from which definitive conclusions on HLA Class I downmodulation on tumors can be drawn, provided that normal cells of the patient are available for comparison. Using this technique, selective downmodulation of individual HLA-A, -B, or -C alleles were found in Burkitt lymphomas, HLA-A and -B alleles in renal cell carcinoma, and HLA-B alleles in a bladder carcinoma and in melanoma (Table I). Selective losses

TABLE I SYNOPSIS OF HLA CLASS I DOWNMODULATION I N HUMAN TUMORS

Tumor Acute lymphoblastic leukemia

HLA Class I downmodulateda (% of tumors)

Allele downmodulatedb

4/44 (9) 2/44 (5) 10144 (23) 21/21 (100) 29/66 (44) 16/64 (25) 1/18 (6) 591185 (32) 1/1 (l00)f 1211364

A,B A B

1/1 (100)f

AAC

Burkitt lymphomaf

3/14 (21) 4/14 (28) 1/14 (7) 1/14 (7)

A l l , Aw69 A l l , A3, Cw7, Cw5 Cw8 A l l , B39

Cervical carcinoma

9/10 (90) 11/67 (16) 11/67 (16)

A,B,C A,B,C, $2-m A2, A?, Bw4, Bw6

Choriocarcinomaf Colorectal carcinoma

212 (100) 69/502 (14) 16/39 (41)

A,B,C AJ,C A, A2, A3

Basal cell carcinoma B-cell lymphoma Bladder carcinoma

Breast carcinoma

Correlation with progressionc

Correlation with myc activationd 1

$2-m

AAC AAC Bw6

23 4 5,6 6

Yes, (Ref. 4) Yes (Ref. 5)

7 8

$2-m

8 7 , B44 A,B,C

Ref.e

No (Ref. 15) Yes (Ref. 13, 14)

N o (Ref. 28)

9-15

c-mycg c-mychzi

16 17,18 19

c-myci

20 21 22

c-mycg

23 24 25-33 27,29,30 (continued)

TABLE I (Continued) ~

Tumor

HLAClass I downmodulatedo (% of tumors)

Allele downmodulatedb

19/140(13) 3/85(4)

B, Bw4 A3

10110 (100) 7/8 (88) 13/83 (16) 1131 (3) 13/60(22) 5/60 (8) 1/60 (2) 16/59 (27) 1/59 (2) 3/59 (5)

$2-m

Correlation with progression.

~-

w

‘9 t~

Laryngeal carcinoma Lung carcinoma (non-small cell)

Lung carcinoma (small cell)

3/3 (100)

11/12 (92)f

A3,C AB,C B Ab,C A

961263 (37) 6/12(50)f 17/39(44) 111

(looy

~~~~~~~

&f.C

27,29,30,32 27 34

35 21 32.36 32 37

Yes

B

A&,C A B

No (c-myc)

38

c-, N-, Lmyc

39 40 40 39 41-44 45

A&,C AJ,C c-, N-, Lmyc

Melanoma

~

Correlation with myc activationd

c-mycgth Eccrine procarcinoma Endometrial carcinoma Gastric carcinoma

~

A,B,C B3, B8, BI3, Bw62, Bw53,/Bw63, Bw56, Bw55 $2-m A,B,C,$2-m

Yes (Ref. 42,43) c-myc

46 47

Neuroblastoma

Ovarian cancer Renal cell carcinomaf

10/10 (100) 414f (100) 416f (67) 516f (83) 116f (16) 7/33 (21) 818 (100) 119 219

319

0 03

-

48 49

N-myc, c-myc N-myc c-myc N-myc

50 51 52 21

53

Includes tumors with 50-100% downmodulation of HLA Class I. only expression of &-microglobulin was determined; in other cases the downmodulation of the loci (A, B, or C) or the individual alleles is listed. This correlation concerns HLA Class I downmodulation with disease progression (disease stage or survival). The correlation of HLA Class I expression with expression of either of the members of the family of myc oncogenes is listed. When no HLA Class I expression is listed in the indicated row, the reference(s) reports activation of myc oncogene(s) in the tumor type and no data on HLA Class I. References. If more than one reference is listed, the data represent a cornhination of the data in the cited references. 1: Pozzi et al., 1990; 2: Baadsgaard et al., 1990; 3: Holden etal., 1983; 4: Moller etal., 1987; 5: Tomita etal., 1990a; 6: Nouri etal., 1990; 7: Elliott etal., 1989; 8: Nouri etal., 1991; 9: Moller etal., 1989; 10: Fleming etal., 1981; 11: Natali et al., 1983b; 12: Rowe and Beverly, 1984; 13: Bhan and DesMarais, 1983; 14: Whitwell et al., 1984; 15: Wintzer et al., 1990; 16: Krief et al., 1989; 17: Kozbor and Croce, 1984; 18: Escot etal., 1986; 19: Anderson et al., 1991; 20: Klein, 1983; 21: Ferguson etal., 1985; 22: Connor and Stern, 1990; 23: Bourhis et al., 1990; 24: Trowsdale et al., 1980; 25: Van den Ingh et al., 1987; 26: Momburg et al., 1986; 27: Momhurg et al., 1989; 28: Moller et al., 1991; 29: Smith et al., 1989a; b; 30: Rees et al., 1988; 31: Durrant et al., 1987; 32: Lopez-Nevot et al., 1989; 33: McDougall et al., 1990; 34: Sugio el al., 1988; Mauumura et al., 1990; Vie1 et al., 1990; Heerdt et al., 1991; Maestro et al., 1991; 35: Holden etal., 1984; 36: Harnmerlingetal., 1987; 37: Esteban etal., 1990; 38: Redondoetal., 1991; 39: Bergh, 1990, for review; 40: Doyle etal., 1985; 41: Nataliet al., 1983a; 42: Brocker et al., 1985; 43: Van Duinen et al., 1988; 44: Ruiter et al., 1984; 45: Versteeg et al., 1988; 1989a; Schrier et al., 1991; 46: Turhitt and Mackie, 1981; Takata etal., 1989; 47: Durso etal., 1991; 48: Whelan etal., 1985; 49: Lampson etal., 1983; 50: Bernards etal., 1986; 51: Versteegetal., 1990; 52: Sugioetal., 1991; 53: Kruse et al., 1992. f Cell line(s). g Elevated expression. Amplification. ' Genomic rearrangement or translocation. J mRNA expression was determined. a

-

A,B,C A,B,C A,B,Ci A,B,Q B44 A,B,Ci AB,C A,B,C A or B A2, A23, B13, B37, 8 4 4 , Bw57IBw58, Bw62

* A,B,C: monomorphic determinant; &-mt

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were also reported for ovarian carcinoma and breast carcinoma in a small series of tumors explored (Wang et al., 1991). Finally, locus-specific analysis of HLA Class I mRNA can be performed by Northern blotting with locus-specific probes. In this way, the B-locus-specificdownmodulation by the c-myc oncogene in melanoma could be confirmed (Versteeg et al., 1989a, 1990). In addition, mRNA analysis is very well capable of discriminating between loss of the heavy chains and loss of &-microglobulin. In this way it was revealed that the loss of W6/32 (anti-HLA Class I) reactivity could be attributed to a loss of &-microglobulin mRNA in the majority of colon carcinomas (Momburg and Koch, 1989) and in a melanoma cell line (Durso et al., 1991). Locus-specific analysis of HLA Class I in human tumors has stressed that loss of HLA Class I antigens can be easily overlooked: the reduction might be below detection level when measured with antibodies specific for all HLA Class I proteins such as W6/32 (anti-backbone) or for p2microglobulin. This is clearly the case when comparing melanoma cell lines: using FACS analysis or immunoprecipitation, we have seen more than 20-fold differences in HLA Class I expression using an HLA-Bspecific antibody, whereas the anti-backbone antibody showed hardly any difference. Also, in the analysis of the tumors with antibodies in tissue sections, this effect becomes apparent: considering the downmodulation in colon carcinoma, only 14% of the tumors were HLA-A-, -B-, or -C-negative, whereas in a locus-specific analysis, 41% of the tumors were negative for at least one HLA-A allele (Table I). In some tumor types, downmodulation of certain loci predominates, e.g., HLA-B in melanoma and lymphoblastic leukemia. In other types, however, loss of all HLA Class I alleles is preponderant. Striking examples of the latter category are choriocarcinoma, neuroblastoma, and small cell lung carcinoma. In the case of the choriocarcinomas, the absence of HLA Class I expression most likely reflects the low HLA expression found in normal embryonal tissue (Bodmer, 1987). The same probably holds for the neuroblastomas and small cell lung carcinomas, both originating from tissues of neuroendocrine origin, which express low levels of HLA Class I (Doyle et al., 1985; Whelan et al., 1985). In these cases the HLA Class I expression in fact reflects a normal situation in the tissues from which the tumor has originated. In other tissues, however, HLA Class I is normally expressed and therefore, in tumors derived from other tissues, the downmodulation should be caused by other (external) factors. There are a number of factors that could account for the downmodulation of HLA Class I in these cases. First, the absence of HLA Class I may be due to a random mutation and the clonality of the HLA-negative phenotype may be just the result of subsequent selection, whereby only cells with

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low MHC Class I expression can survive. Such selective pressure may be the T-cell system, killing cells with good HLA Class I expression, but leaving cells with low HLA Class 1 expression intact. However, other, nonimmunological, selective advantages may go along with low HLA Class I expression, such as, for instance, a growth advantage (Haliotis et al., 1990). Second, altered HLA Class I expression can be considered a phenotypical trait of the tumor cells that cannot be sequestered from other traits. This seems the case in Burkitt lymphomas, where the loss of HLA Class I alleles is almost strictly correlated with the status of other markers such as expression of B-cell differentiation antigens and viral antigens (Andersson et al., 1991). Third, it can be hypothesized that the HLA Class I loss is neither accidental nor a phenotypical trait coinciding with characteristics typical for the tumor cell, but rather a genotypical trait intrinsically coupled to the alterations underlying the oncogenic transformation. The most conspicuous candidates for these alterations are oncogenes or tumor suppressor genes involved in the actual process of oncogenesis. This has clearly been shown for transformation of rodent cells by the adenovirus ElA oncogene: the expression of the oncogene itself is responsible for the MHC Class I downregulation. Also, in the case of human tumors, elevated oncogene expression has shown to be involved in HLA Class I regulation. Remarkable examples are the downmodulation of HLA-B by c-myc in melanomas and the capability of the N-myc oncogene to switch off HLA-A, -B, -C in neuroblastomas. The role of oncogenes in modulation of MHC Class I expression is discussed in the next section.

111. Modulation of MHC Class I Expression by Oncogenes A. ADENOVIRUS ElA The first discovery that transforming genes may affect MHC Class I expression was made several years ago when the reactivity of heteroantisera raised in mice against transformed rat cells was studied (Schrier et al., 1983). The sera raised against nononcogenic adenovirus 5-transformed rat cells recognized an epitope absent on oncogenic adenovirus 12-transformed cells. This epitope turned out to be an as yet unidentified MHC Class I molecule noncovalently linked to &-microglobulin. Also, rat alloantisera detected a dramatic difference between the two types of cells, indicating that MHC Class I expression is switched off in the oncogenic Ad 12-transformed cells, but not in the nononcogenic Ad5-transformed cells. Transformation with the viral ElA oncogene was

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sufficient to establish the downmodulation of the Class I heavy chain mRNA and protein (Schrier et al., 1983; Vaessen et al., 1986). The effect was also seen in murine and human cells transformed by Ad12 (Eager et al., 1985; Vaessen et al., 1986; Vasavada et al., 1986; Grand et al., 1987). Expression of P2-microg1obulin was not affected (Eager et al., 1985, 1989; Lassam and Jay, 1989), although in one study several transformed cell lines showed a somewhat lower expression (Lassam and Jay, 1989). It should be stressed that in early infected cells Ad5 as well as Ad 12 EIA, in cooperation with EIB, were capable of enhancing MHC Class I expression by transcriptional activation (Rosenthal et al., 1985). Therefore, it seems that the downmodulation by Ad12 EIA is only manifest upon transformation of the cell and not in an early state of infection. In a later state of infection, however, another adenovirus protein (19 kDa) encoded by the E3 region of nononcogenic subgroup C viruses (e.g., Ad2 and Ad5) can block cell surface expression of certain MHC Class I alleles by complex formation with de novo synthesized MHC Class I heavy chains, thereby inhibiting their glycosylation and correct processing (Andersson et al., 1985; Burgert and Kvist, 1985; Paabo et al., 1989). Since the E3-encoded 19-kDa protein of type A viruses (e.g., Ad12) does not possess this property (Paabo et al., 1986), it appears that the early viral proteins of the subgroup A on one hand, and subgroup C on the other hand, display different mechanisms to prevent MHC Class I protein expression on the membrane. The precise biological significance of this difference has not yet been clarified, but in both cases the consequence is that the transformed or infected cells evade immune defense by cytotoxic T cells (Bernards et al., 1983; Burgert and Kvist, 1987; Burgert et al., 1987). In case of an infection this evidently will contribute to the persistence of the virus in the host, a typical property of adenoviruses. Interestingly, evasion from CTLs is not only effected by low MHC Class I expression: expression of Ad5 E3 proteins also reduces the expression of the EIA protein by post-transcriptional regulation (Zhang et al., 1991). Since in the investigated cells the expression of MHC Class I (H-2Kd) was not affected, the reduced susceptibility of the Ad5-infected cells to T cells (Zhang et al., 1991) is clearly due to low expression of a viral immunodominant epitope that resides in the EIA protein (Kast et al., 1989; Mullbacher et al., 1989; Urbanelli et al., 1989; Routes et al., 1991). These data altogether show that, dependent on the serotype, adenoviruses use various sophisticated strategies to evade immune surveillance of the host. As far as transformed cells are concerned, it has been found that the downmodulation of MHC Class I by the Ad12 EIA gene is not always apparent. In several established cell lines as well as in cells of certain rat strains the adenovirus EIA gene did not affect, or hardly affected, MHC

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Class I expression (Mellow et al., 1984; Vaessen et al., 1986), indicating that other factors determined by the target cell may either overrule the downmodulation or preclude it. A clear example is y-interferon, a cytokine enhancing MHC Class I expression in Ad12-transformed cells (Eager et al., 1985; Hayashi et al., 1985). Since the downmodulation by Ad12 EIA is regulated at the transcriptional level (Ackrill and Blair, 1988; Lassam and Jay, 1989; Meijer et al., 1989; Tomita et al., 1990b), as is the enhancement by y-interferon, the two agents may either exert their effect via separate regulatory sequences within the MHC Class I promoter/enhancer or act differently on a common target (see Section 1V for further discussion). The level of MHC Class I expression determines the oncogenicity of adenovirus-transformed cells in syngeneic animals. For example, Ad 12transformed cells treated with y-interferon are less oncogenic than their untreated counterparts (Hayashi et al., 1985; Tanaka et al., 1988).Transfection of an exogenous H-2 gene into the oncogenic cells in order to raise the MHC Class I expression results in reduction of their oncogenic potential (Tanaka et al., 1985, 1986).This shows that reexpression of the Class I gene itself, and not some other effect of y-interferon, is responsible for the reversal of oncogenicity. An Ad5-specific MHC Class I-restricted CTL clone recognizing an EIA-encoded peptide has been isolated and shown to be capable of efficiently eliminating Ad5 EIA-induced tumors in nude mice (Kast et al., 1989). Comparable Ad12-specific CTLs have not yet been found, even after enhancement of the MHC Class I expression of the stimulator cells (W. M. Kast, personal communication).Therefore, it cannot be excluded that, in addition to low MHC Class I expression, other factors contribute to the escape of Ad12-transformed cells from the immune defense of the host. Such factors might relate to poor binding of Ad12 ElA peptide to MHC Class I molecules. This is favored by the notion that the amino acid sequence of the major Ad5 MHC Class I-binding peptide (Kast et al., 1989; Kast and Melief, 1991) is significantly different in the Ad12 ElA protein (W. M. Kast, personal communication). Since the oncogenicity of Ad 12-transformed cells can be reversed by transfection of exogenous MHC Class I genes (Tanaka et al., 1985, 1986), it should be assumed that such a peptide presentation defect can be (partially) overcome by high MHC Class I expression.

B. OTHERVIRALONCOGENES Apart from adenoviruses, many other viruses have been shown to affect MHC Class I expression. These include the DNA viruses herpes

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simplex types 1 and 2 (Jennings et al., 1985), hepatitis B (Onji et al., 1987), and several transforming retroviruses: Moloney murine sarcomalmurine leukemia virus (Mo-MSV/MLV) (Flyer et al., 1985; Wilson et al., 1987; Racioppi et al., 1988), Rous sarcoma virus (RSV) (Gogusev et al., 1988), Kirsten murine sarcorna/murine leukemia virus (Ki-MSV/ MLV) (Fontana et al., 1987; Maudsley and Morris, 1989), myoproliferative sarcoma virus (MPSV) (Racioppi et al., 1988), polyoma murine leukemia virus (PyMLV) (Racioppi et al., 1988), and radiation leukemia virus (RadLV) (Meruelo et al., 1978). Of these viruses, the DNA viruses reduce the expression of MHC Class I upon infection. Although the transforming genes of these viruses have been designated, it is not known whether these genes in fact are responsible for downmodulation. In the case of the retroviruses, the effect on MHC Class I expression may be either a downmodulation (Wilson et al., 198’7; Racioppi et al., 1988; Seliger et al., 1988) or an upregulation (Flyer et al., 1985; Fontana et al., 1987; Maudsley and Morris, 1989), dependent on the cell system and conditions used. T h e acute transforming retroviruses harbor a viral oncogene (v-onc), e.g., v-mos, v-src, and v-rm. In principle, MHC Class I expression can be regulated by various viral sequences: the most likely candidates are the viral LTR, a transcriptional enhancer, and the viral oncogene. In several cases the oncogene has been shown to be responsible for the effect. Downregulation has been found for v-my, v-mos (the transforming gene of MoMSV), and v-H-ras (the transforming gene of K-MSV) when transferred in murine 3T3 cells (Seliger et al., 1988; Seliger and Pfizenmaier, 1989). In contrast to the transcriptional regulation of MHC Class I genes by the EZA oncogene, the regulation in 3T3 cells by these retroviral oncogenes seems to be post-transcriptional (Seliger and Pfizenmaier, 1989). Downmodulation by these oncogenes is not seen consistently but may vary dependent on the cell type o r even on the cell clone infected. For instance, in one particular rat thyroid epithelial cell line, downregulation of MHC Class I was found for a combination of two oncogenes, the c-myc oncogene and the polyoma middle T gene, whereas neither of the two genes could do so on its own (Racioppi et al., 1988). In this particular case, the regulation was at the transcriptional level. In another clone of the cell line, only the Harvey murine sarcoma virus (carrying the rus oncogene) could completely abolish MHC Class I expression at the post-transcriptional level (Racioppi et al., 1988). These experiments show that other cell-specific factors, in cooperation with the oncogene, determine the modulation of MHC Class I expression. T h e effect cannot be directly attributed to the oncogene in all cases where the retroviruses affect MHC Class I expression. In fibroblasts, infection with KiMLV could block the stimulation of MHC Class I by y-

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interferon (Maudsley and Morris, 1989). Since KiMLV is a slow leukemia virus and does not carry a viral oncogene, this result suggests that the viral LTR in trans is responsible for the blocking (Maudsley and Morris, 1989). In another set of experiments with Moloney viruses, the Moloney sarcoma virus, carrying its own oncogene v-mos, blocked MHC Class I transcription induced by the Moloney leukemia virus (Flyer et al., 1985; Wilson et ul., 1987). This differential effect on MHC Class I expression might have to do with the biology of the two viruses (Wilson et al,, 1987); that is, proliferation of their host cells is necessary for adequate viral replication. The leukemia viruses, not carrying their own oncogene to stimulate this proliferation, might induce proliferation of specific T cells that may serve as host cells for the virus via autostimulation by the MoMLV-infected T cells. For the latter process high MHC Class I expression is profitable. In contrast, sarcoma viruses, carrying their own oncogene providing the proliferative signal, benefit from evasion of immune destruction by switching off MHC Class I expression at the surface of the infected cell. In conclusion, the effects of the retroviruses and retroviral oncogenes in murine cells on MHC Class I expression are complex. Distinct effects of the retroviral LTRs and the oncogene products have been found, often depending on the cell type as well as on the type of virus. LTRs can either upregulate or downregulate the expression of MHC Class I expression. This may be effectuated by a direct effect of the LTR in cis at the transcriptional level or by an indirect effect; that is, the LTR may squelch factors involved in the regulation of transcription of MHC Class I genes. In contrast, the viral oncogenes, m y , mos, and ras downregulate MHC Class I expression via a mechanism where, in addition to transcriptional regulation, post-transcriptional regulation is involved. C. fos, raJ

AND

rus

In the previous sections we have discussed the effects of viral oncogenes on MHC Class I expression. These effects, in most cases, can be related to a biological advantage for the virus in the infectious cycle. An important question now is whether activation of protooncogenes in normal cells or in transformed cells can also affect MHC Class I expression. In this section we will see that such an effect indeed has been found for the protooncogenesfos, ras, and raf. The effect of the myc oncogenes will be discussed in Section II1,D. 1. fos Expression of the c-fos oncogene was found to correlate with the level of MHC Class I expression in murine tumor cells (Feldman et al., 1988;

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Kushtai et al., 1988). Also, in various leukemic human tumor cell lines, a differentktion-induced increase in c-fos expression often correlated with an increase in HLA Class I expression and vice versa (Barzilay et al., 1987). T h e c-fos gene is activated during fetal development by a sharp raise and fall in expression during a short period at the end of gestation on the day of birth (Muller et al., 1982; Kasik et al., 1987). Furthermore, expression can be induced by external stimuli such as agents that promote proliferation or differentiation in cultured cells (Kaufmann et al., 1987). T h e c-fos protein complexes with another protooncogene product, cjun, and together they form the transcription factor AP- 1, involved in transcriptional activation of many genes (Ransone and Verma, 1990). T h e fos-jun dimer binds to a consensus DNA sequence that is present in murine MHC Class I genes nearby enhancer A (Korber et al., 1988; Singer and Maguire, 1990). With these findings in mind, it is imaginable that an increase in fos expression augments MHC Class I transcription. Indeed, it was found that the low metastatic Lewis lung carcinoma cells expressed constitutive high amounts of c-fos and H-2K, whereas high metastatic cells did not (Kushtai et al., 1988). Transfection of c-fos (or v-fos) into nonexpressor cells elevated the expression of cjun (Kushtai et al., 1990) and MHC Class I, whereas other genes such as c-my, P-actin, and P,-microglobulin were not affected (Kushtai et al., 1988, 1990). T h e fos-transfected clones were less metastatic than the parental cell lines and their tumorigenicity was lower (Kushtai et al., 1990). Despite the apparent correlation between c-fos and MHC Class I expression in tumor cells, there is as yet no evidence that the AP-1 complex is involved in the direct regulation of transcription of MHC Class I genes (see also Section IV). Moreover, in developmental regulation, no effect of c-fos on MHC expression has been noted (Kasik et al., 1987). T h e tumor cells, however, may contain tumor-specific o r even tissue-specific regulatory factors (repressors or activators) that interact with the MHC Class I promoter region, and this process may be affected by the binding of the c-fos protein (AP-1) to a nearby AP-1 consensus sequence. T h e relationship between MHC Class I expression and c-fos holds only for murine cells. Surveying a number of human HLA Class I promoter sequences, we could not find consensus AP- 1 binding motifs. This may explain why upon cytolytic activation of a human T-cell hybridoma, increased c-fos expression did not go along with an increase in HLA expression (Kaufmann et al., 1987). Moreover, differentiation induction and subsequent alteration of c-fos expression in other human cell lines did not correlate with HLA Class I expression (Barzilay et al., 1987), suggesting that in this case HLA Class I expression is modulated as a

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direct consequence of the altered differentiation state rather than being a direct effect of modulation of c-fos expression. 2. ras

As discussed in Section IILB, viral ras genes have opposite effects on MHC Class I expression, dependent on the system studied. A detailed analysis of the precise effect of transfected ras genes, either with or without an activating point mutation, should reveal whether ras plays a role in the regulation of expression of MHC Class I genes. A number of such studies have been performed but no consistent effect of ras genes has been determined. One study (Lu et al., 1991) reports a decrease in MHC Class I expression after transfection of NIH3T3 fibroblasts with K-ras genes carrying point mutations leading to various amino acid substitutions in the p21 ras protein. In transfectants with wild-type ras or ras mutated at positions 10, 33, or 51, H-2 expression was clearly downmodulated and the transfectants were more oncogenic and metastatic than the parental cell line. However, since only mutations at amino acid positions 12, 13, or 61 convey transforming activity to the ras protein and only these mutations have been found in naturally occurring animal and human tumors (Barbacid, 1990; Bos, 1989; Sukumar, 1990), no effect of a transforming ras mutation on oncogenicity via MHC Class I expression has been determined. Similarly, no effect of H-ras transfection on MHC Class I expression was observed in C3H lOT4 cells (Elliott et al., 1989). Although supertransfection of Ad 12 E l A-transformed human embryonic retinal cells with a point-mutated N-ras gene could upregulate HLA Class I expression (Grand et al., 1987),this phenomenon was not found in all clones and no direct correlation between N-ras expression and HLA Class I expression was shown. In human tumors no correlation has been reported between HLA Class I expression and ras mutations; in one report on colon carcinoma an explicit lack of such a correlation was demonstrated for mutations in codon 12 of the K-ras gene (Oliva et al., 1990). In a converse situation, transfection of an MHC Class I gene (H-2Dk) into the nonmetastatic murine T 10 sarcoma resulted in downmodulated endogenous c-K-ras expression and a high metastatic phenotype (Alon et al., 1987).The latter phenomenon is most likely caused by the reexpression of H-2Dk, since evidence from other experimental models suggests that ras activation is correlated with high metastatic potential or with an oncogenic phenotype (Price et al., 1989; Fry et al., 1990; Kinsella et al., 1990; Lowndes et al., 1990). In earlier studies expression of Dk was indeed shown to be correlated with an increased metastatic phenotype

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(Hammerling et al., 1987). Recently, Kast and collaborators (Melief and Kast, 1991) found that transfection of activated ras into nononcogenic adenovirus 5-transformed cells with high MHC Class I expression made these cells highly resistant to an Ad5 EIA-specific CTL in viva The high oncogenicity of these cells is possibly due to an increased production of transforming growth factor-f3 (TGF-f3)as consequence of the ras transfection. TGF-f3 has an antiproliferative immunosuppressive effect (reviewed in Melief, 1991) and a correlation between ras expression and TGF-f3 production has been reported for C3H 10Ti cells (Gingras et al., 1988). ras transfection did not alter MHC Class I expression in C3H 10Ti cells (Elliott et al., 1989). It is concluded that there is no correlation between ras and MHC Class I expression in tumor cells and that the high tumorigenicity of rastransfected cells is caused by other factors, independently of MHC Class I expression. In contrast, MHC Class I1 expression has been shown to be inducible by activated ras in a Burkitt lymphoma cell line (Hume et al., 1987) and in human melanocytes (Albino et al., 1986).This is of particular relevance, since mutated ras peptides are efficiently presented by HLA Class I1 molecules to CD4-positive T helper cells (Jung and Schluesener, 1991; Peace et al., 1991). 3 . raf

The c-raf oncogene (the cellular homolog of the murine sarcoma virus 361 1 oncogene) is activated in several human epithelial cancers (Shimizu et al., 1985; Kasid et al., 1987). It encodes a serinelthreonine kinase involved in signal transduction downstream of the ras product. A human bladder epithelial cell line has been transfected with an activated raf oncogene (v-raf), by which the transfectants gained strongly reduced levels of HLA Class I proteins (Ottesen et al., 1990). Moreover, these tumor cells were more tumorigenic in nude mice than their parental counterparts. The raf-transfected cells contained increased levels of c-myc mRNA, which could account for the decreased HLA Class I expression (see Section 111,D). In principle, this HLA Class I modulation may account for altered immune sensitivity of the tumor cells, since nude mice have natural killer cells. However, the higher tumorigenicity of the raf-transfected cells cannot be explained by the low HLA Class I expression since this renders tumor cells refractile to lysis by NK cells (see below). Since activated m y genes augment tumorigenicity of established cell lines in nude mice (Keath et al., 1984), the effect of the raf oncogene on tumorigenicity is probably due to the high c-my expression that raf induces in the human tumor cells.

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D. myc 1. myc Activation

The myc protooncogene family consists of at least six genes, c-, N-, L, P-, R-,and B-myc (Schwab et al., 1983; Nau et al., 1985; Alt et al., 1986; Ingvarsson et al., 1988). Here, we will focus on c-myc and N-my, because these two genes have been shown to be capable of altering HLA Class I expression. These genes consist of three exons in humans encoded on chromosome 8 (c-my) and chromosome 2 (N-my). Exon 1 is noncoding and exons 2 and 3 code for a nuclear 65- to 6’7-kDaphosphoprotein that complexes with another cellular protein, termed max, to form a DNA binding complex that interacts with the consensus nucleotide sequence CACGTG (Blackwell et al., 1990; Blackwood and Eisenman, 1991). The precise function of the myc proteins is not known, but they play a role in the regulation of the cell cycle, in particular at the onset of proliferation and in transformation and differentiation of certain cell lineages (Cole, 1986, 1991; Liischer and Eisenman, 1990). The mechanism by which these processes are effectuated usually involves elevated expression of the myc gene, probably leading to enhanced rates of transcription of a variety of other cellular genes involved in proliferation and cell growth. c-myc can be activated in many cell types, whereas the expression of N-myc seems restricted to certain lineages derived from the neuronal crest. The expression of N-myc is often correlated with a particular state of differentiation (Thiele et al., 1985; Stanton et al., 1986; Zimmerman et al., 1986). A typical property of myc genes is their capacity to transform primary rodent cells in cooperation with other oncogenes, e.g., an activated rus gene (Land et al., 1983, 1986; Hunter, 1991). The resulting transformed cells are oncogenic in mice and the expression of the c-myc oncogene clearly contributes to the tumorigenic properties of the cells (Keath et al., 1984). Mice that are transgenic for c-myc develop tumors, but the location of the tumor is limited to certain tissues and lymphoid cells (Stewart et al., 1984; Leder et al., 1986). Often, tumor formation occurs in cooperation with other activated oncogenes (Alexander et al., 1989; Strasser et al., 1990; Haupt et al., 1991; Van Lohuizen et al., 1991). Activation of the c-myc oncogene has been found in numerous forms of human cancer. Prominent examples are colon carcinoma (Vie1 et al., 1990) and lung carcinoma (Bergh, 1990). Usually, the activation consists of augmented expression, which is often a result of gene amplification. Activation of c-myc by chromosomal translocations involving the enhancer sequences of immunoglobulin genes occurs in Burkitt lymphoma (Klein, 1983). Also, in other types of B-cell malignancies m y rearrange-

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ments are often involved in concert with activation of the bcl-2 gene (Klein, 1991). T h e N-myc gene is primarily involved in neuroblastoma (Schwab et al., 1983; Seeger et al., 1985; Bernards et al., 1986; Rosen et al., 1986; Garvin et al., 1990) and small cell lung cancer (Nau et al., 1986; Bergh, 1990). T h e gene is often amplified and in neuroblastoma the degree of amplification is inversely correlated with progression-free survival (Seeger e,! al., 1985). In certain cases of neuroblastoma, including neuroepithelioma, c-myc was found to be activated, coinciding with low or absent N-myc expression (Rosolen et al., 1990; Versteeg et al., 1990); this suggests that their expression is mutually exclusive. 2. myc and HLA Class I Remarkably, in neuroblastomas as well as in small cell lung carcinomas, a very low expression of HLA Class I antigens was found (Trowsdale et al., 1980; Lampson et al., 1983; Doyle et al., 1985; Bernards et al., 1986). In addition, in a number of other tumor types with low HLA Class I expression a m y activation has been noted (Table I). These include breast carcinomas (Kozbor and Croce, 1984; Escot et al., 1986; Krief et al., 1989), Burkitt lymphomas (Klein, 1983), cervical carcinomas (Bourhis et d.,1990), colorectal carcinomas (Sugio et al., 1988; Matsumura et al., 1990; Vie1 et al., 1990; Heerdt et al., 1991; Maestro et al., 1991), and melanomas (Versteeg et al., 1988, 1989a; Schrier et al., 1991).For most tumor types, low HLA Class I expression and myc activation were found in independent studies, but in neuroblastoma and melanoma, the two phenomena were found in the same individual tumors (Table I). This suggested the possibility that elevated expression of c-myc or N-myc can regulate HLA Class I expression. This idea was supported by another relevant finding: the c-myc and N-myc genes have demonstrated a functional homology with the adenovirus E l A gene; that is, either of these genes can cooperate with an activated ras oncogene in transforming primary rodent cells (Land et al., 1983, 1986; Ruley, 1983; Schwab et al., 1985). This implies that the myc genes on one hand and the EZA gene on the other might interact with similar target structures in the cell and may have similar functional effects other than the transformation of primary cells per se. One of the peculiar properties of E1A proteins is their capability to downmodulate MHC Class 1 expression along with transformation. T h e question therefore arose whether c-myc or N-myc could also downmodulate MHC Class I expression. A number of independent studies in melanoma and neuroblastoma tumors and cell lines revealed that this is indeed the case. In a panel of melanoma cell lines, an inverse correlation between c-myc expression and HLA Class I expression was found (Versteeg et al., 1988,

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1989a,b; Schrier et al., 1991). In a series of neuroblastoma cell lines, the same phenomenon was found for the N-myc gene (Bernards et al., 1986; Bernards and Lenardo, 1990; Gross et al., 1990; Versteeg et al., 1990; Sugio et al., 1991). Proof for a direct role of c-myc and N-myc in switching off MHC Class I expression came from transfection experiments in which functional myc genes are transfected in tumor lines with low expression of myc, i.e., melanoma cell lines with c-myc (Versteeg et al., 1988, 1989a,b) and a rat neuroblastoma cell line with N-myc (Bernards et al., 1986). Transfected clones with high expression of the myc genes showed low MHC Class I expression at the mRNA level as well at the protein level. &-microglobulin expression was not affected in the melanoma cell lines, but decreased in one of the two transfected rat neuroblastoma cell lines. These experiments show a direct correlation between elevated myc expression and low MHC Class I expression. 3 . Locw-Specific Downmodulation

When the assay for HLA Class I expression was performed with locus-specific tools, surprisingly, only downmodulation of HLA-B genes was noted in melanoma cell lines (Versteeg et al., 1989a). Downmodulation of HLA-A occurred to a much lesser extent and HLA-C mRNA expression was found in only one cell line. Therefore, the downregulation of HLA-C might be allele-specific rather than locus-specific. The low HLA-B expression is not an effect of establishment of the cell lines o r long-term culture, because in two patients whose tumor material was available, the selective abrogation of HLA Class I expression was also found in sections of the original tumor using locus-specific antibodies for histochemistry (Versteeg et al., 1989a). Moreover, the selective loss of HLA-B was also apparent after transfection of c-myc into two different cell lines with low endogenous c-myc expression: in these cases HLA-B8 and HLA-Bw62 proteins were downregulated, whereas expression of HLA-A1 and HLA-A2 remained unaltered (Versteeg et al., 1989a). In various transfected clones, the level of HLA Class I downmodulation precisely correlated with the amount of c-myc protein analyzed on Western blots (L. T. C. Peltenburg, unpublished results). The expression of HLA Class I is regulated at the transcriptional level (see Section 1V) and therefore it seems that the action of c-myc is limited to specific sequences in the promoter of the HLA-B genes that differ in the HLA-A promoter. 4. Downmodulation in Other Tumors In several other human tumors, locus-specific regulation of HLA-B alleles has been found, in particular in bladder carcinoma and renal cell carcinoma (Table I). In other tumor types such as Burkitt lymphoma and

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colorectal carcinoma, several H LA-A alleles are selectively downmodulated (Table I). In none of these cases has a direct correlation with activation of c-myc been reported. In small cell lung carcinomas (Marley et al., 1989) and in renal tumors of the Wilms type (Shaw et al., 1988; Maitland et al., 1989) a correlation between c-myc expression and HLA Class I downmodulation was found; however, in these cases no locus-specific analysis was performed. Reviewing the literature, no inverse correlation between high c-myc expression and HLA Class I expression was found in other forms of human cancer. Lack of such a correlation has been reported for a human breast carcinoma cell line (Minafra et al., 1989), colon carcinoma (Soong et al., 199l), a murine osteosarcoma cell line (Dahllof, 1990), and human non-small cell lung carcinomas (Redondo et al., 1991).A straightforward explanation for the discrepancy might be that the analyses were usually not performed with locus-specific tools and therefore low expression of specific alleles might have been overlooked. An alternative explanation might be that the effects of myc expression on MHC Class I expression are lineage-specific. It can be hypothesized that the regulatory suppressive factors mediating the effect of myc are merely present in certain tissues or certain differentiation lineages. In this context it may be relevant that tumors in which an effect of c-myc or N-myc was often found, i.e., neuroblastoma, small cell lung carcinoma, and melanoma, are all of neuroendocrine origin and we speculate that expression of the suppressive factors is only permitted in these types of cells. Even more specifically, expression may be limited to a particular stage of differentiation o r development at which the cells were arrested during tumorigenesis. This may explain why in two neuroblastoma cell lines with low N-myc expression, HLA Class I expression could not be switched off by transfection of the N-myc gene (Feltner et al., 1989). Another possibility to reconcile the inconsistencies might be that the level of myc protein in the tumor cell lines or in the transfected cell lines is not sufficiently high to bring about the HLA Class I downmodulating effect. Our own experience is that, in the case of c-my, the extent of downmodulation clearly depends on the level of c-myc expression. The same was found for the expression of N-myc in the rat system (Bernards et al., 1986). Neuroblastoma or neuroepithelioma cell lines without expression of N-myc often express c-myc at high levels (Bernards et al., 1986; Feltner et al., 1989; Versteeg et al., 1990). For one particular neuroepithelioma cell line with high c-myc expression, a locus-specific analysis of HLA Class I alleles was performed and, strikingly, HLA-B expression was virtually absent, whereas HLA-A and -C were expressed (Versteeg et al., 1990). An

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interesting observation was made when this cell line was fused to highN-myc-expressing neuroblastoma cell lines without HLA Class I expression. First, N-myc expression was lost due to suppression by a tentative tumor suppressor gene in the neuroepithelioma cell line. Second, HLA-A expression reappeared but HLA-B expression did not, apparently due to the high c-myc expression of the fusion partner (Versteeg et al., 1990). T h e important conclusion that can be drawn from these experiments is that in neuroblastoma, relaxation of HLA Class I expression can occur upon loss of N-myc expression, stressing the regulatory connection between N-myc and HLA Class I. Moreover, it can be concluded that locus-specific downregulation of HLA-B by c-myc is also operative in neuroblastoma, suggesting a mechanism of myc-induced HLA Class I regulation similar to that in melanoma. In many respects the function of c- and N-myc genes is similar. With respect to MHC Class I regulation, however, these genes act differently: N-myc downregulates all HLA loci, whereas c-myc acts only on HLA-B loci. Indeed, differences in transcriptional regulation have been found: N-myc acts via modulation of binding of H2TFl to enhancer A, whereas c-myc exerts its effect through another regulatory sequence in the HLA Class I promoter (see Section IV).

5 . Modulation Interferon Apart from the transfection and fusion experiments described above, myc expression in tumor cells can also be influenced by interferons and/or tumor necrosis factor (TNF) (Dani et al., 1985; Einat et al., 1985; Knight et al., 1985; Resnitzky et al., 1986; Yarden and Kimchi, 1986; Jonak et al., 1987; Kimchi, 1987; Seliger et al., 1988). In one melanoma cell line, a strong transient downmodulation of c-myc after treatment of the cells with y-interferon was found (Versteeg et al., 1988). Similar effects have been seen in other melanoma cell lines and often TNF has an additional effect on abrogation of c-myc expression (Osanto and Jansen, 1992). Since y-interferon and TNF are potent stimulators of HLA Class I expression (Basham et al., 1982; Friedman et al., 1984; Burrone et al., 1985; Israel et al., 1986, 1989a; Pfizenmaier et al., 1987; Marley et al., 1989; Johnson and Pober, 1990), it has been suggested that upregulation of HLA Class I expression could proceed via c-myc downmodulation. On the other hand, c-myc downmodulation is no prerequisite for stimulation of HLA Class I expression: in a number of cell lines interferon acts independently of myc expression (Bernards et al., 1986; Chen et al., 1986; Lomo et al., 1987). Here, y-interferon apparently overrules the effect of myc on HLA Class I expression, probably by greatly increasing the rate of transcription through the interferon response element (IRE) in the

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Class I promoter, an element located adjacent to the enhancer A sequence (see Section IV). Interestingly, the elevation of HLA Class I expression by y-interferon is locus-specific; i.e., the effect on HLA-B alleles is more pronounced than that on HLA-A and -C alleles (Schmidt et al., 1987, 1990; Girdlestone and Milstein, 1988; Hakem et ad., 1989, 1991). Furthermore, differences were found between various B alleles: the effect of y-interferon on HLA-B7 and HLA-Bw64 is much more pronounced than that on other alleles. In some cases, this differential effect could be attributed to subtle nucleotide differences in the IRE sequence of the HLA genes [(Hakemet al., 1991),see also Section IV]. In this way, the allele- or locusspecific effects can be explained by differential binding of transcription factors and/or regulatory factors to the promoter region. In summary, a large body of evidence points to a downregulation of MHC Class I molecules by elevated expression of c-myc or N-myc oncogenes. This phenomenon, however, seems limited to certain tumor types, most of neuroectodermal origin. The downmodulation by c-myc is locus-specific and preferentially HLA-B alleles are affected. The effect of N-myc is more general and involves all HLA loci. Possible mechanisms and biological consequences of the my-HLA relationship will be discussed in the next two sections. IV. Moiecular Mechanism of MHC Class I Reguiation by Oncogenes

A. GENERAL MECHANISMS OF REGULATION Extensive studies on the regulation of MHC Class I expression have clarified that a number of different regulatory mechanisms exist. These various mechanisms account for the similarity in tissue distribution of the various MHC Class I and Class I-like products in the adult animal and for the expression during development. Also, they may explain the modulation of MHC Class I by cytokines, viruses, and oncogenes. In this section, we will summarize the results of recent studies that have begun to elucidate the molecular basis of regulation of MHC Class I expression by modulating factors. We will focus on data obtained on transcriptional regulation of MHC Class I genes, because in most of the model systems studied expression of these antigens seems to be regulated at the mRNA level. 1. DNA Sequence Elements Involved in Transcription

Transcription of eukaryotic genes is normally regulated by DNA sequence elements, located upstream of the coding sequence. The 5’ end

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of several MHC Class I genes has been studied extensively and as a result, a number of important DNA elements have been identified. In most MHC Class I genes, the TATA box promoter element is located 20 bp upstream of the transcription start site. The other common promoter element, the CAAT box, is found 25 bp further upstream. In addition to these general elements, a number of other positive and negative regulatory sites, which in some cases are unique for MHC Class I genes, have been identified. These sequences and their associated trans-acting factors will be described. I n Fig. 1, most of the elements identified in the mouse H-2Kb gene are shown. T h e enhancer A element, also called CRE/region 11, located between 190 and 138 bp upstream of the cap-site is present in most MHC Class I genes (Israel et al., 1986; Kimura et al., 1986; Miyazaki et al., 1986; Baldwin and Sharp, 1987). It appears to have a significant effect on expression of MHC Class I, because removal of this box reduces the expression dramatically, whereas linkage of the enhancer to a heterologous promoter can augment the transcription (Kimura et al., 1986; Miyazaki et al., 1986). The trans-acting factor binding site in this element is 5’-TGGGGATTCCCCA-3’ and is located in the 3’ end of it (Baldwin and Sharp, 1987). Three copies of this perfect dyad fused to an enhancerless promoter can drive efficient transcription of a linked reporter gene (Lenardo et al., 1989). T h e IRE, in combination with the enhancer A box, contributes to the inducibility of MHC Class 1 genes by interferons (Israel et al., 1986; Sugita et al., 1987; Korber et al., 1988). Another enhancer, known as enhancer B, maps just 5‘ to the promoter. This element has been shown to have lower activity in vitro than enhancer A, but its in vivo function is

enhancer B

enhancer A

CAAT

IRE

- 160

-200

-

- 100

TATA

-60

0 bv

bindlng rite for regulatory protelnr: TQQQQATTCCCCA

FIG. 1. Schematic representation of the regulatory elements identified in the 5’-flanking region of MHC Class I genes. The location of these elements is similar in all MHC Class I genes analyzed thus far. IRE: interferon response element; arrow: initiationof transcription. The bold line in enhancer A depicts the position of the most important binding site for regulatory proteins in this element. The numbers indicate the distance (in base pairs) relative to the transcription start site.

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still unclear. Furthermore, several short sequence elements with homology to known transcription factor binding sites are present in the enhancer A region of some MHC Class I genes. Among these are AP-I binding sites, a CAMPresponse element, and a sequence resembling the SV40 enhancer (Singer and Maguire, 1990). However, there is no evidence that these sequences have a functional role in the regulation of MHC expression (Korber et al., 1988; Degols et al., 1991). Additional negatively regulating sequences will be discussed below.

2 . Regulation in Development Expression of MHC Class I genes is developmentally regulated: the mRNA and surface antigens are not detected until the midsomite stage of murine embryogenesis (Ozato et al., 1985). During T-cell development MHC Class I expression is specifically upregulated. Experiments with human leukemic T-cell lines arrested at various stages of development demonstrated that variations in HLA Class I expression resulted from different transcription rates and confirmed a role for enhancer A binding proteins in this developmental process (Zachow and Orr, 1989). Murine embryonal carcinoma F9 cells, which exhibit various properties characteristic of early embryos, do not express appreciable levels of MHC Class I. However, they are expressed after treatment with interferon or induction of differentiation (Miyazaki et al., 1986). The H-2Ld expression in undifferentiated cells seemed to be negatively regulated through sequences in the upstream enhancer A region (Miyazaki et al., 1986). Only in tissues that express MHC Class I products at relatively high levels is nuclear protein binding to the 3' part of enhancer A detected, suggesting an involvement of enhancer A binding activity in controlling developmental expression of MHC Class I antigens in vivo (Burke et al., 1989). Surprisingly, a recent report by the same group showed that mutations in the IRE region, but not in the enhancer A sequence, resulted in an increase in transcription from the H-2Ld promoter in undifferentiated cells (Flanagan et al., 1991). This region could be confined to the central part of the IRE sequence, called the negative regulatory element (NRE). Competition experiments and gel retardation assays with this region indicated that undifferentiated F9 cells contain a titratable negative factor that binds to the NRE and is absent in differentiated cells (Flanagan et al., 1991). 3 . Regulation by Inteferom

Differential screening of a library of human cells treated with cx-interferon resulted in the identification of several induced mRNAs, of which one corresponded to a MHC Class I-encoded message (Friedman et al.,

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1984). A 29-bp sequence present in all Class I genes about 150 nucleotides upstream of the cap site (IRE) (Kimura et al., 1986) is closely homologous to a consensus sequence found by Friedman et al. (Kelly et al., 1985) in the promoters of human genes stimulated by a-interferon. The fact that only a combination of the IRE element and enhancer A can render a heterologous promoter fully responsive to the three types of interferons was shown by placing these boxes either separately or in combination upstream of a promoter-reporter construct (Israel et al., 1986; Sugita et al., 1987).The IRE element, which acts as an inducible enhancer independently of location and orientation, alone can augment the activity of a heterologous promoter only twofold upon stimulation with a-or p-interferon. Enhancer A alone does not stimulate when interferon is added, but exerts a strong synergistic effect (Sugita et al., 1987).The finding that induction of MHC Class I expression is often a dominant regulatory effect is illustrated by the observations that yinterferon is able to override the repression by adenovirus type 12 (Eager et al., 1989; Ackrill and Blair, 1990) and c-myc (Versteeg et al., 1988, 1989a; Seliger and Pfizenmaier, 1989). Indications that induction of HLA proteins by interferon can be a locus-specific phenomenon came from studies using a monoclonal antibody recognizing only a subset of HLA molecules (Burrone et al., 1985). The interferon-induced subpopulation appeared to consist mainly of B-locus products. Additionally, in Molt4 thymomas (Girdlestone and Milstein, 1988) and peripheral blood lymphocytes of normal donors (Abi-Hanna and Wakefield, 1990) locus-specific effects on MHC Class I expression by y-interferon were detected. Lack of K kaugmentation contrary to H-2Db, Kb, and Dk induction by interferon was reported in a leukemia virus-induced murine tumor (Green et al., 1988), pointing to a locus-specific phenomenon in mice as well. Several research groups used HLA-transfected mouse L cells to focus on differential regulation of human HLA Class I expression by y-interferon (Schmidt et al., 1987, 1990; Hakem et al., 1989). Comparison of the IRE sequences of the different alleles showed that small changes in this 5' enhancer element could account for the differential effect (Hakem et al., 1989; Schmidt et al., 1990). Studies using various HLA-CAT reporter constructs and sitedirected mutagenesis of the IRE box showed that two nucleotide differences increased the level of inducibility of HLA-A3 by a- and yinterferon to that of HLA-B7 (Hakem et al., 1991). It should be noted that other sequences may also be involved in regulation by interferons. These include sequences present in the 5' part of enhancer A (Korber et al., 1988), sequences within the HLA coding sequence or at their 3'untranslated end (Schmidt et al., 1990), or sequences constituting an

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exact composition of the binding site of enhancer A o r of the IRE (Schmidt et al., 1990). 4 . Regulation of MHC Class I by Vzruses ’

As described in Section 111, MHC Class I antigens can be modulated by viruses. T h e result, increase or decrease in MHC Class I expression, depends on the type of virus and whether it concerns transformation or infection. For example, infection with Moloney murine leukemia virus can increase the level of MHC Class I proteins as well as p,-microglobulin on the surface of the cells via a trans effect at the transcriptional level (Wilson et al., 1987). Experiments using CAT constructs suggest that the effect of the leukemia virus may be mediated through a sequence somewhere within the 2.1-kb region upstream of the coding region of H-2Kb. Moloney murine sarcoma virus, which has the opposite effect on MHC Class I expression upon infection of murine cells, is believed to exert its downregulating effect on a region located further than 2.1 kb upstream of the H-2Kb coding sequences (Wilson et al., 1987). Adenoviruses exert opposite effects upon infection and transformation on MHC Class I expression as discussed in Section II1,A. Adenovirus type 12 in transformation is capable of downmodulating MHC Class I expression (Schrier et al., 1983). T h e Ad12 transforming gene, EIA, which is responsible for the reduced MHC Class I expression, is shown to act by reducing the rate of transcription of MHC Class I genes (Ackrill and Blair, 1988; Friedman and Ricciardi, 1988; Lassam and Jay, 1989; Meijer et al., 1989). Through which element in the 5’-flanking region the interaction occurs and whether direct repression o r indirect modulation of transcription factors is involved is still a matter of debate. Meijer et al. (1989) demonstrated a regulating effect of Ad12 on the 2-kb 5’-flanking region of the H-2K gene. Kimura et al. (1986) suggested that the K b promoter, but not the enhancer, is downregulated in AdlZ-transformed cells. Another report on regulation of the MHC Class I gene by Ad12 (Katoh et al., 1990) demonstrated that a distal sequence, located between -1837 and -1521 bp relative to the cap site, contributes to the negative regulation by EIA. A positive regulatory element seemed to be localized in the enhancer A/IRE region (Katoh et al., 1990). Specific binding of a nuclear factor to the distal negative regulatory element was detected in gel retardation and DNase I footprint experiments. However, the involvement of this distal region could not be confirmed by others: in fact, association of the presence of a region located between -1.1 and -1.5 kb and downregulation of Ciass I expression by Ad12 was

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observed (E. Blair, personal communication). Moreover, it has been found that binding of H2TFl and NF-KBto the dyad element in enhancer A and the functional activity of this enhancer in CAT assays are reduced in Ad12-transformed baby rat kidney cells (I. Meijer and A. J. van der Eb, personal communication). As both groups used the H-2Kb gene instead of the Kbml gene exploited by Katoh et al. (1990), differences between the haplotypes might explain the discrepancy. However, the promoter region of H-2Kbm1turned out to differ from the Kb promoter by only a single nucleotide deletion outside the most distal putative upstream negative regulatory element (Ozawa et al., 1990),and therefore, it seems unlikely that the discrepancy can be ascribed to differences between the examined haplotypes.

5. The Involvement of trans-Acting Factors To this point we have focused only on the DNA sequences important for positive and negative regulation of MHC Class I genes. We have seen that the enhancer A sequence appears to be the most powerful regulating element. A binding site for multiple factors is located in the 3' part of the enhancer A box and has perfect dyad symmetry (Baldwin and Sharp, 1987, reviewed in Singer and Maguire 1990). The first purified protein binding this palindrome is KBFl (Yano et al., 1987). This 48-kDa protein, isolated from a nuclear extract of mouse thymoma cells, also binds to the enhancer of the P,-microglobulin gene (Israel et aZ., 1987). In undifferentiated embryocarcinoma cells, in which this palindromic sequence shows no enhancer activity, this factor is absent (Israel et al., 1989b), indicating that KBF 1 activity is regulated during differentiation. The second purified protein, called KBF2, which has an apparent molecular weight of 58 kDa, also binds to this sequence (Israel et d., 1989b). In contrast to KBFl it is present in undifferentiated EC cells and its levels are decreased after treatment with differentiation inducing agents (Singer and Maguire, 1990). Next, the dyad element showed binding activity of a distinct factor, H2TF1, which is detectable in a large variety of differentiated mammalian cell lines. This 110-kDa factor also binds to a similar sequence in the 72-bp repeat enhancer element of simian virus 40 (Baldwin and Sharp, 1987; Baldwin et al., 1990). The transcription factor NF-KB,which is constitutively only present in B cells and can be induced by treatment with a tumor-promoting agent (TPA) in non-B cells, is also capable of binding the regulatory sequence in enhancer A (Baldwin and Sharp, 1988). This factor, which was first identified by its ability to bind to an enhancer in the immunoglobulin light chain genes, is in the inactive state found in the cytoplasm bound by a specific inhib-

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itor IKB.Following activation by various stimuli, NF-KBis translocated to the nucleus, apparently by triggering phosphorylation of IKB, which releases NF-KB from its complex (Ghosh et al., 1990). Cloning and sequencing of the cDNAs encoding the DNA binding subunits of the transcription factors NF-KB and KBFl revealed that these domains are probably identical and belong to the family of oncoproteins encoded by the rel oncogene, to which the Drosophila dorsal gene product also belongs (Gilmore, 1990; Kieran et at., 1990; QuilletMary P t al., 1991). Dorsal is known to be involved in controlling the expression of zygotic genes along the dorsal-ventral axis. Like NF-KB, the re1 and dorsal proteins show unusual patterns of subcellular localization and might act as transcription factors. As the nucleotide sequence of a cDNA coding for a putative transcription factor usually gives much information concerning the nature and characteristics of the protein, cloning of cDNAs encoding these factors by screening libraries with the desired binding site was undertaken as an approach. Using an oligonucleotide probe containing the enhancer A binding site to screen a human B-cell expression library, a cDNA clone that encoded a fusion protein recognizing this element was isolated. This cDNA hybridizes to a single copy gene that codes for a 10-kb mRNA, which is expressed in a variety of cells (Singh et al., 1988). Although some of its properties overlap with those of KBFl, H2TF1, and NF-KB, it is unlikely that this gene encodes one of these earlier identified enhancer A binding factors (Baldwin et al., 1990; Fan and Maniatis, 1990). Screening of the same library at lower stringency resulted in two additional cDNAs encoding enhancer A binding proteins (Rustgi et al., 1990). These proteins both contained zinc finger domains, characteristic of DNA binding domains of transcription factors; however, they could not be identified as H2TF1 or NF-KB. It is possible that additional new enhancer A binding proteins have been identified in this way, but it cannot be excluded that proteins binding to this dyad element stem from the same gene family o r result from different degradation products encoded by a limited number of genes. The expression of MHC Class I genes during differentiation and development seems to be regulated at the level of transcription, mainly through proteins binding to the enhancer A sequence. Regulation at the level of transcription also occurs for the expression of Class I-like proteins, which, unlike the classical Class I proteins, have a restricted tissue distribution (Handy et al., 1989; David-Watine et al., 1990). This differential expression pattern may be due to sequence variations in the protein binding site of enhancer A of the Class I-like QZO gene compared with the Class I genes (Handy et al., 1989) or in other protein binding

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regions neighboring the dyad element (David-Watineet al., 1990). It has been demonstrated that mutagenesis of the two involved nucleotides in the putative enhancer A binding site of the QlO gene is capable of restoring the DNA-protein interaction (Handy et al., 1989). As the mouse Class I-like gene QlO is mainly expressed in the liver, factors present in liver extracts binding to regions in the promoter of this gene have been characterized (David-Watine et al., 1990). These studies resulted in the identification of a tissue-specific factor, TA-f, that binds to the TATA box of the QlO and the H-2Kb genes and is found only in liver and kidney. The identification of a factor, with limited tissue distribution, binding specifically to the region of MHC genes containing the general transcription elements, might be very interesting, because the oncogene c-myc is likely to induce a Class I repressing factor that may bind to this region in HLA-B genes (see below). Induction of MHC Class I expression by stimulation of cells with interferon also appears to be regulated through specific tram-acting factors. The action of interferon is effectuated in a complex and diverse manner, as is illustrated by the number of factors Binding to the IRE that have already been identified. These factors, which are either expressed constitutively or induced by interferon, bind identical residues in the DNA and share some structural similarity in the putative DNA binding domain (Shirayoshi et al., 1988; Blanar et al., 1989; Driggers et al., 1990). The first identified members of this gene family, IRF-1 and IRF-2, are involved in the regulation of Class I expression by a-and P-interferon (Miyamoto et al., 1988; Harada et al., 1989), whereas a third identified factor, ICSBP, is preferentially induced by y-interferon (Driggers et al., 1990). With regard to the trans-acting factor data, it is clear that the enhancer A region is by far the most extensively studied region of the MHC Class I enhancer/promoter. Most characterized MHC Class I promoter region binding proteins interact with this element. Completion of the cloning of the cDNAs encoding these proteins should reveal the similarities in nature, tissue distribution, and mechanisms of activation of the different DNA binding factors. Some additional enhancer-like elements present in Class I genes have been described. The enhancer located between the CAAT box and enhancer A in the 5'-flanking region, enhancer B (Kimura et al., 1986; Baldwin and Sharp, 1987; Ganguly et al., 1989), is reported to show a region of protection with extracts of a murine and a human cell line in footprint assays. This binding activity could not be discriminated from binding of the transcription factor AP-1 to this region (Korber et al., 1988). The enhancer-like elements found in introns 3 and 5 of HLA-B7

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(Ganguly et al., 1989) have not been reported to bind a protein in vzvo or vitro. In the flanking sequences of MHC Class I genes several negatively regulating elements have also been detected. One is located in the IRE box and is involved in regulation during differentiation (Flanagan et al., 1991). A distal region located several kilobases upstream appears to be involved in downmodulation of Class I by adenovirus (Katoh et al., 1990; E. Blair, personal communication). Furthermore, a dominant negative regulatory element is present in the PD1 swine Class I gene comprising a silencer as well as an enhancer (Ehrlich et al., 1988; Singer and Maguire, 1990; Weissman and Singer, 1991). Various factors can bind to this region: a labile silencer factor competes with a constitutively expressed stable enhancer factor, thus determining the level of MHC Class I expression (Weissman and Singer, 1991). The precise identity of the proteins binding to the various putative negative regulatory elements has not yet been revealed.

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6. Role for Methylation

T h e 5’ regions of ubiquitously expressed housekeeping genes are mostly linked to so-called CpC islands. These islands have been identified as elements often associated with transcriptional active regions in the genome. The methylation status of these dinucleotides is thought to determine the level of expression of adjacent genes. Studies on the accessibility of Ft’-CpG-rich regions of HLA Class I genes in a lymphoblastoid cell line to methylation-sensitive rare-cutter enzymes showed that these islands are mostly unmethylated in this Class I-expressing cell line (Pontarotti et al., 1988). Induction of H-2K expression upon differentiation of F9 cells is believed to be (partly) regulated by DNA methylation (Tanaka et al., 1983). Although the precise locations of all methylation sites have not been mapped, blocking these sites with 5-azacytidine is correlated with a reduction of H-2K expression in differentiated cells. T h e secreted Class I-like antigen Q10, which is highly homologous to the classical membrane-bound molecules, is synthesized only in the liver. Analysis of DNA from different murine tissues with methylation-sensitive restriction enzymes revealed a unique methylation profile for the QlO gene in the liver (Tanaka et al., 1986). It was, however, difficult to determine exactly which sites were recognized by the enzyme and whether presence or absence of methylation was responsible for the tissue-specific pattern of expression. More conclusive results arose from the treatment of animals with the demethylating agent 5-azacytidine, from studies of the methylation status during the different stages of development and from mice with aberrant QZO expression. These re-

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sults all pointed to a correlation between expression and hypermethylation of the 3'-flanking region of the QlO gene (Tanaka et al., 1986). Whether these findings indicate the presence of a mechanism in which transcription of all MHC Class I genes is regulated by differential levels of methylation, possibly in combination with activity of transcription factors, remains to be determined. The performance of experiments establishing the methylation status of human Class I genes is hampered by the high level of polymorphism of these genes, because mapping of all polymorphic restriction sites and the design of strictly locus-specific probes will be difficult.

B. REGULATION IN TUMOR CELLS 1. Mechanisms As seen before, MHC Class I expression is often downregulated in tumor cells. In many cases the responsible (oncogene-encoded) factor has not been identified. In most cases where the mechanism of downregulation has been studied in more detail, Class I expression turned out to be regulated at the level of transcription, often by inactivation of enhancer A (see below). First, in AKR leukemias absence of H-2Kk molecules is correlated with a decrease of binding of H2TF1 or KBFl to enhancer A (Henseling et al., 1990). Second, the suppression of MHC Class I gene expression by the oncogene N-myc is regulated through enhancer A inactivation (Lenardo et al., 1989). Third, the locus-specific downregulation of HLA Class I mRNA in colorectal carcinoma seems to involve changes in protein binding to the enhancer A region (Soong and Hui, 1991; Soong et al., 1991). Fourth, in the K562 cell line, weak KBFl and NF-KB binding to enhancer A correlating with low HLA Class I expression was found (Blanchet et al., 1991). Other mechanisms for downregulation of Class I expression in tumor tissue have been proposed. The different chromatin structure found in fibrosarcoma cells, which is proposed as a mechanism of downregulation of Class I expression, may still be compatible with changes in the in uivo activity of transcription factors (Maschek et al., 1989a,b).The MHC Class I expression in the erythroleukemia cell line K562 is suggested to be suppressed not by local repressive influences, but by more complex chromatin-related restraints (Maziarz et al., 1990) or by the presence of repressor molecules (Chen et al., 1987). The locus-specific shut-off of MHC Class I genes in murine lymphoma sublines is thought to be determined by cis-dominant regulatory elements (Keeney and Hansen, 1989; Keeney et al., 1989).

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As extensively discussed in the previous section, downregulation of MHC Class I expression by the oncogenes N-myc and c-myc occurs in neuroblastoma and melanoma, respectively. T h e region in the myc genes important for (co)transformation and autosuppression is formed by 85 carboxy-terminal amino acids. This region contains several structural motifs: a basic region, a helix-loop-helix domain, and a leucine zipper, which are often found to be present in transcription factors and are known to be involved in protein dimerization and DNA binding (reviewed in Luscher and Eisenman, 1990; Penn et al., 1990b). These structural homologies prompted the investigators to focus research on myc protein-DNA interactions. The dimerization domain suggested the existence of an interaction between c-myc and other basic regiodhelixloop-helix proteins or the formation of homodimers. However, the presence of myc homodimers could never be demonstrated in vitro (Penn et at., 1990a; Smith et al., 1990; Dang et al., 1991; Prendergast and Ziff, 1991). Recent experiments using a bacterially expressed fusion protein, which contained the carboxy-terminal end of the human c-myc protein, resolved the core sequence to which m y dimers can bind in uitro (Blackwell et al., 1990). Binding to this recognized element 5’-CACGTG-3’ can be inhibited by methylation at the second C-residue (Prendergast and Ziff, 1991). Since c-myc had not been found to homodimerize or to form dimers with known basic regiodhelix-loop-helix proteins, much effort was put into the cloning and identification of myc binding factors. Almost at the same time the cloning of the human (Blackwood and Eisenman, 1991) and mouse (Prendergast et at., 1991) homologs of a basic regiodhelixloop-helix/leucine zipper c-myc binding protein, respectively called Max and Myn, were reported. Both 18-kDa proteins dimerize specifically with c-niyc through their helix-loop-helixlleucine zipper domains, enabling the complex to recognize the known c-myc binding site CACGTG. Coexpression of Myn in a myc + ras focus formation assay augmented the transforming activity of c-myc (Prendergast et al., 1991), indicating that the outcome of transformation by an activated myc oncogene may depend upon the level of the Max or Myn accessory protein in the particular cell line. 2. N-?nyc

N-myc overexpression in rat neuroblastoma tumor cells causes a reduction of Class I mRNA by inactivation of the main enhancer, enhancer A, of the MHC Class I gene (Lenardo et al., 1989). As shown in gel retardation assays, using the enhancer A binding site as a probe, elevated N - m y expression appears to correlate with reduced binding of

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H2TFl to enhancer A in both human neuroblastoma cell lines and rat neuroblastoma transfectants. Because all myc proteins share the basic regiodhelix-loop-helix/leucine zipper motif, indicative of DNA binding properties, N-myc was expected to interact directly with the promoterlenhancer region of the Class I gene. However, Bernards (1991) has shown a possible interference of myc with signal transduction pathways in N-myc-overexpressing neuroblastoma cells. He hypothesized that H2TF1 binding activity could be regulated through a phosphorylation mechanism, although such a mechanism of activation of enhancer A binding proteins has not formally been proven. To induce protein kinase C (PKC) activity, neuroblastoma cells were treated with TPA. As a control for the inducibility of the cells he monitored the induction of NF-KB, which can be induced by TPA in normal non-B cells (see Section IV,A,5). A small increase in the binding activity of H2TFl was found, but, unexpectedly, he noted a deficiency in NF-KB induction by TPA of N-myctransfected cells. Hybridization of Northern blots with probes detecting the different isoforms of PKC demonstrated a suppression of PKC-6 expression by the N-myc product, accompanied by an induction of the 5 isoform. The relationship between these observations was confirmed by showing that introduction of a PKC-y expression vector in N-myc-transfected neuroblastoma cells restored phorbol ester induction of NF-KB and induction of the expression of c-fos. As reversing the effect of N-myc by transfection of PKC type y does not coincide with restoration of H2TFl binding to the Class I enhancer, these results might indicate that alterations in PKC expression are not responsible for the suppression of H2TFl activity. The failure to activate H2TF1, however, could be explained by the fact that the y isoform was used to supertransfect the N-myc-overexpressing rat neuroblastoma cells, whereas the N-myc-modulated type is the 6 isoform. It cannot be excluded that PKC-y has a substrate specificity distinct from the 6 isoform.

3. c-myc Contrary to N-my, which is shown to be capable of suppressing the expression of all MHC Class I loci in neuroblastoma, activation of c-my in human melanoma results in downmodulation of predominantly HLA-B locus products, as was demonstrated in our own laboratory (Versteeg et al., 1989a). Locus-specific downregulation has in addition been reported for a mouse lymphoma model (Keeney et al., 1989), in which regulation appears to take place at the level of transcription. However, the component responsible for the locus-specificshut-off of Class I products has not been identified in this model system. As N-myc and c-myc are both members of the myc family of proteins, our initial studies on the

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mechanism of regulation of HLA Class I by c-myc focused on the enhancer A region influenced by N-myc. The binding of regulatory proteins to this conserved palindromic sequence, present in whole-cell extracts of c-myc transfectants and the original melanoma cell line IGR39D (Versteeg et al., 1988), was determined in vitro. No (inverse) correlation between binding activity of HZTF-1 and c-myc expression could be established in gel retardation assays (Peltenburg et al., 1992a). This indicates that the enhancer A sequence is not involved in downmodulation of HLA Class I genes by c-myc. These findings were confirmed by studying the functional activity of the enhancer A box in transient transfection assays with the CAT gene as a reporter. Using CAT constructs controlled by a minimal fos promoter with and without the HPTFl binding site confirmed the observation that the palindrome in low- and high-c-mycexpressing cell lines is similarly active in inducing transcription (see Table 11). These experiments definitively exclude a role for the enhancer A region in downregulation of HLA-B by c-myc. This conclusion is in agreement with the thus far available DNA nucleotide sequences of HLA-A and HLA-B genes, showing no specific differences among the respective enhancer A regions that could explain the HLA-B-specific downmodulation by c-myc. Indirect evidence suggests a role for another region in the HLA-B gene, which might harbor a negative regulatory element, in downregulation by the c-myc oncogene (Peltenburg et al., 1992a) (see Fig. 2). As discussed above (Section IV,A), other dominant negative regulatory elements have been reported to be involved in regulation of MHC Class I. The locations of these regions are summarized in Fig. 2: first, an element located in the IRE box exerting its effect TABLE 11 TRANSIENT TRANSFECTION OF fos PROMOTER CONSTRUCTS I N MELANOMA CELLS

Plasmid pfosCAT pMHCfosCAT pMoCAT

Number of experiments 4 4

3

70 CAT activity IGR39D

I .6 82.9 53.8

IGR-myc 1.2 40.1 50.3

Note. bfelanoma cell lines 1GRSSD and its c-my transfectant IGR-my (clone 3) (Versteeg et al., 1988) were transiently transfected with pfosCATA56, pMHCfosCATA56 (Lenatdn et al., 1989) and with a plasmid containing the Moloney murine leukemia viris LTR in front of the CAT coding sequence (pMoCAT). Transfections and enzyme assays were performed as described (Lenardo el a/.,1989). with the exception that cell lines were transfected with a mixture of 10 p,g CAT plasmid and 10 pg of a pgalactosidage plasmid (as a control for the transfection effiriency). The average percentage conversion of rhloramphenicol into acetylchloramphenicol obrained with extracts of these transfections, as determined by counting the thin layer plates with a Phospho-Imager. is indicated.

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0 kb

FIG. 2. Schematic drawing of the presumed dominant negative regulatory elements identified in various MHC Class I genes. The location and length of these boxes are illustrated in this hypothetical MHC Class I gene; the length of each box does not necessarily imply that the entire stretch of sequence is involved. Arrow: initiation of transcription; the numbers beneath represent the distance (in kilobases) relative to the transcription start site. 1: regulation by adenovirus (Katoh et al., 1990);2: regulation by adenovirus (G. E. Blair, personal communication; 3: present in PD1 Swine Class I gene (Ehrlich et al., 1988);4: regulation during differentiation(Flanaganet al., 1991);5: regulation of HLA-B by c-my (Peltenburg, 1992);0 :CAAT and TATA elements.

during differentiation (Flanagan et al., 1991); second, two distal elements involved in regulation by adenovirus (Katoh et al., 1990); and finally, an element located far upstream in the swine PD1 gene (Singer and Maguire, 1990; Weissman and Singer, 1991). Other genes have been demonstrated to be downregulated by c-myc: the LFA-1 adhesion molecule (Inghirami et al., 1990) and, at the level of transcription, the mouse pro-alpha 2 (I) collagen (Yang et al., 1991), as well as the human c-neu oncogene (Suen and Hung, 1991). The latter two appear to be regulated by c-myc through a region just upstream of the cap-site of these genes. A parallel can be drawn between regulation of MHC Class I expression and downmodulation of these two genes: the downregulation of HLA-B by the oncogene c-myc seems to involve a dominant regulatory element located in the region surrounding the general transcription elements (Peltenburg et al., 1992a). Whereas the effect on Class I expression induced by N-my is regulated through enhancer A, the effect exerted by c-my on Class I expression appears to be achieved in an entirely different manner, as is suggested by the experiments determining enhancer A activity in human melanoma cells. In this light the observation that expression of the PKC isoform 6, which seems to be suppressed by N-my in neuroblastoma (Bernards, 1991), could not be detected in RNA samples from several different melanoma cell lines may be interesting (L. T. C. Peltenburg, unpublished observation). The only PKC mRNA that could be detected in melanoma cells was of the ct isoform. It was expressed at the same level in a panel of c-my transfectants and the parental cell line. In conclusion, neither inactivation of enhancer A function nor reduction of a special PKC isoform found in N-myc-transfectedneuroblastoma cells was observed in c-myc-transfectedmelanoma cell lines. On the basis

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of their structural homology, a similar MHC Class I regulatory mechanism for N-myc and c-myc could be expected. However, the differences in tissue distribution of the various PKC isoforms and variation in tissuespecific expression of the myc proteins (Jakobovitz et al., 1985; Stanton et al., 1986) argue against an identical function of c-myc and N-myc. V. Biological Consequences of MHC Class I Downmodulation by Oncogenes

So far, it has become clear that MHC Class I expression is affected in many tumors and that oncogenes, in particular N-myc and c-my, can be responsible for this effect. In the last section of this review, we will focus on the consequences of this phenomenon on tumorigenicity of cells with particular reference to the immune response they elicit. Also, we will discuss the relevance of the direct connection between oncogene activation and immune modulation.

A. MHC

A N D PROGRESSION

In animal models, the tumorigenic properties of cells may depend on the expression of particular H-2 alleles. For instance in the case of the T10 sarcoma with downmodulated H-2 alleles, clones expressing both H-2D alleles are metastatic, whereas clones expressing only D" are not metastatic. In contrast, oncogenicity or metastatic capability can be abrogated by transfection of the proper H-2 molecule back into H-2-negative tumor cells. This has been demonstrated for various other tumor systems (Hui et al., 1984; Wallich et al., 1985; Bahler et al., 1987; Plaksin et ul., 1988; Gopas et al., 1989; Porgador et al., 1989; Sturmhofel and Hammerling, 1990). No consistent effect of H-2 modulation on tumorigenicity, however, can be assigned: sometimes the tumorigenicity and metastatic capability is abrogated by elevated H-2 expression, as, for instance, is the case in transfection of H-2Kb in T10, 3LL, or B16 melanoma cells (Wallich et al., 1985; Plaksin et al., 1988; Porgador et al., 1989) and H-2Kk in AKR leukemia cells (Hui et al., 1984). In other cases, however, the metastatic capacity of the cells increases after H-2 transfection. T h e latter has been seen for H-2Dk transfection in T10 cells (Gopas et al., 1989). As has been extensively discussed (Sections I1 and III), in human tumors, many cases of specific downmodulation have been found. However, no data on their effect in vivo are available in the majority ofcases. Only a few publications permit clear conclusions on an effect of HLA Class I expression on progression in patients (see also

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Table I). In a large series of colon carcinomas no correlation between HLA Class I expression and recurrence rate or survival could be found, in contrast to a prognostic role for disease stage or histological grade (Moller et al., 1991). A similar conclusion was drawn from a study on breast carcinoma (Wintzer et al., 1990). In another study on breast carcinoma (Moller et d., 1989) no correlation of HLA Class I expression with tumor type or grade could be established, whereas, conversely, in two other reports such a correlation was found (Bhan and DesMarais, 1983; Whitwell et al., 1984). A positive correlation between low HLA Class I expression and tumor invasiveness or high-grade malignancy was found for bladder carcinoma (Tomita et al., 1990a) and for B-cell lymphomas (Moller et al., 1987). Also, laryngeal carcinomas with low HLA Class I expression had a more aggressive phenotype but not a higher metastatic potential. In addition, the patients with these tumors had a worse prognosis (Esteban et al., 1990). In melanomas, low HLA Class I was correlated with metastasis (Brocker et al., 1985) and poor prognosis in terms of survival (Van Duinen et al., 1988).In the latter case, however, the conclusion has to be interpreted with care, since tumors with high expression of HLA Class I1 always coexpressed HLA Class I, and high HLA Class I1 expression by itself has a bad prognosis (Van Duinen et al., 1988). The latter effect has been attributed to an immunosuppressive effect of HLA Class I1 expression on the proliferation of tumor-specific T cells. Therefore, in evaluating a correlation between progression in vivo and HLA Class I expression, a possible effect of coincident HLA Class I1 expression should be taken into account. In conclusion, the data on an in v i m effect of HLA Class I modulation are sparse. From the available data no clear conclusion can be drawn: in some tumor types, e.g., colon carcinoma, an apparent lack of correlation between HLA Class I expression and malignant potential has been found; in other types, e.g., B-cell lymphomas and melanomas, such correlation could be assigned. B-cell lymphomas as well as melanomas are considered to be particularly immune-reactive because they appear at relatively high incidence in immunosuppressed individuals (Penn, 1988; Witherspoon et al., 1989). It might therefore not be coincidental that low HLA Class I expression in these cases is correlated with poor prognosis: in these tumor types the immune system of the patient may be active in eliminating virally induced or spontaneously arising tumor cells and low HLA Class I expression will largely diminish the efficacy of eradication of newly formed neoplastic cells by cytolytic T cells. Although the relation between “low HLA” and “poor prognosis,” at least from a theoretical point of view, favors a role for T cells in immune surveillance against cancer, it should not be ignored that cells with low MHC Class I

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expression are more refractile to lysis by N K cells than comparable cells with normal or high MHC Class I expression. T h e repercussion of this phenomenon for immune defense mechanisms in cancer patients will be discussed (Section V,F). B. ‘I’ CELLS

The selective downmodulation of particular loci or alleles in a number of human and rodent tumors may provide a hint as to the functional consequences of low MHC Class I expression on tumor cells. Especially in human tumors known to be immunoreactive, such as melanoma, renal cell carcinoma, colon carcinoma, virus-associated cervical carcinomas, and Burkitt lymphomas (Doherty et al., 1984; Melief et al., 1989; Melief, 1991), selective losses of HLA antigens have been found. A straightforward explanation for these allelic losses would be that tumors escape from immunoreactive T cells by downmodulating those HLA alleles that present altered self-peptides or viral peptides recognized by the tumor-specific T cells. In that case, the alleles still present would not be capable of presenting the tumor-specific peptides, or alternatively, T cells reactive with those HLA allele-peptide combinations are not present in the T-cell repertoire. That T cells are involved in the defense against virus-associated tumors was demonstrated by the negative correlation of HLA-AZI and the occurrence of skin cancer (associated with papilloma virus) in renal-transplant patients: none of the 66 cancer patients were HLA-AIZ-positive, compared to 12% in a control group of 631 normal individuals (Bouwes Bavinck et al., 1990, 1991). These data are highly suggestive for a role of HLA-AII in the presentation of human papillomavirus peptides to the immune system of the patients, protecting against the disease. No direct correlation of particular HLA Class I alleles with the occurrence of cancer has been found for other human cancers, but infrequently, HLA Class I-restricted tumor-reactive T cells have been found in melanoma, renal cell carcinoma, and ovarian cancer patients (Anichini et al., 1989; Belldegrun et al., 1989; Wolfel et al., 1989; Degiovanni et al., 1990; Gervois et al., 1990; Crowley et al., 1991; Ioannides et al., 1991a). This indicates that at least tumor-specific or tumor-associated antigens, most likely presented by HLA Class I molecules, are present in the tumor of these patients. In contrast, very effective T-cell responses have been found in animal tumor models. A case in point is the kill of tumors induced by adenovirus 5-transformed cells (Kast et al., 1989). Here, a T-cell clone has been characterized specific for an EIA-encoded peptide presented by H-2Db (Kast and Melief,

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1991) that has the capacity to eradicate large-size tumors inoculated in nude mice in combination with IL-2. When T-cell responses are elicited, a good expression of MHC Class I antigens on the tumor cells is necessary for proper presentation of these antigens. In principle, there are two effective ways for the tumor cell to escape from immune destruction: either downmodulation of the selfantigen or downmodulation of the MHC Class I expression. Both mechanisms have been shown to occur. For instance, the selection of variants negative for the tumor-specific antigen has been demonstrated in a variety of tumor models (Boon et at., 1989; Melief et al., 1989). However, when the tumor antigen is an activated oncogene involved in growth control, the escape will be not effective, since downmodulation will result in growth arrest of the tumor cells. In such cases, the downmodulation of the MHC Class I molecule will be more effective. This has become manifest in Ad 12-transformed cells with strongly downmodulated expression of MHC Class I antigens (see Section 111).These tumors escape from T-cell surveillance since the absence of H-2 precludes a proper proliferation of T cells. This view is underscored by the finding that it has not yet been possible to raise CTLs against Adl2-transformed tumors in mice. I n addition, upregulation of the H-2 expression by treatment of these cells with y-interferon o r by transfection of an exogenous H-2 gene rendered these cells less tumorigenic in syngeneic animals (Hayashi et al., 1985; Tanaka et al., 1985, 1986). By and large, the combined data show an important role for T cells in the immune surveillance against tumor cells. On the other hand, many examples are available where low MHC Class I expression leads to lower tumorigenic or metastatic properties of tumor cells probably due to increased sensitivity to NK cells (see below). Considering this mechanism, the effect of low MHC Class I expression is not evasion from the immune system, but rather elimination of the tumor cells by NK cells. C. OTHERFACTORS INVOLVED IN THE IMMUNE REACTION AGAINST TUMOR CELLS It has been shown in a number of experimental tumor models that the sensitivity of tumor cells to NK cells is determined by the level of expression of MHC Class I antigens on the tumor cells (Bell and Stern, 1985; Karre et al., 1986; Taniguchi et al., 1987; Ljunggren et al., 1988; Algarra et al., 1989; Sturmhofel and Hammerling, 1990; Carbone et al., 1991; Liao et al., 1991). This phenomenon has led to the formulation of the “missing self” hypothesis by Karre and co-workers (see Ljunggren

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and Karre, 1990, for a recent review). Briefly, it says that permanently surveilling N K cells scan somatic cells for MHC Class I expression. If MHC Class I is not present, the N K cell will kill the target. Two mechanisms can be envisaged (Ljunggren and Karre, 1990). First, the “target interference” model, where MHC Class I protects a tentative NK target from interaction with the NK cell. Second, the “effector inhibition” model, in which after interaction of NK cell and target cell and recognition of MHC Class I, the lytic hit is blocked. If MHC Class I is absent the target cell is killed. The latter model requires positive selection of NK cells: some sort of receptor should discriminate between self and nonself (or absent) and education of NK cells to deliver the lytic hit dependent on MHC Class I expression has to occur. This view is corroborated by the finding (Bix et al., 1991; Liao at al., 1991) that mice knocked out for the P2-microglobu1ingene, which do not express MHC Class I molecules on the surface of their cells, do have NK cells, but these are not effective in killing NK-sensitive targets. This suggests that for establishment of a proper N K lineage, education of N K cells through contact with functional MHC Class I molecules is a constraint for correct discrimination of self (autologous MHC Class I) or nonself (allogeneicor no MHC Class I). The advantage of such a surveillance system is that the immune system need not be primed after appearance of a foreign or altered epitope present in the target cell, as in the case of a T-cell response, but will immediately attack and eliminate an aberrant MHC Class I-negative cell. In this view, low MHC Class I expression on a tumor cell is not the result of an escape from immune surveillance (by for instance Class I-restricted T cells), but rather an intrinsic property of the tumor cell. An inverse correlation between Class I levels and N K susceptibility has not been found in all tumors with altered MHC Class I expression (Chevernak and Wolcott, 1988; Nishimura et al., 1988; Gorelik et al., 1990), indicating that factors other than MHC Class I are involved in determining NK sensitivity of a tumor target. Moreover, the effect cannot be attributed to N K cells in all experiments showing an increase in metastatic potential upon augmentation of MHC Class I expression (Gopas et al., 1989; Bertschmann et al., 1990). Therefore, MHC Class I proteins may have other effects than influencing the immunological interaction between tumor cells and immune effector cells. Indeed, it has been shown that increase of H-2Kd expression in transformed rat fibroblasts alters the growth properties of the cells in vitro and may increase their metastatic capability in immunodeficient animals, indicating that nonimmunological factors contribute to the tumorigenic properties of cells (Gattoni-Celli et al., 1989, 1990; Haliotis et al., 1990). These may include signal transduction (Haliotis et al., 1990) and homotypic adhe-

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sion (Gattoni-Celli et al., 1989). The latter factor may affect the social organization and cross-feeding of the tumor cells, ultimately leading to an altered metastatic capacity. A nonimmunological role for MHC Class I has also been found for human cells: a murine MHC Class I gene (H-2Ld) was transfected in either a human colon cancer cell line (Gattoni-Celli et al., 1988) or a human neuroendocrine carcinoma line (Sunday et al., 1989). The colon carcinoma cell line became less tumorigenic in nude mice after transfection, suggesting an effect of the MHC Class I gene independent of the T-cell immune system. On the other hand, an opposite effect of H-2Ld was found when the gene was transfected in the human neuroendocrine tumor. In this case, the cells exhibited an increased growth potential in vztro and were more metastatic in nude mice. This might be due to a decreased susceptibility to NK cells, but other effects of increased MHC Class I H-2Ld expression may also account for the difference. In that case one should assume that the effects of MHC Class I are pleiotropic and that these proteins can have positive as well as negative growth regulatory effects depending on the cell type transfected (Sunday et at., 1989). D. HLA CLASSI MEDIATESEFFECT OF c-my ON NK SENSITIVITY Although these nonimmunological effects of MHC Class I molecules may contribute to growth of tumor cells, the major consequence of modulation of MHC Class I expression on human tumor cells seems to be a genuine immunological one: several lines of evidence suggest that in human tumors modulation of MHC Class I expression alters the NK sensitivity of the tumor cells. First, the level of expression of HLA Class I is inversely correlated with NK sensitivity in several lymphoblastoid cell lines (Storkus et al., 1987; Ohlen et al., 1989; Harel-Bellan et al., 1991). Second, lysis of a number of human tumor cell lines by NK cells can be blocked by antibodies specific for HLA Class I (Lob0 and Spencer, 1989; Maio et al., 1991).These cell lines include 3- and T-lymphoma, small cell lung carcinoma, and melanoma. Third, as most convincing evidence, transfection of HLA Class I genes or P,-microglobulin genes into cell lines completely devoid of HLA Class I expression resulted in NK resistance. This was done for HLA Class I loss mutants of B lymphoblastoid cell lines (Shimizu and DeMars, 1989; Storkus et al., 1989b), for the P,-microglobufin-negative Daudi cell line (Quillet et at., 1988), and for a f3,-microglobulin-negative melanoma cell line (Maio et al., 1991). The effect was dose-dependent; i.e., the effect on NK sensitivity correlated with the expression of the transfected gene. In addition, the identi-

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ty of the gene was important: not all HLA Class I alleles exerted the effect (Storkus ct a!., 1989a) and murine genes had no effect at all in human systems (Storkus et al., 1989b). This again suggests a specific interaction of some sort of receptor structure on N K cells with HLA Class I molecules. In the experiments discussed so pdr, the tumor cells were devoid of the products of all HLA Class I loci. As we have seen in the previous sections, in many tumor cells the regulation of HLA Class I expression is locus-specific or even allele-specific. This means that in these cases the unaffected alleles are still abundantly present at the cell surface. The question now is whether N K cells detect subtle allelic losses o r only gross losses of HLA Class I. We have addressed this question using two melanoma cell lines, one homozygous for HLA-A1 and -Bw62, and the other typed Al, A2, and B8. It was shown that selective downmodulation of either HLA-Bw62 o r HLA-B8 occurred by the c-myc oncogene and that this could render the cell line sensitive to N K cell activity (Versteeg et ul., 1989b). This suggests that NK cells can selectively detect the loss of either Bw62 o r B8, independent of the presence of the HLA-A alleles. However, on the basis of these observations, it cannot be excluded that the c-myr oncogene itself is responsible for the increased susceptibility of the tumor cells, by altering expression of other proteins involved in recognition and kill by N K cells. To investigate this further, we have supertransfected one of the c-myc-transfected cell lines (IGR-myc) with a HLA-B27 gene under control of the strong exogenous MoMLV promoter. HLA-B27-positive clones and control clones were selected and tested for NK sensitivity. T h e result of a typical experiment is shown in Fig. 3, in which NK sensitivity and expression of HLA-B27 of the various clones are represented. It can be seen that myc transfection increases the N K sensitivity of the melanoma cell line IGR39D (IGR-myc). Because HLA-B27 is measured with a B27-specific antibody, not detecting the endogenous HLA-B8, the concomitant downmodulation of HLA-B8 is not seen in this experiment. T h e B27-supertransfected clones (B27 c7, c3, and c4) with high HLA-B27 expression (dark bars) have a strongly reduced N K sensitivity (light bars), comparable to the original IGR39D melanoma cell line. It should be stressed that c-myc expression was similarly high in all depicted transfected cell lines (Peltenburg et al., 1992b). This unequivocally shows that exclusively HLA-B, and not c-myc, is responsible for the alteration of NK sensitivity in this cell line. Moreover it shows that lack of a particular HLA allele (Bw62 in this cell line) is capable of influencing NK sensitivity, independent of the expression of other HLA Class I alleles (HLA-A1 in this case). This indicates that NK cells recognize tumor cells in an HLA Class I-dependent way and to this

myc

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I

I 50

40

.v) v)

30

Y

x

z

20

s

10

IGR39D

myc

gpt-c5

B27-c7 B27-c3 827-c4

cell line fluorescence

%

N K lysis

FIG.3. Effect of HLA-B27 transfection on N K sensitivity of melanoma cell lines. Depicted are the HLA-B27 surface expression (dark bars) versus the percentage of specific lysis of melanoma lines by peripheral blood mononuclear cells of a normal donor (light bars). Briefly, the c-myc-transfected melanoma cell line ICR39D (Versteeg et al., 1988) was supertransfected with a plasmid containing the HLA-B27 genomic clone downstream of a Maloney murine leukemia virus LTR together with a plasmid encoding the selection marker xanthine-guanidine phosphoribosyl transferase (gpt). HLA-B27 expression was determined by flow cytometry (Versteeg et al., 1988) with the HLA-BZ7-specific monoclonal antibody ME1 (Ellis et al., 1982) and is given as arbitrary units of relative fluorescence on a logarithmic scale (left vertical axis, fluorescence). The N K sensitivity of the isolated clones was determined according to Versteeg et al. (1989b). The data are represented as percentage specific lysis (right vertical axis, NK lysis) and are the mean value of a triplicate experiment at an effector: target ratio of 100. IGR39D: parental cell line; myc: IGR-myc clone 3, the c-myc transfectant of IGR39D; gpt-c5: a clone isolated from transfection of IGR-myc with the selection marker gpt; B27-c7, c3, and c4: three HLA-B27-positive transfectants originating from myc transfectant IGR-my.

end use a receptor capable of discerning allelic differences. This view is supported by the recent findings of Storkus et al. (1991), showing that amino acid alterations in the peptide binding groove of an HLA Class I molecule that normally does not protect against N K lysis result in protection against NK lysis when the HLA Class I molecule is transfected in an NK-sensitive target. This strongly suggests that a tentative N K receptor interacts with the peptide binding groove. Putting all data together, protecting and nonprotecting HLA allelic products can be discriminated, HLA-A3, -Aw68, -Aw69, -85, -88,and 827 being protecting alleles

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(Shimizu and DeMars, 1989; Versteeg et al., 1986b; Storkus et al., 1991). I n two independent studies, HLA-A1 and -A2 expression apparently did not protect (Versteeg et al., 1989b; Storkus et al., 1991), whereas in another study these alleles did have at least some effect on NK susceptibility (Shimizu and DeMars, 1989). These results can be reconciled by assuming that other factors, in addition to HLA Class I, may contribute to the eventual susceptibility of a tumor target to lysis by N K cells.

E. OTHER FACTORS DETERMINING N K SUSCEPTIBILITY A cell-type-specific factor involved in determining the NK sensitivity might be the peptide in the groove of the HLA Class I molecule, because alterations of amino acids in the groove were found to affect NK sensitivity (Storkus et d.,1991). Therefore, one might speculate that such a peptide, referred to as N K target structure or NKTS (Storkus et al., 1991), is abundantly present in the cell and that the discrimination of self and nonself, as discussed before, proceeds on the basis of HLA Class I + NKTS. In this model, NK cells will kill the target when either self-HLA Class I is absent, or peptide is absent or competed out of the groove of HLA Class I. A similar situation exists in hybrid resistance in mice, a phenomenon where parental bone marrow from one of the parents is rejected in the F1 by NK cells (Cudkowicz and Bennett, 1971a,b; Cudkowicz and Nakamura, 1983; Bordignon et al., 1985; Sentman et al., 1989). This property is recessively inherited and encoded by the socalled Hh-l locus mapping in the MHC (Cudkowicz and Stimpfling, 1964; Cudkowicz and Nakamura, 1983; Daley and Nakamura, 1984; Rembecki et al., 1990). In humans, a phenomenon reminiscent of hybrid resistance has been described (Ciccone et at., 1990a,b). T h e lysis spectrum of an alloreactive N K clone was tested on phytohemagglutinin blasts of a large family and the phenotypic trait “sensitive to lysis” was inherited in a autosomal recessive manner. T h e locus (EC-I) has been mapped on chromosome 6 within the MHC between the complement cluster and HLA-B, a location similar to the mapping of Hh-I in mice. A large number of genes are localized in that region, among which are Bassociated transcripts (BAT) 1 to 9, TNF-(r and -p,and two heat shock protein genes (Sargent et al., 1989; Spies et al., 1989a,b). One of these latter genes, HSX-70, is-unlike other known heat shock proteinsexpressed at a relatively high basal level (Wu et al., 1987; Simon et al., 1988) and could be a candidate for NKTS. A peptide derived from this protein bound to the groove could give the signal “no kill.” In this model, cells homozygously negative for the EC-I locus will be killed due

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to absence of NKTS. Furthermore, NKTS peptides may not bind to certain HLA Class I alleles explaining why in certain cells HLA Class I does not protect against NK lysis (Dennert et al., 1988; Leiden et al., 1989; Stam et al., 1989; Pena et al., 1990). In this view, not only will defects in peptide processing render cells sensitive to NK cells, but also situations where the NKTS peptide is driven out of the groove due to competition, for instance, with viral or tumor-associated peptides.

VI. Concluding Remarks

In a vast number of animal and human tumors MHC Class I expression is abrogated. Often the downmodulation is locus-specific or allele-specific, suggesting that subtle regulatory mechanisms are involved, most likely at the transcriptional level. It has been shown that downregulation can be effectuated by oncogene activation, in particular by the c-myc and N-myc oncogenes. Loss of MHC Class I alters the sensitivity of tumor cells to T cells and NK cells. Whatever the precise mechanism of NK recognition and kill might be, in all models, downregulation of MHC Class I expression by the myc oncogenes alter the MHC make-up of the cell surface and render the cells sensitive to NK cells. In this way, activation of myc would lead to N K lysis via downmodulation of MHC Class I. Since all melanoma cell lines with high c-my expression have downmodulated HLA-B alleles and expression of these alleles can also be switched off by transfection with c-myc (Versteeg et al., 1988, 1989a), it is concluded that, the low HLA Class I expressionleading to NK sensitivity-is an intrinsic property of tumor cells with activated c-myc and not the result of immune selection. In this way, the coupling between high c-myc expression and low HLA-B expression may serve as a potential mode of immune surveillance by N K cells: cells with activated c-myc will be killed by NK cells through their low HLA-B expression (Fig. 4A). At this point it should be stressed, however, that the myclHLA relationship is not necessarily meant to act as an immune surveillance system, but might be coincidental with a change in some regulatory circuit in the cell that is not understood at present. Whatever the actual rationale for the coupling is, the result is modulation of immune sensitivity of the c-myc-activated tumor cells. Considering a role of this mechanism in immune surveillance against cancer cells, we cannot conclude whether the first line of defense is an attack by T cells o r by N K cells. T h e accomplishment of an appropriate T-cell response has a time lag, and therefore we speculate that in the case of c-myc-activated tumors an attack by NK cells could be a first line of defense. Since the tumor nevertheless has developed in the patient, one should assume that it has

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A c-rnyc activation early event

-

___*

activation o f c-myc

activation

normal cel l

tumor c e l l killed by NK cells, escape from T cells

escape from NK and T c e ll surveillance

B c-myc activation late event

___,

oncogene activation

normal cel l

cl : M H C Class I

activation o f c-myc

tumor c e l l killed by T cells

:

NKTS

k i l l by NK cells, escape from T cells

:

tumor peptide

escape from NK and T c e ll surveillance

L ,

:

accessory molecule

FIG.4. Hypothetical model for the effect of myc activation on the interaction of tumor cells with the immune system. T w o scenarios are conceivable. First, c-myc activation is an early event in tumorigenesis (model A). In this case c-myc activation is involved in transformation of the normal cell and MHC Class I expression (represented by notched circles) is downmodulated. This leads to an NK-sensitive phenotype due to lack of NKTS (solid wedge bound to MHC Class I, see text for explanation). In a later stage of tumor progression, this cell may escape from NK cells by activation of another oncogene (X) and/or the modulation of accessory molecules, which are essential for interaction of the tumor cell with immune effector cells (represented by the open hook linked to the cell surface). In the second scenario, the tumor cell has developed by an oncogene activation not affecting MHC Class I expression, and c-myc activation is a late event involved in tumor progression (model 3).In this case, presentation of a tumor-specific peptide (open wedge) is blocked by downmodulation of MHC Class I by c-myc. The resulting cell escapes from T cells but acquires sensitivity to NK cells. Eventually, this cell may escape as in model A.

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escaped from the N K defense by an as yet unknown mechanism. This may include modulation of factors other than HLA Class I, such as adhesion molecules or other accessory molecules involved in NK cell lysis (Fig. 4A). An alternative mechanism might be that in the case of these c-myc-activated tumors, c-myc activation has been a late step in tumor progression, resulting in an escape from T-cell surveillance (Fig. 4B). Such a mechanism might, for instance, occur during metastasis. In that case the downmodulation of HLA Class I antigens by c-myc still might act as a second-line surveillance, whereby c-myc-activated tumor cells are trapped by NK cells, thus preventing the process of metastasis (Fig. 4B). In both models, the link between c-myc activation and regulation of MHC Class I expression plays an important role in determining the sensitivity of the tumor cells to the immune system of the host. However, it should be kept in mind that the major function of c-myc lies in the regulation of cell growth and differentiation in normal cells. Although the precise biological function of the coupling between c-myc expression and MHC Class I expression in normal cells is not known, this phenomenon may in principle provide a mode of immune surveillance against tumor cells with activated myc genes. ACKNOWLEDGMENTS We thank Wilma Steegenga and Renske Steenbergen for assistance in writing this review and W. Martin Kast and Ingeborg Meijer for stimulating discussions and critical reading of the manuscript. We also acknowledge authors who made available unpublished data as personal communications for this review. This work was supported in part by a grant from the Dutch Cancer Society (KWF), Project Grant IKW 89-11.

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IMMUNOSUPPRESSIVE FACTORS IN HUMAN CANCER Dov Sulitzeanu The Lautenberg Center for General and Tumor Immunology, The Hebrew University, Hadassah Medical School, Jerusalem

I. Introduction 11. Immunosuppression in Cancer-Recent

Evidence 111. Immunosuppressive Factors Produced by Tumor Cells/Cell Lines IV. Well-Characterized Immunosuppressive Molecules A. Transforming Growth Factor p B. Lymphocyte Blastogenesis Inhibitory Factor C. P15E D. Suppressive E-Receptor V. Partially Characterized Immunosuppressive Factors VI. Immunosuppressive Factors in Sera and Effusions VII. Other Immunosuppressive Factors A. Colony-Stimulating Factors B. Acute-Phase Proteins C. Miscellaneous Molecules VIII. Concluding Remarks: Immunosuppressor or Growth-Regulatory Cytokines? References

I. Introduction Patients with advanced cancer are frequently found to exhibit impaired immune responses, as demonstrated by a variety of in vitro reactions (Stutman, 1975; Kamo and Friedman, 1977; Cianciolo and Snyderman, 1983; Aune, 1987; Nelson and Nelson, 1987; Schulof et al., 1987; Cianciolo, 1988; Brunson and Goldfarb, 1989). Early research on immunosuppression in cancer was focused mainly on defective responses at the cellular level: depressed macrophage and polymorphonuclear cell functions (migration, phagocytosis, bactericidal activity); depressed Ig production by B cells; reduced natural killer (NK) cytotoxicity; and, most often, the inability of the patients’ lymphocytes to respond to mitogenic stimuli (lectins, antigens, or alloantigens). More recently, attention has shifted to defects in cytokine release [particularly interleukin-2 (IL2)] and cytokine receptors. The exact mechanism of the immunosuppression is still uncertain, but a multiplicity of factors have been postulated to contribute to its occurrence: suppressor T cells and

247 ADVANCES IN CANCER RESEARCH, VOL. 60

Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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suppressor macrophages, immune complexes, acute phase proteins, tumor-derived suppressor molecules, suppressor substances in sera and effusion fluids of patients, and chemotherapy and irradiation. In this article, I propose to review succinctly recent studies (published mainly during the last 5 years) on immunosuppressive molecules (mostly of human origin) elaborated by tumor cells or found in sera and effusions. It is true that the active molecules have only rarely been purified to homogeneity or identified with certainty. However, progress is being made and we believe that an assessment of the present state of the art may be instructive and also useful in charting further directions of research. II. Immunosuppression in Cancer-Recent

Evidence As mentioned above, a great deal of the early research on immunosuppression in cancer has dealt with the poor proliferative response obtained after treatment of lymphocytes with mitogenic stimuli. The advent of the lymphokine activated killer (LAK) cell era (Rosenberg, 1988) presented immunologists with a new target and prompted a flurry of activity aimed at showing that peripheral blood lymphocytes (PBL) of patients with advanced cancer or of tumor-bearing animals were unable to develop into fully effective LAK (Monson et al., 1987; Shu et al., 1987; Ting et al., 1987; Maccubbin et al., 1989; Favrot et al., 1990) or cytotoxic lymphocytes (CTL) (Shu et al., 1987; Maccubbin et al., 1989). Lymphocytes isolated from tumors [tumor-infiltrating lymphocytes (TIL)] were also found to proliferate poorly and to acquire only limited cytotoxicity (Whiteside et nl., 1988). Interest now appears to have switched to the impaired production of cytokines or of their receptors by PBL of cancer patients (Herman et al., 1985; Monson et al., 1986; Elliot et al., 1987; Mantovani et ul., 1987; Hakim, 1988). However, reports of normal I L 2 production by PBL of patients with malignant disease (Hargett et al., 1985; Wanebo et al., 1986; Eskinasi et al., 1989) and of normal IL-1 production by cells of tumor-bearing mice (Holan and Lipoldova, 1990) may indicate that such impairment does not always account for the observed immunosuppression.

I l l . Immunosuppressive Factors Produced by Tumor Cells/Cell Lines

Culture supernatants of tumor cells of all types and of all species have been found by numerous investigators to contain strongly immunosuppressive factors, as evidenced by their capacity to inhibit a variety of immune reactions (Spector and Friedman, 1983; Aune, 1987; Nelson

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249

and Nelson, 1987; Cianciolo, 1988; Brunson and Goldfarb, 1989): delayed-type hypersensitivity; macrophage accumulation at sites of inflammation; macrophage chemotaxis, phagocytosis, and cytotoxicity; skin graft rejection (Gresser et al., 1975); lymphocyte proliferation in response to mitogenic stimuli; antibody synthesis; generation of LAK, TIL, and CTL cells; and lymphokine production (see details and references in Table I). A multitude of substances derived from human or animal tumors, capable of suppressing immunocyte functions, have been described in the literature. Unfortunately, in most cases, the characterization of the active molecules has been only partial at best; therefore it is not possible to determine to what extent these publications might be overlapping. Only a few of the immunosuppressive factors have been studied in sufficient detail and have been characterized at the molecular level; these will be reviewed first. Other factors, described sporadically in recent years, will then be listed. For references to older work, dating in fact to the beginning of tumor immunology, the reader is referred to the earlier reviews cited at the beginning of this section.

IV. Well-Characterized Immunosuppressive Molecules A. TRANSFORMING GROWTHFACTORp Transforming Growth Factor p (TGF-P) was identified originally by its ability to impart a transformed phenotype to normal fibroblasts. Several closely related molecules constituting the TGF-P family were discovered subsequently (three in mammals). TGF-P is produced by most cells (Wakefield et al., 1987) and has an extraordinarily wide range of biological activities (for recent reviews, see Bascom et al., 1989; Sporn and Roberts, 1989; Barnard et al., 1990; Roberts et al., 1990; Moses et al., 1991). Of particular relevance to this review are its strong inhibitory capacity for epithelial cells of both normal and malignant origin (Barnard et al., 1990) and its potent immunosuppressive activity (Palladino et al., 1990; Lucas et al., 1991). TGF-P inhibits T- and B-cell proliferation (Kehrl et al., 1986a,b, 1989; Wahl et al., 1988a) and expression of B-cell activation markers (Cross and Cambier, 1990); it inhibits LAK and CTL generation (Ranges et al., 1987; Espevik et al., 1988; Grimm et al., 1988; Kasid et al., 1988; Mule et al., 1988; Fontana et al., 1989; Jin et al., 1989; Geller et al., 1991; Smyth et al., 1991; Tada et al., 1991), NK cytolytic activity (Rook et al., 1986; Lucas et al., 1991), and macrophage oxygen metabolism (Tsunawaki et al., 1988). It downregulates Human Leukocyte Antigen (HLA) DR (Czarniecki et al., 1988; Zuber et al., 1988) and

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TABLE I IMMUNOSUPPRESSIVE FACTORSPRODUCED BY TUMOR CELLS/CELL LINES

Tumor Chlorectat cancer; other tumor extracts Various tumor cell supernatants r-cell leukemia

Factor size (kD,) 4.5

ND

88

>SO0

Characteristics and biological activity Acid extract inhibits response to mitogen, alloantigen; PI ( 3 . 0 ; sensitive to trypsin Inhibit mitogenic response of PBL

Hoagland et al. (1986)

Inhibits proliferation response of normal T cells to mitogens, alloantigens; inhibits proliferation of malignant, hemopoietic cells, normal myelomonocytic progenitor cells Inhibits response to Con A and to IL-2; suppress IL-2 production; inhibits response to B-cell mitogen; stable to heat, trypsin; not stable to acid PI 7.9; inhibits response to mitogen; IL-2 production; sensitive to heat at 56°C. pH extremes Inhibits response to mitogen, antigen, alloantigen Inhibits response to mitogens, DTH; delays rejection of syngeneic tumor Inhibits response to mitogen, IL-2; sensitive to heating at 56°C

Santoli et al. (1986)

T-cell leukemia

50,70

Colon cancer

56

Promyelocytic leukemia (;olon carcinoma

ND

H ydatidiform mole

35-50

T-cell leukemia

80,11.5

Suppress T-cell proliferation; highly purified preparations with PIS 3.5 and 7.4, respectively

T-cell leukemia

66

B CLL

<5

Melanoma

225

Stable at 56°C; cytostatic; inhibits response to PHA Inhibits response to PHA, IL-2 production; susceptible to neuraminidase Heat stable (56°C); inhibits LAK cell generation; produced also by gastrointestinal cancer cell line

ND

Reference

Miescher et al. ( 1986)

Shirawaka et al. (1986); Tanaka et a/. (1987) Ebert et 01 ( 1 987) Paietta et al. (1987) Pommier et al. (1987) Bennett et al. (1988); Cowan et al. ( 1989) Montaldo et al. (1988); Ponzoni et al. ( 1988) Abolhassani el al. (1989) Burton P t al. ( 1989) Guillou et al. (1989a,b)

I

(continued)

IMMUNOSUPPRESSIVE FACTORS IN CANCER

25 1

TABLE I (Continued)

Tumor

Factor size (kD,)

Choriocarcinoma

ND

Renal cancer

ND

T-cell line (Jurkat) Lung cancer

ND >150

Characteristics and biological activity

Reference

Inhibits response of T cells to phorbol esther, calcium ionophore, IL-2; also response to lectins, alloantigens; inhibits generation of alloreactive CTL Inhibits response to mitogen, IL-2 production; nondialyzable; sensitive to 56°C Inhibits response to mitogen, alloantigen; inhibits Ig production Inhibits response to PHA, IL-2 production; inhibits IL-2-dependent proliferation of activated lymphocytes; sensitive to trypsin, 60°C

Matsuzaki et al. (1989a,b)

Muraki et al. (1989) Telerman et al. (1989) Wang et al. ( 1989)

Note. ND: no data.

IL2R (Kehrl et al., 1986a), and it delays rejection of heart allografts (F’alladino et al., 1990). TGF-P was identified as a tumor-associated immunosuppressive molecule following a series of studies of immunosuppression in glioblastoma. It had been known for some time that patients with glioblastoma responded poorly in T-cell-mediated immune reactions (see Siepl et al., 1988, for brief summary of early work). Subsequent investigations prompted by these observations led to the demonstration that sera of glioblastoma patients contain an immunosuppressive factor (Brooks et al., 1972). A similar factor, which inhibited IL2-induced proliferation and CTL generation, was found in glioblastoma cell culture supernatants (Fontana et al., 1984) as well as in freshly explanted malignant glioma cells (Roszman et al., 1987). The factor was identified as TGF-P2 (Wrann et al., 1987; Siepl et al., 1988; Bodmer et al., 1989). Further work showed that TGF-P mRNA was found in all tumors tested (Derynk et al., 1987) and that the protein was made by many tumor cells lines: rhabdomyosarcoma (Romeo and Mizel, 1989), colorectal carcinoma (Coffey et al., 1986), breast carcinoma (Knabbe et al., 1987; Arteaga et al., 1990), endometrial carcinoma (Boyd and Kaufman, 1990), prostatic adenocarcinoma (Ikeda et al., 1987), and thyroid carcinoma (Jasani et al., 1990). TGF-P was also demonstrated in ascitic fluids of patients with ovarian

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(Hirte and Clark 1991; Wilson et al., 1991) and breast cancer (Hatzubai and Sulitzeanu, 1992), and in ascitic fluids of mice bearing plasmacytoma (Berg and Lynch, 1991) or hepatoma (Tada et al., 1991). TGF-P is probably responsible for many of the immunosuppressive activities attributed to “suppressor” lymphocytes and macrophages. The role of TGF-P as a mediator of suppressor cell activity is nicely demonstrated by the work of Wahl et al. (1988b), who showed that the immunosuppression induced in rats by injection of GpA streptococcal cell walls was due to TGF-P released by macrophages. B. LYMPHOCYTE BLASTOGENESIS INHIBITORY FACTOR Lymphocyte blastogenesis inhibiting factor (LBIF), a newly identified cytokine produced by the U937 macrophage cell line (Fujiwara and Ellner, 1986; Fujiwara et al., 1987), was purified to homogeneity by Sugimura et al. (1988, 1989). It is a 45-kDa polypeptide chain, with a PI of approximately 4.5, possessing a variety of immunosuppressive activities (Sugimura et al., 1990); it inhibits I L 1 , IL-2, antigen- and lectininduced proliferation, and expression of the IL-2 p75 receptor; it arrests lectin-stimulated T cells at early G1 phase; and it acts on both human and murine lymphocytes. Furthermore, it is cytostatic or cytotoxic for numerous tumor cell lines of various lineages, at very low concentrations-of the order of 10-40 ng/ml. In fact, its growth-inhibitory activity was manifested against a wider range of targets than that of other cytokines tested (TGF-P, I L 1-p, TNF-a, IFN--y, IFN-a). Cytokine LBIF appears to share with TGF-P the ability to act both on lymphocytes and on epithelial cells. These cytokines should therefore be considered both immunosuppressors and growth inhibitors. C. p15E It is well known that infection with retroviruses may cause a marked impairment of immune responsiveness (Snyderman and Cianciolo, 1984). Mathes et al. (1978, 1979) were the first to publish evidence implicating p15E, a transmembrane retroviral envelope protein, as a major mediator of retroviral immunosuppression. Subsequent studies revealed that retroviral structural components and the p15E protein possess a remarkable range of immunosuppressive activities (Cianciolo and Snyderman, 1983; Cianciolo, 1988; Brunson and Goldfarb, 1989): they inhibited lymphocyte blastogenesis, tumor immunity in uiuo, macrophage accumulation in vivo, and monocyte chemotactic response and erythroid colony formation in vitro. Substances crossreacting with p 15E

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253

were demonstrated in murine and human cell lines, in plasma of patients with hematologic disorders (Jacquemin et d., 1984), and also in human malignant effusions. Addition of effusion fluid inhibited the monocyte response to chemoattractants and this inhibition could be reversed by anti-p15E antibodies. Cianciolo et al. (1985) prepared a synthetic, 17 amino acid peptide, homologous to a highly conserved region of the p15E proteins of murine and feline retroviruses, of HTLV I, 11, and 111 and also of a putative protein encoded by an endogenous C-type human retroviral DNA. Conjugates of this peptide inhibited the proliferation of an IL2dependent murine CTL line and of alloantigen-stimulated murine and human lymphocytes. It also inhibited the cytocidal activity of recombinant I L l - a and -p against A 375 melanoma cells (Kleinerman et al., 1987) and the cytotoxicity of human N K cells (Harris et al., 1987). It suppressed the respiratory burst of human monocytes (Harrell et al., 1986) and their ability to produce interferon-y in response to stimulation by staphylococcal enterotoxin A (Ogasawara et al., 1988). Antibodies to CKS- 17 neutralized the capacity of tumor cell culture supernatants to inhibit the production of I L 2 by EL-4 cells stimulated by mitogen (Nelson and Nelson, 1990). Conjugates of CKS-I 7 depressed delayedtype hypersensitivity (DTH) reactions in mice (Nelson et al., 1989) and this effect could be prevented by actively immunizing the mice with conjugated CKS- 17 or by injecting the animals with immunoglobulin of immunized mice. Such immunization could protect not only against the effects of CKS-17 but also against similar effects of tumor products (Nelson et al., 1985, 1989). The CKS-17-like factor produced by tumor cells is reported to be a molecule of -18 kDa (Nelson and Nelson, 1990), but, regrettably this molecule has not been characterized any further. Its relation to the 74kDa protein (Jacquemin et al., 1984) and to the other crossreacting molecule found in dividing cells (Snyderman and Cianciolo, 1984) is not known. Its origin as an endogenous human retrovirus product (Lindvall and Sjogren, 1991) cannot be considered proven, although this possibility must be taken into account, in view of the reported presence of such viruses in human cells (Larsson et al., 1989; Wilkinson et al., 1990). D. SUPPRESSIVE E-RECEPTOR T h e suppressive E-receptor (SER) was isolated by Oh and co-workers (1987a) from malignant effusions derived from patients with several types of cancer (ovarian, lung, head and neck). This immunosuppressive molecule inhibited E rosette formation (hence the name) as well as sever-

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a1 cellular immune responses, both in nitro and in vivo (Oh et al., 1987b, 1988, 1990). These included T-cell proliferation in response to mitogen and alloantigen, Ig synthesis induced in zdro by PWM + PMA, antibody response to T-dependent antigen in nivo, N K function, and polyrnorphonuclear phagocytosis. T h e molecule was identified as a polymeric form of haptoglobin, related immunochemically to neonatal haploglobin. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, SER yielded two polypeptides (38-42 kDa and 17-19 kDa), with N-terminal amino acid sequences identical to those of the f3 and a-2 subunits of adult haptoglobin (Oh et al., 1987a). T h e SER is secreted by macrophages, which, however, do not synthesize haptoglobin. The authors propose that SER may be an oxidized form of plasma haptoglobin, generated by macrophages (Oh et al., 1989). Thus, SER is not a tumor-derived, but rather a tumor-associated, molecule. V. Partially Characterized Immunosuppressive Factors

A list of publications describing recently studied immunosuppressive factors is given in Table I. Despite the meager data available, it seems that a large number of molecules with immunosuppressive activity may be produced by tumor cells, judging from the considerable variation in physicochemical properties reported by the different investigators. Unfortunately, little information is available on the production of analogous substances by nonmalignant cells (Brunson and Goldfarb, 1989). Several such substances, tested mainly for growth-inhibitory activity, are listed in Table 11.

VI. Immunosuppressive Factors in Sera and Effusions

Immunosuppressive factors can be found in normal sea, but their level is increased in cancer sera (Wile, 1989).These factors may contribute to the defective cellular responses of' patients. Thus, cells of patients with lung cancer, which exhibited low reactivity to rnitogens, responded normally when tested in the presence of sera of healthy donors (Hadjipetrou-Kourounakis et af., 1985). Numerous immunosuppressive factors (generally poorly characterized) have been described in sera and effusions of patients (see Table 111 for a list of recent publications). Of these, only SER and the pl5E-related molecule have been studied in greater detail (Section IV).

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IMMUNOSUPPRESSIVE FACTORS I N CANCER

TABLE I1 INHIBITORY FACTORSPRODUCED BY NORMAL TISSUES Human trophoblast cells Human lung cells

Suppression of NK activity

Saji et al. (1987)

Suppression of lympyhocyte blastogenesis, NK activity

Rat liver

Growth inhibitor for several cell types

Human, rat liver cells Normal mouse lung Human fetal liver cells

Inhibition of liver cell proliferation

Baughman and Strohofer (1989) Chapekar et al. (1989) Chen et al. ( 1989) Ikuta et al. ( 1989) Wu et al. (1989)

Growth inhibitor of mouse monocytic leukemia cells Inhibitor of HL-60 cell proliferation

TGF-P (Section IV,A) has been identified in ascitic fluids and further work is likely to show it to be a major contributor to the immunosuppressive activity of effusions. In the course of a systematic fractionation of effusions from patients with breast cancers, aimed at identifying immunosuppressive and growth-inhibitory factors contained therein, several of the fractions were found to contain TGF-P-like molecules, as determined by inhibition tests with anti-TGF-P serum (Hatzubai and Sulitzeanu, 1992). T h e multitude of seemingly different factors described in the literature seem to indicate that effusion fluids (and presumably, also patients’ sera) may be teeming with a variety of cytokines, with immunosuppressive and probably other biological activities. The results of our fractionation studies, which show immunosuppressive and growth-inhibitory activity in many fractions, differing greatly in physicochemical properties, suggest that this assumption is probably correct. VII. Other Immunosuppressive Factors A. COLONY-STIMULATING FACTORS

Tumor cell lines, particularly of mouse, but also of human origin, have often been shown to secrete appreciable amounts of colony-stimulating factors (CSF) (Hardy and Balducci, 1985; see also Ralph et al., 1986: Tweardy et al., 1987’;Evans et al., 1989; Kacinski et al., 1989). This apparently also occurs in vivo, since increased CSF-1 levels can be demonstrated in patients’ sera (Hardy and Balducci, 1985; Kacinsky et al.,

256

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TABLE 111 I M M U N O S U P P R E S S I V E SUBSTANCES IN SERA A N D

ASCITES

Source

Type of cancer

Serum

Stomach

Inhibition of PBL response

Stomach

Inhibition of PHA cytotoxicity

Ly mphocytic leu kemia (rats) Leu kosis, bovine B-CLL

Inhibition of PHA, Con A response

Melanoma

Various Lung, esophageal cancer H&N cancer Ascites

Various

Gastrointestinal

P-8 I5 niastocytoma (mouse) Walker 256 carcinoma (rat) Various

Characteristics and biological activity

Reference Kanayarna et al. (1985) Sugiyama et al. (1987) Strumberg et ul. (1988)

Inhibition of mitogen stimulation, phagocytosis, other immune fuctions Reduction of PHA-induced IL-2 production Inhibition of LAK activation and of PHAinduced cytotoxicity; factor production dependent o n nionocytes Inhibition of lymphocyte blastogenesissmall molecules Suppressor of macrophage phagocytosis

Takamatsu et al. (1988) Burton et al. (1989) Itoh et al. (1989)

Inhibitor of LAK generation

Bugis et al. ( 1990) Medoff et al. ( I 986)

.iO-kDa molecule; pl 3.4; resistant to proteolysis; suppressed response to PHA Inhibition of PHA response and of DTH a 1-acid glycoprotein Molecule smaller than 10 kDa; lipid-like; resistant to proteolysis; inhibited response to Con A Eight hands of suppresssor substances, causing suppression of PHA induced splenocyte proliferation, obtained by preparative PAGE Inhibitor of LAK cell induction

Wile (1989) Zhou et al. (1989)

Fujii et a/. (1987) Cornelius and Normann (1988) Cohen et al. (198s)

Pelton et al. (1991)

1989). Hemopoietic alterations have indeed been seen in both human and experimental cancer, including increased production of granulocytes and macrophages, and decreased B cells, thymocytes, and NK cells (Fu et al., 1991). Although the colony-stimulating factors are not immunosuppressive by themselves, they stimulate proliferation and differentiation of hemopoietic cells and induce the appearance of mac-

IMMUNOSUPPRESSIVE FACTORS I N CANCER

257

rophages possessing immunosuppressive activity (Hardy and Balducci, 1985; Tsuchiya et al., 1988; Young et al., 1989; Fu et al., 1990, 1991). Daily injections of granulocyte macrophage-CSF into mice caused immunologic alterations similar to those noted in tumor-bearing animals. The macrophage-associated suppression was attributed to cell-to-cell contact as well as to release of PGE,, since the effect could be partially reversed by indomethacin (Fu et al., 1991). B. ACUTE-PHASE PROTEINS

Acute-phase proteins (haptoglobin, al-acid glycoprotein, ceruloplasmin, C-reactive protein, and others) are produced by the liver in response to inflammation and are found in increased amounts in sera of patients with cancer (briefly reviewed by Samak et al., 1982). Some of them were found to depress lymphocyte proliferation. Synthesisof acute phase proteins is mediated by a number of cytokines, which may be produced directly by the tumor cells (e.g., hepatocyte-stimulatingfactor 111, Baumann and Wong, 1989) or indirectly, by tumor products acting on macrophages (Evans et al., 1991). C. MISCELLANEOUS MOLECULES

A number of additional molecules have been studied as potentially immunosuppressive: gangliosides (Krishnaraj et al., 1982; Ladisch et al., 1983; Robb, 1986), prostaglandins (Aune, 1987; also see Droller et al., 1978; Chouaib et al., 1985; Kicza and Szkaradkiewicz, 1986; Leung, 1989), 1-methyl-adenosine(Takano et al., 1986),and uric acid and uracyl (Sami et al., 1986). However, interest in such molecules has been rather marginal and therefore they are mentioned only briefly here. VIII. Concluding Remarks: lmmunosuppressor or Growth-Regulatory Cytokines?

There can be no doubt that tumor cells release into the culture medium (and most likely also in vim) a fairly large number of cytokines with immunosuppressive activity. Unfortunately, in the vast majority of cases, the identification of these cytokines (some of which might be as yet unknown) has lagged far behind the description of their immunosuppressive properties. Given the technical difficulties involved in isolating the tiny amounts likely to be present in the culture supernatants or body fluids and the apparent multitude of factors, this is not unexpected. However, this is a challenge which, one hopes, will be met, since the

258

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presence of these cytokines in body fluids cannot fail to affect the hosttumor relationships. A tumor-derived cytokine likely to draw increasing attention as a cancer-associated immunosuppressive molecule is TGF-P. Many tumors have already been shown to secrete this highly active immunosuppressive molecule in vitro and although in uivo secretion has been little studied so far, one can expect it to be as widespread as in vitro secretion. TGF-P is a major suppressor factor in ovarian cancer ascites (Hirte and Clark, 1991), where it is quite likely to be derived, at least in part, from malignant cells. TGF-f3 may well be a major contributor to the impairment of immune functions (decreased DTH and reduced proliferation on phytohemagglutinin (PHA) or alloantigen stimulation-Wiebke et al., 1988; Hank et al., 1990; Favrot et al., 1990) seen to develop in patients undergoing IL-2 therapy. Interleukin-2 is a potent inducer of LAK cells and these have been shown to release appreciable amounts of TGF-P (Kasid et al., 1988; Larisch-Bloch et al., 1990), which most probably inhibits further LAK cell development as well as other immune reactivities. Interestingly, unlike most other cells, which secrete latent TGF-P, the LAK cells appear to secrete a biologically active form of the molecule, which nevertheless is attached to a large carrier protein (LarischBloch et af., 1990). Activation could occur, however, in the culture supernatant, through the action of proteolytic enzymes (Kehrl et al., 1989). Many immunologists appear to incriminate tumor-derived (or tumorinduced) immunosuppression as a major contributor to the failure of immunotherapy to affect the course of the disease (Mule et al., 1988; Guillou et al., 1989,; Barba et a/., 1989). Immunosuppression has indeed been proposed as the reason why LAK cell treatment is so effective in eliminating clinical metastases in mice, yet so disappointingly ineffective in man. T h e difference, according to Guillou et at. (1989b), is due to the source of the LAK cells, which are prepared from healthy syngeneic cells in animal experiments, but are prepared from autologous (and presumably suppressed) lymphocytes, for use in patients. Yet, Hirte and Clark (1991) suggest that LAK cells can be induced by IL-2 in patients with cancer-associated effusions, despite the presence of inhibitors in the effusions. A dif€icult, though highly important, question is the extent to which immunosuppression might be responsible for the escape of the developing tumor from immune control and, ultimately, for the growth and spread of the tumor. Experimental evidence implicating tumor-induced immunosuppression in the development of the malignant process is not lacking. Thus, Mullen et al. (1985) showed that tumor-induced immu-

IMMUNOSUPPRESSIVE FACTORS IN CANCER

259

nosuppression may permit growth of a secondary tumor, whereas TorreAmione et al. (1990) demonstrated that transfection with TGF-P cDNA enabled an otherwise immunogenic tumor to grow progressively in mice. Yet, it is unlikely that these elegant experimental models (particularly the highly immunogenic UV-induced tumor employed by TorreAmione et al.) resemble the vast majority of human cancers. There is still a great deal of uncertainty regarding the actual role immunity plays in the control of human malignancy (Sulitzeanu, 1985; Brunson and Goldfarb, 1989), and until this point is settled, the role of immunosuppression will also remain in doubt. Individual opinions will vary and they are more likely to rest on beliefs than on hard scientific data. Nevertheless, the immunosuppression that accompanies tumor development is bound to affect the patient at least indirectly, by the predisposition to infections, which complicate the treatment and can be a major cause of mortality. A well-studied and illuminating example of the likely relationship among malignancy, immunosuppression, and infection can be found in multiple myeloma. Patients with multiple myeloma are aWicted with a severe Bcell deficiency, resulting in an inability to make primary antibody responses, which, in turn, is responsible for the morbidity and mortality due to recurrent bacterial infections. A similar deficiency is exhibited by mice with the related malignancy plasmacytoma (Jacobson and ZollaF’azner, 1986). A very interesting work (Berg and Lynch, 1991) has shown recently that B cells of mice with plasmacytoma differ from normal B cells in the expression of several surface markers (e.g., decreased surface IgM, transferrin receptor, and CD23 receptor). This difference was traced to TGF-P, which is secreted in very large amounts by the plasmacytoma cells. It now remains to be seen whether an analogous situation exists in the human disease, namely excess TGF-P secretion by the multiple myeloma cells, resulting in immunosuppression and consequent susceptibility to infection. It must be stressed that, although investigators appear to assume that the immunosuppressive factors in sera and effusions are actually tumor products, this assumption seems unwarranted. Thus, TGF-j3 is produced by all cells, whereas SER and the factor described by Sheid and Boyce (1984) are macrophage products. The 74-kDa molecule that crossreacts with p15E is also present, albeit in smaller amounts, in normal sera (Jacquemin et al., 1984). An inhibitor of LAK cell generation demonstrated in sera of patients with head and neck cancer was also found in control (normal) sera (Bugis etal., 1990).There is no evidence, in fact, that any of the factors studied thus far are exclusively tumor products.

Isolation p r o c e d u r e A m m o n i u m sulfate precipitation (% SdtLl~dtlOll)

DEAE-sephacel Chromatography (mM T r i s bufier)

1

25-50

100-200

2

29-50

250-500

5

250-500

1.5 5 1.5

Fraction No.

3

50-75

Growth inhibition

Concentration

(mg/rnl) 5" 2.5 0.5

Immunosuppression

++

++ ++ +++ ++

++ +

A315

HT29

+++

++

+++ ++ +++ ++

++ ++

+ + ++ ++ +++ ++

Nule. Fractions were prepared by precipitation with ammonium sulfate, followed by ion-exchange chromatography on DEAE-sephacel. They were tested for immunosuppressive activity (ability to inhibit PHA-induced lymphocyte proliferation) and for growth inhibition (inhibition of A375 nielanoma and HTP9 colon carcinoma cells). The differences in the isolation procedure and the differences in biological activity suggest that the activity of the three fractions was probably due to different cytokines. Percentage immunosuppression/growth inhibition is indicated as follows: + + +, >60%; + +, 30-60%; +, 20-30%; -, less than 20%. a Growth-inhibitory activity of this fraction against A375 (but not against HTPL))cells was abolished by anti-TGF-P I serum, suggesting that the fraction contained at least t w o different inhibitors.

IMMUNOSUPPRESSIVE FACTORS IN CANCER

26 1

At a more theoretical level, one might ask whether tumors can be expected to produce substances that specifically downregulate the immune system? One might invoke, perhaps, selection pressures leading, in the course of a minievolutionary process, to the survival of cells capable of neutralizing the immune defenses, an argument that has, of course, a strong teleological flavor. It appears more plausible, on the other hand, to assume that the “immunosuppressive cytokines” produced by the tumor may be simply growth-regulatory substances, which, while inhibiting growth and differentiation of lymphocytes, may act similarly on many other cell types. One such cytokine is already well known-TGF-P, a classical growth inhibitor that is also a strong immunosuppressor molecule. The less well-known lymphocyte inhibitor, LBIF, was similarly found to possess wide-ranging growth-inhibitory activity. Work in progress in our laboratory (Hatzubai and Sulitzeanu, 1992) suggests that effusions from patients with breast cancer contain a rather large number of cytokines (as judged from the activity of fractions obtained by a variety of techniques) with both growth-inhibitory and immunosuppressive activity (Table IV). Not surprisingly, one of these is TGF-P. Finally, the release of growth-inhibitory substances by tumors may provide a ready explanation for what has been a rather enigmatic phenomenon thus far, namely the outbreak of metastases that sometimes follow the surgical removal of a primary tumor (Prehn, 1991). Removal of the primary tumor could simply free susceptible metastatic foci from the growth control exerted by growth-inhibiting cytokines derived from the primary tumor, thus allowing the metastases to grow. Examples of autocrine growth control abound in the literature. Thus, growth of chronic myeloid leukemia cells is inhibited by endogenous tumor necrosis factor (Duncombe et al., 1989), whereas growth of breast cancer cells is inhibited by the TGF-f3 they secrete (Knabbe et al., 1987; Arteaga et al., 1990). More complex growth-regulatory loops may originate in the colony-stimulating factors released by tumor cells, which induce the appearance of suppressor macrophages. Cytokines released by the latter have been demonstrated to be toxic for tumor cells, singly (Lachman et al., 1986; Lovett et al., 1986), or in various combinations (Morinaga et al., 1989; Maekawa et al., 1990).Thus, all that would be required to account for the outbreak of metastases would be a difference in susceptibility to negative growth control, between the primary and the metastatic cell. T h e possibility that metastatic foci might be controlled in this fashion seems worth investigating, since positive results might have therapeutic implications.

262

DOV SULITZEANU

ACKNOWLEDGMENTS I gratefully acknowledge the support of The Society of Research Associates of the Lautenberg Center, the Concern Foundation of Los Angeles, and the WakefernlShoprite Endowment for Basic Research in Cancer Biology and Immunology. Special thanks are due to Ms. Marcella Wachtel for assistance in the preparation of this manuscript.

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LYSOSOMES, LYSOSOMAL ENZYMES, AND CANCER Michael J. Boyer and Ian F. Tannock Departments of Medicine and Medical Biophysics, Ontario Cancer Institute and University of Toronto, Toronto, Canada M4X 1K9

I. Introduction 11. Methods for Studying Lysosomes 111. Lysosomal Enzyme Activity in Tumors

IV. Lysosomes, Tumor Invasion, and Metastasis V. Lysosomes and Tumor Microenvironment A. Lysosomes and Hypoxia B. Lysosomes and Extracellular pH VI. Lysosomes and Anticancer Therapy A. Lysosomes and Radiation B. Lysosornes and Hyperthermia C. Lysosomes and Drugs VII. Conclusion References

I. Introduction Lysosomes are membrane-enclosed cytoplasmic organelles which posses an acidic interior that contains many hydrolytic enzymes. Their major function is thought to be the degradation of macromolecules, which may be cellular or foreign in origin. Although some disagreement exists, lysosomes are probably formed by budding from the Golgi apparatus (Holtzman, 1989). Lysosomal hydrolases are synthesized and glycosylated in the rough endoplasmic reticulum. The oligosaccharides that are added to the protein moieties of lysosomal enzymes contain a high proportion of mannose 6-phosphate residues; these residues are essential for the sorting and uptake of newly synthesized enzymes by lysosomes (Dahms et al., 1989). Newly formed lysosomes containing hydrolytic enzymes that have not yet taken part in intracellular digestion are termed primary lysosomes. Damage to the cell by these highly effective degradative enzymes is prevented by their packaging within the lysosome. Most of the hydrolases have maximum activity in the acidic pH range (Holtzman, 1989), and the intralysosomal pH is maintained between 4.0 and 5.0, by the action of a proton translocating ATPase (Schneider, 1981). For macromolecular digestion to occur, substrates destined for degradation must be delivered to the lysosome. This takes place by fu269 ADVANCES IN CANCER RESEARCH, VOL. 60

Copyright 6 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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sion of the lysosome with an endosome (in the case of extracellular material) or an autophagic vacuole (for intracellular substrates). After fusion of a lysosome with a phagocytic body, the resulting structure, containing both enzymes and materials that are undergoing digestion, is termed a secondary lysosome. Lysosomes have long been postulated to play a role in the malignant process. Aspects of carcinogenesis (Allison, 1969), invasion and metastasis (Poole, 1973), and shrinkage of neoplasms in response to therapy (Gullino and Lanzerotti, 1972) have all been attributed to lysosomes or their products. In addition, lysosomes or their contents may represent a possible target for therapeutic manipulation; de Duve first proposed using lysosomes as the target of anticancer therapy shortly after their discovery (de Duve, 1969), although to date no therapy aimed specifically at lysosomes has been developed and used in cancer patients. For such therapy to be tumor specific, a difference between the lysosomes or lysosomal enzymes of normal and neoplastic tissue is required. Alternatively, differences in the microenvironment that might influence lysosomal activity must exist between tumors and normal tissue. This chapter will summarize available information about properties of lysosomes and their enzymes that may be relevant to malignancy. II. Methods for Studying Lysosornes

Lysosomes may be studied by several methods, including isolation by ultracentrifugation, assay of enzyme activity, histochemical techniques, and electron microscopy. Furthermore, measurements of the intralysosomal pH can be made within living cells. Traditionally centrifugation in a sucrose gradient has been used to prepare subcellular fractions and thus isolate organelles. However, since the density of lysosomes is similar to that of other organelles, such as mitochondria, separation of these organelles is difficult. Techniques have been developed that cause changes in lysosomal density and thus allow improved purification (Leighton et al., 1968).In uiuo loading of lysosomes with compounds such as the nonionic detergent Triton WR-1339, dextran-500, iron, and colloidal gold has been used for this purpose. Unfortunately, the introduction of foreign material into lysosomes may alter some lysosomal properties with elevation in the activities of some enzymes and changes in lipid and cholesterol content (Leighton et al., 1968; Dean, 1975). To avoid this problem, metrizamide gradients have been used to isolate unmodified lysosomes (Wattiaux et al., 1978). Methods for assaying the activity of almost all the known lysosomal enzymes (Barrett and Heath, 1977) exist. Activity can be measured both

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for enzymes confined within the lysosome (sedimentable activity) and for those that have been released into the cytoplasm (free or unsedimentable activity). Changes in the proportion of enzyme activity that is free have been used as a measure of lysosomal membrane integrity (Wattiaux and Wattiaux-de Coninck, 1984). Measurement of the activity of lysosomal enzymes in tumors is limited by contamination with stromal elements and host effector cells such as macrophages. These cells are rich in lysosomes so that differences observed between tumors and normal tissues may be a reflection of changes in the number of these host cells rather than a tumor-related phenomenon. Histochemical methods can overcome this problem and provide information on the distribution of enzymes within tissue. Histochemical techniques for several of the lysosomal enzymes exist, including acid phosphatase, N-acetyl-P-glucosaminidase, a-mannosidase, pgalactosidase, cathepsin B, and nonspecific esterases (Bitensky and Chayen, 1977; Walker, 1984; Weiss at al., 1990). Intralysosomal pH is most commonly measured by fluorescinated dextran (FD), which is endocytosed and accumulates in lysosomes (Ohkuma and Poole, 1978). Fluorescence intensity at 495 nm is dependent on the ionization status of a COOH/C00- group, and is thus pH dependent, whereas intensity at 450 nm is independent of pH. Using the ratio of fluorescent emission at excitation of 495 nm to that obtained by excitation at 450 nm, a measure of pH that is independent of the amount of FD in the lysosomes is obtained. Other fluorescent probes are taken u p into lysosomes and fluorescence microscopy following loading with lucifer yellow may be used to study lysosomal rupture (Miller et al., 1983).

111. Lysosomal Enzyme Activity in Tumors

T h e specific activity of several lysosomal enzymes in tumors is elevated compared to that in the normal tissue from which the tumors are derived (Fishman and Anlyan, 1947; Boyland et al., 1957; Conchie and Levvy, 1957; Watts and Goldberg, 1969; Poole et al., 1978; Recklies et al., 1980; Bernacki et al., 1985). Table I summarizes the results of studies that have investigated the activity of lysosomal enzymes in human tumors. A limitation of many of these investigations is the failure to identify whether tumor cells or host cells are the source of the enzymes. In the one study that addressed this problem, no correlation was detected between activity of cathepsins B or D and neutrophil or macrophage infiltration of the tumor, suggesting that the tumor cells were the source of the enzyme activity (Abecassis et al., 1984). In addition, levels of ex-

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TABLE I AC~IVITY OF LYSOSOMAL ENZYMES I N HUMAN TUMORS COMPARED TO THE NORMAL TISSUE OF ORIGIN

Enzyme

Tumor 'Y Pe

Acid phosphatase

Breast

a-Mannosidase

Breast Colon

Arylsulfatase

Bladder

P-Galactosidase

Breast Colon

P-Glucuronidase

Bladder Breast Breast Cervix Colon

Lung

Ovary

Renal Cathepsin B

Breast Breast Gastric Colon

Comment

Ref.

Increased activity compared to normal tissue Increased activity compared to normal tissue Increased activity compared to normal tissue Increased activity compared to normal bladder mucosa Increased activity compared to normal tissue Increased activity compared to normal tissue Increased activity compared to normal bladder mucosa Increased activity compared to normal tissue Increased activity compared to normal tissue Increased activity in tumor homogenates Reversal in ratio of A & B isoenzymes in tumors Increased activity in tumor homogenates; change in biochemical characteristics of B isoenzyme Increased activity in tumor hornogenates; highest activity in poorly differentiated tumors Change in ratio of A & B isoenzymes Increased extracellular excretion Increased activity in tumor homogenates Increased activity in tumor homogenates Increased activity in tumor homogenates; activity correlated with local spread and venous invasion

Bosmann and Hall (1 974) Bosmann and Hall (1974) Bosmann and Hall ( 1974) Boyland et al. (1957) Bosmann and Hall (1974) Bosmann and Hall ( 1 974) Boyland rt at. (1 957) Fishman and Anlyan (1 947) Bosmann and Hall (1974) Watts and Goldberg (1969) Brattain et al. ( 1 977) Narita et at. ( I 983)

Chaterjee rt al. ( 1982)

Okochi et al. (1979) Bernacki et al. (1985) Abecassis et (11. ( 1984) Vasishta et al. (1985) Durdey et al. ( 1985)

(continued)

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TABLE I (Continued)

Enzyme

Tumor ‘YPe

Colon

Cathepsin D

Breast Gastric Ovary

Cathepsin L

a

Colon

Comment

Ref.

Increased activity in tumor homogenates; inverse relationship with Dukes’ stage Increased activity in tumor homogenates Increased activity in tumor homogenates Increased activity in tumor homogenates Increased activity in tumor homogenates; inverse relationship with Dukes’ stage

Sheahan et al. (1989)

Abecassis et al. ( 1984)

Vasishta et al. ( 1985)

Scambia et al. (1991)

Sheahan et al. (1989)

P-N-Acetylglucosaminidase.

pression of mRNA for cathepsins B and L have been measured in tumor and normal tissues (Chauhan et al., 1991; Murnane et al., 1991). Increased levels of expression of cathepsin L mRNA have been detected in tumors of the testis, kidney, colon, and lung, when compared to normal tissues (Chauhan etal., 1991).Similar studies with cathepsin B mRNA in human colon carcinoma have demonstrated an increase in expression in samples of carcinoma when compared to normal colonic mucosa from the same patient (Murnane et al., 1991).These studies are also limited by a lack of information concerning the cellular origin of the mRNA (tumor versus stromal cells). Changes in enzyme activities have also been studied in experimental tumors in animals. In the rat, total activities of cathepsin D, acid ribonuclease, and acid deoxyribonuclease were found to be elevated in hepatomas compared to normal liver (Deckers-Passau et al., 1957), whereas in murine skin tumors, acid phosphatase, P-glucuronidase, and cathepsin D were increased compared to normal skin (Shamberger and Rudolph, 1967). Elevation in the serum level of lysosomal enzymes has been reported in both cancer patients and tumor-bearing animals. Rats with Reuber H35 hepatoma and mice with L1210 leukemia have elevated levels of several lysosomal giycosidases, including an eightfold increase in 6-Nacetylglucosaminidase (Bosmann et al., 1975). Elevation of serum and

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urine levels of P-N-acetylglucosaminidaseand P-glucuronidase has been reported in humans with hepatic, gastrointestinal, and gynecological malignancies (Boyland et al., 1955; Wollen and Turner, 1965; Lo and Kritchevsky, 1978; Chamut et al., 1980; Reglero et al., 1980; Nagdsue et al., 1982). It is not known whether the increased enzyme activity originates from the tumor itself or from normal tissues that are modified by the presence of tumor. Lysosomal enzyme activity has been estimated in different regions of tumors. Cells at the periphery of transplanted murine tumors have higher intrmellular “catheptic activity” than central cells (Sylven and Malmgren, 1957). By contrast, in interstitial fluid the activities of acid phosphatase, f3-glucuronidase,arylsulfatase, and cathepsins are highest at the center and fall toward the periphery of several transplanted murine tumors (Sylven, 1968). Enzyme activity may be higher in central regions because of cell death and necrosis (Weiss, 1978), whereas increased vascularity near the periphery of the tumors may allow the clearance of such enzymes from interstitial fluid (Sylven, 1968). A few investigators have attempted to determine the cellular origin of lysosomal enzymes in tumor homogenates or interstitial fluid. The cathepsin B activity in rabbit V2 carcinoma has been shown by immunocytochemical techniques to arise from fibroblasts and leukocytes at the invading edge of the tumor rather than from tumor cells (Graf et al., 1981; Baici et al., 1984). By contrast, quantitative analysis of the pattern of lysosomal enzymes in transplanted murine tumors (Sylven, 1968) showed a distinct distribution from that observed in experimental granulomata (Niemi and Sylvh, 1968), which comprised predominantly macrophages; this suggests that the lysosomal enzymes in tumor interstitial fluid are the product of tumor cells rather than of host macrophages. Ryan et al. (1985) have used centrifugal elutriation followed by trypan blue dye exclusion to isolate viable tumor cells from tumor cell suspensions. They found that over 90% of the cathepsin B activity in suspensions from eight rodent tumor lines of five histological types arose from viable tumor cells. These experiments, together with those describing the activity pattern of cathepsins from human tumors described above, suggest strongly that many types of tumor cell do have elevated levels of activity of lysosomal enzymes. The following explanations have been proposed to account for the increased activity of lysosomal enzymes in tumors (Poole, 1973; Allison, 1974). (a) Rapidly proliferating cells are known to have greater lysosomal enzyme activity than their normal counterparts (Levvy, 1956; Franklin, 1962; Adams, 1963; Watanabe and Fishman, 1964). (b) A high rate of pinocytosis has been demonstrated in some tumors (Easty et al.,

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1964; Ghose et al., 1962), and stimulation of pinocytosis may lead to elevations in lysosomal hydrolase levels (Cohn and Fedorko, 1969). (c) Elevation of intralysosomal pH has been observed following rar transformation of murine and human fibroblast lines (Jiang et al., 1990): differences in intralysosomal pH between tumors and normal tissues could account for differences in enzyme activity both intracellularly and in the interstitial fluid. (d) Since tumors contain dying and necrotic cells, changes in enzyme levels in the interstitial fluid might be due partly to leakage from these damaged cells. Experiments that assign relative weights to these mechanisms can and should be undertaken: lysosomal pH can be measured in tumors and normal tissues using fluorescent dyes. Also, lysosomal activity can be studied in large and small tumors of rodents, with and without necrosis, and among a series of tumors with different abilities to take up dyes by pinocytosis. IV. Lysosomes, Tumor Invasion, and Metastasis

In order for tumors to invade and metastasize, destruction of the basement membrane and other elements of the extracellular matrix must occur (Tryggvason et al., 1987).Several proteases play a role in this process, including cathepsins. Other proteolytic enzymes that may be required for invasion and metastasis include plasminogen activator, collagenases (especially type IV), and proteoglycanases (Tryggvason et d., 1987). The association between tumor invasion and lysosomal enzyme activity has been investigated extensively. The ability of cathepsins B, D, and L to degrade elements of the basement membrane has been studied in several experimental systems (Briozzo et al., 1988; Lah et al., 1989; Maciewicz et at., 1989; Guinec et al., 1990; Weiss et al., 1990). Although the pH optimum of most lysosomal enzymes lies in the acidic range, some enzymes, for example, cathepsins B or D, show appreciable activity at neutral pH (Woessner, 1967; Burleigh et al., 1974; Lah et al., 1989). These lysosomal enzymes may be active extracellularly under the microenvironmental conditions found in tumors and also in some normal tissues (Wike-Hooley et al., 1984). The relationship between lysosomal enzyme activity and invasiveness has been studied in ovarian cancer. Invasiveness has been demonstrated to be associated with increased levels of cathepsin B (Blackwood et al., 1965)and cytoplasmic P-glucuronidase (Watts and Goldberg, 1969).The activity of P-N-acetylglucosaminidasewas found to be elevated in tumors that were poorly differentiated, but not in those that were well differentiated (Chatterjee et al., 1982). This enzyme has been shown to play a major role in the in vitro degradation of glucosamine in the extracellular

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matrix by a human ovarian cancer cell line (Niedbala et al., 1987a,b). In addition, the ability of different ovarian carcinoma cell lines to degrade extracellular matrix correlated with their metastatic aggressiveness in the patients of origin (Niedbala ~t al., 1987a). Data exist suggesting that lysosomal enzymes are associated with invasiveness and metastatic potential in some experimental tumors. Activities of P-glucuronidase, P-N-acetylglucosaminidase, and cathepsin D in homogenates of experimental tumors have been shown to vary with the mode of tumor growth, with higher levels detected in murine tumors grown subcutaneously (invasively) than in the same tumor lines grown in an ascitic form (Conchie and Levvy, 1957; Wollen and Turner, 1965; Carr, 1965). Increases in levels of several lysosomal enzymes (P-N-acetylglucosaminidase, P-glucuronidase, acid phosphatase, 6-galactosidase, 01mannosidase, and P-glucosidase) were found to be temporally associated with the development of pulmonary metastases from transplanted primary Lewis lung tumors (Dobrossy et al., 1980). Cathepsin B activity and the activity of many glycosidases have also been found to be elevated in a metastatic variant of the B 16 melanoma line compared to the less metastatic parental cell line (Bosmann et al., 1973; Dobrossy et al., 1981; Sloane et ul., 1981, 1982). T h e subcellular localization of cathepsin B, cathepsin L, and 6-Nacetylglucosaminidase was found to change from lysosomes to the plasma membrane in tumor lines with high metastatic potential (Sloane et ul., 1986; Rozhin et al., 1989); a related finding has been noted in human cervix, with cathepsin B localized to the plasma membrane in squamous carcinoma cells but not in normal cervical epithelium (Pietras and Roberts, 1981). In transplanted bladder tumors in nude mice, active cathepsin B was found to be localized to the plasma membrane of the invasive bladder carcinoma cell line EJ (Weiss et al., 1990). By contrast, both normal bladder epithelium and a noninvasive bladder carcinoma cell line had cathepsin B confined to lysosomes. Furthermore, plasma membrane fractions isolated from the invasive line (but not from the noninvasive cell line) had the ability to degrade purified laminin (Weiss et al., 1990). In addition to changes in the activity and localization of hydrolases in malignant or metastatic cells, alterations in endogenous inhibitors of lysosomal cysteine proteinases have been observed. T h e major endogenous cysteine proteinase inhibitors (CPI) are the stefins and cystatins, although kininogens may also inhibit cysteine proteinases in plasma (Barrett, 1987; Sloane et al., 1990). Decreases in the activities of lowmolecular-weight CPI have been found to be associated with enhanced

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metastatic potential of B 16 melanoma in an experimental metastasis assay (Rozhin et al., 1990).It is not known which of the CPI was altered in these experiments, but there is evidence that stefin A may be altered in malignancy. Stefin A mRNA is reduced during the progression from skin papillomas to carcinoma, and reduced staining for stefin A has been noted in human malignant tumors (Rinne et al., 1984; Kyllonen et al., 1984; Howley-Nelson et al., 1988). Further studies are required to confirm these findings and to demonstrate that the association is causal. The major excreted protein of transformed mouse fibroblasts is cathepsin L (Denhardt et al., 1986; Gal and Gottesman, 1986; Portnoy et al., 1986). Augmented procathepsin L excretion into medium has been correlated with transfection of thefos oncogene into rat fibroblasts (Taniguchi et al., 1990) and with enhanced metastatic potential of rm-transformed murine fibroblasts (Denhardt et al., 1987). Furthermore, inhibitors of cathepsin L prevented invasion by murine mammary carcinoma lines in an amnion invasion assay (Yagel et al., 1989). In this study, however, metalloproteases, such as collagenase, were more important for invasion than the cathepsins, and the role of the cathepsins (B and L) was proposed to be that of activators for these enzymes (Yagel et al., 1989). Experimental studies suggest an association between cathepsin D and metastasis. Rat 3Y 1-Ad12cells (a transformed cell line that forms tumors in athymic mice and secretes no cathepsin D) were transfected with a construct causing overexpression of human cathepsin D (Garcia et al., 1990). The resulting stable transfectants exhibited an increase in malignant phenotype as measured by higher growth rate and overgrowth in culture, and greater metastatic ability in an in uivo experimental metastasis assay. Whether the enhanced metastatic behavior was due to the proteolytic activity of cathepsin D or to another mechanism remains unknown. Several reports have described tumor content of cathepsin D as a prognostic factor in node negative breast carcinoma in women. In three of these studies (Spyratos et al., 1989; Thorpe et al., 1989; Tandon et al., 1990) higher levels of cathepsin D or its precursor, measured by radioimmunoassay, correlated with reduced disease-free and overall survival. Potential explanations include the activity of cathepsin D as a mitogen, as reported in estrogen-deprived MCF-7 cells (Rochefort et al., 1987), and ability of the enzyme to stimulate invasion and metastasis of tumor cells. By contrast, Henry et al. (1990), using an immunocytochemical method, found the opposite relationship; time to relapse and overall survival were both longer in patients whose tumors displayed positive staining for cathepsin D.

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V. Lysosomes and Tumor Microenvironment

T h e microenvironment within some regions of tumors differs from that of normal tissues. As a consequence of inadequate blood supply, regions of both hypoxia and acidity are known to develop (Wike-Hooley rt al., 1984). Limited vascular supply to tumors may lead to resistance to radiation therapy (because of hypoxia); it may also lead to resistance to some anticancer drugs because of limited penetration of drugs to poorly perfused tumor regions, or because of decreased drug sensitivity of slowly proliferating cells in these regions. There is thus a specific need for new therapies directed toward cells in nutrient-deprived regions of solid tumors (Tannock and Rotin, 1989). In considering lysosomes as possible targets of anticancer therapy, it is therefore important to review what is known about lysosomal function under acidic or hypoxic conditions such as may occur within the microenvironment of solid tumors.

‘4.LYSOSOMES AND HYPOXIA Changes in the activity of lysosomal enzymes have long been recognized as a consequence of ischemia (de Duve and Beaufay, 1959). The effects of hypoxia on lysosomes have been examined primarily in the heart and liver, because of a postulated role for lysosomal enzymes in ischemic injury of these organs; we are not aware of information relating directly to tumors. Of central importance is whether changes in lysosomal enzyme activity are the primary cause of irreversible cellular damage, or whether they are nonspecific changes which occur in cells that are damaged as the result of some other fundamental cellular alteration in response to ischemia. T h e major method that has been used to examine the response of lysosomes to hypoxia has been the measurement of the free (or unsedimentable) activity of lysosomal enzymes in the cytoplasm and the activity of enzymes confined to the lysosomes themselves (sedimentable or latent activity). Increases in the proportion of free activity represent lability or loss of integrity of the lysosomal membrane (Wattiaux and Wattiaux-de Coninck, 1984). Furthermore,the timing of these changes in relation to other markers of cell damage can be examined. In both the liver and the heart of experimental animals, ischemia results in an increase in the free activity of lysosomal enzymes. The precise pattern of change depends on the duration of ischemia and reperfusion. In the liver, it appears that lysosomal changes do not precede the development of irreversible damage to the cell, and thus are probably not important in causing this damage (Wattiaux and Wattiaux-de Coninck, 1981, 1984). The situation

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in the heart is less clear (Wildenthal, 1978). Using a combined immunohistochemical and biochemical approach, changes in the activity and localization of cathepsin D have been demonstrated before structural damage to the cell is evident (Decker et al., 1977; Wildenthal et al., 1978); these observations raise the possibility that in the heart, lysosomal enzyme release may play a role in the pathogenesis of ischemic damage, although other primary damaging events can occur in cells (e.g., those caused by lethal doses of radiation) long before structural damage is observed. Other experiments have suggested that lysosomal rupture is not a primary cause of cell death. Using hypoxia and substrate depletion of cultured fetal mouse hearts as a model of ischemia, lysosomal changes were not demonstrated until recovery after hypoxia commenced (Ingwall et al., 1975). Furthermore, in a different model of ischemia, no changes in the lysosomes of autolyzing heart were demonstrated until after irreversible necrosis was established (Lesch, 1977). The relevance of the above results to hypoxia in tumors is unclear. The data have been obtained from experimental models where hypoxia is induced rapidly in hepatic or cardiac tissue and allowed to continue for variable periods of time (usually 1 to 2 hr). Although some regions within tumors may become hypoxic acutely due to temporary cessation in blood flow, other regions are exposed to longer duration of hypoxia because of limited diffusion of oxygen from functioning blood vessels (Tannock, 1972). The effects of chronic hypoxia on lysosomes and their enzymes may be quite different from those of acute hypoxia, and experiments should be undertaken to investigate the effects of varying duration of hypoxia on lysosomal function in malignant cells. We have examined the effect of 6 hr of hypoxia on the activity of cathepsin L in the EMT 6 mouse mammary sarcoma cell line. This exposure does not lead to cell death, as measured by a colony forming assay, and no difference in enzyme activity was observed between the hypoxic cells and normal controls (Table 11).Experiments of this type could usefully be extended to study the activity of other enzymes after varying periods of exposure to hypoxia, with and without inhibitors. Such results should be correlated with estimates of survival of the cells using a colony-forming assay.

B. LYSOSOMES AND EXTRACELLULAR pH T h e mean extracellular pH (pH,) in solid tumors is lower than that in normal tissues, and values in the range pH 6.0-6.5 have been recorded quite frequently when measurements were made with microelectrodes (Wike-Hooley et al., 1984). Although much is known about the effects of changes in lysosomal pH on lysosomal function, little is known of the

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TABLE I1 CATHESPIN La I N CONTROL E M T 6 CELLSA N D I N THOSE INCUBATEDUNDER HYPOXIC OR ACIDIC CONDITIONS FOR SIXHOURS A N D THEN E X T R A C T E D W I T H 0.1% TRITON x-100

ACI IVITY

OF

Activity of cathepsin L

Group Aerobic

203 t 14.0

Hypoxic pH, 7.4

209 t 24.8 213 t 47.1

pH, 6.0

209

P = 0.65

P 2

=

0.85

15.3

Note. T h e substrate used was Z-Phe-Arg-N Mec (obtained from T h e Peptide Institute, Osaka, Japan) and the procedure of Barrett and Kirschke (1981) was followed. KO correction was made for cathepsin B activity, a proportion of which is also detected by this assay. Results are the means of five (hypoxic) or six (pH 6) measurements ? standard deviation and are compared using Student's I test. Hypoxia was generated by continuous gassing of a stirred suspension of tells with nitrogen as previously described (Mohindra and Rauth, 1976). T h e level of pH, varied by less than 0.1 unit over the course of the experiment. * Expressed in units of fluorescence per LOO mg cell protein.

influence of alterations in environmental pH. Decreasing pH, to 6.6 or less for 90 min has been shown to increase lysosomal enzyme activity, but only at temperatures above 42°C (Overgaard and Skovgaard Poulsen, 1977; Keech and Wills, 1979). At 37"C, pH had no influence on enzyme activity. Studies in our laboratory with the EMT6 mouse mammary sarcoma cell line have failed to detect any change in activity of cathepsin L following a 6-hr incubation at pH 6 (Table 11). This exposure did not lead to cell death as measured by colony formation. Two other groups have examined the effects of lowering extracellular pH on the activity of lysosomal enzymes. Lie et al. (1973) found the activity of acid phosphatase in human fibroblasts in culture to be normal at pH 6.8. Increased activity was noted as the pH of the culture medium was raised to 7.7. In the other study, the more metastatic of two different rat sarcoma cell lines was found to release larger amounts of cathepsin B at pH 6.5 than its nonmetastatic counterpart (Krepela et al., 1989). Unfortunately, cell viability in this study was assessed only by morphological criteria, and the higher levels of cathepsin B in the extracellular medium may simply have been a result of greater sensitivity of the metastatic subline to acidic conditions, with greater cell death and enzyme release after 24 hr of incubation at pH 6.5. Little is known of the relationship between pHe and intralysosomal pH. I n the only study that has been performed to examine this question,

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28 1

as pH, rose from 5.0 to 8.5, intralysosomal pH also rose from 5.0 to '1.0 (Reijngoud et al., 1976). However, the nonphysiological levels of pH, used in these experiments make it difficult to assess the significance of these findings. Experiments should be performed to examine the relationship among pH,, cytoplasmic pH, and lysosomal pH, within the range of pH, (6.0-7.5) found in solid tumors. Evidence for release of lysosomal enzymes should be sought in the same experiments, and related to changes in cell survival. Such experiments would provide useful information relating to the effects of the acidic microenvironment of tumors on lysosomes and their possible role in promoting cell death.

VI. Lysosomes and Anticancer Therapy A. LYSOSOMES AND

RADIATION

There is extensive evidence that the major site of radiation-induced damage in cells is the nucleus, with the target being DNA. However, lysosomal enzymes might enhance the effects of radiation on cellular structures, if they are released from lysosomes following radiation. The effect of ionizing radiation on lysosomal enzyme activity has been investigated both for tissues in vivo and for cells and isolated lysosomes in vitro. Following radiation of normal tissues in vivo, increases in both the specific activity and the proportion of unsedimentable activity of several lysosomal enzymes have been noted (Watkins, 1973). Typically, radiation doses of 7 to 10 Gy have been used in these experiments, although elevation in enzyme activity in the rat duodenum has been demonstrated with as little as 4 Gy (Hartiala et al., 1961). Increases in enzyme activity have been observed, commencing from 2 to 24 hr postirradiation, and lasting for 5 to 6 days (Watkins, 1973). The precise pattern of change depends on the enzyme that is measured and the tissue of origin. Increases in activity have been demonstrated for acid phosphatase, acid deoxyribonuclease, and cathepsin B in the rat liver (Goutier-Pirotte and Goutier, 1962; Kocmierska-Grodzka and Goutier, 197 1): P-gluccuronidase, ribonuclease and acid phosphatase in the rat spleen (Roth and Eichel, 1959; Roth et al., 1962); and P-glucuronidase and acid phosphatase in the small intestine of the rat (Hartiala et al., 1961; McFadyen and Baker, 1968). Interpretation of some of these data is complicated by the loss of other cellular proteins as a consequence of radiation; this may give rise to apparent elevations in the specific activity of acid hydrolases, which may have slower turnover than other cellular proteins (Gottlieb et al., 1964). This is a particular problem in radiosensitive tissues such as

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the spleen, which may lose up to 80% of its wet weight during the 3 days following 7.8 Gy whole-body irradiation (Kurnick et al., 1959). Few experiments have directly assessed the influence of radiation dose on the subsequent elevation in enzyme activity. In one such experiment, mouse mammary carcinomas were irradiated in vivo with 5, 10, or 70 Gy in a single dose (Paris and Brandes, 1971). As the dose of radiation increased, elevation in enzyme activity was detected earlier, reached higher peak values, and persisted for a greater length of time. These changes were noted for both the specific activity of whole homogenates and for the unsedimentable fraction. It remains uncertain whether these data reflect primary effects of radiation on lysosomes or are due to greater and earlier release of lysosomal enzymes following higher rates of cell death with increasing doses of radiation. Irradiation of isolated lysosomes in vitro may result in the release of enzymes (Desai et al., 1964; Harris, 1966a,b) and also, under some conditions in their inactivation (Desai et af., 1964; Watkins, 1970). These effects are only observed, however, after very high doses of radiation of 100 Gy or above (Desai et al., 1964; Sottocasa et al., 1965), and are therefore unlikely to be of relevance to antitumor effects of therapeutic radiation. In an attempt to increase the effect of irradiation on tumors, labilizers of lysosomal membranes, such as vitamin A (Weissman, 1964),have been used in combination with radiation. Increased necrosis of mouse mammary gland carcinomas (measured by electron microscopy) was demonstrated following treatment in vivo with the combination of vitamin A and radiation compared to radiation alone (Brandes et al., 1967). Another study has demonstrated that the enhancement of tumor control by the combination of vitamin A and radiation is mediated by an immune effect. In an antigenic fibrosarcoma of the mouse, intraperitoneal injection of vitamin A prior to radiation therapy resulted in a 15-20% decrease in the mean dose of radiation required for tumor control (Tannock et al., 1972). However, there was no effect on normal tissue toxicity, or in immune-suppressed mice. This effect could be due to an enhanced immune response against viable cells that survived radiation, due to more rapid degradation of competing antigenic targets on lethally damaged cells; or to an effect of vitamin A acting as an immunological adjuvant, leading to nonspecific stimulation of the immune response Uurin and Tannock, 1972). On the basis of current information, it is not clear whether the elevations in lysosomal enzyme activity observed after radiation play a role in tissue damage. Most studies of the effects of irradiation on lysosomes either in cells in vitro o r in tissues in vivo have not related the observed

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changes to cell death. Consequently, it is uncertain whether the elevations in lysosomal activity that occur following radiation either cause o r add to cell and tissue damage, o r are the result of them. Further experiments designed to examine the timing of changes in lysosomal integrity and enzyme activity, particularly shortly after radiation, are required to resolve this question. Most mammalian cells remain morphologically intact for many hours after exposure to radiation and then die while attempting mitosis. Experiments should be carried out to study lysosomal enzyme activity in relation to these events. Finally, experiments using inhibitors of lysosomal enzymes may demonstrate whether these enzymes enhance damage caused by radiation. B. LYSOSOMES AND HYPERTHERMIA T h e mechanisms by which heat treatment leads to cell death remain unknown. One mechanism that has been postulated to contribute to cell death is the effect of elevated temperature on the lysosomal membrane and lysosomal enzymes (Overgaard, 1977; Hume et al., 1978). Using histochemical techniques, increases in the activity of acid phosphatase have been demonstrated following exposure of both HeLa cells (Keech and Wills, 1979) and normal spleen (Hume et al., 1978) to temperatures above 42" C. I n addition, at temperatures above 42.5"C an immediate increase in lysosomal membrane permeability, lasting for at least 4 hr, was detected (Hume et al., 1978). In transplanted C3H mouse mammary adenocarcinoma exposed to hyperthermia both in vitro and in vivo, temperatures of 41.7 to 43.5"C resulted in increased activity of both acid phosphatase and P-glucuronidase (Barratt and Wills, 1979). Ultrastructural investigations have also demonstrated increases in lysosomal number in the first few hours following hyperthermia (Overgaard, 1976). Other investigations (Tamulevicius and Streffer, 1983), in which the activity of lysosomal enzymes in liver and spleen have been measured after hyperthermia, have failed to confirm the above findings. This discrepancy may be due to the different techniques used to assess lysosomal enzymes (direct measurements of enzyme activity versus histochemistry). Additional studies are needed to define further the alterations in lysosomal enzyme activity that may take place after hyperthermia. These studies should focus on the timing of changes in order to determine whether they may be the cause of cell damage or simply a reflection of it, and should be correlated with changes in cell survival as measured by a clonogenic assay. It will also be important to determine whether there are differences in lysosomal enzyme activity in cells of malignant and normal tissues exposed to hyperthermia.

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C. LYSOSOMES AND DRUGS Although none of the anticancer agents in clinical use today is believed to kill tumor cells through an effect on lysosomes, there is some evidence that lysosomes may play a part in the uptake, processing, or activity of some compounds. For example, the cytotoxicity of bleomycin to cells in tissue culture appears to be enhanced by agents that disrupt lysosomal function (Lazo et al., 1990). Increased toxicity of bleomycin was noted when the drug was given in combination with lysosomotropic amines such as ammonium chloride, the ionophore monensin, and the calcium channel antagonists verapamil and diltiazem. Increased cell killing was not due to increased intracellular accumulation of bleomycin, nor via the induction of increased single-strand DNA breaks. Although all of the above agents have lysosomal effects, most also have other sites of activity, and further evidence is required to confirm that the demonstrated increase in bleomycin cytotoxicity is being mediated by effects on the lysosomal compartment. A possible role for the lysosomal-endosomal compartment in the development of a multidrug resistance phenotype has been proposed (Sehested et al., 1987). Both doxorubicin and daunorubicin have been shown to accumulate in lysosomes, probably because they are weak bases (Noel et al., 1978); vinca alkaloids are also bases and might be expected to show similar accumulation in lysosomes. Sehested et al. (1987) have shown that Ehrlich ascites tumor cells resistant to anthracyclines and vinca alkaloids have an increased rate of membrane turnover that is accompanied by an increase in trafficking of vesicles between the plasma membrane and the lysosomal-endosomal compartment. This process has been proposed as a possible basis for the active drug extrusion noted in resistant cells. However, a larger body of evidence implicates the 170kDa P-glycoprotein as an active membrane-based extrusion pump for these drugs, and overexpression of P-glycoprotein appears to be the more common cause of multidrug resistance. Some cells have been reported to become resistant to multiple drugs, however, without evidence for overexpression of P-glycoprotein [or of altered activity of topoisomerase I1 (Endicott and Ling, 1989)] and drug extrusion via lysosomes or endosomes might contribute to drug resistance in some of these cells. Potentiation of the activity of anthracyclines and vinca alkaloids in resistant cell lines by lysosomotropic compounds that are known to elevate lysosomal pH has been cited as evidence that lysosomes may be involved in multiple drug resistance (Klohs and Steinkampf, 1986, 1988; Shiraishi et al., 1986; Zamora and Beck, 1986).These agents also have

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other effects [including binding to P-glycoprotein (Endicott and Ling, 1989)]and may act on other acidified organelles such as the trans Golgi network (TGN) to elevate pH. The TGN comprises acidified vesicles that are destined for secretion and it has been postulated that drug may accumulate here prior to efflux from the cell. On the basis of the differential effects of methylamine or ammonium chloride (which neither elevate pH in the TGN nor potentiate the action of anthracyclines in resistant cells) and other lysosomotropic agents (which can cause both of these effects), the TGN has been proposed as an alternate site of drug sequestration and efflux in multidrug resistant cells (Klohs and Seinkampf, 1988). The bulk of evidence suggests that P-glycoprotein associated with the plasma membrane is the main mechanism for drug efflux in multidrug resistant cells (Endicott and Ling, 1989).Whether the lysosomal or other acidified compartments play some role in adding to the effects of Pglycoprotein remains unclear. This mechanism should be investigated in atypical multidrug resistant cells that have not been shown to express Pglycoprotein, as well as cells that show the more usual multiple drug resistance phenotype. Useful experiments would isolate lysosomes from treated cells and assess the drug concentration within them; concentration of drugs in lysosomes from resistant but not from drug-sensitive wild-type cells would provide evidence for a role of lysosomes in the development of resistance. A novel approach to cell killing that involves utilizing lysosomal enzymes has been proposed by Firestone (Firestone and Pisano, 1979). A group of compounds termed lysosomotropic detergents has been synthesized; it is postulated that these noncharged molecules diffuse into cells and then are concentrated within lysosomes because of the pH gradient across the lysosomal membrane. Once inside lysosomes, they become protonated (and hence charged) due to the low intralysosomal pH, allowing a continuous gradient for drug entry into lysosomes. In the charged form, the compounds have detergent properties and, when sufficient accumulation has occurred, they could dissolve the lysosomal membrane, releasing lysosomal enzymes into the cytoplasm. These enzymes could then degrade cellular structures and result in cell death. The slightly lower pH found within some regions of tumors is probably associated with a lower cytoplasmic pH, and could result in enhanced toxicity based on the low pH optimum of most lysosomal enzymes. Previous attempts to labilize the lysosomal membrane using vitamin A did not directly result in enhanced cell killing, although an immunemediated effect was seen (Tannock et al., 1972). This suggests that release of lysosomal enzymes into the cytoplasm induced by vitamin A

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(Weissman, 1964) may not be sufficient to result in cell killing. By contrast, in preliminary experiments, N-dodecylimidazole (NDI), a lysosomotropic detergent, has been shown to be cytotoxic, and the cell killing was mediated by lysosomal enzymes (Miller et al., 1983; Wilson et al., 1987).Others have reported conflicting results, however, suggesting that the action of NDI is not mediated by lysosomes or their enzymes (Forster et al., 1987). Unpublished data from our laboratory have shown that NDI kills cells in tissue culture with increased activity at low extracellular pH, but that these effects are most likely mediated by interaction of the drug with the cell membrane. VII. Conclusion

Understanding of the role that lysosomes play in malignancy is limited by a lack of information from appropriately designed experiments. An association has been demonstrated between the metastatic potential of some experimental tumors and the activity of cathepsins B and L. Furthermore, increases in the activity of lysosomal enzymes have been demonstrated consistently in untreated tumors when compared to normal tissue. T h e cause of these elevations in activity is unknown, and doubt still exists about the cellular origin of the enzymes being measured. Although further investigation is required to increase our understanding of the biology of lysosomes in tumors, differences between malignant and normal tissues offer the potential for therapeutic exploitation. Changes in activity of lysosomal enzymes have been documented following exposure of cells o r tissues to heat or radiation, but the significance of these changes in relation to cell death remains unknown; alterations in lysosomal enzymes may be simply a marker of injury mediated by another mechanism. Currently there is no anticancer therapy that is aimed primarily at lysosomes, although lysosomes may play a role in the uptake, processing, o r efflux of some cytotoxic agents and might provide mechanisms leading to some forms of drug resistance. In order to develo p anticancer therapy rationally, differences between the properties of lysosomes in malignant and normal tissues need to be demonstrated and understood. ACKNOWLEDGMENTS Experimental work described in this paper was supported by the Medical Research Council of Canada. and by a grant (CA5 1033) from the National Institutes of Health. Dr. Boyer is supported by a Medical Research Council of Canada Fellowship.

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INDEX

A P-N-Acetylglucosaminidase,lysosomes and, 272-276 Acid phosphatase, lysosomes and, 272274,276,280-281, 283 Acidic fibroblast growth factor multigene family, 35 early studies, 6 ligand binding, 24-27 mitogens, 2-3, 5 multiple forms, 17-18, 20-21 Acidity, lysosomes and, 278, 281, 285 Acute lymphoblastic leukemia MHC Class I expression and, 190 soft tissue tumors and, 106 Acute-phase proteins, immunosuppression and, 257 Adenocarcinoma, genomic instability and, 147 Adenovirus MHC Class I expression and, 224 cancer, 189-190 modulation by oncogenes, 195-197, 202,204 regulation by oncogenes, 211-212, 216, 221 multidrug resistance gene and, 172 soft tissue tumors and, 102-103 Aflatoxin genomic instability and, 146 soft tissue tumors and, 101-102 Age genomic instability and, 126 MHC Class I expression and, 182 Alleles genomic instability and, 123, 142-144

MHC Class I expression and, 231 cancer, 186, 188, 190-195 downmodulation, 222, 224, 228-230 modulation by oncogenes, 196, 205206, 208 protein tyrosine kinase GFRs and development, 50-52.55-57 structure, 45 tumorigenesis, 64 soft tissue tumors and, 85, 101, 103 Amino acids FGF receptor multigene family and, 2, 35 alternative splicing, 15 characterization, 8-14 Drosophila, 30-31 mitogens, 3, 5 multiple forms, 18-19, 21-23 signal transduction, 32 immunosuppression and, 253-254 MHC Class I expression and, 218 cancer, 186-187 downmodulation, 229-230 modulation by oncogenes, 197, 201 multidrug resistance gene and, 160, 175 protein tyrosine kinase GFRs and, 5859,63 Ammonium chloride, lysosornes and, 284-285 Amplification genomic instability and, 126, 141-142 MHC Class I expression and, 181, 203 multidrug resistance gene and, 160 protein tyrosine kinase GFRs and, 64-65 soft tissue tumors and, 99, 104-105 Anemia, protein tyrosine kinase GFRs and, 52-53

293

294

INDEX

Aneuploidy, genomic instability and, 122, 124, 135-136 Angiogenesis FGF receptor multigene family and, 23, 35 protein tyrosine kinase GFRs and, 50 Anthracyclines, lysosomes and, 284-285 Antibodies, see ulso Monoclonal antibodies FGF receptor multigene family and, 8 genomic instability and, 124, 133 immunosuppression and, 253-254, 259 MHC Class I expression and, 190, 194, 205, 227-228 Anticancer drugs lysosomes and, 278, 284-286 multidrug resistance gene and, 158159, 166, 176 Antigens immunosuppression and, 252 Iysosomes and, 282 MHC Class I expression and, 182, 233 cancer, 184-189, 195 modulation by oncogenes, 204 regulation by oncogenes, 208. 210, 216 multidrug resistance gene and, 162, 171 soft tissue tumors and, 100, 102 AP-1 MHC Class I expression and, 200, 210, 215 multidrug resistance gene and, 173-174 APC gene, soft tissue tumors and, 81-82, 100,109 Arsenite, multidrug resistance gene and, 170 Arylsulfatase, lysosomes and, 272, 274 Aspergillus nrdulans, genomic instability and, 132 Ataxia telangiesctasia, genomic instability and, 136 AI’P multidrug resistance gene and, 159160, 162-165 protein tyrosine kinase GFRs and, 63 ATP-binding cassette (ABC), multidrug resistance gene and, 162-163 ATPase, lvsosomes and, 269 Autosomes genomic instability and, 132 MHC Class 1 expression and, 230

protein tyrosine kinase GFRs and, 66 soft tissue tumors and, 80-81, 83, 8687 wl,protein tyrosine kinase GFRs and, 63, 65-66

B B cells genomic instability and, 124, 133, 137, 139 immunosuppression and, 247, 249, 256, 259 MHC Class I expression and, 195, 203, 213-214, 219, 223 Bacteria, immunosuppression and, 259 Basic fibroblast growth factor multigene family, 34-35 early studies, 6 ligand binding, 24-27 mitogens, 2-3, 5 multiple forms, 17-21 BAT @-associated transcripts), MHC Class I expression and, 230 bcr, soft tissue tumors and, 88-89, 107 Beckwith-Wiedemann syndrome, soft tissue tumors and, 85, 103 bek, protein tyrosine kinase GFRs and, 64 Benign tumors genomic instability and, 124 soft tissue tumors and, 76-77, 80, 8587,94 Bladder carcinoma lysosomes and, 276 MHC Class I expression and, 190, 205, 223 Bloom’s syndrome, genomic instability and, 126, 136 Bone marrow MHC CIass I expression and, 230 rnultidrug resistance gene and, 168 Breast cancer immunosuppression and, 251-252, 255, 260-261 lysosomes and, 277 MHC Class I expression and, 194, 204, 206, 223 multidrug resistance gene and, 166, 169 protein tyrosine kinase GFRs and, 6465

295

INDEX

soft tissue tumors and, 78, 80, 82, 101, 104-105

Burkitt’s lymphoma MHC Class I expression and, 190, 195, 202-203, 205

soft tissue tumors and, 88

C c-abl, soft tissue tumors and, 88-89, 107 c-elk, soft tissue tumors and, 95 c-erbB-2, soft tissue tumors and, 104-105 c-fos FGF receptor multigene family and, 31 MHC Class I expression and, 200-201, 219

c-jun, MHC Class I expression and, 200 c-kit, protein tyrosine kinase GFRs and, 46, 51-56, 60

c-myc FGF receptor multigene family and, 31 MHC Class I expression and, 183, 231233

downmodulation, 222-230 modulation by oncogenes, 198, 202207

regulation by oncogenes, 211, 215, 219-222

multidrug resistance gene and, 175 soft tissue tumors and, 88, 104-105 a,!c.MHC Class I expression and, 202 Calcium FGF receptor multigene family and, 31 lysosomes and, 284 Carcinogenesis genomic instability and, 126 lysosomes and, 270 MHC Class I expression and, 187-188 soft tissue tumors and, 78, 83 Cathespins, lysosomes and, 271-277, 279281, 286 cbfl, genomic instability and, 136 cDNA FGF receptor multigene family and, 23, 7, 23, 30 alternative splicing, 15, 25-26 characterization, 8-9, 11 multiple forms, 17-23 immunosuppression and, 259

MHC Class I expression and, 214 multidrug resistance gene and, 160, 162, 175

protein tyrosine kinase GFRs and, 4647

soft tissue tumors and, 100, 102, 108 Centrioles, genomic instability and, 134 Centromeres, genomic instability and, 134-135

Cervical carcinoma, MHC Class I expression and, 204, 224 Chemotaxis FGF receptor multigene family and, 34 immunosuppression and, 249, 252 protein tyrosine kinase GFRs and, 54 Chemotherapy genomic instability and, 131, 145 immunosuppression and, 248 multidrug resistance gene and, 157158, 166-170, 174

soft tissue tumors and, 104-105 Chloramphenicol acetyltransferase (CAT) MHC Class I expression and, 211-213, 220

multidrug resistance gene and, 170, 175 Chondrosarcomas, soft tissue tumors and, 99-100

Choriocarcinoma, MHC Class I expression and, 194 Chromatids, genomic instability and, 134, 138

Chromatin, MHC Class I expression and, 217

Chromosomes FGF receptor multigene family and, 3 genomic instability and, 121-127, 144, 147

mechanisms, 131-141 MHC Class I expression and, 185, 203, 230

protein tyrosine kinase GFRs and, 51, 66

soft tissue tumors and, 78 abnormalities, 89-100 cloning, 107-108 gene amplification, 104-105 predictors of behavior, 105 ras gene, 87 susceptibility, 81, 84-85 tumor suppressor genes, 100-104

296

INDEX

Chronic myelogenous leukemia genomic instability and, 139-140 multidrug resistance gene and, 166, 174 soft tissue tumors and, 88-90, 101, 108 Chronic myeloid leukemia immunosuppression and, 261 protein tyrosine kinase GFRs and, 65 Clones FGF receptor multigene family and, 23,23-25 characterization, 8-9, 11 Drosophila, 30-31 multiple forms, 18, 20, 23 genomic instability and, 132, 136, 142143 MHC Class 1 expression and cancer, 184, 188 downmodulation, 222, 224, 228, 230 modulation by oncogenes, 197-198, 200-201,205 regulation by oncogenes, 214, 218 multidrug resistance gene and, 159-163 soft tissue tumors and, 78, 110 chromosomal abnormalities, 88, 9192 gene amplification. 105 ras gene, 87 susceptibility, 81, 84-85 translocation breakpoints, 107-109 tumor suppressor genes, 100, 103 Colchicine, multidrug resistance gene and, 158, 170 Colon cancer genomic instability and, 126 lysosomes and, 273 MHC Class I expression and, 194 downmodulation, 223-224, 227 modulation by oncogenes, 201, 203204, 206 regulation by oncogenes, 217 multidrug resistance gene and, 171, 174 soft tissue tumors and, 80-81, 101, 10Y Colony-stimulating factor 1, protein tyrosine kinase GFRs and, 45, 49, 62 Colony-stimulating factors, immunosuppression and, 255-257, 261 Colorectal carcinoma, immunosuppression and, 251 Cross-linking, FGF receptor multigene family and, 6-7, 17-18

Cyclic AMP MHC Class I expression and, 210 multidrug resistance gene and, 170, 175 Cysteine proteinase inhibitors, lysosomes and, 276-277 Cytokines, immunosuppression and, 247, 252,255,257-261 Cytoplasm lysosomes and, 269, 271, 275, 278, 281, 285 MHC Class I expression and, 213 multidrug resistance gene and, 159 Cytotoxic drugs, multidrug resistance gene and, 158-159, 168 Cytotoxic T cells immunosuppression and, 248-249, 25 1, 253 MHC Class I expression and cancer, 183-184, 188 downmodulation, 225 modulation by oncogenes, 196-197, 202 Cytotoxicity immunosuppression and, 253 lysosomes and, 284-285

D Daunomycin, multidrug resistance gene and, 170 Daunorubicin, lysosomes and, 284 Delayed-type hypersensitivity reactions, immunosuppression and, 253, 258 Deletion genomic instability and, 139, 141 MHC Class 1 expression and, 182, 213 multidrug resistance gene and, 160 protein tyrosine kinase GFRs and, 62 DER, protein tyrosine kinase GFRs and, 56-57 Desmoids, soft tissue tumors and, 8182 Development genomic instability and, 124, 142 MHC Class I expression and, 210 protein tyrosine kinase GFRs and, 4950 mutants of Drosophila, 56-60 mutants of mouse, 50-56

297

INDEX

Diabetic retinopathy, FGF receptor multigene family and, 4, 35 Differentiation FGF receptor multigene family and, 2, 4, 32, 34 genomic instability and, 124, 133, 137, 140 immunosuppression and, 256, 261 MHC Class I expression and, 181-183, 195 modulation by oncogenes, 200-203, 206 regulation by oncogenes, 210, 213215, 221 protein tyrosine kinase GFRs and, 45, 60, 64 soft tissue tumors and, 76, 78, 86, 93, 104, 110 Dimerization FGF receptor multigene family and, 32 MHC Class I expression and, 218 protein tyrosine kinase GFRs and, 56 DNA FGF receptor multigene family and, 5, 11, 18 genomic instability and, 124-125, 145, 147 mechanisms, 129-142 quantitative considerations, 144-145 immunosuppression and, 253 lysosomes and, 281, 284 MHC Class I expression and, 183 modulation by oncogenes, 197-198, 200 regulation by oncogenes, 208-210, 213-216 regulation in tumor cells, 218-220 multidrug resistance gene and, 173 protein tyrosine kinase GFRs and, 6364 soft tissue tumors and, 80, 87, 102, 108 chromosomal abnormalities, 89, 92, 94,99 gene amplification, 104-105 predictors of behavior, 105-106 DNA ligases, genomic instability and, 136, 138 DNA polymerase, genomic instability and, 127-129, 147

DNA replication genomic instability and, 127-130, 132, 134, 136, 138, 144 MHC Class I expression and, 187 dnaQ, genomic instability and, 129 N-Dodecyclimidazole,lysosomes and, 286 Downmodulation, MHC Class I expression and, 231, 233 biological consequences, 222-231 cancer, 189-195 modulation by oncogenes, 196-199, 204-208 regulation by oncogenes, 212, 217-220 Downregulation, immunosuppression and, 261 Doxorubicin lysosomes and, 284 multidrug resistance gene and, 158159,167-168 Drosophila FGF receptor multigene family and, 30-31 genomic instability and, 135, 147 MHC Class I expression and, 214 protein tyrosine kinase GFRs and, 45, 49 Drosophila melanogaster genomic instability and, 135 protein tyrosine kinase GFRs and, 46, 49-50,56-60

E EIA, MHC Class I expression and, 212, 224 cancer, 189-190, 195 modulation by oncogenes, 195-197, 201, 204 Effector cells, MHC Class I expression and, 182, 185, 187, 189, 226 Electrophoresis, soft tissue tumors and, 94, 99, 108 ellipse, protein tyrosine kinase GFRs and, 56-57 Embryogenesis FGF receptor multigene family and, 45, 8, 29 genomic instability and, 121 MHC Class I expression and, 201, 210

INDEX

protein tyrosine kinase GFRs and, 45, 49-50.52-58 soft tissue tumors and, 87, 97, 100, 104 Embryonal carcinoma cells, MHC Class I expression and, 210, 213 Endonuclease, genomic instability and, 132, 138 Endoplasmic reticulum, multidrug resistance gene and, 162 Endothelial cells FGF receptor multigene family and, 34, 6, 8, 24, 28 multidrug resistance gene and, 165 soft tissue tumors and, 76 Enhancers MHC Class I expression and modulation by oncogenes, 197, 203, 207-208 regulation by oncogenes, 209-216 regulation in tumor cells, 217, 219221 multidrug resistance gene and, 173 a v , protein tyrosine kinase GFRs and, 62 Environment genomic instability and, 124, 132, 145146 soft tissue tumors and, 81 Enzymes, see also Lysosomal enzymes genomic instability and, 129, 131, 138, 147 immunosuppression and, 258 MHC Class I expression and, 216 E P H , protein tyrosine kinase GFRs and, 65-66 Epidemiology, genomic instability and, 131, 147 Epidermal growth factor FGF receptor multigene family and, 9 protein tyrosine kinase GFRs and, 45, 49,56,60-61,64 Epigenetic DNA modification, genomic instability and, 140-141 Epithelial cells FGF receptor multigene family and, 6 immunosuppression and, 249-252 lysosomes and, 276 MHC Class I expression and, 198, 202 multidrug resistance gene and, 165, 171 protein tyrosine kinase GFRs and, 57 soft tissue tumors and, 95

Epitopes, MHC Class I expression and, 187, 195-196 Eschm'chia coli, genomic instability and, 128-132, 138-139 estl, genomic instability and, 136 Eukaryotes genomic instability and, 129-130, 135136, 142 MHC Class I expression and, 208 multidrug resistance gene and, 162, 172 Exonuclease, genomic instability and, 129 Extracellular matrix, lysosomes and, 275276,280 Extraskeletal osteosarcoma, soft tissue tumors and, 76-78 Eye, protein tyrosine kinase GFRs and, 57-58

F Familial adenomatous polyposis, soft tissue tumors and, 80-81 Fanconi's anemia, genomic instability and, 136 Fat, soft tissue tumors and, 76, 86, 94 FGF receptor, protein tyrosine kinase GFRs and, 46,64 FGF receptor multigene family, 2, 33-35 alternative splicing, mRNA, 28-29 characterization, 11-15 FGFR 1,8-11 Drosophila, 30-31 early studies, 6-7 expression, 28-30 FGFR 1,8-15.34 alternative splicing, 28-29 Drosophila, 30-31 expression, 29-30 ligand binding, 24-27 multiple forms, 15-23 signal transduction, 32 FGFR 2, 11-15, 19,23 alternative splicing, 28 expression, 29-30 ligand binding, 24-28 multiple forms, 20-23 signal transduction, 32 FGFR 3, 11-14, 23-24 expression, 29-30 signal transduction, 32

INDEX

FGFK 4, 11-15, 23, 31 expression, 29-30 ligand binding, 27 ligand binding, 24-25 alternative splicing, 25-27 specificities, 27-28 mitogens, 2-5 multiple forms, 23 alternative splicing, 15-16 FGFK I, 15-19 FGFR 2,20-23 nomenclature, 7-8 signal transduction, 31-33 Fibroblast growth factor receptor multigene family, see FGF receptor multigene family Fibroblasts genomic instability and, 126 immunosuppression and, 249 lysosomes and, 274-275, 277,280, 282 MHC Class I expression and, 201, 226 protein tyrosine kinase GFRs and, 62 soft tissue tumors and, 76-77, 83, 87, 100, 109-110 Fibromatosis, soft tissue tumors and, 8082, 100 Fibrosarcoma lysosomes and, 282 MHC Class I expression and, 189, 217 soft tissue tumors and, 76, 78, 80, 87 chromosomal abnormalities, 95, 99100

tumor suppressor genes, 100 flb, protein tyrosine kinase GFRs and, 5657 flg, protein tyrosine kinase GFRs and, 64 Fluorescence, lysosomes and, 271 fmr-1, genomic instability and, 140-141 fos

lysosomes and, 277 MHC Class I expression and, 199, 220 Fragile X syndrome, genomic instability and, 140-141 Free radicals, genomic instability and, 145-147

G gag, protein tyrosine kinase GFRs and, 62

GAP, see GTPase-activating protein

299

Gardner syndrome, soft tissue tumors and, 81-82 Gene expression genomic instability and, 124, 140-141 MHC Class I, see MHC Class I expression, myc oncogene activation and multidrug resistance gene and, 157-169 Genes FGF receptor multigene family and, see FGF receptormultigene family protein tyrosine kinase GFRs and, 5859, 64-66 soft tissue tumors and, 78-79, 109-110 chromosomal abnormalities, 88 cloning, 107-108 gene amplification, 104-105 predictors of behavior, 105 ra.s gene, 87-88 susceptibility, 85 tumor suppressor genes, 102-103 Genomic instability, tumor progression and, 121-124, 145-147 definitions, 124-126 implications, 126-127 mechanisms, 141-142 checkpoints, 133-134 chromosomes, 136 DNA damage, 130-132 DNA replication, 127-129 epigenetic DNA modification, 140141 immunoglobulin gene hypermutation, 133 ploidy, 134-136 postreplicative mismatch correction, 130 recombination, 137-140 quantitative considerations, 142-145 Genotype genomic instability and, 146 multidrug resistance gene and, 167 protein tyrosine kinase GFRs and, 54 glz, soft tissue tumors and, 89, 92, 94, 104 Glial tumors, genomic instability and, 123 Glioma cells, immunosuppression and, 249, 252 f3-Glucuronidase, lysosomes and, 272, 274-276,281,283 Glycoprotein MHC Class I expression and, 185

300

INDEX

multidrug resistance gene and, 159 protein tyrosine kinase GFRs and, 51, 59 soft tissue tumors and, 106 Glycosylation lysosomes and, 269 MHC Class I expression and, 196 multidrug resistance gene and, 160, 175 protein tyrosine kinase GFRs and, 43 Granulocytes, immunosuppression and, 256 GTPase-activating protein (GAP) FGF receptor multigene family and, 32-33 soft tissue tumors and, 84

H H-2 region, MHC Class I expression and, 190 downmodulation, 222, 224-225, 227 modulation by oncogenes, 197, 201 regulation by oncogenes, 209, 212, 215217 Harvey murine sarcoma virus, MHC Class I expression and, 198 Heart, lysosomes and, 278-279 Heat shock, multidrug resistance gene and, 169-170, 174 Heat shock proteins, MHC Class 1 expression and, 187, 230 Hematopoiesis immunosuppression and, 256 MHC Class I expression and, 189 protein tyrosine kinase GFRs and, 50, 52-53 Heparin, FGF receptor multigene family and, 3, 5, 7-8 Hepatitis B virus, MHC Class I expression and, 198 Hepatoblastoma, soft tissue tumors and, 103 Hepatocellular carcinoma genomic instability and, 146-147 soft tissue tumors and, 101 Hepatoma immunosuppression and, 252 lysosomes and, 273

Herpes simplex virus genomic instability and, 128 MHC Class I expression and, 197-198 Heterozygosity genomic instability and, 123, 138, 141143, 146 loss of, genomic instability and, 127, 137-138, 143-146 H2F1, MHC Class I expression and, 213214, 217, 219-220 Hh-1, MHC Class I expression and, 230 Histochemistry lysosomes and, 283 MHC Class I expression and, 190, 205 Histones genomic instability and, 135 MHC Class I expression and, 187 HLA immunosuppression and, 249 MHC Class I expression and, 231, 233 cancer, 184, 186-187, 190, 194-195 downmodulation, 222-224, 227-231 modulation by oncogenes, 200-207 regulation by oncogenes, 210-211, 215 regulation in tumor cells, 217, 219221 soft tissue tumors and, 80 Homology FGF receptor multigene family and, 23, 5, 19, 23, 31 genomic instability and, 135-139 immunosuppression and, 253 MHC Class I expression and cancer, 185-186 modulation by oncogenes, 204 regulation by oncogenes, 210-211, 216, 218, 222 multidrug resistance gene and, 162 protein tyrosine kinase GFRs and, 49, 56 soft tissue tumors and, 76, 84, 103 Homozygosity, genomic instability and, 123, 138, 141-143, 146 Hormones, multidrug resistance gene and, 171 hprt mutants, genomic instability and, 139 hst, FGF receptor multigene family and, 5-6, 24, 27, 35

INDEX

Human multidrug resistance gene, see Multidrug resistance gene, human Human soft tissue tumors, see Soft tissue tumors, human Hybridoma, MHC Class I expression and, 200 Hybrids FGF receptor multigene family and, 5, 8, 11, 29 MHC Class I expression and, 214, 219, 230 protein tyrosine kinase GFRs and, 49, 54, 56, 65-66 soft tissue tumors and, 87, 97-98, 101, 104, 108 Hydrogen, multidrug resistance gene and, 164 Hydrogen peroxide, genomic instability and, 131 Hydrolysis, FGF receptor multigene family and, 31, 33 Hydrophobicity, multidrug resistance gene and, 158, 160, 162-163 8-Hydroxyguanine, genomic instability and, 128, 132 Hypermutation, genomic instability and, 133 Hyperthermia, lysosomes and, 283 Hypoxia, lysosomes and, 278-280

I Immortalization, soft tissue tumors and, 77, 83, 100-101 Immune defense, MHC Class I expression and, 182-183, 231-233 cancer, 183-189 downmodulation, 224-227 modulation by oncogenes, 197 Immune response, MHC Class I expression and, 222 Immunocytochemistry, lysosomes and, 274,277 Immunogenicity immunosuppression and, 259 MHC Class I expression and, 187-189 Immunoglobulin FGF receptor multigene family and, 23, 34-35

30 1

alternative splicing, 28-29 characterization, 8, 13, 15 Drosophila, 31 ligand binding, 24-28 multiple forms, 17-21 genomic instability and, 124, 133, 138139 immunosuppression and, 247, 259, 263 MHC Class I expression and, 203, 213 soft tissue tumors and, 107 Immunohistochemistry, multidrug resistance gene and, 171 Immunology, genomic instability and, 133 Immunoprecipitation, MHC Class I expression and, 185, 194 Immunosuppression, 247-248, 254-255 acute-phase proteins, 257 colony-stimulating factors, 255-257 growth-regulatory cytokines, 257-261 lymphocyte blastogenesis inhibitory factor, 252 MHC Class I expression and, 202, 223 P15E, 252-253 recent evidence, 248 soft tissue tumors and, 79 suppressive E-receptor, 253-254 transforming growth factor+, 249, 251252 tumor cells, 248-251 Immunotherapy, multidrug resistance gene and, 157 in situ hybridization FGF receptor multigene family and, 29 protein tyrosine kinase GFRs and, 49, 54 soft tissue tumors and, 98-99 Inflammation genomic instability and, 145-147 immunosuppression and, 249, 257 Inheritance, MHC Class I expression and, 230 Inherited syndromes, genomic instability and, 136 Inhibitors FGF receptor multigene family and, 2, 4, 32, 34 genomic instability and, 134, 145-146 immunosuppression and, 248-249, 251-256, 258, 260-261

302

INDEX

lysosomes and, 276, 279, 283 MHC Class I expression and, 196, 213214, 218, 226 multidrug resistance gene and, 167-171 soft tissue tumors and, 95, 101, 106 Instability, genomic, tumor progression and, see Genomic instability, tumor progression and Insulin FGF receptor multigene family and, 8-9 protein tyrosine kinase GFRs and, 46, 62 imt-1, soft tissue tumors and, 89, 92 Interferon immunosuppression and, 253 MHC Class I expression and, 207-212, 215 a-Interferon, MHC Class I expression and, 210-211, 215 7-Interferon, MHC Class 1 expression and, 197, 207-208, 211,215,225 Interferon response element, MHC Class I expression and, 207-212, 215-216, 220 Interleukin-I FGF receptor multigene family and, 3 immunosuppressron and, 247-248, 251-253, 258 Interleukin-2 immunosuppression and, 247-248, 251-253, 258 MHC Class I expression and, 183, 225 lodination, FGF receptor multigene family and, 6, 25 Iron, genomic instability and, 131 Ischemia, lysosomes and, 278-279

J Jun, MHC Class

I expression and, 200

K K-rm, MHC Class 1 expression and, 201 KARI gene, genomic instability and, 135 Karyotypy genomic instability and, 122-124, 147

soft tissue tumors and, 88-89, 91-92, 94-95,97,99-100 KBFl, MHC Class I expression and, 213214, 217 Keratinocyte growth factor multigene family, 5-6, 11, 20, 35 alternative splicing, 28 ligand binding, 24-27 Kidney, multidrug resistance gene and, 165-166, 170 Kinesins, genomic instability and, 135 Kinetochores, genomic instability and, 135 Kirsten rnurine leukemia virus, MHC Class 1 expression and, 198 Kirsten sarcoma leukemia virus, MHC Class I expression and, 198

L Lectin, immunosuppression and, 252 Leiomyomas, characterization of chromosomal abnormalities, 91-94 susceptibility, 81, 85-87 Leiomyosarcomas, characterization of, 76, 87, 99, 101-102, 105 Leukemia acute lymphoblastic MHC Class I expression and, 190 soft tissue tumors and, 106 chronic myelogenous genomic instability and, 139-140 multidrug resistance gene and, 166, 174 soft tissue tumors and, 88-90, 101, 108 chronic myeloid immunosuppression and, 261 protein tyrosine kinase GFRs and, 65 genomic instability and, 125 lysosomes and, 273 MHC Class I expression and, 194, 210, 217 multidrug resistance gene and, 166167, 169, 174 soft tissue tumors and, 82, 88, 107109 Leukemia viruses, MHC Class I expression and, 198-199, 211, 222

303

INDEX

Li-Fraumeni syndrome, soft tissue tumors and, 80, 82-83 Ligands FGF receptor multigene family and, 31, 34-35 alternative splicing, 29 binding, 24-28 characterization, 8 early studies, 6 mitogens, 5 multiple forms, 19 protein tyrosine kinase GFRs and, 43, 45 development, 49-51,53-54.58-59 structure, 45-49 tumorigenesis, 60-62, 64-65 Linkage, soft tissue tumors and, 82, 85, 98 Lipids, multidrug resistance gene and, 164 Lipomas, characterization of, 76 chromosomal abnormalities, 89-91, 9394 susceptibility, 81, 85-86 Liposarcomas, characterization of, 76, 87, 102, 108 chromosomal abnormalities, 93-95 gene amplification, 104-105 Liposomes, multidrug resistance gene and, 159 Liver genomic instability and, 132 immunosuppression and, 257 lysosomes and, 278, 281, 283 MHC Class I expression and, 215-216 multidrug resistance gene and, 165166, 169, 172 Long terminal repeats, MHC Class I expression and, 198-199 Loss of heterozygosity, genomic instability and, 127, 137-138, 143-146 Lung cancer genomic instability and, 144 MHC Class I expression and, 190, 194 downmodulation, 227 modulation by oncogenes, 200, 203204, 206 multidrug resistance gene and, 166167 soft tissue tumors and, 101-102

Lymphoblastoid cells, MHC Class I expression and, 216, 227 Lymphocyte blastogenesis inhibitory factor, immunosuppression and, 252, 258, 261 Lymphocytes genomic instability and, 126, 139 immunosuppression and, 247-249, 252-253,257-258,261 MHC Class I expression and, 182-184, 211 Lymphoid cells, MHC Class I expression and, 203 Lymphokine-activated killer cells immunosuppression and, 248-249, 259 MHC Class I expression and, 183 Lymphokines, immunosuppression and, 249 Lymphoma, see also Burkitt’s lymphoma genomic instability and, 139 MHC Class 1 expression and, 189-190, 217,219, 223,227 multidrug resistance gene and, 166, 168-169 protein tyrosine kinase GFRs and, 63 soft tissue tumors and, 107-108 Lysosomal enzymes, 269-277, 286 anticancer therapy, 281-283, 285-286 tumor microenvironment. 278-279 Lysosomes, 269-270, 286 anticancer therapy drugs, 284-286 hyperthermia, 283 radiation, 28 1-283 enzyme activity in tumors, 271-275 methods for study, 270-271 tumor invasion, 275-277 tumor microenvironment, 278 hypoxia, 278-279 pH, 279-281

M Macrophages immunosuppression and, 247-249, 252, 254, 256, 259, 261 lysosomes and, 271

304

INDEX

Major histocompatibility complex Class I expression, see MHC Class I expression Malignancy genomic instability and, 122, 127, 131, 143, 145

immunosuppression and, 248-249, 251, 253,258-259

lysosomes and, 270, 274, 277, 286 MHC Class 1 expression and, 182, 190, 203, 223

multidrug resistance gene and, 167 soft tissue tumors and, 76-78, 81, 85, 93, 105

Malignant fibrous histiocytomas, soft tissue tumors and, 76, 78, 105, 109 chromosomal abnormalities, 95, 99 tumor suppressor genes, 101-102 Malignant peripheral nerve sheath tumor (MPNST), soft tissue tumors and, 7677, 84, 95, 100, 104

Mast cell growth factor, protein tyrosine kinase GFRs and, 51-54 mdr regulation of expression, 169-176 transporter, 160, 162-163 MDRl regulation of expression, 169-175 transporter, 162, 165-168 mdr-1, soft tissue tumors and, 106 Mechanisms of genomic instability, tumor progression and, see G n o m i c instability, tumor progression and Melanocytes FGF receptor multigene family and, 24 MHC Class I expression and, 202 protein tyrosine kinase GFRs and, 5154, 60, 66 Melanogenesis, protein tyrosine kinase GFRs and, 50 Melanoma immunosuppression and, 253 lysosomes and, 276-277 MHC Class I expression and cancer, 184, 188-190, 194-195 downmodulation, 222-224, 227-228 modulation by oncogenes, 204-207 regulation by oncogenes, 218, 22022 1

protein tyrosine kinase GFRs and, 66

Mesenchyme cells, soft tissue tumors and, 77,94, 108-109

Mesotheliomas, characterization of, 7677,79

Messenger RNA, see mRNA MET receptors, protein tyrosine kinase GFRs and, 46,63 Metastasis genomic instability and, 123, 126 immunosuppression and, 258, 261 lysosomes and, 275-277, 280, 286 MHC Class I expression and, 233 downmodulation, 222-223, 225, 227 modulatiori by oncogenes, 200-201 multidrug resistance gene and, 157-158 Methylation genomic instability and, 130, 140-141 MHC Class I expression and, 216-217 soft tissue tumors and, 94 MHC Class I expression, myc oncogene activation and, 181-183, 231-233 cancer immune defense, 183-189 tumors, 189-195 downmodulation, 222 HLA, 227-230 immune reaction, 225-227 NK susceptibility, 230-231 progression, 222-224 T cells, 224-225 modulation adenovirus E I A , 195-197 199-201 myc, 203-208 raf, 199, 201-202 r a , 199,202

~OS,

viral oncogenes, 197-199 regulation mechanisms, 208-217 tumor cells, 217-222 &Microglobulin, MHC Class I expression and cancer, 185-186, 194 downmodulation, 226-227 modulation by oncogenes, 195-196, 200,205-206

regulation by oncogenes, 212-213 Microtubules, genomic instability and, 134-135

Mitochondria, genomic instability and, 132

305

INDEX

Mitogens FGF receptor multigene family and, 26,24,33-34 immunosuppression and, 247-248, 253-254 lysosomes and, 277 protein tyrosine kinase GFRs and, 53 Mitosis genomic instability and, 133-135, 144 lysosomes and, 283 soft tissue tumors and, 103 Mitotic index, soft tissue tumors and, 88 Moloney murine leukemia virus, MHC Class I expression and, 198-199, 212, 228 Moloney murine sarcoma virus, MHC Class I expression and, 198, 212 Monoclonal antibodies MHC Class I expression and, 211 multidrug resistance gene and, 165, 171 soft tissue tumors and, 106 Monocytes, immunosuppression and, 253 mos, MHC Class I expression and, 199 mRNA FGF receptor multigene family and, 2, 23,29,33-34 alternative splicing, 15, 28-29 early studies, 6 mitogens, 5 multiple forms, 18-20 immunosuppression and, 251 lysosomes and, 273, 277 MHC Class I expression and cancer, 190, 194 modulation by oncogenes, 196, 202, 205 regulation by oncogenes, 208, 210, 214 regulation in tumor cells, 217-218, 22 I protein tyrosine kinase GFRs and, 49, 52,66 Multidrug resistance lysosomes and, 284-285 soft tissue tumors and, 105-107 Multidrug resistance gene, human, 176 regulation of expression, 169 development, 171 P-glycoprotein, 175-176 promoters, 171- 174

protein kinase, 170 RNA, 169-170 tumor progression, 174-175 transporter, 157-158 clones, 159-163 drug efflux pump, 159 gene expression, 165-167 inhibitors, 168-169 mechanism of action, 163-165 in vitro model systems, 158-159 in vivo model systems, 167-168 Multiple myeloma, multidrug resistance gene and, 168 Mutation FGF receptor multigene family and, 23, 33,35 genomic instability and, 123-124, 126127, 145-147 mechanisms, 128-134, 136, 138-142 quantitative considerations, 142-145 MHC Class I expression and, 181, 201202, 210, 215 protein tyrosine kinase GFRs and development, 50-60 genetics, 66 tumorigenesis, 60-62, 64-65 soft tissue tumors and rm gene, 87-88 susceptibility, 80-84 tumor suppressor genes, 100-101, 103 mutD, genomic instability and, 129 m a , genomic instability and, 132 mYC MHC Class I expression and, 231-233 modulation by oncogenes, 199, 203208 regulation by oncogenes, 218-219 oncogene activation, MHC Class I expression, see MHC Class1 expression, myc oncogene activation and soft tissue tumors and, 77, 109 myf, soft tissue tumors and, 110 Myxoid tumors, soft tissue tumors and, 94, 100

N N-myc MHC Class I expression and, 222, 231

306

INDEX

modulation by oncogenes, 203-208 regulation by oncogenes, 217-222 soft tissue tumors and, 104-105 N-704 MHC Class 1 expression and, 201 Natural killer cells immunosuppression and, 247, 249, 253-256 MHC Class I expression and, 182, 231233 cancer, 183, 185, 187 downmodulation, 224-231 modulation by oncogenes, 202 Negative regulatory element, MHC Class I expression and, 210, 213, 216, 220 Neoplasia genomic instability and, 124 lysosomes and, 270 MHC Class I expression and, 182-183, 223 protein tyrosine kinase GFRs and, 65 soft tissue tumors and, 77, 80, 82, 88, 92, 107 Neovascularization. FGF receptor multigene family and, 35 Neu, protein tyrosine kinase GFRs and, 61 NEU, protein tyrosine kinase GFRs and, 63-66 Neuroblastoma MHC Class I expression and, 194-195 modulation by oncogenes, 204-207 regulation by oncogenes, 218-219, 221 multidrug resistance gene and, 166, 171, 174 Neutrophils, lysosomes and, 271 NFI genes, soft tissue tumors and, 100, 109 NF-KB,MHC Class I expression and, 213-214, 217, 219 Nucleosides genomic instability and, 132, 142-143 multidrug resistance gene and, 158 Nucleosomes, genomic instability and, 134 Nucleotides genomic instability and, 127-129, 139 MHC Class I expression and modulation by oncogenes, 203, 208 regulation by oncogenes, 211, 213215,220 multidrug resistance gene and, 165, 172

0 Oncogenes, see also Protooncogenes FGF receptor multigene family and, 11 genomic instability and, 137, 140, 142, 146 lysosomes and, 277 MHC Class I expression and, see MHC Class 1 expression, myc oncogene activation and multidrug resistance gene and, 174-175 protein tyrosine kinase GFRs and, 60, 62-66 soft tissue tumors and, 77, 87, 101, 105, 109 Orphan receptors, protein tyrosine kinase GFRs and, 46 Osteosarcoma, characterization of, 84, 101-102 Ovarian cancer immunosuppression and, 251, 258 lysosomes and, 276 MHC Class 1 expression and, 194, 224 multidrug resistance gene and, 166, 169 protein tyrosine kinase GFRs and, 65 Oxidative DNA damage, genomic instability and, 130-132 Oxygen genomic instability and, 145-147 lysosomes and, 279

P P53 genomic instability and, 123 multidrug resistance gene and, 174-175 soft tissue tumors and, 77, 82-83, 100103, 109 P-gl ycoprotein lysosomes and, 284-285 multidrug resistance gene and regulation of expression, 170-171, 175-176 transporter, 159-161, 163-168 soft tissue tumors and, 106-107 patch, protein tyrosine kinase GFRs and, 50, 54-56 P15E, immunosuppression and, 252-254, 259

INDEX

Peptides FGF receptor multigene family and, 3, 8-9, 15, 30, 32 MHC Class I expression and, 182 cancer, 184, 186-188 downmodulation, 224, 229-231 modulation by oncogenes, 197, 202 multidrug resistance gene and, 162, 171 p g f , multidrug resistance gene and, 171172, 174 PH FGF receptor multigene family and, 31 genomic instability and, 145 lysosomes and, 271, 275 microenvironment, 279-281 therapy, 284-286 multidrug resistance gene and, 158 Phagocytosis immunosuppression and, 249, 254 lysosomes and, 270 Phenotype FGF receptor multigene family and, 2, 4, 32 genomic instability and, 122-123, 126127, 145-146 mechanisms, 131-134, 141 quantitative considerations, 142-145 immunosuppression and, 249 lysosomes and, 277, 284, 285 MHC Class I expression and, 194-195, 201, 223,230 multidrug resistance gene and, 160 protein tyrosine kinase GFRs and, 45 development, 50-56, 59 tumorigenesis, 65 soft tissue tumors and, 77, 80, 85 Phospholipase C, FGF receptor multigene family and, 32-33 Phosphorylation FGF receptor multigene family and, 3133 MHC Class I expression and, 214, 219 multidrug resistance gene and, 175176 protein tyrosine kinase GFRs and, 43, 62 Ph ytohemagglutinin genomic instability and, 126 immunosuppression and, 258 MHC Class I expression and, 230

307

Plasma membrane lysosomes and, 276, 284-285 multidrug resistance gene and, 159160, 163-164 Plasmacytoma, immunosuppression and, 252,259 Platelet-derived growth factor, FGF receptor multigene family and, 33 Platelet-derived growth factor-a, protein tyrosine kinase GFRs and, 45, 54-55 Platelet-derived growth factor+ FGF receptor multigene family and, 9, 13 protein tyrosine kinase GFRs and, 45, 50 Ploidy genomic instability and, 125-126, 134136 soft tissue tumors and, 105-106 Polymerase chain reaction FGF receptor multigene family and, 11, 13, 19 multidrug resistance gene and, 168 protein tyrosine kinase GFRs and, 46 soft tissue tumors and, 87, 103 Polymerization, genomic instability and, 128 Polymorphism, MHC Class I expression and, 185-186, 217 Polypeptide mitogens, FGF receptor multigene family and, 2-5 Polypeptides genomic instability and, 129 immunosuppression and, 252, 254 multidrug resistance gene and, 162 Positive regulatory element, MHC Class I expression and, 212 Post-transcriptional regulation, MHC Class 1 expression and, 196, 198-199 Post-translational regulation, multidrug resistance gene and, 169, 175-176 Progesterone, multidrug resistance gene and, 165, 171 Prokaryotes, genomic instability and, 129130 Pr,oliferation hmunosuppression and, 248-249, 252-254,256-257 lysosomes and, 274, 278 MHC Class I expression and, 181-183, 199-200,203

308

INDEX

Promoters MHC Class I expression and, 190 modulation by oncogenes, 200, 205, 208 regulation by oncogenes, 209, 215, 219 multidrug resistance gene and, 171175 Prostate cancer, genomic instability and, 146-147 Protein FGF receptor multigene family and, 23-27 characterization, 8-15 early studies, 6 mitogens, 3, 5 multiple forms, 15, 18-20, 22-23 signal transduction, 31-33 genomic instability and, 125, 130, 134136, 140 immunosuppression and, 248, 251-253, 257-258 lysosomes and, 269, 277, 281 MHC Class I expression and, 181-183 cancer, 184-188, 190, 194 downmodulation, 227-228, 230 modulation by oncogenes, 196, 201206 regulation by oncogenes, 210-215 regulation in tumor cells, 218-220. 222 multidrug resistance gene and, 160, 162, 165, 170, 174 protein tyrosine kinase GFRs and, 43, 66 development, 52-53, 57-60 structure, 45-47 tumorigenesis, 61-65 soft tissue tumors and, 77 chromosomal abnormalities, 95, 99 susceptibility, 84-85 tumor suppressor genes, 101-103 Protein kinase FGF receptor multigene family and, 9 niultidrug resistance gene and, 170, 175-176 Protein kinase C, MHC Class I expression and, 219, 221-222 Protein tyrosine kinase, soft tissue tumors and, 107

Protein tyrosine kinase GFRs, 43-45 development, 49-50 mutants of Drosophilu rnelunogmter, 56-60 mutants of mouse, 50-56 dominant white (W)spotting, 50, 52-53, 55-56 genetics, 65-66 structure, 45-49 tumorigenesis, 60-61 cellular oncogenes, 63-65 retroviral oncogenes, 61-62 Proteolysis immunosuppression and, 258 lysosomes and, 275, 277 Protooncogenes MHC Class I expression and, 181, 199200, 203 protein tyrosine kinase GFRs and, 64, 66 soft tissue tumors and, 89, 94-95, 104, 108

Q QlO, MHC Class 1 expression and, 214217 Qa-Tla genes, MHC Class I expression and. 185-186

R R7 cell, protein tyrosine kinasc GFRs and, 57-60 R8 cell, protein tyrosine kinase GFRs and, 60 RAD mutants, genomic instability and, 132 Radiation genomic instability and, 131, 145 MHC Class I expression and, 187-188 Radiation therapy, lysosomes and, 278, 281-283 Radiothaerapy, soft tissue tumors and, 78 raf, MHC Class I expression and, 199, 202 rapl, genomic instability and, 136 ras

MHC Class I expression and, 187, 198203, 218

309

INDEX

soft tissue tumors and, 77, 84, 87-88, 100,109 RAS, multidrug resistance gene and, 174 RBI, soft tissue tumors and, 85, 98, 100, 102-103, 105 Receptor protein tyrosine kinases, see Protein tyrosine kinase GFRs Recombination FGF receptor multigene family and, 3 genomic instability and, 123-125, 127, 146 mechanisms, 134, 137-140, 142 quantitative considerations, 144 soft tissue tumors and, 103 rel, MHC Class I expression and, 214 Renal cell carcinoma, MHC Class I expression and, 205, 224 Renal tumors, MHC Class I expression and, 206 Replication genomic instability and, 127-130, 132, 134, 136, 138, 142,144 MHC Class I expression and, 199 RET, protein tyrosine kinase GFRs and, 63-64 Retina, protein tyrosine kinase GFRs and, 57 Retinoblastoma characterization of, 84-85, 102 genomic instability and, 127, 146 Retrovirus immunosuppression and, 252-253 MHC Class I expression and, 198-199 protein tyrosine kinase GFRs and, 51, 60-62 soft tissue tumors and, 77 Reverse genetics, soft tissue tumors and, 108 .fp, protein tyrosine kinase GFRs and, 63 Rhabdom yosarcomas characterization of, 76-77, 104, 106, 108, 110 chromosomal abnormalities, 97-99 rm gene, 87-88 susceptibility, 82, 85 tumor suppressor genes, 101, 103 immunosuppression and, 251 Ribosomes, protein tyrosine kinase GFRs and, 63

RNA MHC Class I expression and, 221 multidrug resistance gene and, 160, 165-171 RNA polymerase, genomic instability and, 132 Rous sarcoma virus MHC Class I expression and, 198 multidrug resistance gene and, 175

S Sarcoma viruses, MHC Class I expression and, 198-199 Sarcomas, see also Soft tissue tumors, human lysosomes and, 279-280 MHC Class I expression and, 190, 201, 222 multidrug resistance gene and, 166 sld, protein tyrosine kinase GFRs and, 52-53 SDS-PAGE, immunosuppression and, 254 Self-antigens, MHC Class I expression and, 182, 189, 196, 225 Sequences FGF receptor multigene family and, 23, 34-35 alternative splicing, 28 characterization, 8, 11-13 Drosophila, 30-31 ligand binding, 24-27 mitogens, 3, 5 multiple forms, 17-21, 23 genomic instability and, 124-125 mechanisms, 127, 136, 139, 141-142 MHC Class I expression and cancer, 186-187, 190 modulation by oncogenes, 197-198, 200,203,205,208 regulation by oncogenes, 208-214, 216 regulation in tumor cells, 218, 220 multidrug resistance gene and, 160, 162, 171,173 protein tyrosine kinase GFRs and, 46, 52, 61-62, 64-65 SER, immunosuppression and, 254, 259 sevenless, protein tyrosine kinase GFRs and, 57-60

310

INDEX

Signal transduction FGF receptor multigene family and, 3134 MHC Class I expression and, 202, 219, 226 protein tyrosine kinase GFRs and, 43, 45, 60 soft tissue tumors and, 99 Simian virus 40 genomic instability and, 135 MHC Class I expression and, 210, 213 multidrug resistance gene and, 175 soft tissue tumors and, 100, 102 Skin cancer genomic instability and, 132 MHC Class I expression and, 222 Soft tissue tumors, human, 75-78 chromosomal abnormalities, 88-89, 99100 leiom yomas, 91-93 lipomas, 89-91 liposarcomas, 93-95 malignant fibrous histiocytomas, 99 rhabdomyosarcomas, 97-99 synovial sarcomas, 95-96 cloning, 107-109 etiology, 78-80 future, 109-110 gene amplification, 104-105 predictors of behavior, 105-107 rm gene, 87-88 susceptibility, 80 Beckwith- Wiedemann syndrome, 85 benign tumors, 85-87 fibromatosis, 80-82 Li-Fraurneni syndrome, 82-83 retinoblastoma, 84-85 Von Recklinghausen’s neurofibromatosis, 83-84 tumor suppressor genes, 100-104 Spindle pole body, genomic instability and, 135 Spleen, lysosomes and, 281-283 STE6, multidrug resistance gene and 162 steet, protein tyrosine kinase GFRs and, 50-52 Stefins, lysosomes and, 276-277 Stem cell factor, protein tyrosine kinase GFRs and, 51-54

Steroids, multidrug resistance gene and, 165 Suppressive E-receptor, immunosuppression and, 253-254 Susceptibility to soft tissue tumors, 80-87 syn, genomic instability and, 129 Synovial sarcomas, characterization of, 95-96, 102, 108

T T cell lymphoma genomic instability and, 139 protein tyrosine kinase GFRs and, 63 T cell receptors genomic instability and, 138-139 soft tissue tumors and, 107 T cells genomic instability and, 124, 137, 139 immunosuppression and, 247, 249, 251-252, 254 MHC Class I expression and, 182, 231, 233 cancer, 183-184, 187, 189, 195 downmodulation, 223-227 modulation by oncogenes, 196, 199200,202 regulation by oncogenes, 210 TI0 cells, MHC Class I expression and, 222 Target interference, MHC Class I expression and, 226 Telomeres genomic instability and, 134, 136 MHC Class I expression and, 185 6-Thioguanine, genomic instability and, 126-127 TIMP, soft tissue tumors and, 95-96 Tissue specificity FGF receptor multigene family and, 25, 28-30 genomic instability and, 140 MHC Class I expression and, 200, 215216, 222 soft tissue tumors and, 110 Topoisomerases, genomic instability and, I34

INDEX

torpedo, protein tyrosine kinase GFRs and, 56-57 Toxic injury, multidrug resistance gene and, 169-170 Toxins, genomic instability and, 134 trans-acting factors, MHC Class I expression and, 209, 213-216 Trans Colgi network, lysosomes and, 285 Transcription FGF receptor multigene family and, 2, 23,33-34 alternative splicing, 28 Drosqphila, 29-30 mitogens, 5 multiple forms, 8, 18-19 signal transduction, 31 genomic instability and, 132, 140-141 MHC Class 1 expression and, 183, 189 modulation by oncogenes, 196-200, 203,207-208 regulation by oncogenes, 208-210, 212-217 multidrug resistance gene and, 169-174 protein tyrosine kinase GFRs and, 57, 64-66 soft tissue tumors and, 102, 108, 110 Transfection genomic instability and, 141 immunosuppression and, 259 lysosomes and, 277 MHC Class I expression and, 231 downmodulation, 222, 225, 227-228 modulation by oncogenes, 197, 200202,205-206 regulation by oncogenes, 219-221 multidrug resistance gene and, 170 protein tyrosine kinase GFRs and, 6364 soft tissue tumors and, 77, 87, 108 Transforming growth factor+, immunosuppression and, 249, 251-252, 255,258-259,261 Translation, multidrug resistance gene and, 160, 172 Translocation genomic instability and, 139 MHC Class I expression and, 203 multidrug resistance gene and, 160 soft tissue tumors and, 78, 109

311

chromosomal abnormalities, 88-97, 99- 100 cloning, 107-109 Transmembrane domains, multidrug resistance gene and, 160-162 trk, protein tyrosine kinase GFRs and, 4649, 61, 63 Tropomyosin, protein tyrosine kinase GFRs and, 47, 63 Tu, protein tyrosine kinase GFRs and, 6566 Tubulin, genomic instability and, 135 Tumor-infiltrating lymphocytes (TIL) immunosuppression and, 248-249 MHC Class I expression and, 183-184 Tumor necrosis factor immunosuppression and, 261 MHC Class I expression and, 207, 230 Tumor progression genomic instability and, see Genomic instability, tumor progression and multidrug resistance gene and, 174-175 Tumor-promoting agent, MHC Class I expression and, 213, 219 Tumor suppressor genes genomic instability and, 123, 137, 140142, 146 MHC Class 1 expression and, 181-182, 195 multidrug resistance gene and, 174-175 soft tissue tumors and, 78, 82, 100-104 Tumorigenesis, protein tyrosine kinase GFRs and, 45,60-66 Tumors FGF receptor multigene family and, 4,35 immunosuppression and, 248-255, 257-259,261 lysosomes and, 270-271, 286 enzymes, 271-275 microenvironment, 278- 28 1 therapy, 282, 284 MHC Class I expression and, 181-183, 231-233 cancer, 184-185, 187-195 downmodulation, 222-228, 230-231 modulation by oncogenes, 197, 199203,205-208 regulation by oncogenes, 211, 217222

312

INDEX

multidrug resistance gene and, 166167, 169 Tyrosine kinase, FGF receptor multigene family and, 30,32 characterization, 8-9, 13 ligand binding, 24 multiple forms, 23 Tyrosine kinase, protein, see Protein tyrosine kinase

U Ultraviolet light genomic instability and, 131-132 immunosuppression and, 259 MHC Class I expression and, 187-188

v v-fmr, protein tyrosine kinase GFRs and,

62 v-kit, protein tyrosine kinase GFRs and, 62 v-mos, MHC Class I expression and, 198 v-myc, MHC Class I expression and, 198 v-mf, MHC Class I expression and, 202 Vascularization, FGF receptor multigene family and, 3-4 Verapamil lysosomes and, 284 multidrug resistance gene and, 168-169 Very short patch repair, genomic instability and, 130 Vinhlastine, multidrug resistance gene and, 158-159, 168, 172 Vincristine, multidrug resistance gene and, 168, 170 VAD therapy, 168 Viruses immunosuppression and, 253

MHC Class 1 expression and, 195-199, 212-214, 224 Vitamin A, lysosomes and, 282, 285 Von Recklinghausen’s neurofibromatosis, soft tissue tumors and; 83-84

W Wilm’s tumor multidrug resistance gene and, 166-167 soft tissue tumors and, 85, 103

X Xenobiotics MHC Class I expression and, 187 multidrug resistance gene and, 165 Xenopus, FGF receptor multigene family and, 4-5,9 Xenopus h i s , protein tyrosine kinase GFRs and, 46 Xeroderma pigmentosum genomic instability and, 132 soft tissue tumors and, 80 Xaphophorus, protein tyrosine kinase GFRs and, 65-66 X I S T , genomic instability and, 141 Xmrk, protein tyrosine kinase GFRs and, 66

Y Yeast genomic instability and, 133-136, 143, 147 multidrug resistance gene and, 162 Yeast artificial chromosomes, soft tissue tumors and, 108