Advances in Cancer Research Volume 53

ADVANCES IN CANCER RESEARCH VOLUME 53

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ADVANCES IN CANCER RESEARCH Edited by

GEORGE F. VANDE WOUDE NCI-Frederick Cancer Research Facility Frederick, Maryland

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

Volume 53

ACADEMIC PRESS, INC. Harcourt Brace Jovanovlch, Publirhers

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COPYRIGHT 0 1989 BY ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

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CONTENTS

CONTRIBUTORS TO VOLUME5 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

Serum-inducible Genes

BARRETTJ. ROLLINS AND CHARLES D. STILES I. Introduction .................. 11. History: The .......................... 111. Biochemistry: The Role of Transcription and Translation in the Mitogenic Response to Serum .............. IV. Molecular Biology: Serum-Inducible Genes and Their Products . . . . . . . . . . . . . . V. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Addendum.. . . . . . . .... ................ References . . . . . . . . .... ................

1 3 5

8 21 22 25

Malaria, Epstein-Barr Virus, and the Genesis of Lymphomas CHRISTINE I. 11. 111. IV. V.

A. FACER AND J. H.L. PLAYFAIR

Introduction ................................ The Epstein-Barr Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Burkitt’s Lymphoma Malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Malaria and the Immune System

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

34 46

68

A Review of Kaposi’s Sarcoma JANE

I. 11. 111. IV.

ARMES

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Features and Epidemiology of Kaposi’s Sarcoma ..................... Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell of Origin of KS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V

73 73 76 76

vi

CONTENTS

Neoplasm or Hyperplasia? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. KS . VI . Etiology of KS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

79

80 85 85

The Relationship between MHC Antigen Expression and Metastasis JACOB

GOPAS.BRACHARAGER.ZISMAN. MENASHEBAR.ELI. GUNTERJ . HAMMERLING. AND SHRAGA SEGAL

89 I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. MHC Antigens. Tumorigenicity. and Metastasis in Animal Models . . . . . . . . . . . 91 111. MHC Regulation of Antitumor Immunity by Cytotoxic T Cells . . . . . . . . . . . . . . . 93 97 I v. MHC Regulation of Antitumor Immunity by NK Cells ...................... V. MHC Antigens. Tbmorigenicity. and Metastasis in Man ..................... 103 VI . Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Genetics of Tumor Susceptibility in the Mouse: MHC and Non-MHC Genes

P. DEMANT. L . c. J . M . OOMEN.

AND

M . OUDSHOORN-SNOEK

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Site of Action of Tumor Susceptibility Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Biology of Tumor Susceptibility Genes IV. Genetic Definition of lhmor Susceptibi V. Major Histocompatibility Complex-Structure and Function . . . . VI . Susceptibility to Epithelial Tumors and the Role of MHC .................... VII . Tumor Susceptibility Genes: Molecular and Cellular Perspective . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

117 119

150 169 170

Perspectives on the Role of MHC Antigens

BRUCEE . ELLIOTT.DOUGLAS A . CARLOW. ANNA-MARIE RODRICKS. AND ANDREWWADE I. I1 . I11. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Basis of Tumor Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Biologyof MHC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MHC Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

161

182 186 191

CONTENTS V. VI. VII. VIII. IX.

X.

MHC Expression in Malignancy.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence for a Role of MHC Antigens in Malignancy ....................... Proposed Function of MHC in Malignancy ....... Regulation of Altered Class I MHC Expression in Malignancy. . . . . . . . . . . . . . . . Organ- and Tissue-Specific Effects on Immune Surveillance and Tumor Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii 193 200 209 220 229 232 234

Antioxidants-Carcinogenic and Chemopreventive Properties

NOBUYUKI ITO I. 11. 111. IV. V. VI. VII.

INDEX

AND

MASAOHIROSE

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tumorigenic Effects of Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Histopathological Characteristics of Antioxidant-Induced Tumors . . . . . . . . . . . . . Possible Mechanisms of Action of BHA in Forestomach Tumorigenesis . . . . . . . . . Modification of Carcinogenesis by Antioxidants Evaluation of Antioxidants as Human Hazards or Chemopreventers of Human Carcinogenesis Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . .......

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247 249 258 269 270 288 291 293 303

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CONTRIBUTORS TO VOLUME 53 Numbers in parentheses indicate the pages on which the authors' contributions begin.

JANEARMES,Institute of Cancer Research, Chester Beatty Laboratories, London, England, SW3 6JB (73) MENASHEBAR-ELI,Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheua 84 105, Israel (89) DOUGLASA . CARLOW,Mount Sinai Hospital Research Institute, Toronto, Ontario, Canada, M5G 1 x 5 (181) I? DEMANT,Division of Molecular Genetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, T h e Netherlands (117) BRUCEE. ELLIOTT,Division of Cancer Research, Department of Pathology, Queen's University, Kingston, Ontario, Canada, K7L 3N6 (181) CHRISTINEA. FACER,Department of Haematology, T h e London Hospital Medical College, London, England, El 2AD ( 3 3 ) JACOB GOPAS,T h e Institute of Oncology, Soroku Medical Center, and Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gunbn University of the Negev, Beer-Sheva 84 105, Israel (89) GUNTERJ. HAMMERLING, Institute f o r Immunology and Genetics, German Cancer Research Center, 0-6900 Heidelberg, Federal Republic of Germany (89) MASAOHIROSE,First Department of Pathology, Nagoya City University, Medical School, Mizuho-cho, Mizuho-ku, Nagoya 467, Japan (247) NOBUYUKI ITO, First Department of Pathology, Nagoya City University, Medical School, Mizuho-cho, Mizuho-ku, Nagoya 467,Jafian (247) L. C. J. M . OOMEN,Division of Molecular Genetics, T h e Netherlands Cancer Institute, 1066 CX Amsterdam, T h e Netherlands (117) M. OUDSHOORN-SNOEK, Division of Molecular Genetics, T h e Netherlands Cancer Institute, 1066 CX Amsterdam, T h e Netherlands (117) ix

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CONTRIBUTORS TO VOLUME 59

J. H. L. PLAYFAIR, Department of Immunology, University College and Middlesex School of Medicine, London, England, W1P 9PG ( 3 3 ) BRACHARACER-ZISMAN,Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Shew 84 105, Israel (89) ANNA-MARIE RODRICKS, Department of Oncology Research, Toronto General Hospital, Toronto, Ontario, Canada, M I G 2C4 (181) BARRETTJ . ROLLINS,Dimkion of Medicine, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 (1) SHRAGASEGAL,Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Shew 84 105, Israel (89) CHARLESD. STILES,Dimkion of Cellular and Molecular Biology, DanaFarber Cancer Institute, and Department of Microbiology and Molecular Genetics, Haruard Medical School, Boston, Massachusetts 02115 (1) ANDREWWADE, M I R Office, Faculty of Medicine, University of Calgay , Calgay,Alberta, Canada, T 2 N 1N4 (181)

SERUM-INDUCIBLE GENES Barrett J. Rollins' and Charles D. Stilest 'Oivision 01 Medicine. Dana-Farber Cancer Institute, Harvard Medical school, Boston, Massachusetts 02115 tDivision of Cellular and Molecular Biology, Dana-Fartmr Cancer Institute. and Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

I. Introduction 11. History: The Mitogenic Response to Serum A. Serum Requirement for Animal Cell Growth in Vitro B. Two Stages in the Mitogenic Response to Serum: Competence and Progression C. The Functional Components of Serum 111. Biochemistry: The Role of Transcription and Translation in the Mitogenic Response to Serum A. Relatively Minor Quantitative Transcriptional Changes in the Mitogenic Response to Serum B. Serum-Induced Transcription of Proliferation-Specific mRNA C. Requirement of Gene Expression for the Establishment of Competence IV. Molecular Biology: Serum-Inducible Genes and Their Products A. Proliferation-Related Proteins B. Growth Factor-Inducible mRNAs Detected by Differential Screening of Gene and cDNA Libraries C. Growth Factor-Inducible mRNAs Derived from Identified Genes D. Other Serum-Inducible mRNAs V. Perspectives VI. Addendum References

I. Introduction

Eagle, Temin, Holley, and others share in the important discovery that serum contains growth factors that are required for animal cell proliferation in vitro. The mitogenic response to these serum growth factors depends on transcription of unique-sequence genes and translation of their cognate mRNAs. Paradoxically, there is little change in the rate of transcription or translation during cell growth. Likewise there is little change in the complexity of mRNA during transit through the cell cycle. This paradox has been resolved by the observation that serum growth factors stimulate expression of a small number of vary labile messenger RNAs in their target cells. Specific gene sequences induced by serum growth factors have been identified. Of the many growth factors contained in serum, four have been exten1 ADVANCES IN CANCER RESEARCH, VOL. 53

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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sively studied in the context of the cell cycle and oncogene expression. These are platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin like growth factors (IGF), and transforming growth factor p (TGF-P). This review will focus on genes for which expression is regulated by these four agents. Such genes can be grouped into two sets. The first consists of genes that respond to growth factors as a secondary consequence of transit through the cell cycle. For convenience, our laboratory refers to these as “progression genes.” The abundance of progression mRNAs fluctuates in cycling cells, and their induction is blocked when growth factors are added together with protein synthesis inhibitors such as cycloheximide. By these criteria, the progression genes encode familiar proteins such as the histones, thymidine kinase, and dihydrofolate reductase. Some cellular protooncogenes such as p53 and c-myb may also be contained within this group. The expression of other serum-inducible genes is clearly not dictated by cell cycle traverse. These have been termed “immediate early genes” by Nathans and colleagues and “cell division cycle genes” by Baserga. Our own laboratory refers to them as “competence genes”-a term that will be used hereafter only through force of habit. This set of genes is defined by superinduction of their expression in the presence of a growth factor plus a protein synthesis inhibitor. This review is primarily concerned with the structure and function of this set of genes. At least three cellular protooncogenes (c-myc, c-fos, and c-jun) are members of the competence gene family. The proteins encoded by these genes appear to function as intracellular mediators of the mitogenic response to growth factors such as PDGF. Other competence genes encode nuclear proteins that may be functionally related to these protooncogenes, although a direct link to cell cycle control and neoplasia has yet to be established. Still other competence genes encode proteins that cannot possibly function as intracellular mediators of growth factor action for the simple reason that they are secretory proteins. These proteins may play a systemic role in growth factor physiology. This review draws heavily on data generated by the examination of competence gene expression in density-inhibited murine connective tissue cells such as 3T3 cells. However, the central features of competence gene expression transcend cell and tissue boundaries. The response of lymphocytes to plant lectins such as concanavalin A (Con A) is especially instructive, and some of these data are reviewed. The molecular biology of the mechanisms that underlie competence gene induction is a rapidly developing area of investigation that is beyond the scope of this article. Readers are directed to several excellent reviews on signal transduction (Nishizuka, 1986; Rozengurt, 1986; Berridge, 1987).

SERUM-INDUCIBLE G E N E S

3

II. History: The Mitogenic Response to Serum

A. SERUMREQUIREMENTFOR ANIMALCELLGROWTHin Vitro In the late 1940s and 1950s, the growth requirements of animal cells in culture were explored systematically by Eagle, Fisher, Puck, and others. Their studies led to a disappointing, but inescapable conclusion, which was formally described by Eagle (1955). No matter how enriched and complex the nutrient medium may be, animal cells could not proliferate unless a small amount of animal protein, typically blood serum, was present in the medium. The extent of cellular proliferation, and the final saturation density that normal fibroblasts attain in culture was shown to be proportional to the concentration of serum in the medium (Holley and Kiernan, 1968; Clarke et al., 1970). When normal fibroblasts in culture were deprived of serum for one or more days, they entered a state of Go growth arrest. Addition of high concentrations of serum to these cells induced them to leave the Go state, progress through the G, phase of the cell cycle, replicate their DNA, and divide (Todaro et al., 1965; Burk, 1966, 1970; Wiebel and Baserga, 1969; Clarke et al., 1970; Dulbecco, 1970). The induction of DNA synthesis by serum occurred after a well-defined lag time of 12-16 hr after serum treatment. The precise length of this G , lag phase was cell type-specific, but was completely independent of the concentration of serum to which the cells were exposed (Temin, 1971; Brooks, 1975). It appeared that serum was inducing a metabolic change in cells that required 12-16 hr before it resulted in DNA synthesis. What was the nature of the metabolic change? The two opposing hypotheses were (1) that cells in low concentrations of serum were nutritionally starved, and exposure to high concentrations of serum simply replenished the cells’ nutrient pools, allowing them to resume growth; and (2) that serum induced the synthesis of a relatively short-lived macromolecular substance, the accumulation of which was necessary for cells to pass through G , into S phase (Pardee, 1974). Transient exposure, or “pulse” experiments argued against the first hypothesis. If quiescent cells in low concentrations of serum were exposed to high serum concentrations for only 3 hr and then placed back into low serum, the cells still underwent DNA synthesis after the obligatory 12-hr delay (Todaro et al., 1965; Wiebel and Baserga, 1969; Burk, 1970; Temin, 1971). Rather than replenishing nutrients, serum induced a metabolic change that persisted in the absence of continuous exposure to serum. Furthermore, cells were committed to this metabolic change after a relatively brief exposure to serum.

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BARRETT J. ROLLINS A N D CHARLES D . STILES

B. Two STAGESI N THE MITOGENICRESPONSETO SERUM:COMPETENCE AND PROGRESSION Attempts to isolate the mitogenic activity contained in serum had been fraught with failure for many years. Fractionation of serum usually led to attenuation or loss of activity (Paul et al., 1971; Pierson and Temin, 1972), suggesting that serum contained multiple factors that acted synergistically to induce proliferation. The first step toward solving this problem came from the observation of Balk (1971) that while chick fibroblasts grew rapidly in serum, they grew poorly in platelet-depleted plasma. In fact, a heated extract of purified platelets could be shown to reconstitute serum activity when added back to platelet-free plasma (Kohler and Lipton, 1974; Ross et a l . , 1974; Rutherford and Ross, 1976). The activity in platelet extracts was subsequently identified as PDGF (Antoniades et al., 1979; Heldin et al., 1979). The discovery that platelet extract plus plasma could reconstitute serum carried a corollary observation that platelet extract alone or plasma alone were not mitogenic. Experiments in the late 1970s demonstrated that these two components of serum work sequentially on their target cells. Plateletderived growth factor exerted its effects first, and by itself induced a state termed competence (Pledger et al., 1977). When competent cells were exposed to platelet-poor plasma (PPP), they progressed through the cell cycle (Pledger et al., 1977, 1978; Vogel et al., 1978). Cells not previously treated with PDGF (i.e., not competent) would not respond to plasma. This two-step mitogenic sequence was initially discovered in fibroblasts. It has a direct parallel, however, in the mitogenic activation of resting T lymphocytes. If Go-arrested T cells are treated only with lectin (Con A or phytohemagglutinin, PHA), antigen, phorbol esters, or anti-T-cell receptor antibodies, they do not proliferate. While not sufficient to induce proliferation, these stimuli are necessary, and they all induce the appearance of the receptor for interleukin 2 (IL-2) on the T-cell surface (Robb et al., 1981; Cantrell and Smith, 1983, 1984; Stern and Smith, 1986). At a critical level of IL-2 receptor expression, binding of IL-2 to its receptors will induce cells to enter S phase (Leonard et al., 1982; Smith et al., 1983; Cantrell and Smith, 1984; Stern and Smith, 1986). Thus the initial exposure to lectin, antigen, phorbol ester, or anti-T-cell receptor is analogous to the induction of competence in fibroblasts by PDGF. Subsequent exposure to IL-2 is analogous to the cell cycle progression induced in competent fibroblasts by plasma. Just as treatment of fibroblasts with plasma alone does not induce cell cycle progression, treatment of noncompetent T cells with IL-2 alone does not .lead to DNA synthesis (Stern and Smith, 1986). Extensive similarities in the patterns of gene expression between competent fibroblasts and T lymphocytes will be described in detail later.

SERUM-INDUCIBLE GENES

5

C. THE FUNCTIONAL COMPONENTS OF SERUM The studies of Pledger et al. (1977)and Vogel et al. (1978)established that PDGF regulated the initial event in the mitogenic response of 3T3 cells, namely competence. The same studies indicated, however, that treating cells with PDGF alone was not sufficient for eliciting an optimal mitogenic response. Other factors contained in the PPP fraction of blood regulated progression of PDGF-treated cells through G, and into the S phase of the cell cycle. Subsequent work showed that the active components in plasma were the IGF’s and EGF or an EGF-like agent such as TGF-ol (Stiles et al., 1979). The history, biochemistry, and biology of PDGF (Ross et al., 1986), EGF and TGF-a (Carpenter and Cohen, 1979; Derynck et al., 1984; Lee et al., 1985), and the IGF’s (Blundell and Humbel, 1980) have been reviewed. For fibroblasts, the last functional component of serum to be identified was TGF-P (Massague, 1987; Sporn et al., 1986). Studies by Assoian et al. (1984) and Roberts et al. (1985) showed that TGF-P cooperated with PDGF and the progression factors (EGF and IGF’s) to promote anchorage-independent fibroblast growth. Later studies from the laboratories of Moses, Sporn, and others (reviewed in Massague, 1987; Sporn et al., 1986) showed that TGF-P would also inhibit the growth of most nonfibroblast cell types.

Ill. Biochemistry: The Role of Transcription and Translation in the Mitogenic Response to Serum

A. RELATIVELY MINORQUANTITATIVE TRANSCRIPTIONAL CHANGES IN THE MITOGENIC RESPONSE TO SERUM An extensive literature has documented alterations in macromolecular synthesis in response to serum. Many cell types, from fibroblasts to lymphocytes, have been shown to increase their rate of protein synthesis during the G, lag period (Lieberman and Ove, 1962; Todaro et al., 1965; Stanners and Becker, 1971; Ahearn et al., 1974; Johnson et al., 1974). Much of this increase appears due to the ability of serum to chase cytoplasmic mRNA from the unbound state into polyribosomes (Levine et al., 1965; Stanners and Becker, 1971; Rudland, 1974). In addition, however, the overall rate of RNA synthesis increases during the G, lag, and does so before the increase in protein synthesis occurs (Lieberman and Ove, 1962; Lieberman et al., 1963a; Levine et al., 1965; Todaro et al., 1965; Burk, 1970; Abelson et al., 1974; Johnson et al., 1974, 1975). The degree of increased RNA synthesis is the same even if cells are exposed to serum for a 3-hr pulse (Todaro et al., 1965; Burk, 1970; Johnson et al., 1974). The details of the RNA response to

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serum are complex. The general increase in cytoplasmic RNA in stimulated cells is primarily due to an increase in ribosomal (rRNA) synthesis, but the relative increase in mRNA is much greater than that of rRNA (Johnson et al., 1974). The appearance of increased amounts of mRNA in the cytoplasm is due to an increased rate of processing and transfer of mRNA out of the nucleus, since the rate of hnRNA synthesis does not alter after serum treatment (Mauck and Green, 1973; Rovera et al., 1974; Johnson et al., 1975). Are these increases in the rate of protein and RNA synthesis necessary in order for cells to progress into S phase? This question has been answered by experiments using metabolic inhibitors. Addition of cycloheximide or puromycin to serum-stimulated cells prevents DNA synthesis (Harris, 1959; Powell, 1962; Lieberman and Ove, 1962; Terasima and Yasukawa, 1966; Kim et al., 1968; Wiebel and Baserga, 1969; Temin, 1971; Brooks, 1977). Inhibition of DNA synthesis occurs only if the inhibitors are added during the G , lag phase. They are no longer effective if the cells have already entered S phase (Mueller et al., 1962; Brooks, 1977). Similarly, treatment of cells with actinomycin D or 5,6-dichloro-a-~ribofuranosyl-benzimidazole (DRB) during the G, lag phase inhibits entry into S phase (Harris, 1959; Lieberman et al., 1963a,b; Baserga et al., 1965; Temin, 1971). There is also a genetic basis for the assertion that mRNA synthesis is required for progression into S phase. A temperature-sensitive cell cycle mutant of baby hamster kidney (BHK) cells has been described that does not synthesize DNA after serum stimulation at the restrictive temperature. This phenotype is caused by a temperature-sensitive mutation of RNA polymerase I1 (Rossini and Baserga, 1978). Furthermore, normal cell cycle responses to serum can be reinstated by microinjection of wildtype polymerase I1 into the nuclei of these mutant cells (Waechter et n l . , 1984). The metabolic inhibitor studies also suggest that the mRNAs and cognate proteins that are required for S-phase progression are unstable. Otherwise, their resynthesis during G, would not be necessary. Over 20 years ago, this hypothesis was tested by treating Chinese hamster ovary cells with serum followed by actinomycin D and cycloheximide. If cycloheximide is removed early after serum stimulation, these cells can still go on to synthesize DNA. By progressively delaying the removal of cycloheximide and determining the time at which DNA synthesis no longer occurs, the half-life of growthessential mRNAs can be estimated. This value was 2.9 hr (Tobey et n l . , 1966). Since cycloheximide alone can also inhibit RNA synthesis (Schniederman et aZ., 1971), the growth-essential product with a half-life of 50% of patients dead within a year of onset.

D. IMMUNOSUPPRESSION-ASSOCIATED KS This category includes patients who are immunosuppressed for reasons other than HIV infection. In one series (Penn, 1979), KS accounted for 3%of 600 malignancies among renal transplant recipients, an incidence of 150 times that seen in a normal Western population. The patients are usually younger than those who develop sporadic KS (mean age of onset is in the fourth decade), and their KS is closely related to the onset of their immunosuppressive therapy. Skin lesions are commonly seen in this form of KS, as is visceral involvement. Death is more often related to KS in this category of the disease than in sporadic KS. Immunosuppression-associated KS is most effectively treat-

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ed by removal or reduction of immunosuppressive therapy, if the immunosuppression is iatrogenic. This may result in regression of the tumor. In other cases chemotherapy or radiotherapy are sometimes successful. Ill. Pathology

Kaposi’s sarcoma has two major components: abnormal endothelial structures and spindle cell aggregations. Both are present in most lesions, but the proportion of either component can vary enormously. Histologically, KS is seen in three stages: patch, plaque, and nodular. The patch stage is probably the earliest and is the least cellular. It is composed of interlacing networks of thin-walled, dilated vessels with irregular outlines, which are lined by flattened endothelium. Spindle cell aggregations are few, and mitoses and cellular atypia are not seen. The plaque stage is more cellular and may represent a progression from the patch stage. The abnormal vasculature is still a predominant feature, but there are also collections of spindle cells. If the lesion is in the skin, it usually extends deeper in the dermis than the patch lesions. Again there are no mitoses nor cellular atypia. Many biopsy samples taken from AIDS patients show the patch and plaque morphology (Francis et al., 1986) (Fig. l ) , but these early lesions have also been reported in nonepidemic KS (Ackerman, 1979). In the nodular variety of KS (Figs. 2 and 3), there are well-defined aggregates of spindle cells and the tumor is much more cellular. Very small spaces, sometimes containing erythrocytes, are surrounded by spindle cells, and there is extravasation of erythrocytes among the spindle cells. Often the nodule is surrounded by a few thin-walled, dilated vessels reminiscent of the abnormal vessels seen in the patch and plaque stages. It is not clear whether nodular KS develops from patch and plaque lesions, or arises de novo. Usually the spindle cells do not show mitoses or cellular atypia. However, there is a histological variant, composed of spindle cells with a high mitotic rate and with cellular and nuclear pleomorphism. It is seen particularly in the locally aggressive, endemic form of KS. Postmortem findings from patients with disseminated KS (e.g., AIDSrelated), show that common sites of involvement other than skin are the lymph nodes, gastrointestinal tract, lungs, liver, and spleen. The histological patterns in these organs are similar to those described before. IV. Cell of Origin of KS

Kaposi’s sarcoma is thought to be a tumor of endothelial cells, mainly because of the presence of abnormal vessels within the tumor. Immu-

A REVIEW OF KAPOSI’S SARCOMA

FIG.2. Nodular KS. x 128.

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FIG.3. Nodular KS as seen in Fig. 2, to illustrate well-defined border and surrounding abnormal vasculature. x 1280.

nohistochemical and ultrastructural techniques have been used to try to ascertain the nature of KS cells, and to attempt to determine whether KS is of vascular or lymphatic origin. Beckstead et al. (1985)used a combination of immunohistochemical, enzyme histochemical, and lectin-binding techniques to compare the profile of staining in KS lesions to that of normal vascular or lymphatic endothelium. Only the abnormal endothelial component stained reproducibly, and this had a profile more similar to normal lymphatic than vascular endothelium. However, the spindle cells failed to stain completely, or showed only patchy, weak staining with two of their seven endothelial markers. Jones et al. (1986) also concluded that the endothelial components of KS lesions had immunohistochemical properties shared with lymphatic endothelium. However, this conclusion was based on the rather negative evidence of the absence of staining of KS with the relevant antibodies, as seen in lymphatic endothelial cells, as opposed to the presence of staining with these markers in vascular endothelium. The spindle cell component did not stain consistently with their panel of endothelial markers. Alternatively, an immunohistochemical study by Rutgers et al. (1986) concluded that both the spindle and endothelial component were of vascular

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rather than lymphatic origin. Another study (Suzuki et al., 1986)found that KS tissue stains intensely with antibodies against HLA-A, B, and C antigens, as do normal blood vessels, whereas normal lymphatics do not. An ultrastructural marker of vascular endothelium is the Weibal-Palade body. This has not been seen in the spindle cells of KS (McNutt et al., 1983). In spite of these studies, which are consistent with an endothelial origin of KS, no conclusive evidence for the origin of the cells has yet been produced. One important problem in these studies is that most histochemical markers used for identifying endothelium have not been directly raised from endothelial-cell antigens. As a result their labeling specificity is poor. Other markers, such as the antibody to Von Willebrand factor, are known to be unreliable markers of endothelial cells (Beckstead et al., 1985), as the sensitivity varies substantially, depending on the processing of the tissue. Until a precise panel of endothelial-cell markers is available, the exact cellular origin of KS is difficult to define. An important possibility yet to be ruled out is that the excessive vascularity may represent overstimulation of normal vessels by angiogenic factors produced by abnormal neighboring cells. V. KS-Neoplasm

or Hyperplasia?

The concept of KS as a malignant neoplasm has been disputed and the hypothesis put forward that the early lesions, at least, are hyperplastic (Costa and Rabson, 1983; Brooks, 1986). The main reasons for this hypothesis are explained here. Kaposi’s sarcoma is commonly related to some form of immunosuppression (e.g., HIV infection and iatrogenic immunosuppression in transplant recipients). An interesting feature of KS is its ability to regress, particularly when the immune status returns toward normal. Regression is an unusual feature of any other neoplasm, and nowhere does it occur as consistently as in KS. Regression is more likely to occur in a lesion that is hyperplastic rather than truly neoplastic. In the former, growth is not completely autonomous, and regression may be explained by either the removal of the external stimulus to proliferation or the reimposition of a state of growth inhibition. Most solid malignant neoplasms begin as a primary tumor from which metastases subsequently develop. There are some peculiarities of KS that point to the disease being of multifocal origin from the outset. Lesions often occur simultaneously, in crops, and late-occurring lesions during the course of the disease may have the histological appearances of early lesions (i.e., the abnormal endothelial component predominates) (Reynolds et al., 1965). “Metastatic” lesions in the disseminated form of the disease do not occur at typical sites for metastases seen in any other tumor. Foci of KS occur at

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multiple sites along the bowel mucosa or submucosa, but not the serosa where most secondary gut tumors occur, and pulmonary lesions are also atypical. In uitro propagation of KS biopsy material has led to an outgrowth of cells that express some endothelial-cell markers and have been shown to contain chromosomal rearrangements (Delli-Bovi et al., 1986). These rearrangements, though clonal for one culture, differ from those of other KS cultures, even when the cultures originate from the same biopsy specimen. Such heterogeneity suggests that the tumor has not developed by a clonal expansion of a single cell, and therefore may be hyperplastic. Another indication that KS is a hyperplastic lesion consists of reports suggesting the presence of abnormal endothelial proliferation in clinically normal skin of AIDS patients (Schwartz et al., 1984; De Dobbeleer et al., 1987). In one study (De Dobbeleer et al., 1987), normal-looking skin samples were taken from four AIDS patients. Two of the patients had KS elsewhere at the time of biopsy and one developed KS 10 months later. The ultrastructure of the clinically normal skin closely resembled early KS lesions. Normal-looking skin from age-matched controls did not show any KSlike features. Whereas all the aforementioned points are consistent with KS being a hyperplastic proliferation, in some clinical forms of KS, such as locally aggressive endemic KS, the disease certainly behaves in a malignant fashion. A hypothesis to unify the extremes of behavior of the disease could be that there is one or many initial stimuli to endothelial proliferation that, when removed, may lead to regression of the tumor. If continued, this may result in a final progression of some cellular elements to true malignancy, with growth independent from the initiating stimuli. VI. Etiology of KS

The etiology of KS has not been clearly defined. However, many peculiarities of the tumor do indicate mechanisms that may be important.

A. AN INFECTIOUS AGENT An infectious agent associated with KS has been proposed for some time. Initially it was based on the geographic distribution of KS in Africa. Kaposi’s sarcoma is concentrated into a tumor belt in central sub-Saharan Africa, where it is one of the most commonly occurring tumors. The incidence falls off sharply north of the Sahara and more gradually toward the east and south. McHardy et al. (1984) reported an endemic KS in the West Nile district of

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TABLE I AIDS CASESREPORTED TO THE CENTERSFOR DISEASE CONTROL THROUGH MARCH 4, 1985, ACCORDING TO RISKGROUP“

Group Homosexual men Intravenous drug users Haitians Patients with hemophilia Sexual contacts of members of high-risk group Transfusion recipients Children “No identified risk or uncharacteristic” Total

Total number of patients

Number of patients with KSb

Percentage with KS

6293 1478 280 62 68

2264 63 29 1 3

36.0 4.3 10.4 1.6 4.4

104 104 308

2 7 48

1.9 6.7 15.6

8697

2417

27.8

Listed according to risk group. From Haverkos et al. (1985). KS, Kaposi’s sarcoma.

Uganda, where most cases were seen in people who lived above an altitude of 853 m. However, KS was not seen everywhere above this altitude and most cases were found in very close proximity to each other, although in sparsely populated areas. They also reported space-time clustering of cases, which has previously been reported in endemic KS (Owor and Hutt, 1977; Bland et al., 1977), although this did not reach statistical significance If one considers epidemic, HIV-associated KS, it is much more common among the homosexual population than any other risk group (Table I) (Haverkos et al., 1985). Among homosexuals with AIDS, 36% develop KS; however, only 1.6%of hemophiliacs with AIDS develop KS. In addition, there has been a decline in the proportion of AIDS cases presenting with KS in the face of an increase in the number of AIDS patients (Des Jarlais et al., 1987). These factors would suggest that there is a separate agent, other than HIV, which may be transmitted by sexual activity and hence is particularly likely to affect the homosexual population. The declining incidence of KS may parallel the recent decline of other sexually transmitted diseases seen in homosexual males such as gonorrhea and syphilis. In Africa HIV infection is frequently transmitted by heterosexual contact. It is therefore interesting to note that atypical, aggressive KS is a common manifestation of African AIDS. Also, this form of African KS affects women much more commonly than the conventional, endemic KS, and this may reflect the importance of their

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increased contact to a sexually transmitted agent responsible for KS, other than HIV. These epidemiological studies have led to many experiments that have attempted to identify the proposed infectious agent. In early experiments (Giraldo et al., 1972), herpes-type particles were observed in five of eight tissue culture lines derived from different cases of African KS. However, this study has a number of serious drawbacks. The cultivated cells were heterogeneous, with cell types only defined on morphological grounds, raising the probability that fibroblasts and macrophages from normal adjacent skin were also being propagated. Furthermore, the cell cultures underwent morphological changes after 2-3 months propagation and only then were virus particles observed; hence contamination of cultures was a real possibility. Glaser et al. (1977) proceeded to characterize the virus found in one of these cultured lines and found it to have some properties shared with human cytomegalovirus (HCMV). Despite the limitations of such studies, these experiments, along with seroepidemiological studies showing a high incidence of CMV antibodies in European and American KS patients (Giraldo et al., 1975, 1978), set the trend for the association of CMV infection with KS. Later, Giraldo et al. (1980) identified the CMV genome in KS tissue biopsy samples using DNADNA reassociation kinetics and in situ hybridization, using purified virion DNA as a probe. However, it was later discovered (Riiger et al., 1984) that CMV DNA has sequences homologous to parts of the human genome, raising the possibility that these early CMV probes were hybridizing to such human DNA sequences. Evidence to support this hypothesis was reported by Ruger et al. (1984) using Southern blot analysis of KS tissue and hybridization to cloned CMV probes, which lacked the sequences homologous to normal human DNA. Most tumors examined did not contain detectable CMV DNA. The viral DNAs that were detected in some KS tissues were at concentrations that did not exceed the amounts sometimes seen in non-KS tissue in generalized CMV infection. Other workers who searched for CMV DNA using similar cloned probes (Delli-Bovi et al., 1986) did show hybridization in some samples of KS tissue, but hybridization also occurred in non-KS tissue taken from the same patients. There are problems in studying the relationship of CMV to KS, in that CMV infection is very common in the normal adult population (58% of pregnant white females were found to be antibody-positive in a study by Stern and Tucker, 1973), and people who are immunosuppressed have an especially high incidence of CMV reactivation and reinfection. It is also known that homosexual men have an extremely high prevalence of CMV

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antibody of up to 94% (Drew et al., 1981).As KS itself is more prevalent in immunosuppressed populations, rigorous controls are needed in any experiment that attempts to show that CMV is causally linked to KS, rather than found by chance alone. At present it appears that the initial studies that suggested a causal relationship between CMV and KS have not been substantiated. Other viruses have been considered, such as Epstein-Barr virus (Giraldo et al., 1975), hepatitis B virus (Siddiqui, 1983; Delli-Bovi et al., 1986), and HIV itself (Delli-Bovi et al., 1986), but again there is little evidence for any causal relationship. It seems that the infectious agent is proving elusive, yet the weight of epidemiological evidence for an infective etiology is too great to dismiss. An entirely new, undiscovered “Kaposi’s sarcoma virus” should be considered.

B. ANCIOCENICFACTORS An interesting aspect of KS is its close relationship with immunosuppression. Reduced immunosurveillance. alone is not enough to explain why KS is so common among immunosuppressed patients. If this was purely the case, one would expect the occurrence of KS to be overshadowed by more common tumors seen in the general population, such as lung and colonic cancers in the West, and hepatoma and bladder cancers in Africa. One has to consider if the immune imbalance itselfin some way positively selects for the development of KS. There is mounting evidence that endothelial-cell function is closely related to cells of the immune system. Supernatant from in uitro-activated T cells can induce HLA-DR antigen expression on endothelial cells (Stastny and Nunez, 1981). Mitogen- and antigen-stimulated T cells are known to release a lymphokine that either directly or indirectly induces angiogenesis (Auerbach and Sidky, 1979). Activated lymphoid cell lines produce a soluble factor that causes inhibition of endothelial-cell migration (Cohen et al., 1982). Macrophages activated either in uiuo or in uitro produce a soluble factor that causes neovascularization in the guinea pig cornea (Polverini et al., 1977). Some of these soluble factors responsible for angiogenesis have been identified (e.g., heparin and prostaglandin El); others have not yet been isolated, and it is likely that angiogenesis is only one of their many functions. So far, the interaction between immune cells and endothelial cells appears intricate and complicated. However, it seems that some molecules involved in the regulation of the immune system have additional effects on angiogenesis. It may be that the immune system imbalance seen during immunosuppression- is responsible for the increased circulation of factors with

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angiogenic properties, or alternatively the loss of molecules with inhibitory effects on angiogenesis. Increased angiogenesis would account for this histological appearance of KS.

C. ONCOGENES The DNA extracted from KS lesions has been examined for the presence of oncogenic sequences. The first report of transforming sequences in KS DNA was by Lo and Liotta (1984). They extracted DNA from KS in subcutaneous tissue and lymph nodes from a homosexual man with extensive KS, and transfected the DNA into NIH-3T3 cells. The transformed colonies were found to contain human repetitive DNA sequences, and the DNA from these primary transformants could be used to generate secondary transformants in NIH-3T3 cells. Both primary and secondary transformants produced angiosarcomatous tumors when injected into nude mice. The DNA from transformed clones were probed for various known oncogenes (rasH, rasN, rasK, v-sis, v-src, v-fes) by Southern blot, but hybridization was not found. Hence the possibility of a new transforming oncogene present in KS was suggested. No further demonstration of this transforming element was reported until very recently (Delli-Bovi and Basilico, 1987; Delli-Bovi et al., 1987). In these studies, DNA from KS biopsy samples from several AIDS patients were transfected into NIH-3T3 cells. A single transformed focus resulted from one case only. This contained human repetitive DNA sequences, and DNA from this clone was capable of producing secondary transformants and tumors in nude mice. The transforming sequence was found to be 11 kb long, from which two mRNA molecules (1.2 and 3.2 kb) were transcribed. The protein translated from the 1 .%kb transcript was shown to have transforming properties and could promote growth in NIH-3T3 cells. The amino acid sequence showed significant homology to basic and acidic fibroblast growth factor; such growth factors are known to have strongly angiogenic properties. Hence, it was postulated that this new KS oncogene could produce an autocrine growth factor that stimulated angiogenesis. However, a serious drawback to this study was the inability to detect this KS oncogene in the original KS necropsy material. Therefore, there is a real chance that this transforming gene was generated through the transfection procedure itself. It also should be noted that only one among several KS specimens was capable of producing a transforming colony in NIH-3T3 cells. Further comparison of the transforming sequence from the KS necropsy specimen has shown homology with a new oncogene (hst) isolated from human stomach cancer and normal stomach mucosa (Sakamoto et ul., 1986; Taira et ul., 1987). However, it is also possible that the hst oncogene is produced during the transfection process, especially as one of the hst

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oncogene sequences was isolated during the transfection of NIH-3T3 cells with DNA prepared from normal stomach mucosa.

D. HORMONAL INFLUENCE

A feature of KS that is not seen in any other sarcoma is its predominance in males. This predominance is seen in most epidemiological categories, but far less so in children. This may suggest some hormonal effect on the disease process. How exactly this control is effected is unknown, and it cannot be entirely due to protection by estrogens, as the proportion of mature females who develop KS before and after the age of the menopause is the same for males in the corresponding age groups (Templeton, 1972). One would have expected an increase of KS in postmenopausal women if estrogens have a protective effect. Reversal of hormone profile has been tried with estrogen therapy, with little success (Templeton, 1972). VII. Conclusion

Even with recent advances in molecular and cellular biology, the exact mechanisms involved in the etiology and pathogenesis of KS are poorly understood. The production of reliable markers to the pathological KS cell would be of use especially in the examination of tissue sections and in tissue cultures. A reproducible culture system would be invaluable to detect individual agents important in the growth of the tumor. It may be that the suggested etiological mechanisms are of unequal importance in the different epidemiological and clinical types of KS, and perhaps KS is the common end point to a variety of initiating processes.

ACKNOWLEDGMENTS I would like to thank Professors A. Bayley and R. Weiss, and Drs D. Venter, C. Marshall, J. McKeating, and J. Weber for their help and advice in preparing this text, and the Cancer Research Campaign (United Kingdom) for financial support.

REFERENCES Ackerman, A. B. (1979). Am. J . Dermutopathol. 1, 165-172. Auerbach, R., and Sidky, Y. A. (1979). J . Zmmunol. 123, 751-754. Bayley, A. C., Downing, R. G . , Cheingsong-Popov, R., Tedder, R. S., Dalgleish, A. G . , and Weiss, R. A. (1985). Lancet i, 359-361. Beckstead, J. H . , Wood, G . S . , and Fletcher, V. (1985). Am. J . Pathol. 119, 294-300. Biggar, R. J . , Horm, J., Fraumeni, J. F., Jr., Greene, M. M., and Goedert, J. J. (1984).J . Natl. Cancer Inst. 73, 89-94. Bland, J. M., Mutoka, C . , and Hutt, M . S. R. (1977). East Afr. J . Med. Res. 4, 47-53.

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Brooks, J. J. (1986). Lancet ii, 1309-1311. Cohen, M. C., Picciano, P. T., Douglas, W. J . . Yoshida, T., Kreutzer, D. L., and Cohen, S. B. (1982). Science 215, 301-303. Costa, J., and Rabson, A. S. (1983). Lancet i, 58. De Dobbeleer, G., Godfrine, S., Andr6, J., Ledoux, M.,and DeMaubeuge, J. (1987). J. Cutaneous Patho!. 14, 154-157. Delli-Bovi, P., and Basilico, C. (1987). Proc. Natl. Acad. Sci. USA 84, 5660-5664. Delli-Bovi, P., Donti, E., Knowles, D. M., 11, Friedman-Kien, A., Luciw, P. A,, Dina, D., Dalla-Favera, R., and Basilico, C. (1986). Cancer Res. 46, 6333-6338. Delli-Bovi, P., Curatola, A. M., Kern, F. G., Greco, A,, Ittmann, M., and Basilico, C. (1987). Cell 50, 729-737. Des Jarlais, D. C., Stoneburner, R., Thomas, P., and Friedman, S. R. (1987). Lancet ii, 10241025. DiGiovanna, J. J., and Safai, B. (1981). Am. J. Med. 71, 779-783. Drew, W. L., Mintz, L., Miner, R . C., Sands, M.,and Ketterer, B. (1981).J. Infect. Dis. 143, 188-192. Francis, N. D., Parkin, J. M., Weber, J., and Boylston, A. W. (1986).J. Clin. Pathol. 39,469474. Giraldo, G., Beth, E., Coeur, P., Vogel, C. L., and Dhru, D. S. (1972).J. Natl. Cancer lnst. 49, 1495-1507. Giraldo, G., Beth, E., Kourilsky, F. M., Henle, W., Henle, G., Mike, V., Huraux, J. M., Anderson, H. K., Gharbi, M. R., Kyalwazi, S. K., and Puissant, A. (1975). Int. J. Cancer 15, 839-848. Giraldo, G., Beth, E., Henle, W., Henle, G., Mike, V., Safai, B., Huraux, J. M., McHardy, J., and De-The, G. (1978). lnt. J. Cancer 22, 126-131. Giraldo, G., Beth, E., and Huang, E . 4 . (1980). Int. J. Cancer 26, 23-29. Glaser, R., Ceder, L., St. Jeor, S., Michelson-Fiske, S., and Haquenau, F. (1977). J. Natl. Cancer lnst. 59, 55-60. Haverkos, H. W., Drotman, D. P., and Morgan, M. (1985). N. Engl. J. Med. 312, 1518. Jones, R . , Spaull, J.. Spry, C . , and Wilson Jones, E. (1986). J. Clin. Pathol. 39, 742-749. Kaposi, M. (1872). Arch. Dermutol. Syph. 4, 265-273. Lo, S.-C., and Liotta, L. A. (1984). Am. J. Pathol. 118, 7-13. McHardy, J., Williams, E. H., Geser, A., De-The, G., Beth, E., and Giraldo, G. (1984).Znt. J. Cancer 33, 203-212. McNutt, N. S., Fletcher, V., and Conant, M. A. (1983).Am. J. Pathol. 111, 62-77. Owor, R . , and Hutt, M . S. R. (1977). East Afr. J. Med. Res. 4, 55-57. Penn, I. (1979). Transplantation 27, 8-11. Polverini, P. J., Cotran, R . S., Gimbrone, M. A., Jr., and Unanue, E. R. (1977). Nature (London) 269, 804-806. Reynolds, W. A., Winkelmann, R. K., and Soule, E. H. (1965). Medicine 44, 419-441. Riiger, R . , Burmester, G. R., Kalden, J. R., Bienzle, U.,Braun, R., Safai, B., Sterry, W., and Fleckenstein, B. (1984). “Acquired Immune Deficiency Syndrome,” pp. 127-137. Alan R. Liss, New York. Rutgers, J. L., Wieczorek, R., Bonetti, F., Kaplan, K. L., Posnett, D. N., Friedman-Kim, A. E., and Knowles, D. M., I1 (1986). Am. J. Pathol. 122, 493-499. Sakamoto, H., Mori, M., Taira, M., Yoshida, T., Matsukawa, S., Shimizu, K., Sekiquchi, M., Terada, M., and Sugimura, T. (1986). Proc. Natl. Acad. Sci. USA 83, 3997-4001. Schwartz, J. L., Muhlbauer, J. E., and Steigbigel, R. T. (1984).J. Am. Acad. Dermatol. 11, 377-380. Siddiqui, A. (1983). Proc. Natl. Acad. Sci. USA 80, 4861-4864.

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Stastny, P., and Nunez, G . (1981). In “Transplantationand Clinical Immunology” (J. L. Touraine et al., eds.), pp. 132-139. Excerpta Med. Found., Amsterdam. Stem, H . , and Tucker, S. M . (1973). Br. Med. J . ii, 268-270. Suzuki, Y., Hashimoto, K., Crissman, J., Kanzaki, T., and Nishiyama, S. (1986).J . Cutaneous Pathol. 13, 408-419. Taira, M . , Yoshida, T., Miyagawa, K., Sakamoto, H . , Terada, M., and Sugimura, T. (1987). Proc. Natl. Acad. Sci. USA 84, 2980-2984. Templeton, A. C . (1972). Cancer (Phihdelphia) 30, 854-867.

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THE RELATIONSHIP BETWEEN MHC ANTIGEN EXPRESSION AND METASTASIS Jacob Gopas,’t Bracha Rager-Zisman,t Menashe Bar-Eii,t Gunter J. HBmmerling,* and Shraga Segalt *Instituteof Oncology, Soroka Medical Center tbpartment of Microbiology and Immunology, Faculty of HeaRh Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84 105, Israel $Institute for Immunology and Genetics, German Cancer Research Center, D-6900 Heidelberg. Federal Republic of Germany

I. Introduction 11. MHC Antigens, Tumorigenicity, and Metastasis in Animal Models 111. MHC Regulation of Antitumor Immunity by Cytotoxic T Cells

IV. MHC Regulation of Antitumor Immunity by NK Cells V. MHC Antigens, Tumorigenicity, and Metastasis in Human Beings VI. Summary and Conclusions References

I. Introduction

The elucidation of the structure of the products of the major histocompatibility complex (MHC) and the understanding of their crucial role in regulating the immune response have been the subject of intensive research over the last 20 years. The murine (H-2K, -D, and -L) and the human (HLA-A, -B, and -C) MHC class I molecules are expressed on the surface of almost all somatic cells as heterodimers composed of a 45,000-Da polymorphic glycoprotein noncovalently associated with an 11,600-Da invariable polypeptide, &-microglobulin (&m) (Nathenson et al., 1981; Kimball and Coligan, 1983; Bjorkman et al., 1987). These molecules are the classical transplantation antigens that mediate allogeneic tissue graft rejection (Snell et al., 1976), and they play a crucial role in the cellular immune response by functioning as the antigen-presenting molecules that enable cytotoxic T lymphocytes (CTL) to discriminate between foreign (nonself) antigens from self antigens. A foreign antigen, whether viral, tumor, or chemically modified, must be associated with the self-MHC molecule, on the cell surface in order to be recognized by CTL as a nonself antigen, a phenomenon known as MHC-restricted recognition, first described by Zinkernagel and Doherty (1979). In contrast, other class I-related molecules, encoded by the Qa-2,3 and 89 ADVANCES IN CANCER RESEARCH, VOL. 53

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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the Tla regions, are less polymorphic; their expression is limited to certain tissues and their function is largely unknown (Old and Stockert, 1977; Mellor et al., 1984). MHC class I1 genes code for the immune response-associated antigens (Ia) called I-A and I-E in the mouse and HLA-DP, -DQ, and -DR in human beings. These molecules are transmembrane heterodimers consisting of a 35-kDa a chain noncovalently associated with a 29-kDa p chain (Kaufman et al., 1984). The Ia molecules are highly polymorphic and are expressed primarily on the surface of B lymphocytes, macrophages, dendritic cells, and certain epithelial and nervous system cells. These molecules are recognized by antigen-specific receptors of helper T cells and regulate the proper interaction of the different subsets of cells involved in the development of the immune response to foreign antigens (Benacerraf, 1981; Hood et al., 1985). Both categories of molecules possess a common feature: they serve as a password between two cells of an individual, allowing the cells to recognize each other as belonging to the same organism, and thus enabling them to cooperate by virtue of their identity at the level of either class I or class I1 gene products. Specific self-recognition of at least one of these products in conjunction with nonspecific complementary interactions (Edwards, 1978; Staunton et a l . , 1988) are needed for the transmission of a specific message from one subpopulation to another. Major histocompatibility complex molecules have been also described as part of more general phenomena, involving such aspects as MHC-associated adhesion, homing, and contact inhibition (Dausset and Contu, 1980; Scofield et al., 1982), interaction with hormones (Allison et al., 1988) and viruses (Inada and Mims, 1984), as well as in association with cell membrane hormone and growth factor receptors (Due et al., 1986; Hosoi et a l . , 1988), transmembrane-regulated protein kinases (Curry et a l . , 1984; Newel et n l . , 1988), and membrane-associated filaments (Feltkamp et al., 1987), for all of which the common denominator is cell communication. Despite the considerable progress made in our understanding of the function of the MHC, its role in cancer and metastasis is still very unclear. The difficulty in understanding the role played by MHC products in malignancy stems from our present lack of understanding of the role that MHC molecules play in regulating cell-cell communication between the tumor cell and its environment both in immunological terms and beyond its immunological implications. The occurrence of metastases reflects the disruption of normal intercellular signals that allows the spread and proliferation of malignant cells and is one of the most urgent problems in the management of cancer. The ability of malignant cells to disseminate from a locally growing tumor and to form secondary lesions at near or distant sites is considered the most life-threatening aspect of cancer.

MHC

ANTIGEN EXPRESSION AND METASTASIS

91

Metastatic formation is a multistep, highly selective process; the fraction of cells with metastatic potential in primary tumors is variable, but usually small (50 kb. Physical mapping of the interval between two genes, mapped meiotically, necessitated a laborious procedure of chromosome walking, using a noninterrupted set of cosmids containing overlapping segments of DNA. The development of methods for analysis and cloning of large segments of DNA allows far more efficient joining of reference points on the chromosome linkage map by defined DNA fragments. The method developed by Burke et a2. (1987) uses the yeast artificial chromosome system (YAC), which contains yeast genes serving as markers for insertion of exogenous DNA as well as sequences for autonomous replication, and for centromere function and generation of telomeres. This method has yielded cloned fragments of human DNA up to 460 kb long. V. Major Histocompatibility Cornplex-Structure

and Function

A. MHC STRUCTURE The MHC of mouse (H-2)and human (HLA) (located on chromosome 17 and 6, respectively) are among the most extensively studied segments of mammalian genome. They contain a number of genes of several classes (Fig. 2). The class I and class 11 genes encode cell surface glycoproteins involved in immune response through presentation of self and nonself antigens to T lymphocytes. Class I genes code for transmembrane polypeptide chains with molecular weight of 40,000-49,000; these molecules are noncovalently associated with a smaller polypeptide, microglo globulin (P2m; MW 12,000) encoded by a gene on another chromosome (2 in mouse, 15 in human). The class I genes encoding the classical transplantation antigens, which are present on almost all somatic cells, map to the K and D regions (Fig. 2). Another large group of class I genes is located in the Qa-Tla region. In the HLA complex the transplantation antigens are encoded by loci A, B , and C . Nonclassical class I genes, equivalents of the murine Qa-T2a region genes, map telomeric of the A locus (Orr and De Mars, 1981). Class I1 genes map into a region between K and D in mice, and centromeric of the class I loci in

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GENES

REQION

K

I

s

D

0.

Tla

CLASS

I

I1

111

I

I

I

FIG 2. Schematic map of the murine MHC (H-2 complex) on chromosome 17. The number of genes varies with haplotype. Most variation is observed for the class I genes. In the D region the number of genes varies from 1 (H-26) to 5 (H-29, in the Qa region from 1 (H-2f) to 10 (H-26). 21A, 21B, 21-Hydroxylase A and B; TNF, tumor necrosis factor.

humans. Class I1 genes code for membrane-bound glycoproteins consisting of two noncovalently-associated polypeptide chains, a and p, of MW 33,oO0-35,000 and 26,000-29,OoO, respectively. Molecular cloning has revealed the organization of the mouse MHC. Two mouse haplotypes were studied in detail (H-2d of BALBJc and H-2b of C57BL/10) (Weiss et al., 1984; Fisher et al., 1985). The MHC class I genes occur in families of 30-40 per haplotype, and the majority maps telomeric of the D region into the Qa-Tla region (Margulies et al., 1982; Winoto et al., 1983). Different haplotypes vary in number of class I genes. This is caused by duplications and deletions resulting most likely from unequal crossing over, which is facilitated by a great number of homologous genes in the MHC region. For instance, the Dd region of the H-2” haplotype contains five class I genes (H-2Dd, H-2Dzd, H-2D3”, H-2D4“, and H-2Ld, in contrast to only one class I gene in the H-2b haplotype (H-2Db). Only two antigens encoded by the Dd region are identified (i.e., H-2Dd and H-2Ld), although serological data (Ivanyi and Demant, 1979) and studies with cytotoxic T lymphocytes of Mann and Forman (1988) suggest the presence of another Dend-encoded class I molecule. A large variability in the number of class I genes in the Q a region of the murine MHC has been observed. This region contains from 1 (H-21) to 10 (H-2b, C57BL/10) class I genes (Eastman O’Neill et al., 1986). In contrast to the K - and D-region-encoded antigens that are expressed on almost all somatic cells, the membrane-bound products of the Qa-Tla region display a more limited tissue distribution; they are predominantly expressed on subpopulations of the hematopoietic cell lineage. The Qa-2 polypeptide encoded by the Q a region utilizes a different form of membrane attachment as compared to the H-2K and H-2D antigens, Qa-2, like Thy-1 and T r y p a n o s o m variant surface glycoprotein, is anchored to the cell membrane via a covalent linkage with phosphatidylinositol (Stiernberg et al., 1987). Telomeric of the mouse Qa region, a new subfamily of class I genes has been characterized, consisting of two or three members. One of them, Mbl, shows 60% nucleotide identity with other class I genes (Singer et al., 1988).

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The H-2K and H-2D regions are extremely polymorphic: >50 alleles of the H-2K locus and 30 alleles of the H-2D locus have been found in laboratory and wild-mouse populations (Klein and Figueroa, 1981). In contrast, the class I-like genes mapping to the right of the H-2D region exhibit only limited polymorphism. It is believed that gene conversion mechanisms contribute significantly to the generation of polymorphism of the MHC. The changes in nucleotide sequence found in spontaneous mutations of H-2K and H-2D genes indicate that all mutations until now can be explained as exchange of sequence (gene conversion) with other polymorphic and nonpolymorphic genes from the K, D , and Qa-Tla regions by intergenic recombination (Mellor et al., 1983; Geliebter et al., 1986). This hypothesis is supported by the finding that certain H-2K and -D antigens share serological epitopes with Qa-Tla-region-encoded products (Ivanyi et al., 1982; Cook et al., 1983; Figueroa et al., 1983; Sharrow et al., 1984; Oudshoorn-Snoek et al., 1984). The polymorphism of class I1 genes is also likely generated by gene conversion events (Widera and Flavell, 1984). Genes encoding complement factors C4, C2, FB, and the C4-like Slp protein (Chaplin, 1985), the gene for the isoenzyme Neu-1 (Figueroa et al., 1982), 21-hydroxylase genes (White et al., 1984a), and genes encoding tumor necrosis factor a and P (TNF-a, TNF-P; Miiller et al., 1987) were also mapped within the stretch of 1.5 c M of the H-2 complex. Although these genes are unrelated to the class I and I1 genes, their location is conserved between the species and their location in the MHC might not be completely coincidental, The Ss protein was identified as the fourth component of the complement (Me0 et al., 1975; Lachmann et al., 1975; Curman et al., 1975). The C4 and the related Slp serum protein (Shrefler, 1976) consist of three polypeptide chains with molecular weights of -200,000, 75,000, and 83,000 (Roos et al., 1978). The complement components C2 and factor B (FB) encoded by Sregion genes exhibit structural polymorphism (Roos and Demant, 1982; Takahashi et al., 1984). The C2 and FB proteins are serine proteinases consisting of a single chain (MW 100,000 and 95,000, respectively). The 21-hydroxylase belongs to the cytochrome P-450 family and is involved in synthesis of cortisol. Its deficiency in the human leads to congenital adrenal hyperplasia (White et al., 198413). The two tumor necrosis factors TNF-a and -P, are involved in destruction of tumor cells and virally infected cells (for reviews see Butler and Cerami, 1986; Old, 1985). A gene located in the S-H-2D interval has been described, which is transcribed in B cells and macrophages (Tsuge et al., 1987). Between the C4 and FB genes of both H-2 and HLA complexes, another novel gene was identified that has an unusual periodic structure, is widely transcribed, and is not homologous to the other genes in the complex (Levi-Strauss et al., 1988). Murine leukemia virus sequences are present within the murine MHC; two viral sequences, Tlevl and Tlev2,

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are found in the Tla region in certain haplotypes (Meruelo et al., 1984; Pampeno and Meruelo, 1986).

B. INTERACTIONS OF NON-MHCA N D MHC GENES Interactions between MHC genes and genes on other chromosomes have not yet been extensively analyzed. However, there is ample evidence that they do exist, and their significance for the biological effects of the MHC is at present probably underestimated. The known examples concern the effect of non-MHC-linked genes on the expression or function of the products of all three classes of MHC genes. The products of class I genes associate with the Pzm chain, which is encoded by a nonlinked gene. In the mouse, detectability of some Qa specificities is affected by the Pzm allele. The specificity Qa-9 is expressed on the cell surface in a high amount in the presence of a b allele and in a low amount in the presence of an a allele of Pzm (Sutton et al., 1983). An even more dramatic effect of Pzm has been seen with Qa-11. Until now, this Qa-Tlaregion specificity has been detected only in strains with a b allele of Pzm (van de Meugheuvel et al., 1985).In a complementation test, F, hybrids between two Qa-ll-negative strains, one with Qa-ll-positive haplotype but a allele of Pzm, and the other with Qa-ll-negative haplotype and b allele of Pzm, are Qa-ll-positive (Oudshoorn-Snoek et al., 1988). Conceivably, allelic forms of Pzm molecules may a e c t conformation of the class I molecules with which they associate. Whether this modification affects the function of class I molecules in antigen presentation is not known. Presently, unidentified nonMHC genes have been shown to affect the viral specificity recognized by cytotoxic T lymphocytes, even if the presenting class I molecule was the same (Plata et al., 1987). Expression of class I1 antigens on the cell surface is affected by non-MHC genes, too. This has been demonstrated in families with severe combined immunodeficiency (SCID), where a non-HLA-linked gene determined the deficient expression of class II molecules on lymphocytes of affected persons (de Preval et al., 1985).As the studies of the immune response in the mouse are usually carried out with congenic strains with different H - 2 haplotypes on the same genetic background, the effects of non-MHC genes may often escape attention. In humans, the heterogeneity of populations and differences in age and immunopathological history of tested persons are poorly defined, and therefore the influence of non-MHC genes on functions of MHC products cannot be analyzed effectively. A partial deficiency of C4 protein in plasma, caused by a non-HLA gene, has been documented in several generations of a large pedigree (Muir et ul,, 1984). This deficiency was characterized by decreased plasma levels of C4

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without any signs of increased consumption or structural alterations. The inheritance was dominant. In order to obtain insight into the presently little-known but biologically relevant interactions of MHC and non-MHC genes, we decided to study in our laboratory the control of expression of H-2 class I11 genes by non-H-2linked genes. The various allelic structural and regulatory variants of C4 and S l p genes offer a very good possibility to analyze the interactions of different alleles of C4 and SZp with non-H-2-linked genes. This study (Bruisten and Demant, 1989) revealed that differences in plasma levels of C4 and Slp caused by non-H-2-linked genes are often at least as large as those caused by the allelic S-region regulatory variants. The non-H-2 genes act mainly at pretranslational level, and in the case of Slp, the low levels caused by nonH-2-linked genes cannot be corrected by testosterone treatment. Several non-H-2 genes are involved and their effects are often haplotype-specific. The similarity of the differences seen in these experiments to the genetic observations in humans (Muir et al., 1984) suggests that the regulation of C4 and Slp by non-H-2-linked genes may serve also as a paradigm for many instances of interactions of HLA and non-HLA genes. The well-characterized molecular and hormonal mechanisms of regulation of C4 and Slp in the mouse (Nonaka et al., 1986; Stavenhagen et al., 1987), and production of strains congenic for non-H-2-regulatory genes (Bruisten and Demant, 1989) offer possibilities for molecular and genetic characterization of the non-H-2regulatory factors, and later for identification of homologous genes in humans. C. FUNCTION OF CLASS I A N D CLASSI1 PRODUCTS

The function of the class I and I1 antigens encoded by the MHC is to present foreign antigens to T cells. Class I antigens are involved in antigen recognition by cytotoxic T lymphocytes (CTL) and class I1 molecules by T helper cells. Foreign antigens (e.g., viral antigens, tumor-associated antigens) are presented by both types of MHC molecules in an MHC“restricted” manner. Effector lymphocytes recognize foreign antigens only in association with MHC molecules identical to the MHC molecules on the antigen-presenting cells, thus T cells can respond to the foreign antigen only in the context of self MHC. The nature of the interaction between MHC molecule, foreign antigen, and T-cell receptor (TCR) is not yet completely understood. Evidence is accumulating that the MHC molecules are presenting the foreign antigen in a processed or degraded form. Class I1 antigens were found to bind immunodominant peptides (Babbitt et al., 1985; Buus et al., 1986; Guillet et al., 1987). Class I molecules presenting influenza virus antigens appeared to be associated with fragments of virus-produced nu-

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CLASS I MOLECULE

TARGET CELL

PLASMA MEMBRANE

CD3 FIG.3. Schematic interpretation of the interaction of a cytotoxic T cell recognizing its target. The T-cell receptor (TCR) recognizes the antigen-derived peptide, presented by the polymorphic part of the class I molecule, while CD8 is postulated to interact with monomorphic determinants in the class I proteins. (Adapted from Parnes, 1986, with permission.)

cleoprotein and not with the cell membrane-bound viral antigen (Townsend et al., 1986).This finding was surprising, since several reports exist supporting the hypothesis of association or interaction of MHC antigens and membrane-bound viral antigens (Schrader et al., 1975; Blank and Lilly, 1977; Kvist et al., 1978; Zarling et al., 1978; Senik and Neauport-Sautes, 1979). Some authors, however, reported negative results (Gomard et al., 1978; Fox and Weissmann, 1979). Our electron-microscopic observations also indicated that when nonspecific cocapping is prevented, no close association is detectable between MuLV or MTV viral antigens, and class I MHC molecules on the cell surface (Calafat et al., 1981). The recently accomplished resolution of the crystal structure of the HLA-A2 molecule (Bjorkman et al., 1987) opens new perspectives for understanding the antigen-presenting capacity of MHC molecules. A class I molecule consists of three external domains cil, ciz, cig, a transmembrane part, and a cytoplasmic tail (for a schematic interpretation, see Fig. 3). On top of the surface molecule, facing away from the membrane, a deep groove runs between two long ci helices derived from the cil and ci2 domains of the molecule. The data strongly suggest that this groove is the binding site for antigens. The groove is expected to accommodate peptides of 8-20 amino acids in length. The crystal structure of HLA-A2 presents an excellent model for a MHC molecule that

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binds an antigen-derived peptide and in this way presents the foreign antigen of the TCR. The same model can theoretically be applied to class I1 antigens (Brown et aZ., 1988) that already have been shown to bind peptide molecules. Except for MHC molecules presenting antigen peptides to the TCR, several other molecules are involved in the cell-cell interactions during T-cell antigen recognition. T-cell differentiation antigens are believed to play an important role in addition to the TCR. CD4 (L3T4)-positive cells, mostly helper cells, are restricted by class I1 MHC proteins, while CD8 (Lyt-2) positive cells, mostly cytotoxic cells, are restricted by class I MHC proteins. It is hypothesized that CD4 and CD8 may be receptors for monomorphic determinants on class I1 and class I MHC molecules, respectively (reviewed in Parnes, 1986; see Fig. 3). The TCR itself is associated with CD3, a T-cellspecific protein that might be important for signal transduction. Although the role for class I and class I1 MHC molecules in antigen presentation is well established, the function for the products of the class I genes mapping in the Qa-Tla region and their human homologs (class IV antigens) is not yet clear. A locus controlling the cleavage rate of preimplantation embryos in the mouse has been mapped in the Q a region, and it was suggested that the Qa-2 antigen is the product of this Ped (preimplantationembryo development) gene (Warner et aZ., 1987). It was further suggested that alloreactive T cells bearing the TCR y6 chains recognize relatively nonpolymorphic antigenic determinants mapping to the H-2D, Qa. or TZa regions (Matis et al., 1987). D. MHC PHENOTYPE OF TUMOR CELLS Several types of variant MHC phenotypes have been observed in tumors. In some established mouse tumor transplantation lines or in oitro tumor cell lines, unexpected class I, H-2-like, specificities normally found only in other haplotypes have been reported (Garrido et al., 1976; Martin et aZ., 1977; Schmidt and Festenstein, 1980), and it has been speculated that the appearance of foreign H-2 class I antigens on tumors may provide the means by which the immune system can bring them under control. However, since the tumor cell lines used in the aforementioned studies are readily transplantable in syngeneic recipients, the reported antigenic change is not necessarily the target for immune reaction against tumors. The published data on “extra” specificities should be interpreted with caution, since in some cases antisera reacting with apparently foreign H-2 antigens on tumor cells were found in subsequent tests to react also with lymphocytes of the tumor host strain (Flaherty and Rinchik, 1978; Robinson and Schirrmacher, 1979). In another case, apparently alien antigens were found to be due to contamination of the tumor line (Robinson et al., 1981), or to previously unre-

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cognized mutation of an H-2 gene in the host strain (Vogel et al., 1988). All the reported “extra” specificities were detected on tumor lines that were maintained for a long time by transplantation or in uitro. Our data on primary AKR leukemias, that were tested with an extensive panel of alloantibodies and monoclonal antibodies, showed that alien antigens do not occur at all or only very rarely on these tumors (Oudshoorn-Snoek and Demant, 1983).Cross-reactions between H-2K and Qa-Tla region products (Ivanyi et al., 1982; Cook et al., 1983; Figueroa et al., 1983; Oudshoorn-Snoek et al., 1984; Sharrow et al., 1984) suggest that expression of a normally silent QaTla-region gene in tumor cells may generate an antigen reactive with H-2Kand/or H-2D-specific antibodies, and thus might be responsible for some cases of apparent “extra” specificities. The known products of the less polymorphic class I genes of the Qa-Tla region have been detected on tumor cells of mouse strains that do not express these antigens on normal cells (Old et al., 1963; Rosenson et al., 1981; Flaherty et a l . , 1982). The anomalously expressed Qa-2 antigens and some Tla gene products could not be distinguished biochemically from the antigens in those strains that normally express these molecules (Michaelson et al., 1983a,b). Firm evidence for expression of aberrant MHC class I antigens due to formation of new class I genes during the malignant process was obtained in the studies of UV-induced tumors. Unique class I antigens, not found on normal C3H tissue, are expressed on the UV-induced C3H fibrosarcoma 1591 (Philips et al., 1985; McMillan et a l . , 1985). The genes for the three identified aberrant class I products were cloned and sequenced, giving for the first time an insight into the molecular basis of expression of “alien” MHC specificities (Linsk et al., 1986). These novel class I genes have been found in the UV-induced C3H fibrosacroma 1591, which expresses at least three unique MHC class I antigens. Two of the genes are very homologous to each other and resemble the H-2Ld gene, while the third gene is a mosaic and possesses characteristics of H-2Kk gene. The novel genes are likely derived by recombination from the endogenous class I genes of the C3H mouse (Linsk et al., 1986). A special class of tumors with anomalous MHC phenotype are the teratocarcinomas, formed from murine embryonal carcinoma (EC) cells. They do not express MHC class I antigens (Artzt and Jacob, 1974). However, if differentiation is induced, MHC class I expression is observed (Croce et al., 1981; Morello et al., 1982). Rejection of teratocarcinoma lines transplanted into allogeneic recipients is often due to incompatibilities of K and D regions of the H-2 gene complex (Johnson et a l . , 1983). Preimmunization of hosts with L cells transformed by H-2Kb- or H-2D1>-containingcosmids leads to induction of radioresistant immunity against PCC3 (129/SvSL, H-2b)

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teratocarcinoma cells (Demant and Oudshoorn-Snoek, 1985; Moser et al., 1985). PCCS teratocarcinoma cells injected in (C3H x C57BL/6)F1 hybrids grow and develop tumors in all hosts. If, however, Kh or Dh mutants are used to make the hybrid hosts, a higher resistance of PCC3 teratocarcinoma growth was observed, indicating the close relationship of the antigenic products of the EC cells and the H-2K and H-2D antigens (Demant and Oudshoorn-Snoek, 1985; Moser et al., 1986). These results suggest that EC cells of PCCS teratocarcinoma express antigenic molecules similar to the H-2Kb and H-2Db molecules. Class I-like structures have been identified on the cell surface of E C cells (Stern et al., 1986; Demant and OudshoornSnoek, 1985; Kvist et al., 1979), although the molecular nature of these antigens has still to be clarified. Several reports demonstrate quantitative changes in class I expression on tumors cells. A well-studied model is the AKR thymus-derived lymphoma. A marked increase in H-2k expression on thymocytes of most AKR lymphomas has been observed by several investigators (Chazan and Haran-Ghera, 1976; Kawashima et al., 1976; Zielinski et al., 1981). Large variations in expression levels were described for established AKR-derived cell lines, as well as for primary tumors (Schmidt et al., 1982, 1985; Oudshoorn-Snoek and Demant, 1983, 1986). In primary spontaneous tumors, elevated H-2K and H-2D expression could be correlated with the degree of MuLV expression (Oudshoorn-Snoek and Demant, 1986). Several reports indicate involvement of viruses and oncogenes in the regulation of MHC expression: class I antigens were found to be switched off after cell transformation by the oncogenic adenovirus-12 in contrast to transformation by the nononcogenic adenovirus-5 (Schrier et al., 1983). Rat cells expressing Ad5 E l a subregion are highly susceptible to cytotoxic T cells, and are only oncogenic in immunodeficient animals, whereas cells expressing Ad12 E l a have a low expression of class I antigens, and reduced susceptibility to T killer cells, and hence are oncogenic (Bernards et al., 1983). Gene transfer of N-myc to a human neuroblastoma cell line causes overexpression of the N-myc gene product paralleled by a reduction of MHC class I antigens (Bernards et al., 1986). Comparable results were obtained in c-myc-transfected melanoma cell lines (reviewed in Bernards, 1987). Enhancement of MHC expression by oncogenes is also observed. In a human B-cell line defective for class I1 antigens, transfection with v-H-ras or N-ras genes increased expression of class I1 antigens specifically but not of class I antigens (Hume et al., 1987). However, the opposite relationship, namely influence of MHC antigen on oncogene expression, was reported as well. Transfection of the H-2Dk gene into T10, Dk-negative cloned sarcoma cell lines not only leads to expression of H-2Dk but also to reduction of the expression of the Ki-ras oncogene, while transfection with H-2Kh had no effect (Alon et al., 1987).

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Other examples of phenotypic M HC alterations were observed when primary tumor and metastases of methylcholanthrene (MC)-induced tumors (De Baetselier et al., 1980), or metastatic and nonmetastatic cloned cell lines of Lewis lung carcinoma were compared (Eisenbach et al., 1983). Moreover, a number of chemically induced primary fibrosarcomas appear to be MHC class I-deficient (Hammerling et al., 1987). For further discussion see Section V,E. Studies of human malignancies have also shown the occurrence of alterations of class I and class I1 phenotype. In Burkitt's lymphoma, specific downregulation of HLA-A-11 antigen expression has been observed (Masucci et al., 1987). The general class I1 phenotype of human lymphatic malignancies is identical to the phenotype belonging to the normal cells at the corresponding stage of differentiation (Radka et al., 1986). However, B-cell lymphomas frequently fail to express the complete set of class I1 antigens. A significant correlation between deficient class I1 antigen expression and high-grade malignancy with poor prognosis was observed (Momburg et al., 1987). Malignant melanoma provides an example of aberrant class I1 expression in nonlymphoid cells. Primary tumor cells, as well as their metastases and derived cell lines frequently express class I1 determinants (Wilson et al., 1979), and especially the DR subset of class 11 molecules (Winchester and Kunkel, 1980). The majority of melanoma cell lines express DP class I1 molecules as well (Pollack et al., 1983).

E. BIOLOGICAL IMPORTANCE OF ALTEREDMHC EXPRESSION Thymocytes infected with radiation-induced leukemia virus (RadLV) show increase of H-2 antigens (Meruelo et al., 1978). On RadLV-infected thymocytes H-2K molecules were significantly increased in cells of susceptible and resistant mice, whereas H-2D antigen increase was only found on thymocytes from resistant strains. It was proposed that increased H-2D expression plays a role in resistance to leukemia because it facilitates elimination of virus-infected cells by CTL (Meruelo, 1980). Alterations of MHC phenotype have been reported to be associated with the metastatic properties of the tumor cells in several models. D e Baetselier et al. (1980) found differences in the expression of H - 2 parental haplotypes between a local F, MC-induced tumor and its descendant pulmonary metastases. Cells isolated from lung metastases expressed both parental haplotypes (i.e., H-26 and H-2'9, whereas cells isolated from the local tumor expressed only the H-2b haplotype. Cell lines cloned from this tumor showed similar correlation of H-2 expression and metastatic properties (Kat-

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zav et al., 1983). The metastatic phenotype was found to be determined by the H-2Dk antigen, since in uiuo immunoselection of one such clone, IE7, that expresses H-2Db and H-2Dk showed that loss of class I expression abolished the metastatic potency of the cell clone. Immunoselection for H-2Dk-positive, H-2Db-negative cells, led even to increased metastatic capacity of the cell line (Katzav et ul., 1984). Transfection of the highly metastatic H-2Db/H-2Dk cells with cloned H-2K genes (Kb, Kk) reduced their tumorigenicity and abolished the formation of metastases in syngeneic mice, while the transfection of nonmetastatic cells of H-2Db phenotype with cloned H-2Dk genes resulted in shifting the cells to the metastatic phenotype (Wallich et al., 1985). Imbalance between expression of K- and D-region products was also found on cloned cell lines of Lewis lung carcinoma (H-29 (Eisenbach et al., 1983), which could be correlated with the metastatic properties of those cells. Not the absolute expression of Kb or Db glycoproteins, but the decrease in the K/D ratio was linked to the metastatic potential of the cloned cell lines. The biological significance of the large differences in absolute expression levels of MHC antigens is not yet clear. The modified antigenic profile of tumor cells might affect the T-cell surveillance of the tumor and hence its growth. For instance, the aggressiveness of SJL/J lymphomas was found to be correlated with the absence of the H-2DS antigens (Rosloniec et al., 1984). Cell lines derived from simian virus 40 (SV40)-transformed C3H fibroblasts that had been adapted to in uiuo growth, demonstrated that the oncogenic potential correlated with lack of H-2Kk expression. This alteration was due to mutation of the H-2Kk gene, although no integration of SV40 in this gene was observed (Rogers et al., 1983). In uitro studies have indeed shown a correlation between the immune response and quantitative variations of H-2 expression on target cells (Plata et al., 1981; Schmidt and Festenstein, 1982). In both studies impaired recognition and killing by specific cytotoxic T cells was associated with reduced levels of the relevant class I antigens. Since MHC class I antigens are necessary to present foreign antigens to CTL, the lack of the required restriction elements will impair or prevent the presentation of the particular tumor antigen to the immune system of the host. In this way the tumor cells may escape immune surveillance. This hypothesis is supported by the absent or reduced class I expression in various experimental tumor systems described earlier, and by the finding of reduced or nondetectable levels of class I antigen expression in certain malignant human tumors (review in Hammerling et al., 1987). More direct evidence comes from the experiments in which class I antigens are reexpressed by introduction of the respective genes by DNA-mediated gene transfer. For instance, the virus-induced AKR leukemia cell line

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K36, lacking H-2Kk expression, is highly tumorigenic in syngeneic AKR mice. Transfection of the H-2Kk gene resulted in expression of H-2Kk on cell surface as well as rejection of the tumor cells by the host (Hui et al., 1984). In primary AKR lymphomas, however, increased expression of H-2K and H-2D antigens was observed as a general phenomenon, and it did not obviously impair their growth (Oudshoorn-Snoek and Demant, 1986). This contradiction is probably explained by the different immune reactivity of the hosts, because with primary tumors the immune system has already failed to prevent the establishment and growth of the tumor. Since AKR mice have an impaired immune response (Green, 1984), MHC expression might not be very relevant for tumor protection. Alternatively, the discrepancy between the results obtained with transfectants and primary tumors may be a matter of balance between defense mechanisms by cytotoxic T cells and natural killer (NK) cells (see later). Finally, the higher expression of H-2K and H-2D antigens in primary lymphomas with high MuLV expression might be a secondary concomitant effect of genetic changes associated with high production of virus. The selective advantage for tumor growth conferred by these changes (MuLV incorporations) may be greater than the disadvantage of immunological vulnerability due to higher levels of H-2KID expression. Other class I transfection experiments as well demonstrated alteration of host-versus-tumor behavior, indicting a role for the immune system in tumor defense mechanisms. Tanaka et al. (1985)showed loss of oncogenicity due to reexpression of MHC class I proteins after DNA-mediated gene transfer of H-2Ld into highly tumorigenic adenovirus-12-transformed cells with impaired class I expression. Reintroduction of H-2Kk of Kb antigens in T10 sarcoma cells by DNAmediated gene transfer changed the metastatic phenotype to nonmetastatic in immunocompetent hosts, while in immunosuppressed mice the cell line was still metastatic (Wallich et al., 1985).In the same system introduction of the H-2Dk gene in an H-2Dk-deficient tumor clone resulted in shifting the phenotype from nonmetastatic to metastatic. Interestingly, this was paralleled by reduction of the expression of the cellular Ki-ras oncogene. These results suggest that the mechanism of metastatic potential is not a direct consequence of class I expression, but that MHC antigens regulate Ki-nus oncogene expression, which may determine the metastatic phenotype (Alon

et al., 1987). Transfection and expression of an allogeneic class I gene (H-2Kb) into a KkDd sarcoma, however, did not reduce the tumorigenicity of this tumor in syngeneic mice, suggesting that the presence of an “alien” alloantigen is insufficient for immune surveillance and tumor rejection (Cole et al., 1987). Apart from the role of CTL in immune surveillance, an alternative anti-

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tumor immune defense strategy involving MHC products was proposed by Karre et al. (1986).They showed that loss of H-2 class I expression correlates with reduced malignancy. Lymphoma variants expressing low levels of H-2 are rejected, whereas high-H-2 expressors grow in syngeneic hosts. Since low-H-2 variants were NK-sensitive, it was suggested that NK cells are the effector cells in the immune defense that an unspecifically kill tumor cells lacking the host’s own MHC antigens that had escaped immune surveillance by CTL. From the different observations on the relationships between expression of H-2 class I products and growth behavior of various tumors, we can conclude that several mechanisms that often counteract each other might operate. The actual relationship between MHC expression and the effectiveness of immune response against tumors still has to be elucidated. In addition, the altered expression of MHC antigens can affect the behavior of tumor cells also nonimmunologically-through regulation of oncogene expression.

F. MHC A N D MuLV-INDUCEDLYMPHOMAGENESIS The resistance to tumor induction by MuLV is controlled by multiple genes. Some of these genes have been mapped to the MHC of the mouse (Lilly et al., 1964; Meruelo et al., 1977; Lonai and Haran-Ghera, 1980; Zijlstra and Melief, 1986). Several H-2-linked resistance genes were mapped for different types of MuLV, and assigned to several regions of the H-2 complex (reviewed in Zijlstra and Melief, 1986). The underlying mechanisms are supposed to be at least in part of immunological nature. The immune-response genes (class I1 genes) in the I region of the MHC are found to regulate the antibody response against MuLV virions (DebrB et al., 1980; Vlug et d.,1981) and MuLV antigens on MuLV-infected (tumor) cells (Aoki et al., 1968; Sato et al., 1973). These antibody responses are an important factor in resistance against some MuLV-induced lymphomas. The influence of class I antigens on susceptibility or resistance is related to the ability of the class I antigen in question to present the processed viral antigen to cytotoxic T cells. In some instances H-2 influences the relative proportion of MCF-induced T- and B-cell lymphomas among infected mice. The H-2 Z-A region influences the development of early T-cell lymphomas. Susceptible strains develop the early T lymphomas. A great part of the resistant strains, however, develop B-cell lymphomas later in life (Zijlstra et al., 1984; Vasmel et al., 1988). Immune T-cell response differences regulated by MHC class I1 I-A genes were proposed to be responsible for this effect (Vasmel et aZ., 1988).

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VI. Susceptibility to Epithelial Tumors and the Role of MHC

A. GENETICSOF LUNGTUMORSUSCEPTIBILITY Since the original observation by Tyzzer (1907) that different families of mice exhibit different incidences of lung tumors, considerable effort has been paid to the analysis of the genes involved (reviewed in Heston, 1966). The study of the genetics of lung tumors was greatly enhanced when inbred strains of mice were developed and introduced, particularly when it was shown that susceptibility to spontaneous lung tumor development differed widely between certain strains. When lung tumors were induced with carcinogens, the inbred strains retained their relative rank order positions in degree of susceptibility they showed for spontaneous lung tumorigenesis (for reviews see, e.g., Stewart, 1959; Heston, 1966). Another observation that has been proven to be of great practical significance in the work on the genetics of lung tumors was that the degree of susceptibility of a particular strain could be measured by counting the number of induced lung tumors in each individual mouse. The average number per strain could be correlated with the incidence of induced tumors or the incidence of spontaneous tumors. All these properties of the lung tumor of the mouse have made it an experimental system of great value in the study of the genetics of tumorigenesis. Using various combinations of inbred strains, it has become well established that susceptibility to spontaneous as well as carcinogen-induced lung tumorigenesis is governed by multiple genes (for references see Table I), although in some studies (using the strain combinations A-C57BL and ABALB), only a single gene appeared to be involved (Bittner, 1938; Andervont, 1937, 1938a; Bloom and Falconer, 1964; Malkinson and Beer, 1983). The susceptibility is reflected by the number of mice with tumors or, in those cases where carcinogen treatment results in appearance of tumors in all mice, by the number of tumors per mouse and the time of appearance of tumors. As can be deduced from Table V, the A strain is in all instances tested the most susceptible of all inbred strains, whereas the C57BL strain is in almost all cases the most resistant. All other strains are classified between these two extremes with varying positions for individual strains, depending on the experimental scheme used. In the most extensive series of strains studied (van der Valk, 1981), apart from the strain differences listed in Table V, some other interesting observations were made. The strain A with MTV appeared to have more lung tumors than the A subline without MTV. Second, in strains BALB/c and A2G, males appeared to be more susceptible than females, whereas such a sex-related difference was not found in the other strains. This suggests that the genome of a particular strain may also

TABLE V STRAIN DIFFERENCES I N LUNGTUMORSUSCEPTIBILITY BETWEEN INBRED STRAINS OF MICE' Tumorinducing agentb DBA (sc) MC (iv) Urethane (ip) Urethane (ip) None Urethane (ip) ENU (ip) None ENU (transplacental) None

Urethane (ip)

Relative strain susceptibilityc

Hybrids studiedd

References

A > C > I, C3H, Y > M, D, C57BL A > C > Y, I, C3H > C57BL, L A, Bagg albino, NH, CBA > DBA, FA, FB A, KL, JU, RIII, CBA, C57BL A > 020, GR > CBA, C3H > DBA, C57BL A > 020, GR > CBA, C3H > DBA, C57BL A, Swiss, C3H, NZW, DBA12, C57BL16 C3H, LP, C57BL110, 129, DBA/2, CBA, C3H.K SWR, AKR, C57BL/6, C57L, DBA/2

None None None F, (15), Be1 (l),Bc2 (1) None Fi (10).F, (1), Bcl (1) None F, (8) None

Andervont (1938b) Shimkin (1940) Shapiro and Kirschbaum (1951) Bloom and Falconer (1964) Bentvelzen and Szalay (1966) Bentvelzen and Szalay (1966) Rice (1973) Smith et al. (1973) Diwan and Meier (1974)

A, MAS, BALB/c, ACR, A2G, 020, OIR, STS, GRS, RIII, LIS, WLL, TSI, CBA, LTS, DD, C3H, C57BL/MHe, BIR, BIMA, DBA/He, DBA/Li, C57BL/Li, C57P A, A.BY, SWR > SS, BALBlc, LS, 129, RIIIS > C57BL/6, HS, B10. D2(58N), BlO.A, C57BL/10, DBA/2, C3H, NZB, C57L, AKR, C57BR, C57BL16-bg

None

van der Valk (1981)

Fi (5), F, (2), Bcl (2)

Malkinson and Beer (1983)

Only those studies in which four or more different inbred strains were tested are included. DBA, 1,2,5,6-Dibenzanthracene;MC, methylcholanthrene; ENU, N-ethyl-N-nitrosourea. Route ofadministration is given in parentheses. Strain ranking in the order of decreasing susceptibility; > indicates that a substantial difference between successive (groups of) strains has been observed according to authors. F, and/or F, and/or backcross (Bc) hybrids studied; number of different crosses given in parentheses. a

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determine whether or not sex-related effects on lung tumorigenesis occur. Associations have been also found between certain specific mutations and susceptibility to lung tumors (Heston, 1957). However, most of these mutations (lethal yellow, dwarf, obese, etc.) exhibit multiple gross phenotypic effects, and therefore it is not clear whether their effects on tumorigenesis are direct or secondary. In view of the finding (Oomen et al., 1989) that chemical induction of lung tumors can be modified by simultaneous administration of glucocorticoid hormone (see later), it is interesting to note that two of these mutations (dwarf and obese) affect hormonal metabolism. The genetics of susceptibility to chemically induced lung tumors has been subsequently studied using an extensive series of RIS between the A/J (susceptible) and C57BL/6J (resistant) strains (Malkinson et al., 1985). The results indicate that at least three genes are involved and thus confirm the existence of a multigene control of lung tumorigenesis. However, the authors could not identify these postulated genes. Ryan et al., (1987), using the same series of RIS, present evidence that the murine Kras-2 gene (or a closely linked genetic element) is one of the genetic factors influencing lung tumor susceptibility. In addition, they show that the allelic variation revealed by restriction-fragment-length polymorphism using a Kras-2 probe, correlates also in individual (C57BL/6J x A/J)F, and backcross mice and in 14 inbred strains with susceptibility or resistance to lung tumor induction. These data, together with the finding of Stowers et a1. (1987)that chemically induced lung tumors in mice contain a mutated transforming Kras-2 gene, strongly implicate the Kras-2 gene, which is located on chromosome 6, as one of the factors involved in susceptibility to lung tumorigenesis in the mouse. Thus, as was shown earlier for the H-2 complex (see later), this is the second example of a relationship between polymorphism of a gene and susceptibility to lung tumors.

B. DIFFERENT LUNGTUMOR TYPES In the mouse two major lung tumor types, alveolar and papillary, can be found. Histologically the papillary type is characterized by a papillary structure and growth into alveoli, bronchioli, and possibly bronchi, whereas the tumors of the alveolar type grow merely along the preexisting septa1 framework. Sometimes tumors of a mixed type occur, especially in older mice. They may represent a transition from the alveolar to the papillary type. The two main tumor types have been reported to differ in their biological behavior (for review see Kauffman et al., 1979):the papillary tumors appear to be more malignant than the alveolar tumors. The morphological characteristics of tumor cells, as revealed ultrastructurally, also differ between the tumor types: cells of alveolar tumors are similar to mature alveolar type 11 cells, whereas cells from papillary tumors are more similar to fetal (pre)alveolar

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type I1 cells (Rehm et al., 1988). The distinctive characteristics comprise cell and nuclear shape, the number of mature lamellar bodies, the number of microvilli, the nature of the glycogen deposits, and the occurrence of primary cilia. A difference between tumor types in glucocorticoid receptor has also been found; cells of papillary tumors show specific nuclear localization of glucocorticoid receptors, while these receptors are not found in alveolar tumor cells (Beer and Malkinson, 1984). Despite these important differences in behavior and cellular characteristics, both tumor types are believed to originate from alveolar type I1 cells (Rehm et al., 1988). Both tumor types can occur spontaneously as well as after induction with carcinogens; in the latter case mice often produce tumors of both types. In the genetic studies discussed in the previous section, lung tumorigenesis was evaluated without considering the tumor type(s) involved. It has, however, become apparent that the alveolar and papillary lung tumors occur in variable proportions in different inbred strains (Witschi, 1985; Beer and Malkinson, 1985) and H-2 congenic strains (Oomen et al., 1983), and hence their development is under different genetic control. In discussing the genetics of lung tumor susceptibility in mice, it is therefore important to take into account the particular tumor types encountered, as they may represent either (a) tumors derived from alveolar type I1 cells at different stages of differentiation or (b) tumors that have distinct differentiation potentials, although they were derived from similar alveolar type I1 cells.

C. SITE OF ACTION OF GENESAFFECTING LUNGTUMORS

The genes involved in lung tumorigenesis appear to act predominantly at the level of the target tissue rather than systemically (e.g., by affecting the immune response). Induction of tumors in lungs transplanted from susceptible and resistant strains into their F, hybrids have shown that susceptibility to carcinogen-induced tumorigenesis in three different strain combinations tested, resides mainly at the target cell level (Shapiro and Kirschbaum, 1951; Heston and Dunn, 1951; Heston and Steffee, 1957; Bentvelzen and Szalay, 1966). Allophenic mice, produced from fused blastomeres of strains susceptible and resistant for lung tumors, contain subpopulations of cells originating from each parental strain in most or all of their tissues. However, the lung tumors found in these allophenic mice were composed overwhelmingly of cells of the susceptible strain (Mintz et al., 1971). Formation of tumors containing almost solely the cells of the susceptible strain within the context of otherwise mosaic lung has been considered as a striking evidence of target cell-localized expression of susceptibility-controlling genes.

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Several other studies have also indicated a relationship between properties intrinsic to the lung cells and susceptibility to lung tumorigenesis. Differences in proliferation of alveolar type I1 cells between strains were reported to correlate with susceptibility to carcinogen-induced lung tumorigenesis; alveolar type I1 cells of the most susceptible strain had the highest labeling index (Thaete et al., 1986). Previous studies using partly different strain combinations, however, did not find such correlation, but instead a correlation between the magnitude of the rebound in alveolar cell proliferation after carcinogen administration and lung tumor susceptibility was found (Shimkin et al., 1969; de Munter et al., 1979). A correlation between the number of putative target cells (alveolar type I1 cells) and lung tumor incidence and multiplicity in mice treated at adult age with varying doses of urethane was also reported (Dourson and O’Flaherty, 1982; O’Flaherty and Dourson, 1982). In a study on prenatal tumor induction by exposure of fetal mouse lung to ENU on different gestation days, a correlation between the resulting number of induced lung tumors and the total number of peripheral epithelial cells in cycle at the time of exposure was found. Furthermore the number of lung tumors per 106 cells in cycle was greatest when fetuses were exposed to ENU on days 15 and 16, as compared to day 17, 18, or 19 of pregnancy (Kauffman, 1976). Mice treated prenatally with ENU exhibit relatively more papillary tumors when the carcinogen is applied at early fetal age (day 10 of pregnancy) than when the treatment is given at a later stage of fetal life (day 15 of pregnancy) (Branstetter et al., 1988). These studies show that number, proliferation, and differentiation stage of target cells may be important factors in the genetically determined susceptibility to carcinogen-induced lung tumorigenesis. Together with the results obtained in lung transplantation studies and in allophenic mice (see earlier), this indicates that the genes involved in lung tumorigenesis indeed act primarily at the target cell level. D. MHC GENESAND LUNGTUMORSUSCEPTIBILITY The finding of Smith and Walford (1978) and Faraldo et al. (1979) that genes in or closely linked to the H-2complex are involved in lung tumorigenesis was the first example of allelic differences in polymorphic genes associated with susceptibility or resistance to lung tumors. Since then these original observations have been extended and the relationship between the H-2 complex and lung tumorigenesis has been firmly established (Table VI). In untreated mice (Smith and Walford, 1978; Faraldo et al., 1979) as well as mice treated with DMN (den Engelse et al., 1981), prenatally or postnatally with ENU (Oomen et al., 1983, 1988, respectively), urethane, and 4-nitro-

TABLE VI H-2 HAPLOTYPE AND LUNGTUMOR RESISTANCE

Background strain A

C57BL/lO

Tumor-inducing agent6

CONGENICSTRAINSOF MICE"

Haplotype-related relative susceptibility High

Intermediate

Low

References

None 4 NQO (sc)

Papillary NSc

Smith and Walford (1978) Miyashita and Moriwaki (1987)

Urethane (sc)

NS

Miyashita and Moriwaki (1987)

None None

Papillary Papillary

Smith and Walford (1978) Faraldo et al. (1979)

DMN (drinking water) ENU (transplacental)

Alveolar

den Engelse et al. (1981)

ENU (ip) C3H

Tumor type

IN

None

Alveolar, papillary Alveolar, papillary Papillary

h2, b4 a, i5

h2

h4, b b h4, b h4, b, i5 b

Oomen et al. (1983) Oomen et al. (1988) Smith and Walford (1978)

Only those studies in which significant differences between H-2 congenic strains were found are included. 4 NQO, 4-Nitroquinoline 1-oxide; DMN, dimethylnitrosamine; ENU, N-ethyl-N-nitrosourea. Route of administration given in parentheses. Not specified. a

b

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AL.

quinoline I-oxide (Miyashita and Moriwaki, 1987), lung tumor development is influenced by the H - 2 haplotype. Furthermore, in all these different experimental systems the H-2" haplotype is always associated with susceptibility and H-2" with resistance. Transplacental induction of lung tumors by ENU in the backcross progeny from a cross between H-2" and H-2" congenic mice, confirmed the H - 2 linkage of this influence and showed that H-2 does not operate through a maternal effect (Oomen and Demant, in preparation). However, in none of the studies just cited was it possible to assign unequivocally the H-2-related effects of a particular region of the H-2 complex. In all studies in which appropriate H - 2 recombinant strains were used (Faraldo et a l . , 1979; Oomen et al., 1983; Miyashita and Moriwaki, 1987; Ooinen et al., 1988), the results indicate involvement of more than one H - 2 gene. This is hardly surprising. First, the H - 2 complex contains several groups of structurally and functionally related genes of the same type derived by gene duplications (see Section V,A), and these related genes may affect tumorigenesis through the same mechanism. Second, the different classes of genes in the H - 2 complex might affect different steps in the neoplastic process. Thus several H - 2 genes might influence lung tumorigenesis either through the same or different mechanisms. Experiments studying the influence of H - 2 on different stages of tumorigenesis are required to elucidate the specific role of individual MHC genes. The two lung tumor types (alveolar and papillary) are differently influenced by H - 2 in mice from H - 2 congenic strains on the C57BL/10 background, treated either prenatally (Oomen et aZ., 1983) or postnatally (Oomen et d., 1988)with the carcinogen ENU. After prenatal treatment incidence and number of alveolar tumors was influenced by H - 2 haplotype. For papillary tumors, mean size but not incidence or number were haplotype-related, and this H - 2 effect on size of papillary tumors has been due to an H - 2 associated decrease in growth rate of papillary tumors, which probably sets in after 2 months of age (Oomen et al., 1983). In postnatally treated mice we showed that time of appearance and incidence of alveolar versus papillary tumors differ markedly in strains €3lO.A(2R)and BlO.A(5R),whereas no such differences were found in strains €310, BlO.A, and BlO.A(4R).Since the cells in alveolar and papillary lung tumors are similar to two distinct differentiation stages of alveolar type I1 cells (see the previous section), these findings indicate that H - 2 genes effect differentially certain specific steps of neoplastic development in the lung.

E. MECHANISMSOF MHC EFFECTSON LUNGTUMORICENESIS The mechanisms whereby the genes of the H - 2 complex influence lung tumorigenesis are still unexplained. Involvement of the immune system has

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to be considered because of the well-known function of the MHC in regulation of the immune response (see Section V,C). The H-2-associated effects on tumorigenesis in tumors with a viral etiology (i.e., leukemias) are mainly due to haplotype-related differences in immunological defense mechanisms against the antigens encoded by the inducing virus (Zijlstra and Melief, 1986). However, for several reasons it is unlikely that the H-2 effects on lung tumorigenesis are exclusively or even predominantly immunological. First, in contrast to virally induced tumors, lung tumors are believed to be weakly antigenic (Shimkin and Stoner, 1975), and no viral etiology of lung tumors was as yet indicated. Second, thymus-dependent immunological defense mechanisms do not seem to play a major role in lung tumorigenesis, since athymic nude mice treated with carcinogen after birth (Stutman, 1974) or transplacentally (Anderson et al., 1978) did not display more lung tumors than their normal littermates. Studies on the effect of neonatal thymectomy on carcinogen-induced lung tumors are conflicting and inconclusive (for review see Shimkin and Stoner, 1975). Third, involvement of non-T-cell antitumor defense by macrophages or NK cells is also not demonstrated, since susceptibility to lung tumors is not affected in mice carrying the bg (beige) mutation, which diminishes considerably NK-cell activity (Malkinson and Beer, 1983). Apart from its function in the immune response, various effects of H-2 pointing to its influence on hormonally regulated phenomena were reported (Ivanyi et al., 1969; Ivanyi, 1975; Mickova and Ivanyi, 1975; Lafuse and Edidin, 1980). The best studied has been the H-2 influence on glucocorticoid-induced cleft palate in embryos (for references see Bonner and Tyan, 1983; Demant, 1985). In addition, H-2 influences the levels of glucocorticoid receptor in lung (reviewed in Goldman and Katsumata, 1986). It has been proposed that H-2 influences susceptibility to tumorigenesis also through hormonal mechanisms (Demant, 1986). The possible significance of hormonal mechanisms in H-2 effects on tuinorigenesis is suggested by the H-2 influence on mammary tumor induction by prolactin without involvement of MTV observed by Muhlbock and Dux (1981).This finding has been recently confirmed and extended in our laboratory (Ropcke et al., 1987; see also Section VI, H). Recently we have obtained evidence that the influence of the H-2 complex on lung tumor susceptibility may to a considerable extent be related to H-2 influence on glucocorticoid hormone affects on target cells (see later). Glucocorticoid hormone is the major factor regulating prenatal development and postnatal functioning of lung epithelium. The differentiation and functional development of the lung is regulated by endogenous glucocorticoid hormones (for reviews see Ballard, 1983; Smith, 1984) and involves epithelial-mesenchymal interactions (Chen and Little, 1987; Smith, 1984). A

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Mesenchyrnal

-

Epithelial Interactions

Glucocorticoid

-

FPF

+ +

Testosterone

1-

Alveolar type II cell

+

Thyroxin

Surfactant FIG. 4. Multihormonal regulation of fetal lung cell maturation.

major feature of lung maturation in the fetus and newborn is the production and secretion of surfactant by alveolar epithelial type 11 cells. Surfactant, composed mainly of phospholipids and proteins, is a surface-active material that covers the surface of the alveoli, reduces the surface tension at the airwater interface, and prevents the alveoli from collapse at expiration. Glucocorticoid acts on fibroblasts to synthesize and release the fibroblast pneumocyte factor (FPF), which stimulates the alveolar type I1 cells to synthesize and release surfactant (Smith, 1979; Post et al., 1984; Post and Smith, 1984; Torday et al., 1985) (Fig. 4). In addition to glucocorticoids, other hormones influence this process as well; they can either depress (androgens and insulin) or enhance (thyroxin) the fibroblast-mediated glucocorticoid effect on alveolar type 11 cells (Torday, 1975; Carlson et al., 1984; Smith and Sabry, 1983) (Fig. 4). These hormonal effects on lung maturation involving epithelial-mesenchymal interactions have been determined in an in vitro system, but their in uivo counterparts have been described as well (for reviews see Ballard, 1983; Smith, 1984). A very large number of studies in a variety of species, including human beings, has shown that administration of glucocorticoids to the immature fetus results in acceleration of lung maturation, which includes enhanced morphological maturation as well as enhanced production of surfactant. Likewise, the stimulating effect of thyroid hormones on fetal lung maturation has also been found to be effective in uiuo in rabbits and rats. The opposing effect of both androgens and insulin on the stimulating effect of glucocorticoid on lung maturation has been observed in the fetus as well. In several species, including humans, the sex of the fetus appears to have an important influence on the rate at which the fetal lung matures and on its

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response to hormonal manipulation of this process. Prematurely born human male infants are at higher risk of developing respiratory distress syndrome (due to lung immaturity) than are female infants of similar gestational age, and prenatal glucocorticoid treatment of lung immaturity benefits only female fetuses, whereas male fetuses do not respond to therapy (for review see Torday and Nielsen, 1987). Experimental evidence obtained in other species strongly suggests that the sex difference in fetal lung maturation is mediated by androgens. With respect to insulin it has been shown that human infants born to mothers with insulin-dependent diabetes are at elevated risk of developing respiratory distress syndrome. Some observations suggest that fetal hyperinsulinemia may be related to the increased incidence of lung immaturity and that insulin might block the stimulating effect of glucocorticoid on lung maturation (Tsai et al., 1981). These findings indicate that hormonal regulation of the features of the mesenchymal-epithelial interactions during lung maturation revealed by in uitro studies, all have their in uiuo counterparts. Apart from the fibroblast-mediated effect on epithelium, the glucocorticoid hormones can also act directly (Post et al., 1984) on fetal alveolar type I1 cells, which contain glucocorticoid receptors (Beer et al., 1984). Glucocorticoid is also the main factor stimulating the generation and differentiation of alveolar spaces (Kauffman, 1977). We investigated whether the H - 2 effects on lung tumor susceptibility (see earlier) might be related to H - 2 influence on these hormonal effects (Oomen et al., 1989). We found that H - 2 influences the enhancing effect of glucocorticoid treatment on lung differentiation. The stimulatory effect of prenatal glucocorticoid treatment on the development of alveolar space in fetal lung is significantly affected by H - 2 haplotype: the increase in alveolar space is several times higher in strain B10 (H-2") than in strain BIO.A (H-2"). We also found that when carcinogen and glucocorticoid hormone are administered simultaneously to mouse embryos, this hormone treatment influences ENUinduced lung tumorigenesis (Oomen et al., 1989). The effect of glucocorticoid treatment is lung tumor type-specific; it affects the papillary tumors but not the alveolar tumors. The number (multiplicity) of papillary tumors is significantly affected by the hormone treatment, and the effect of treatment is influenced by H - 2 haplotype: in strain B10 (H-2") the mean number of papillary tumors is increased, whereas a decrease occurs in mice from the BIO.A (H-2") strain (Oomen et al., 1989). Both the alveolar and papillary tumors are believed to originate from the alveolar type 11 cell, but alveolar tumor cells resemble mature alveolar type I1 cells, while papillary tumor cells are more similar to fetal alveolar type I1 cells (Rehm et al., 1988). Fetal alveolar type I1 cells are likely to be susceptible to direct glucocorticoid action because they have, like the papillary tumor cells, specific nuclear glucocorticoid receptors (Beer et al., 1984; Beer

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and Malkinson, 1984), in contrast to mature alveolar type I1 cells and cells from alveolar tumors, which lack these receptors (Beer and Malkinson, 1984; Beer et al., 1983). It is likely that H - 2 affects susceptibility of immature fetal alveolar type II cells to direct or indirect glucocorticoid hormone effects, and that glucocorticoid-induced changes in differentiation state of fetal alveolar type I1 cells alter also their susceptibility to chemical carcinogenesis. The observation that these effects eventually alter the generation of papillary but not of alveolar tumors suggests that the type of the lung tumor is determined by the differentiation stage of the lung alveolar type I1 cell at the time of initiation, or shortly thereafter. Taken together these findings suggest that the H - 2 complex affects one or more steps in lung organogenesis and tumorigenesis through influence on hormonal regulation of cell differentiation. Since it is possible to study in vitro the functions of alveolar type I1 cells and of fetal lung fibroblasts separately (see earlier and Fig. 4), these techniques can be used to study the specific cellular and molecular processes where H - 2 genes affect differentiation and tumorigenesis.

F. MHC EFFECTSON TUMORIGENESIS I N SMALL INTESTINE We have frequently observed tumors of the small intestine in mice from H - 2 congenic strains on the C57BL/10 background treated prenatally or postnatally with the carcinogen ENU. These tumors were found to be adenocarcinomas of the epithelium, in which histologically different tumor cells resembling the four cell types of the normal intestinal epithelium (i.e., villus columnar, mucous, enteroendocrine, and Paneth cells) were present (Oomen et al., 1984). Since these different cells of the normal intestinal epithelium are believed to originate from common stem cells (Cheng and Leblond, 1974), the observed tumors seem to be derived from these stem cells. In the studies on the role of H - 2 in lung tumorigenesis discussed earlier, we observed also a relationship between H - 2 genes and susceptibility to the carcinogen-induced tumorigenesis in the small intestine. In mice from H - 2 congenic strains on the C57BL/10 background, treated postnatally with ENU, intestinal tumorigenesis is influenced by H - 2 haplotype. As Fig. 5 shows, the mean number of tumors per animal is significantly different between several of the H - 2 congenic strains tested (Oomen et al., 1988). The strain B10.A(2R) is highly susceptible and differs from the relatively resistant strains BlO.A(5R), BlO, and BlO.A(4R) in tumor incidence and number of tumors, while strain B1O.A is intermediate. Strain BlO.A(2R) takes an extreme position also with respect to the location of tumors in the small intestines: in this strain the majority of tumors is

GENETICS OF TUMOR SUSCEPTIBILITY IN THE MOUSE

4.5

161

mean number of tumors

4 3.5 3

2.5 2 1.5 1

BlO.A(PR)

BIO.A

BlO.A(BR)

610

BlO.A(4R)

FIG.5. Number and distribution of tumors along the small intestine of mice from five H-2 congenic strains on the C57BL/10 background treated postnatally with the carcinogen N-ethylN-nitrosourea. For each strain the mean number of tumors per tumor-bearing mouse (vertical axis; combined for females and males) found in the proximal (diagonal lines) and distal 20-cm segment (cross-hatching) of the small intestine is given.

located in the proximal part of the small intestine (duodenum and part of jejunum), whereas in strain BlO.A(4R)most tumors were found in the distal part (part of jejunum and ileum). In strains BlO.A, BlO.A(5R), and B10 the tumors are distributed more evenly along the small intestine (Fig. 5). This is, to our knowledge, the first example of a genetic influence on the location of a certain type of tumor in an organ. These findings may be related to the fact that the small intestine is a longitudinally specialized organ with the proximal and distal part having in many respects very different functions in the digestive process. Different haplotypes may have separate and diverse effects on maturation and function of the two parts of the small intestine, and hence influence also the appearance of tumors in each of them separately. Thus, the H-2 complex affects several parameters of tumorigenesis in the small intestine in congenic strains on the C57BL/10 background: tumor incidence, mean number of tumors per mouse, and the location of tumors along the small intestine. Because the intestine, like lung, is derived from embryonal foregut, and because differentiation and functional development of the small intestine is regulated by glucocorticoid hormone (Smith and Zinman, 1982; Henning, 1986), we investigated whether these H - 2 effects on tumorigenesis in the small intestine are influenced by glucocorticoid treatment. We found that a concomitant prenatal glucocorticoid treatment affects prenatally ENUinduced tumorigenesis in the intestine. Both the number of ENU-induced

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tumors and their location in the small intestine were significantly affected by glucocorticoid treatment, and both effects were influenced by H - 2 haplotype. In strain BlO (H-2") the number of tumors was increased in males and decreased in females, while no effect of hormone treatment on tumor numbers has been seen in B1O.A mice or either sex. The location of the tumors in B10 and BIO.A mice treated prenatally with ENU was not different, but the concomitant glucocorticoid treatment affected it in strain BIO.A (H-2"), where hormone treatment resulted in a shift toward the proximal part of the intestine (Oomen et al., 1989). Thus a concomitant glucocorticoid treatment affects in a H - 2 haplotype-specific manner not only prenatally induced lung tumor development (see earlier), but also prenatal tumorigenesis in the intestine. We propose that the parallel effects of the H-2 genes on differentiation and tumorigenesis in the two developmentally related organs, lung and small intestine, observed in our experiments, may reflect a more general effect of the M HC on hormonal regulation of differentiation of epithelial tissues. These results may also offer a starting point to approach the problem of the relationship between the differentiation stage of target cells and their susceptibility to tumorigenesis.

G. MHC EFFECTSON TUMORIGENESIS I N LIVER The effects of H - 2 on tumorigenesis in the liver, be it spontaneously or chemically induced, have been studied in H - 2 congenic lines on the A, C3H, and C57BL/10 background. Smith and Walford (1978) showed that in mice on the A background spontaneous liver tumor incidence in males was affected by H - 2 haplotype. In mice from H - 2 congenic strains on the C3H background, known to be prone to liver tumor development, an H - 2 influence on spontaneous liver tumorigenesis was suggested also (Smith and Walford, 1978), but this finding could not be confirmed in another study (den Engelse et al., 1981). In both aforementioned studies H - 2 congenic strains on the C57BL/10 background were also included, but in none of these strains did a significant percentage of mice (males nor females) develop liver tumors, whether carcinogen was applied or not. In contrast, we have shown that a postnatal ENU treatment can induce a moderate to fairly high incidence of liver tumors in these strains, especially in males (Oomen et al., 1988). For two types of liver parenchymal tumors, hepatocellular adenomas and hepatocellular carcinomas (the latter tumors frequently give rise to metastases), we found that in males the H - 2 haplotype markedly influences their occurrence. For both liver tumor types, BlO.A(2R) proved to be the most susceptible strain, BlO.A(5R) the most resistant. The other strains tested, BlO.A, BlO.A(4R), and B10, were intermediate. For females no

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differences between strains were found, either for hepatocellular adenomas or for hepatocellular carcinomas. Together these findings indicate that genes in or closely linked to the H - 2 complex are also involved in liver tumorigenesis. Whether their effect is observed depends, however, on the rest of the genome and the experimental system used. H. MHC

AND

MAMMARY TUMOR SUSCEPTIBILITY

The first evidence for the role of the mouse MHC in susceptibility to mammary tumors has been obtained by Muhlbock and Dux (1974) using H - 2 congenic strains on C57BL/ lOScSn background and C3H-MTV. The standard induction procedure in their experiments consisted of foster-nursing newborn mice on MTV-producing females and, after weaning, force-breeding the young females to provide appropriate hormonal stimulation for the mammary gland. The tests of the B10 strain and of 11 H - 2 congenic strains revealed that they differ widely in susceptibility, the strain B10 (H-2b) being the most resistant, the strains BlO.A(SR)( H - F ) being the most susceptible. The other strains were intermediate, forming a continuous range between the most susceptible and the most resistant strain (Muhlbock and Dux, 1974, 1981). Tests of F, hybrids between B10 and BlO.A(SR) revealed that the H-2-linked susceptibility is a dominant trait. As several recombinant haplotypes were present in this group of strains, it was possible to ascertain that the main genetic factors responsible for susceptibility map most likely into the central regions of H - 2 , between I - A and -D, and also to the right of S. In a separate test (Dux, 1983)influence of the TZa region has been demonstrated on C57BL/6 genetic background (Tlu" conferring relative resistance compared to TZub), but not on A strain background. In contrast to the clear evidence for the role of the H - 2 complex in susceptibility to mammary tumors, tests of congenic strains on B10 background differing at n o n - M H C histocompatibility loci H - 1 , H-3, H 4 , H - 7 , H-8, H - 9 , H - 1 2 , and H - 1 3 did not reveal any effect of these genes (A. Dux, unpublished observations). Subsequently, the role of the H - 2 complex in susceptibility to C3H-MTVinduced mammary tumors has been demonstrated by the same authors also on other genetic backgrounds. Differences in susceptibility were found between strains C3H (H-2'9 and C3H.Bl0 (H-2"), BALBlcHeA ( H - 2 9 and B A L B I c - H - ~(H-2b), ~ and 020/A ( H - 2 9 and 020.Q (previously named OIR, H-29). These tests confirm the linkage of mammary tumor susceptibility with the MHC. That non-MHC genes also play an important role has been revealed by the tests of strains sharing the same haplotype, H-29: DBA/A, 020.Q, and C57BLILiA-H-24 (formerly BIR). The latter strain was very resistant, while DBA and 0 2 0 . Q were relatively susceptible. While in all experiments just discussed C3H-MTV has been used, a series

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of experiments using DBA-MTV and GR-MTV revealed a complex interaction between the H-2 haplotype and MTV type in determining tumor susceptibility. The strains 0 2 0 and 020.Q without exogenous MTV both produce a moderate number of mammary tumors at a relatively high age. Infection with CSH-MTV leads to an increase in the tumor incidence and earlier appearance of tumors in 0 2 0 mice, while the strain 0 2 0 . Q is relatively resistant. On the other hand, infection with DBA-MTV leads to a reverse picture: a high tumor incidence and an early appearance of tumors in 020.Q mice, while the strain 0 2 0 is relatively resistant. Thus, the two haplotypes, H-2pz and H-24, have different effects on MTV-induced tumorigenesis depending on the type of MTV. The C57BLILiA-H-2'1 (BIR) mice are, similarly to 0 2 0 . Q (H-2'9 mice, more susceptible to DBA-MTV and GR-MTV than to C3H-MTV. The B10 mice (H-2b)are resistant to C3HMTV but susceptible to GR-MTV, while the congenic BlO.A(5R) mice (H-2i5) are equally susceptible to CSH-MTV and GR-MTV. However, the susceptibility is dependent not only on type of MTV, but also on the method of hormonal stimulation: the strain C57BL/LiA is very resistant to C3HMTV-induced tumors when force-breeding is used, but very susceptible with hypophyseal isografts as the source of hormonal stimulation. The H-2 congenic strains do not differ in the number, structure, or expression of endogenous MTV proviruses (Long et al., 1980). The H-2-linked susceptibility to MTV-induced mammary tumors probably reflects the effect of H-2 on immune response against the MTV. Blair et al. (1983) demonstrated that H-2 genotype influences plasma levels of MTV. Dux and Deinant (1987) showed that the effects of H-2 on susceptibility to CSH-MTVinduced mammary tumors are systemic (see Section 11), in contrast to the direct effects of non-MHC susceptibility genes on the susceptibility of mammary gland itself (for review see Dux, 1981). The H-2 complex influences also the susceptibility to hormonally induced mammary tumors in mice that are free of infectious MTV. The tumors are induced by hypophyseal isografts placed under kidney capsule. The isografts are severed from the direct blood supply from the hypothalamus, and thus the hypophyseal cells are freed from control by hypothalamic hormones. As a result, the hypophyseal cells proliferate and produce prolactin continuously, and possibly also other hormones as well, which stimulates proliferative and secretory activity of mammary epithelium (for review see Boot et n l . , 1981). In many strains, this stimulation leads to the appearance of mammary adenoacanthomas, in contrast to adenocarcinomas, which form the largest proportion of MTV-induced mammary tumors. The cells in these hormonally induced tumors, similarly to mammary epithelium of mice without infectious MTV, do not produce detectable MTV proteins (P. C. Hageman, personal communication); also, the transcripts of endogenous

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MTV are present in very small amount, if at all (Ashley et al., 1980). Muhlbock and Dux (1971, 1981)demonstrated that H-2 genes influence the incidence and time of appearance of hormonally induced mammary tumors in C57BL/10 congenic strains. The strain pattern of relative susceptibility and resistance to this method of tumor induction differed from that for C3HMTV-induced tumors. Ropcke et al. (1987) have confirmed and extended this finding, and found that the congenic strains differ not only in the incidence and time of appearance of mammary tumors, but also in the behavior of the grafted hypophysis. The size of the hypophyseal graft differed highly significantly between the congenic strains, and so did the concentration of estrogen receptors in the hypophyseal graft. There has been no obvious correlation between the susceptibility to mammary tumor induction and these two H-2-influenced traits. However, analysis of additional data (Ropcke and Demant, in preparation) reveals that while in the strains that are relatively susceptible to mammary tumor induction no correlation exists between mammary susceptibility and size of the hypophyseal isografts, in strains that are more resistant to tumor induction, a significant correlation between the two parameters exists, because females with large grafts are more likely to produce tumors. Limited numbers of tests failed to indicate that plasma prolactin levels correlate with the appearance of the hormonally induced tumors (Van der Gugten et al., 1985; Ropcke et al., 1987), and Nagasawa et al. (1976) suggested that susceptibility of mammary gland to hormonal induction of tumors correlates with the proliferative and differentiative response of the gland to hormonal stimulation. Data from our laboratory suggest that the main mechanism of H-%linked susceptibility to hormonal induction of mammary tumors resides probably in the mammary gland, but that in the relatively resistant strains also the second effect of H-2, namely the effect on the growth rate of the hypophyseal graft, influences the tumorigenesis. The molecular mechanisms of these H-2 effects remain to be elucidated. Little is known about the non-MHC genes influencing the susceptibility to virally and hormonally induced mammary tumors. There is a difference in the susceptibility to C3H-MTV due to non-MHC genes between several H-2&inbred strains: C57BL/LiA (very resistant), B10 (resistant), BIMA (intermediate), and C3H. B10 and BALB. B10 (relatively susceptible) Muhlbock and Dux, 1981). The role of non-MHC genes was demonstrated also using RIS produced from the strains BALB/cBy (susceptible) and C57BL/6By (resistant) by Bailey (1971). Of the seven C x B RIS tested, one was resistant, one was more susceptible than BALB/cBy, and the rest were intermediate. This indicates involvement of two or more genes in the difference in susceptibility between BALB/cBy and C57BL/6By (Dux et al., 1978). Very large differences in susceptibility of inbred mouse strains have been

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known to exist (for review see Hageman et aZ., 1981), and the genes involved affect mainly the susceptibility of the mammary gland itself rather than the systemic factors; that is, they are true tumor susceptibility genes (Dux, 1981). Tests using RIS did not until now lead to identification of these genes, because of the multigenic nature of the strain differences. The three series of RCS prepared in our laboratory each involve one parental strain that is susceptible and one that is resistant to mammary tumorigenesis (BALB/ cHe-STS/A, C3H/Sn-C57BL/lOScSn, and 020/A-BlO.O20/Dem, respectively). Their application might contribute to identification of the genetic factors involved in mammary tumorigenesis.

I. MHC

AND

TUMORIGENESIS IN EPITHELIAL ORGANS-SUMMARY

More than 70%of tumors in humans are of epithelial origin as compared to 8% leukemias (Silverberg and Lubera, 1986). The study of the genetics of susceptibility to tumorigenesis in organs like lung, mammary gland, liver, and small intestine can help to assess how the specific risk of a number of common types of cancer is associated with certain genes. Such studies also have theoretical importance. The epithelial cells of these organs carry out very different functions, but the regulation of their development, maturation, and function exhibits common principal features, namely finely tuned multihormonal regulation, which is partly mediated by modulatory effects of mesenchyme. Therefore, these organs provide the opportunity to study the common features of the relationship between differentiation and susceptibility to oncogenesis. The data on MHC influence on tumor susceptibility in the epithelial organs-lung, small intestine, liver, and mammary gland-indicate that several different effects of the H - 2 gene complex are operating. The experiments with mammary tumorigenesis yield several types of effects, depending on the induction scheme. In virally induced tumors, systemic effect of H - 2 predominates (Dux and Demant, 1987), and the haplotype effects are specific for the type of MTV used. This suggests that H - 2 genes influence immune response against MTV antigens on virions or cells. Other effects are seen with hormonally induced mammary tumors (Miihlbock and Dux, 1981), which do not produce MTV proteins in any appreciable amount. The H - 2 genotype affects not only the incidence and time of appearance of mammary tumors, but also the behavior of the heterotopic hypophyseal isograft used to induce the tumors (Ropcke et d., 1987).The growth of the isograft under the kidney capsule correlates in resistant strains with the appearance of tumors. Besides the possible immunological effects, the H - 2 apparently influences the formation of hormonally induced tumors through two mechanisms-one

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that might affect the response of mammary gland to hormonal stimulation, and the other that likely influences the hormonal stimulus itself. In addition, H-2 genotype appears to influence the levels of estrogen hormone receptors in the transplanted hypophysis (Ropcke et al., 1987). These data illustrate that the H-2 complex can affect several nonimmunological processes, some of which may be relevant for development of tumors. Gronberg et al. (1983),have shown that mice of H-2 congenic strains differ in susceptibility to epithelial tumors of skin and to lymphomas after peroral treatment with DMBA. There has been no correlation with NK-cell activity. Koizumi et al. (1987) described H-2-linked differences in Ah receptor levels and Ah inducibility by P-naphthoflavone, but because of differences in the strains tested in the two studies, it is not clear to what extent the results of Gronberg et al. (1983) might be due to differences in metabolic processing of DMBA. In the studies on tumor induction with the directly acting carcinogenmutagen ENU, the need for metabolic activation of the carcinogen is avoided. Polymorphism of genes influencing such metabolic steps, which per se are not related in any way to the neoplastic transformation itself, will therefore not influence the results. Because ENU induces tumors in a variety of organs, the effects of the same genes on tumorigenesis in different organs may be analyzed. The results obtained in H-2 congenic strains on C57BL/lOScSn background (Oomen et al., 1988, and unpublished observations) indicate that the MHC affects susceptibility of lung, small intestine, and liver to ENU carcinogenesis. It has been proposed that many of the effects of H-2 on tumorigenesis are due to nonimmunological biological functions of H-2 (Demant, 1986). The effectiveness of experimental modification of prenatal E NU-induced tumorigenesis by glucocorticoid treatment and the influence of H-2 genotype on the effects of this hormonal manipulation indicate that MHC influences the susceptibility to chemical carcinogenesis through effects on hormonal regulation of cell differentiation (Oomen et al., 1989). The hormonal regulation of function of lung epithelium persists throughout the life cycle. Therefore, the effects of MHC on this regulation might possibly also affect postnatally induced tumors. These observations raise two questions of considerable theoretical and practical interest: (1) what products of the H - 2 gene complex are involved and how do they operate; and (2) what is the mechanism of these effects of MHC on susceptibility to tumor induction? The nonimmunological effects of the MHC may be due to the class I or class I1 genes, one or more genes of the heterogeneous group of class 111 genes, or presently yet unknown genes. The class I and class I1 genes have been shown to associate with various hormone or growth factor receptors on cell membranes (Schreiber et al., 1984; Due et al., 1986; Kitur et al., 1987),

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or proteins inside the cell (Anderson et al., 1985; see also Section V). The intracellular binding of class I or class I1 antigens to biologically important molecules might disturb their metabolism, function, or secretion (for discussion see Parham, 1988). The latter mechanism might be related to the insulin-dependent diabetes mellitus in transgenic mice expressing class I (Allison et a l . , 1988) or class I1 (Sarvetnick et a l . , 1988; Lo et al., 1988) molecules in pancreatic p cells. The discoveries of two unexpected genes with unknown functions in the S region of the H-2 complex and the mapping of the two genes for tumor necrosis factor between S and H-2D (see Section V) suggest that some biological effects of H-2 might be due to genes other than class I or class 11. This question can be resolved by mapping the studied effects to specific regions of the H-2 gene complex and by identifying subsequently the relevant genes by transfection or transgenesis. How do the nonimmunological effects of MHC genes affect susceptibility of cells to tumorigenesis? The available data suggest that the H-2-linked genes affect the susceptibility of cells to regulation of their differentiation state by hormones, especially glucocorticoids. The differentiation state of the cells determines their susceptibility to neoplastic transformation. This has been demonstrated in a variety of experiments using transformation by oncogenes (for review see Klein and Klein, 1986). The results of these studies suggest that, in the spectrum of the possible differentiation stages of a cell, only certain stages-the “differentiation window”-allow the transformation by the oncogene. The factors influencing the outcome of the in vitrotransformation experiments appear to operate after the action of the oncogene product (Klein and Klein, 1986). The alteration of susceptibility to tumorigenesis by the effects of H-2 on hormone susceptibility might be brought about through modification of function of cell surface hormone receptors (see earlier), or modulation of signal transduction, possibly through altered glucocorticoid effects on phospholipase A, (Irvine, 1982), which is an important enzyme in arachidonic acid metabolism (Burgoyne et al., 1987). Another possibility is H-2 influence on expression of oncogenes (see Section 111,A). Glucocorticoids have also been shown to inhibit the tumor promotion (Slaga, 1980), and it would be interesting to investigate whether this effect is influenced by H-2. The study of the relationships between hormonally regulated cell differentiation and susceptibility to tumorigenesis, and the role of MHC genes therein, offers the possibilities of analyzing well-defined host factors that regulate the behavior of the cells. These factors are the ones involved in normal regulation of development and function of various tissues in mammals, and therefore the results of such studies would likely be applicable to actual processes of tumorigenesis. In addition, these studies may provide a better insight into the nonimmunological functions of the MHC, and into

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their evolutionary relationship with other MHC functions. Such information would benefit also the understanding of the relationship between the M HC and susceptibility to various diseases. VII. Tumor Susceptibility Genes: Molecular and Cellular Perspective

The discussion of tumor susceptibility genes in the preceding sections has been necessarily selective and limited in extent. Nevertheless, the present state of knowledge and technology warrants the proposition that, in addition to previous achievements, the long-standing promise of contribution of genetic studies of cancer to the understanding of basic processes in neoplasia will be made true also in the near future. This proposition is based on several premises. 1. The possibility now exists of genetic and molecular identification of tumor susceptibility genes. The use of RCS offers a rational perspective of genetic definition and mapping of a number of tumor susceptibility genes. The current advances in manipulation and cloning of large fragments of DNA and progress in physical mapping of genomic DNA make the cloning of the genes with known meiotic map position more feasible than before. The combination of the genetic and molecular approaches may become a powerful tool for actual molecular isolation of tumor susceptibility genes, which have been escaping identification for such a long time. 2. A better insight into the biological nature of the effects of tumor susceptibility genes will allow a more appropriate and purposeful choice of experimental models. By studying those genes that affect the susceptibility of the cell to tumorigenesis, a link of genetic studies with other relevant issues of neoplastic transformation can be made. A more precise understanding of individual stages of the neoplastic process offers better possibilities for identification of the specific steps at which the tumor susceptibility genes operate. Recognition that the tumor susceptibility genes generally affect the postinitiation stages of tumorigenesis is the first step along this path. A better definition of differentiation stages of normal and tumor cells, and better experimental possibilities of their manipulation, will contribute to the understanding of the “differentiation window” for tumorigenic action of oncogenes. 3. Advances have been made in our understanding of the molecular nature of the neoplastic process. Identification of numerous oncogenes, protooncogenes, and tumor suppression genes offers a host of possibilities of characterization and experimental manipulation of normal or tumor cells, which can be used to study the mechanisms of action of tumor susceptibility

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genes. In the preceding period these advances have led to the recognition that genetic changes indeed lie at the basis of the neoplastic transformation, an understanding that the genes involved are not specific “cancer genes” but rather genes involved in a variety of normal functions of the cell, and that each of these genes contributes to only one or a few of the several steps required to change a normal cell into a neoplastic cell. Thus, the apparent a priori principal differences among oncogenes, tumor suppression genes, and tumor susceptibility genes will in many cases disappear. We propose that the three groups of genes overlap and interact to a considerable extent. The action of oncogenes and tumor suppression genes can be understood only when the critical substrates for the action of their products are identified. The demonstration of genetic linkage of lung tumor susceptibility with the protooncogene Kras-2 (Ryan et al., 1987), the effect of the H-2Dk gene on the expression of the Kras-2 gene (Alon et al., 1987), and evidence for a genetically determined preference for certain retrovirus integration sites in tumors (Mucenski et al., 1988) indicate close interactions between tumor susceptibility genes and oncogenes. The analysis of interactions with the known oncogenes and tumor suppression genes is one of the main tasks in the study of tumor susceptibility genes. Cloning of tumor susceptibility genes will considerably advance the possibility of experimental study of these interactions.

ACKNOWLEDGMENTS We thank Dr. A. Dux for careful reading of Section VI and many useful comments, Dr. M. A. van der Valk for discussions and suggestions in the course of preparation of the manuscript, and Mrs. M. Sonne and Mrs. T. van Diepen for unrelentingly efficient and attentive typing and reediting the manuscript.

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PERSPECTIVES ON THE ROLE OF MHC ANTIGENS IN NORMAL AND MALIGNANT CELL DEVELOPMENT Bruce E. Elliott,* Douglas A. Carlow,t Anna-Marie Rodricks,* and Andrew Wade5 'Division of Cancer Research. Department of Pathology, Queen's University. Kingston. Ontario, Canada. K7L 3N6 tMounf Sinai Hospital Research Institute, Toronto, Ontario, Canada M5G 1X5 +Department of Oncology Research. Toronto General Hospital, Toronto. Ontario, Canada, M5G 2C4 SMlR Office, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada, T2N 1N4

I. Introduction 11. Basis of Tumor Immunology

A. Immunosurveillance Theory of Cancer: Current Concepts B. The Nature of Tumor Rejection Antigens 111. The Biology of M H C A. The MHC Gene Family B. Expression of MHC in Normal Tissue IV. MHC Function A. The MHC-Antigen-T-cell Interaction B. T-cell Adhesion Molecules (CAM) C. Nonimmunological Role of MHC V. MHC Expression in Malignancy A. Clinical Cancer B. Animal Tumor Models VI. Evidence for a Role of M H C Antigens in Malignancy A. Influence of Oncogenes and Oncogenic Viruses B. Gene Transfection Strategies VII. Proposed Function of M H C in Malignancy A. Transplantation Studies and Relevance to MHC and Cancer B. Role of MHC in NK Recognition and Natural Resistance VIII. Regulation of Altered Class I MHC Expression in Malignancy A. General Features of M H C Gene Regulation B. Regulation of Class I M H C in Malignancy IX. Organ- and Tissue-Specific Effects on Immune Surveillance and Tumor Progression A. Compartmentalization of the Immune System B. Influence of Tissue Site on Tumor Growth and Metastasis: Implications for Studies on Tumor Immunogenicity X. Concluding Remarks References

I. Introduction

Over the past decade, the concept that increased expression of class I major histocompatibility complex (M HC)' antigens facilitates T-cell immune 'Abbreviations: Ad, adenovirus; APC, antigen-presenting cell; P2m, &-microglobulin; BL, Burkitt's lymphoma; CAT, chlorainphenacol acetyltransferase; Con A, concanavalin A; EBV,

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defense against cancer has received much attention and some significant support. Another recent development is that in certain tumor systems (e.g., lymphomas and melanomas), augmented expression of class I MHC antigens appears to be associated with increased growth and metastasis, and acquired resistance to natural killer (NK) cells. Depending on the tumor system, augmentation of class I MHC expression could facilitate, compromise, or be of no consequence in progressive malignant disease. The purpose of the present article is (a) to provide an update on the basic properties of MHC biology and function, (b) to review the controversial literature on the involvement of MHC in host antitumor defense, (c)to discuss possible mechanisms of MHC regulation in normal and malignant cells, and (d) to present a new perspective, namely the influence of tissue microenvironment and of tissue-specific lymphocyte migration on host-tumor immune interactions. There are many extensive reviews on the role of class I MHC antigens in malignancy (Doherty et al., 1984; Festenstein and Labeta, 1987; Linsk and Goodenow, 1986; Vogel et d., 1987; Tanaka et al., 1988b; Hammerling et al., 1987a,b). It is our intention in this article to examine those properties of MHC molecules in normal cells that may be relevant to malignancy, to raise important questions for future study, and to emphasize a cautious approach in extrapolating to human malignancies. II. Basis of Tumor Immunology

Two critical issues in tumor immunology have remained controversial despite extensive investigation. The first contention is that immunosurveillance is an active process in defense against cancer, and the second is that tumor-specific antigens exist. Since current views on the putative role of MHC in malignancy are influenced by our understanding of these issues, they are briefly reviewed here.

A. IMMUNOSURVEILLANCE THEORY OF CANCER: CURRENT CONCEPTS Since the demonstration by Gorer (1938) that genes encoding major transplantation antigens control susceptibility to tumor transplants, most tumor transplantation studies were carried out in syngeneic systems. Initially, the use of chemically or virally induced tumors predominated (Gross, 1943; Old Epstein-Barr virus; IL, interleukin; IFN, interferon; LGL, large granular leukocyte; LAK, lyniphokine-activated killers; MC, methylcholanthrene; MHC, major histocompatibility c o n plex; mHA, minor histocompatibility antigen; MNNG, N‘-methyl-”-nitro N-nitrosoguanidille; MuMTV, murine mammary tumor virus; NK, natural killers; PKC, protein kinase C; SV40, simian virus 40; Tc, cytotoxic T lymphocyte; Th, helper T lymphocyte; TPA, 12-0tetradecanoylphorhol-13-acetate;UV, ultraviolet.

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and Boyse, 1964; Klein, 1973). These tumor systems, against which strong immunity in syngeneic hosts could be demonstrated (Hellstrom and Hellstrom, 1969), formed the context in which the immunosurveillance theory of cancer developed. The theory states that malignancies express foreign determinants that can be potential targets of the immune system (Burnet, 1964). However, there are two main criticisms of the immunosurveillance theory. The first criticism concerns the existence of tumor-specific antigens in human malignancy. The roots of serious skepticism arose with reports of Foley (1953), and Prehn (1975), that animal tumors arising spontaneously (i.e., in the absence of known carcinogens) did not display detectable immunogenicity. In addition, it was shown by Stutman (1981) and Parker et al. (1982) that athymic nulnu mice and normal nu/+ littermates develop spontaneous tumors at similar frequencies. The height of skepticism was expressed by Hewitt et al. (1976), who were the first to elaborate in detail on this theme, publishing a series of studies showing that spontaneously arising tumors when transplanted into syngeneic hosts elicited no detectable immunity. In this study, inbred mice from the same colony in which the tumor arose were used, to avoid genetic drift (Bailey, 1982). On the basis of these findings, Hewitt argued that such spontaneously arising tumors “are the only appropriate models of human cancer.” Epidemiological evidence clearly shows that the majority of human cancers are induced by physical and chemical carcinogens (Doll, 1980). Because transplantation experiments cannot be done in humans, it is not known whether human tumors express tumor-specific antigens analogous to experimentally induced animal tumors. Moreover, it is not clear at present how spontaneous animal tumors relate to human malignancy. The second criticism concerns the immunopotentiation of autologous tumor growth during carcinogenesis in v i m . Prehn (1977) pointed out that certain types of immunostimulation could result in reduced latency and enhanced tumor growth, a finding not predicted by the immunosurveillance theory. Indeed, a correlative analysis revealed that tumors with higher immunogenicity often had shorter latencies (Prehn and Bartlett, 1987). Two mechanisms have been proposed to explain this phenomenon. Helper T cell (Th) subsets may be stimulated by the tumor, followed by local release of growth-promoting lymphokines, or certain antigenic epitopes on tumors might stimulate immunosuppression or tolerance in tumor-bearing hosts. Thus, immune responses if elicited are not necessarily detrimental to the development of a neoplasm. Animal systems that have received much interest as possible models of certain human malignancies include ultraviolet-induced fibrosarcomas (Hostetler and Kripkie, 1988), and viral-induced leukemias [e.g., radiation leukemia virus (Meruelo, 1979) and Simian virus 40 (SV40) (Pan et al.,

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1987)l. The antigens expressed on such tumors may be representative of corresponding human malignancies such as melanomas (Mukherji and MacAlister, 1983) and human T-cell lymphotropic virus type I (HTLV-1)associated T-cell leukemias (Stoolman and Rosen, 1983; Levine et a l . , 1988), respectively. Immunosurveillance against such tumors is likely to occur, but the final outcome may depend on the nature of the immune response elicited, and the ability of the tumor to generate variants resistant to immunological restraint (Boon, 1983; Urban et al., 1982; Dennis et al., 1981). In summary, the views on immunosurveillance in cancer remain highly polarized and, over time, the consensus of opinion has swung several times from one extreme to another. The possibility that non-T mechanisms such as NK cells (see Section VII,B) are involved has further complicated the issue. Nevertheless, certain experimentally induced animal tumor systems have proved useful for studies on tumor rejection antigens.

B. THE NATUREOF TUMOR REJECTIONANTIGENS In contrast to the MHC-encoded transplantation antigens, the molecular identification of tumor-specific rejection antigens has eluded most biochemical and genetic analyses. Essentially every putative tumor-specific antigen described in human or animal malignancies was found to be expressed on normal adult or fetal tissue (Old, 1981).There is no evidence that any of these antigens can be effectively targeted by the immune system. Three candidates for the elusive tumor-specific molecules have been described in experimentally induced animal tumor systems (Srivastava and Old, 1988; Schreiber et al., 1988; De Plaen et al., 1988). Preliminary molecular characterization of these structures has been carried out and is summarized here. One group includes the tumor-specific antigens of chemically induced rodent tumors (Srivastava and Old, 1988). An important feature of these antigens is their extensive diversity. In a series of 25 independently derived tumors, no cross-reactivity was found (Basombrio, 1970); two or more sarcomas induced in the same mouse, or induced by different carcinogens, have been suggested to have distinct antigens (Basombrio, 1970; Globerson and Feldman, 1964). Studies by Srivastava and Old (1988) have led to the proposal that this class of polymorphic tumor rejection antigens belongs to a family of surface glycoproteins, designated gp 96. The gp 96 proteins represent a family of molecules encoded by a single gene that is located on chromosome 10 (mouse) (P. K. Srivastava, M. Kozak, and L. J. Old, unpublished observations) and is expressed in both malignant and normal tissue. These molecules can be isolated from carcinogen-induced tumors and appear to carry immunodominant determinants (Srivastava et a l . , 1987).

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Partial sequence analysis has revealed no tumor-specific nucleotide polymorphisms, although modifications in the amino acid sequence of restricted regions of the molecule have not been excluded. Further work is necessary to resolve the polymorphisms at the molecular level and to prove, using gene transfection strategies, that g p 96 molecules can confer tumor antigenicityhmmunogenicity . A second type of murine tumor rejection antigen includes those induced by mutagenesis in an already malignant cell. This process, known as tumor xenogenization, results in the induction of immunogenic variants bearing unique antigens capable of stimulating a strong cytolytic T-cell response (Wolfel et al., 1987). One such antigen, called P91A glycoprotein, has been characterized by D e Plaen et al. (1988). This molecule carries antigenic determinants induced by N’-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and has been partially characterized on immunogenic variants of the mastocytoma P815 (Wolfel et al., 1987). The gene encoding P91A has been transfected into the parental P815 tumor and shown to confer immunogenicity and susceptibility to cytotoxic T lymphocytes (Tc). The P91A gene lacks homology with any known gene, and its nucleotide sequence differs from the wild-type gene in untreated tumor cells by a single nucleotide. These results clearly show that point mutations induced by certain mutagens can lead to the expression of novel antigens on tumor cells. Whether the P91 antigen is related to gp 96 antigens, which are found on already-established tumors that have not undergone subsequent mutagen treatment, remains unresolved. Nevertheless, both the P91 and gp 96 antigen systems provide an interesting approach to examine the molecular interactions that can determine tumor immunogenicity. A third antigen system that has received much attention is the ultravioletinduced sarcoma antigens (Schreiber et al., 1988). A detailed molecular analysis of one such tumor antigen, 1591, has illustrated the difficulties in the tumor antigen field. After extensive investigation, it was found that the 1591 tumor expressed three novel MHC class I molecules (Stauss et al., 1986), in addition to the two normal k haplotype MHC class I molecules (Philipps et al., 1985). These results suggested that new MHC-like molecules were generated in the process of ultraviolet-induced transformation. Unexpectedly, the genes encoding two of the molecules, 149 and 166, were 100% homologous in their encoding regions to L4 and D4 (Linsk et aE., 1986; Lee et al., 1988). The third molecule, 216, was clearly distinct from K 4 (G. Jay, personal communication), and no strain carrying the 216 gene has yet been identified. This finding raises the possibility that the 149 and 216 genes were not the result of mutational changes during tumorigenesis but represented allogeneic class I genes derived from residual heterozygosity (Bailey, 1982), or from genetic contamination of the C3H animal that gave rise to the

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1591 tumor. Previous analyses suggested that genetic drift or genetic contamination of the present-day C3H strain in which 1591 was induced was unlikely in that (a) eight other tumor induced in the same experiment and in the same group of mice did not express these antigens, (b) seven polymorphic isoenzymes showed the typical C3H strain-specific pattern in the 1591 tumor, (c) the tumor expressed all known Kk- and Dk-specific epitopes, and (d) the novel antigens were expressed on both 1591 tumor cells from the first transplant generation and solid 1591 tumors that had never been passaged in uitro (Philipps et al., 1985). Because no DNA from autochthonous nonmalignant tissues was available, a direct comparison of the three novel 1591 genes with DNA from normal tissue of the animal bearing the original tumor was not possible (Schreiber et al., 1988). This study underlies the importance of comparing putative tumor antigen genes with control DNA from the host of tumor origin. A key question in animal tumor systems is whether a gene encoding a particular tumor antigen is the result of somatic mutation or is already present on normal cells of the animal from which the tumor was isolated originally. An answer to this question may provide important insight into the nature of putative tumor-specific antigens in human malignancy. Ill. The Biology of MHC

While scientists have been struggling with the identity of tumor rejection antigens, class I MHC molecules have received increasing attention as the self-component of the antigen-MHC complex recognized by T cells. A brief review of the basic biology of MHC molecules relevant to cancer is now presented.

A. THE MHC GENEFAMILY The MHC is a multigene family, -3000 kb in size, located on chromosome 17 in the mouse (Steinmetz and Hood, 1983; Flavell et a l . , 1986) and chromosome 6 in humans (Wake, 1986;Trowsdale, 1987). Given the scope of this review, only the salient features of class I MHC gene organization are discussed. [See Maziarz et al. (1988) and Koller et al. (1987, 1988) for more extensive reviews.] In the mouse, the MHC consists of 25-35 class I loci, depending on the mouse strain, grouped into four regions, designated H - 2 K , H - 2 D , Qa, and Tla. The organization of the human MHC genes bears many similarities to mouse (Fig. 1). At least 17 HLA loci have been identified, including HLA-A, -B, and -C (Koller et al., 1987) analogous to H - 2 K , -Dand -L in mouse. In addition, several nonclassical class I HLA genes similar to the murine Qa-Tla genes have also been identified, three of which have

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CLASS I1 I

DP -

DX Bd

1

DR BBBBa

-

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CLASS I I

BC

II

E

A

I

I I

CLASS I

FIG.1. Organization of the genes in the human (HLA) and mouse (H-2) MHC. Each box represents a single locus. Expressed and nonexpressed genes are indicated by closed and open symbols, respectively. The number of loci in each region depends on the haplotype. A number of class I genes downstream from HLA-A have been recently described. Homologous genes in the mouse are indicated by dashed lines. The H-2 map shown here represents the BALB/c (H-29 haplotype (Steinmetz et al., 1981). In contrast, C3H ( H - 2 k ) and B10 (H-2b) haplotypes have only one gene at the D region and no L gene. The Tla region consists of only 10 genes in the B10 haplotype and is completely deleted in the C3H haplotype (Mellor et 01.. 1984; R. Goodenow, personal communication).

been shown to be expressed (Koller et al., 1987, 1988; Shimizu et al., 1988; Geraghty et al., 1987). Class I MHC genes encode a 45-kDa glycoprotein that is noncovalently linked to P,-microglobulin (P2m), a 12-kDa peptide encoded by genes on chromosome 2 in mouse and chromosome 12 in humans. Analysis of the gene sequences from mice of the H-2* (Kvist et al., 1983; Lalanne et al., 1983), H-2b (Weiss et al., 1983), and H-2k (Arnold et al., 1984; Watts et al., 1987) haplotypes indicates a fairly high conservation of primary sequence within both introns and exons (Steinmetz et al., 1981; Weiss et al., 1984; Lalanne et al., 1988). Data from both human and mouse systems have shown that class I genes consist of eight exons that encode a leader sequence, three extracellular domains (al, az,a3),a transmembrane domain, and three cytoplasmic domains (Malissen et al., 1982; Hood et al., 1983; Koller et al., 1987). There are some exceptions to this general organization, particularly with certain nonclassical genes. For example, QlO lacks a trans-

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membrane domain (Cosman et al., 1982), and HLA-6.0 lacks the cytoplasmic domain (Geraghty et al., 1987). An important feature of the MHC gene family is the wide range of allelic polymorphisms at various MHC loci (Hunkapiller and Hood, 1986). In the mouse, -50 class I K, D , and L alleles have been identified (Hood et al., 1983). A similar range of MHC polymorphisms has been described in the HLA-A, -B,and -C, genes (Parham et a l . , 1988b; Parham, 1987). In contrast, the Qa and Tla regions show very little polymorphism. Analysis of nucleotide sequence has shown that alleles can differ by one to >lo0 substitutions and the antigenic differences are equally variable (Parham et al., 1988a). In the class I genes, the variable regions have similar locations in various alleles of the same locus within and between species. For example, HLA-A alleles (e.g., A l , A3.1, A3.2, A l l ) have closer homology to chimpanzee ChLA-A locus alleles such as Ch25, than with the HLA-B or HLA-C alleles (Lawlor et al., 1988). Similarly, HLA-B has closer homology to ChLAB , and HLA-C, to ChLA-C. At the level of amino acid sequence, distinct variable regions of MHC class I molecules are located in the cil and ci2 domains, whereas the cig domain remains highly conserved (Parham et al., 1988a). The variabilities observed relate to point substitutions and recombinational events in the corresponding genes. Analysis of HLA-A, -B,and -C genes indicate that genetic exchange between alleles of the same locus has contributed more to the generation of diversity than genetic exchange events between alleles at different loci (Parham et al., 1988a,b). However the role of exchange events between genes at different loci could also occur, as has been hypothesized for shared residues between the H-2K" gene and certain Qa-Tla genes (Nathenson et al., 1986). The latter mechanism does not appear to have major impact in generating HLA-A, -B, and -C diversities. Major histocompatibility complex polymorphisms have provided a basis for our understanding of the molecular nature of immunological (i.e., MHCantigen interactions) and nonimmunological functions of MHC molecules (see Section IV).

B. EXPRESSION OF MHC

IN

NORMALTISSUE

In order to assess whether MHC antigens are abnormally expressed during malignancy, an understanding of the patterns of MHC antigen expression during ontogeny, in normal tissue, and during normal cell development is essential. 1. Tissue Distribution

Class I MHC antigens are expressed on virtually all adult tissues, though at varying levels (Klein, 1975). The organ with the highest MHC antigen

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expression is the spleen, followed by other lymphoid organs. The lowest expression is on muscle and nervous tissue. Liver MHC antigen expression is primarily associated with Kupffer cells and, to a much lesser degree, with parenchymal cells. The primary distribution of “classical” class I MHC antigens in the gut, glands, lung, and kidney is on epithelial-derived cells. In addition, vascular endothelial cells also express MHC antigens. In contrast to class I MHC molecules, class I1 molecules exhibit a more restricted tissue distribution mainly on B cells, some T cells, and specialized antigen-presenting cells (APC; Klein, 1975). Most tissues exhibit augmentation of both class I and class I1 MHC antigens in response to various lymphokines, including a-and y-interferon (IFNa, IFNy) and certain growth factors (Collins et al., 1984). Allograft rejection and graft-versus-host disease can also trigger expression of class I and class I1 MHC antigens in various tissues, for example, skin and kidney (de Waal et al., 1983; Wadgymar et al., 1984). Since many epithelial, endothelial, and lymphoreticular cell subsets have antigen-presenting function (Kaye et al., 1985; Tzehoval et al., 1983), these cell types could facilitate local presentation of antigens, lymphocyte triggering, and homing. In addition to the tissue-specific pattern of class I and class I1 MHC expression, there are also quantitative differences in the pattern of locusspecific gene products expressed. In the mouse, the H-2K gene product is generally expressed at higher levels than H-2D or -L gene products, as a result of subtle differences in the biosynthetic pathways (Le and Doyle, 1982) (see Section VIII). Class I1 molecules (DR, DP, and DQ in the human), though expressed on all normal or Epstein-Barr virus (EBV)-induced B-cell lines, exhibit noncoordinate patterns of expression in certain nonlymphoreticular tissues (e.g., gastric epithelial cells, lung bronchiole epithelium, and melanocytes; Natali et al., 1986; Rognum et al., 1987; Sakai et al., 1987). In some cases, DP-, DQ-, or DR-negative cells can express increased levels of class I1 antigens following IFNy treatment (Collins et al., 1984). The noncoordinate expression of class I and class I1 MHC molecules on certain cell subtypes could significantly influence the antigen-presenting potential of these cells.

2 . Ontogeny In contrast to adult tissue, class I MHC antigens are not expressed during early stages of embryogenesis [i.e., before day 2 (P2m) or day 10 (class I MHC)] in the mouse (Ozato et al., 1985; Flavell et al., 1986). For this reason, class I MHC expression is considered to be developmentally regulated (see later). During this period the intact embryo is still responsive to IFN treatment (Ozato et al., 1985). However, MHC genes isolated from teratocarcinomas, which lack MHC antigens, are expressed after transfection into L cells, confirming that the genes are functional (Holmes and

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Parham, 1985). Lala and colleagues (1983a,b) have carefully examined histologically the distribution of class I MHC antigens in the early embryo using a sensitive radioimmunoassay. They reported that the first tissues to exhibit detectable class I MHC antigens were late morulae and early blastocytes. However, at the late-blastocyte stage MHC antigens are no longer detectable. This stage-specific loss or masking of MHC antigens corresponds to the time of implantation, providing a possible explanation for the lack of immunogenicity of the semiallogenic conceptus (Lala et al., 1983b). By day 10 of gestation, fetal MHC antigens are again detectable in the spongiotrophoblast, which forms the outer layer of the placenta, closest to the uterine wall. The labyrinth that forms the inner layer remains negative (Raghupathy et al., 1983). Injection of monoclonal antibodies against class I MHC antigens (Chatterjee-Hasrouni and Lala, 1982) has localized fetal MHC determinants in direct contact with the maternal spinal arteries. Thus class I MHC antigens are clearly expressed in early embryos at or near the fetal-maternal interface. The expression of “nonclassical” class I MHC mRNAs by Northern blotting with probes that can identify exons encoding the transmembrane domain of different class I antigens has been examined (Wegmann, 1987; Hunzinger, 1987).By this approach, certain fetal Tla gene products, including T1 (and/or T l l ) , T3 (and/or T13), and T7,were observed in 10-day to 14day placentas. Further studies are required to determine which mRNA species are translated and expressed on the cell surface, and what cell types are involved. A different nonclassical MHC gene, the QlO MHC molecule, is produced by the visceral endoderm (as early as 10 days gestation) (Stein et al., 1986), and by adult liver cells (Mellor et al., 1984). Because the QlO gene lacks a transmembrane domain, its product is secreted. The function of the T1, T3, T7, and QlO molecules is unknown. Some investigators have proposed that they may be involved in immune suppression in the region of the maternal-fetal barrier (Lala et al., 1983b; Clark et al., 1986). The ontogeny studies raise the question whether aberrant expression of nonclassical class I mRNA species occurs during malignancy, and may provide a possible insight into the mechanisms of deregulation of MHC expression in tumors.

3. Differentiution and Cell Cycle-Specijlc Expression Although MHC antigens are widely expressed, their cell surface density is highly variable. In general, more differentiated cells within the same lineage tend to express a greater density of MHC antigens. For example, lymphocytes in the medulla of the thymus are less differentiated and tend to express a lower density of MHC antigens than T cells in the cortex of the thymus, which are more differentiated, or in the periphery (Klein, 1975). In certain nonlymphoid systems, such as transitional bladder epithelium, a gradient of

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increasing @,m expression, an indicator of class I surface expression, is observed on more differentiated cells compared to cells in the basal (stem cell) layer (G. Dotsikas, B. E. Elliott, and W. Mackillop, unpublished observations). Although class I MHC is not a definitive marker of differentiation, quantitative changes have been observed to correlate with different stages of maturation in certain cell lineages. It is also possible that class I MHC density is cell cycle-dependent. Studies by Matsui et al. (1986b) have indicated that class I MHC antigen expression on phytohemagglutinin-activated T cells and antigen-activated Tcell clones increases in GI phase and decreased in G,, whereas class I1 antigen density increases in G, (Matsui et al., 1986a,b). These researchers were the first to express the changes in terms of surface antigen density, using a flow cytometry approach, not influenced by synchrony induction methods or fixation. These findings are consistent with reports that tumor target cells are often more sensitive to T-cell-specific lysis in G , than in G, phases (Leneva and Svet-Moldavsky, 1974). Since cells at different stages of maturation have distinct cell cycle characteristics, differentiation-associated changes in MHC expression could also be cell cycle-related. IV. MHC Function

Although the most clearly understood function of class I MHC molecules is as a self-component recognized by MHC-restricted T cells, nonimmunological roles have also been proposed. Both concepts are briefly reviewed in this section to provide a basis for later discussions on MHC function in malignancy (Section VII).

A. THE MHC-ANTIGEN-T-CELLINTERACTION

The question of what portion of the MHC molecule determines its association with antigen is important in understanding the nature of T-cell recognition of tumor-associated antigens as well as escape of tumor cells from the immune system. Molecular analysis of MHC structure-function relationships were first carried out in allogeneic systems. Experiments involving hybrid molecules created by exon exchange between intraspecies class I genomic clones demonstrated that the two external domains of the class I molecule, aland a,, contain the polymorphic determinants recognized by most allospecific T-cell clones (Stroynowski et al., 1985; Arnold et a l . , 1985; Ajitkumar et a l . , 1988). The highly conserved a3 domain does not directly interact with antigen or the T-cell receptor. Exon-shuffling experiments in which murine a1and a2 domains are linked to the human a3 domain have shown that ag can influence recognition of antigenic determinants located

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within a land a2 domains (Maziarz et at., 1988). Using X-ray crystallography, Bjorkman et al. (1988) have shown that antigenic peptides bind to a cleft between the a1and a2 domains. Analogous approaches are likely to be very informative in determining the molecular nature of the epitopes of the M HC-antigen complex on tumor cells recognized by the T-cell immune system. B. T-CELLADHESION MOLECULES (CAM) In addition to MHC-antigen and T-cell receptor interactions, new evidence has shown molecules such as CD2, CD4, and CD8 on T-cell subsets appear to function as CAM in the binding of antigen-specific T cells to target antigen-bearing cells (Bierer and Burakoff, 1988; Bierer et al., 1988). These workers have demonstrated interactions between CD8 and the a3 domain of class I (Ratnofsky et al., 1987), CD4 and class I1 (Sleckman et al., 1987), and CD2 and LFA-3 (Bierer et al., 1989). In addition, LFA-1 was shown to interact with I-CAM-1 (Marlin and Springer, 1987). The general approach (for CD2, CD4, and CD8 molecules) was to transfect genes encoding each “receptor” into T-cell hybridomas, and to test for binding to purified ligand incorporated into liposomes and enhancement of an antigen-specific response, interleukin 2 (IL-2) secretion (Bierer and Burakoff, 1988). The possibility that the class I-CD8 reaction can occur on non-T cells was demonstrated by Norment et al. (1988). These workers showed direct binding of human CD8 molecules in transfected Chinese hamster ovary cells to class I MHC molecules on human B cells. Binding was proportional to the amount of CD8 expressed, and was specifically inhibited with anti-CD8 and anticlass I MHC monoclonal antibodies. These results confirm that CD8 can interact with class I MHC molecules independent of the T-cell receptor. The antigen-independent adhesion reactions described here could facilitate subsequent antigen-specific recognition events in low-affinity, low-avidity receptor-target interactions, as might occur in certain malignancies. C. NONIMMUNOLOGICAL ROLE

OF

MHC

In addition to the immunological role of MHC molecules just described, it is also possible that MHC has nonimmunological functions. Two such putative roles are discussed here. First, it has been proposed that class I MHC antigens are involved as CAM in many cell-cell interactions, similar to those described for CD8 on T cells in Section IV,B (Edidin, 1983, 1986). It has been suggested that MHC molecules may have primitive recognition functions similar to fusion molecules in self-nonself discrimination as is displayed by tunicates (Scofield et al., 1982a,b). This possibility is an area for future investigation.

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Second, there is accumulating evidence that class I MHC molecules might be functionally associated with a variety of peptide hormone receptors, including insulin (Simonsen et al., 1985), glucagon (Edidin, 1986), epidermal growth factor (Schreiber et al., 1984) and y-endorphin (Claas et al., 1986). Two main sources of evidence have been provided to support an association of receptor molecules with class I MHC heavy chains. Coprecipitation of HLA molecules and insulin receptor with and without crosslinking agents has been demonstrated (Phillips et al., 1986). Inhibition of receptor function occurs when receptor-bearing cells are incubated with both ligand and antiHLA antibodies (Schreiber et al., 1984).Although the functional significance of associations of class I MHC molecules with certain surface receptors is not known, it has been proposed that class I molecules might facilitate local concentration of ligand (Edidin, 1988). Barber et al. (1988) have examined the molecular association of class I heavy chains with P2m and other surface structures. They have shown that (1) dissociation of P2m to form “free” class I heavy chains can occur once the cell surface location of class I heavy chains has been attained; (2) dissociation from P2m can result in a conformational change in epitopes on the a1and a2 domains of surface-expressed class I heavy chains; and (3) there are differences in the epitopes expressed on the C-terminus domain of free and p,m-associated class I molecules (detected by antibodies specific for the Cterminal regions). These findings raise the possibility that the altered confirmation of the molecule delivers a signal to the intracellular environment. Barber et al. (1988) and others (Edidin, 1988) have proposed that free class I MHC chains can interact with hormone receptors as well as other self or nonself molecules in a manner similar to the association with P2m, thereby influencing many cellular functions. The formal proof and functional significance of this hypothesis remain to be demonstrated. Although there is no direct evidence that MHC plays a nonimmunological role in malignancy, this possibility offers a new avenue for investigation. V. MHC Expression in Malignancy

Correlative studies on MHC expression in human malignancies are important because they provide a link between experimental studies and clinical relevance. Information from correlative studies has been helpful in the interpretation of results from animal tumor models.

A. CLINICAL CANCER There is now a vast literature describing the expression of HLA-A, -B, -C, and -D antigens in human malignancy (Doherty et al., 1984; Tanaka et al.,

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1988a; Bernards, 1987; Hammerling et al., 1987a,b). However, there are several problems in interpreting the clinical data summarized here. (a) Absolute comparisons among the different reports are difficult because of the nonquantitative, subjective nature of the techniques used. (b) Essentially all studies have involved frozen sections stained by immunofluorescence or iinmunoperoxidase. Frozen sections make histological localization of antigens more difficult than paraffin-embedded tissues, and virtually preclude the possibility of retrospective and follow-up studies of individual cancer cases. S. Ferrone (personal communication) has produced monoclonal antiHLA antibodies that react with fixed tissues; such antibodies are likely to become widely used in this field. (c) With few exceptions, in all the studies reported so far, the class I-specific antibodies used did not distinguish among HLA-A, -B, and -C subregion products. Rees et al. (1988) have used specific antibodies to detect loss of polymorphic A2 and Bw4 antigens in colon carcinoma in patients typed for the respective specificities. In addition, the recent availability of a nonpolymorphic HLA-B-specific antibody (S. Ferrone, personal communication) and of HLA-A, -B, and -C-specific oligonucleotide probes (Davidson et al., 1985) makes it now possible to detect noncoordinate expression of certain class I HLA genes in any haplotype. (d) Finally, because the technique is nonquantitative with sensitivity levels difficult to define, expression of trace levels of MHC antigens could be below the threshold level of detectability, yet might be functionally relevant. Given these limitations, a representative sample of the literature in which identical antibodies (i.e., the HLA framework antibody W6/32, the Pzmspecific antibody BBM1, and the HLA-DR-specific antibody L243) are used is shown in Table I. Analysis of MHC levels on certain tumors has suggested a decreased level of MHC expression compared to normal tissue: examples are infiltrating ductal carcinomas (Natali et al., 1983, 1986), basal cell carcinomas (Turbitt and Mackie, 1981; Holden et al., 1983), and mucinous colorectal carcinomas (van den Ingh et al., 1987). Very few of these data have been analyzed in a statistically rigorous manner. In one study of colorectal carcinoma (Momburg et al., 1986), a significant correlation between loss of class I MHC and degree of dedifferentiation (as assessed by morphology) was found. N o correlation with the stage of tumor progression was observed, and many metastases were found not to be different in their MHC phenotype compared to the primary tumor. In contrast to class I HLA molecules, locus-specific class I1 (DR, DP, DQ) molecules can readily be distinguished by monoclonal antibodies against nonpolymorphic determinants. Certain B-cell lymphomas have been shown to lack at least one of the three HLA-D subregion products. In certain cases, such as colorectal carcinoma (Momburg et al., 1986) and lung carcinoma

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TABLE I SUMMARY OF COMPARISONS OF MHC ANTIGENS ON BENIGN AND MALIGNANT HUMAN TISSUES4 Proportion of tumors lacking (N = total number examined) Histological diagnosis

HLA-A, -B, -C

Pem

HLA-DR

Reference6

Breast (carcinoma) Ductal infiltrating Medullary Lobular

53% (58) 0% (7) 78% (9)

22% (32) 0% (7) 20% (5)

73% (52) 0% (7) 67% (9)

1 1 2 1 1-3

Other (tubular, mucoid) Benign mammary lesions Squamous-cell carcinoma Keratoacanthoma Basal cell carcinoma Basal cell papilloma Eccrine porocarcinoma Benign eccrine poroma Colorectal carcinoma Mucinous Nonmucinous Colorectal adenoma Gastric carcinoma Gastric epithelium Bladder carcinoma Bladder urothelium Endometrial carcinoma Endometrium Lung Small-cell lung carcinoma Non-small-cell lung carcinoma Normal lung epithelium Malignant melanoma Normal melanocytes B-Cell lymphoma Burkitt's lymphoma

67%

(6)

0% (16)*

NTc NT NT NT NT NT 75% (8) 0% (lo)* 0% (3) NT NT NT NT 50% (8) 0% (13)* 100% (3) NT NT (3)* NT 28% (36) 0% (8)* 44% (66) ( A l l y 100% (5)* 0%

NT

67%

0% (16)*

50%

(6)

0% (6) 100% (21) 83% (6) 50% (lo)* 100% (5)* 75% (8) 0% (lo)* 0% (3) NT NT 32% (185p 0% (17p NT NT 100% (3) 81% (32) 28% (77) 0% (3)* 0% (log)* 25% (36) NT 89% (26) NT

(6)

0% (16)*

2

NT

4

NT NT NT NT NT

4 4, 5 4

5 5

75% (8) 90% (10) 0% (3) 47% (15) 0% (15) NT NT 75% (8) 0% (13)*

NT NT NT

11 12 12

NT NT 44% (36) 100%

NT NT

6 6, 7