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

GENETICS OF TUMOR SUSCEPTIBILITY IN THE MOUSE: MHC AND NON-MHC GENES P. Demant, L. C. J. M. Oomen, and M. Oudshoorn-Snoek Division of Molecular Genetics, The Netherlands Cancer Instiute, 1066 CX Amsterdam, The Netherlands

I. Introduction 11. Site of Action of Tumor Susceptibility Genes 111. Biology of Tumor Susceptibility Genes A. Oncogenes, Tumor Suppression Genes, and Tumor Susceptibility B. Tumor Susceptibility Genes and the Multistage Process of Neoplastic Development IV. Genetic Definition of Tumor Susceptibility Genes A. Multigenic Determination of Tumor Susceptibility B. Recombinant Inbred Strains C. Recombinant Congenic Strains D. Quantitative and Statistical Considerations E. From Genetic Mapping to Molecular Isolation of Genes V. Major Histocompatibility Complex-Structure and Function A. MHC Structure B. Interactions of non-MHC and MHC Genes C. Function of Class I and Class I1 Products D. MHC Phenotype of Tumor Cells E. Biological Importance of Altered MHC Expression F. MHC and MuLV-Induced Lymphomagenesis VI. Susceptibility to Epithelial Tumors and the Role of MHC A. Genetics of Lung Tumor Susceptibility B. Different Lung Tumor Types C. Site of Action of Genes Affecting Lung Tumors D. MHC Genes and Lung Tumor Susceptibility E. Mechanisms of MHC Effects on Lung Tumorigenesis F. MHC Effects on Tumorigenesis in Small Intestine G. MHC Effects on Tumorigenesis in Liver H. MHC and Mammary Tumor Susceptibility I. MHC and Tumorigenesis in Epithelial Organs-Summary VII. Tumor Susceptibility Genes: Molecular and Cellular Perspective References

I. Introduction

The role of genetic factors in tumor susceptibility in humans was originally recognized in rare instances of familial Occurrence of tumors of a certain type (for review see Schneider et al., 1986). Inbred strains of mice, developed in the first half of this century, also exhibit strain-specific susceptibility for 117 ADVANCES IN CANCER RESEARCH, VOL. 53

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certain types of tumors (for reviews see Heston, 1963; Murphy, 1966).These observations in humans and mice stimulated efforts to identify the genetic factors responsible for tumor susceptibility, and to elucidate the mechanisms of their action. The ensuing genetic studies in mice and humans contributed evidence for some of the basic concepts in tumorigenesis: the viral etiology of tumors revealed by the role of “milk factor” in induction of mammary tumors in mice (Bittner, 1936), the evidence for tumor suppression genes (“anti-oncogenes”) resulting from analysis of the familial form of retinoblastoma (Knudson, 1985), the concept of the oncogenic effect of the integrated provirus, based on male autosomal transmission of mammary tumor virus or MTV (Bentvelzen, 1968). These developments indicate also the potential of future genetic studies to contribute to the understanding of the neoplastic process, since the mechanisms of action of most tumor susceptibility genes remain unknown. A considerable proportion of the genetic studies in the past decades has been directed toward the role of the major histocompatibility complex (MHC) in tumor susceptibility. This trend was started by the finding of Lilly et al. (1964) that H-2 haplotype influences susceptibility to Gross virusinduced leukemias in the mouse and has been fueled by the development and wide availability of H-2 congenic strains (Snell, 1958) and by the rapid accumulation of the knowledge about the structure and function of MHC. These studies provided a considerable body of information about the immune response against viruses and virally infected cells, especially in relation to leukemia. In this review we shall compare the general biological characteristics of tumor susceptibility genes and discuss the methods for their identification, particularly a novel genetic tool, the recombinant congenic strains (RCS), which may be used to identify the presently elusive non-MHC tumor susceptibility genes. Then we shall present a brief overview of the structure and function of the MHC of the mouse and its role in tumorigenesis. In the discussion of the genetics of susceptibility to specific types of tumors we shall concentrate on the tumors of epithelial origin. We shall review MHC and, where known, also non-MHC genes controlling susceptibility of mice to tumors of epithelial origin in mammary gland, lung, small intestine, and liver, and discuss the potential contribution of these studies to the understanding of the neoplastic process. Study of these tumors is of considerable importance, because the genetics of susceptibility to their induction is less well known than that of murine leukemias, and because the majority of human tumors is of epithelial origin. The genetic factors in leukemogenesis have been extensively reviewed elsewhere (e.g., Lilly and Mayer, 1980; Meruelo and Bach, 1983; Kozak, 1985). We shall argue that, contrary to the prevailing belief, a considerable part of the effects of the MHC on the

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susceptibility to nonvirally induced tumors in epithelial organs is due to nonimmunological effects of the MHC, namely to the MHC effects on hormonal control of epithelial differentiation and function. What contributions to the understanding, manipulation, or prevention of cancer can be expected from the study of the genetics of tumor susceptibility? Obviously, the tumor susceptibility genes are likely to form a very heterogeneous family, and it is not possible to predict the actual mechanisms of action of individual tumor susceptibility genes or their normal function. However, by putting the question in reverse and asking what class of tumor susceptibility genes we have to investigate if we want to gain insight into certain aspects of the neoplastic process, it is possible to optimize the choice of the experimental genetic system. II. Site of Action of Tumor Susceptibility Genes

The site of action of tumor susceptibility genes is important in these considerations. A gene that affects susceptibility of the target tissue to tumorigenesis is possibly involved in cellular functions relevant to certain aspects of the neoplastic development. In contrast, a gene that affects tumor development through extracellular systemic factors need not be involved in the actual neoplastic process. For example, immune-response genes affect tumorigenesis through immunological elimination of tumor cells. The site of action of tumor susceptibility genes has been studied in carcinogen induced lung tumors (Shapiro and Kirschbaum, 1951; Heston and Dunn, 1951; Heston and Steffee, 1957; Bentvelzen and Szalay, 1966), castration-induced adrenal tumors (Huseby, 1951), hormonally induced testicular tumors (Trentin and Gardner, 1958), virally induced mammary tumors (Dux and Miihlbock, 1968; Dux, 1981), and chemically induced leukemias (Ishizaka and Lilly, 1987). These tests compared tumorigenesis in the organs of a susceptible and a resistant strain in situ with tumorigenesis in these target organs or tissues when they were transplanted into F, hybrids of the two strains. In all these studies the difference in tumor induction between the organs of susceptible and resistant strains was manifest also when they were transplanted into F, hybrid hosts, indicating that the tumor susceptibility genes affect mainly the target organ itself, and only to a lesser extent or not at all the systemic factors of the host organism. The inbred mouse strains used in these studies differed from each other at a very large number of genes. In cases in which a tumor susceptibility gene is identified, it is possible to study specifically whether this gene operates systemically or affects the target tissue. This has been tested for the MHC genes affecting the susceptibility to MTV-induced mammary tumors by Dux

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and Demant (1987), who compared appearance of MTV-induced tumors in mammary glands in the strain C57BL/10 (H-2h, resistant) and its congenic partner strain BlO.A(5R) (H-2fi5,susceptible), and in mammary glands of these two strains transplanted into the F, hybrid hosts. In this experiment, the rate of tumor development differed between C57BL/10 and BlO.A(5R) females, but in the F, host the transplanted mammary glands of the two strains were equally susceptible. This indicates that the MHC affects development of MTV-induced mammary tumors through systemic factors, rather than affecting the susceptibility of the target tissue. This MHC effect on MTV-induced mammary tumors is likely to be similar to many instances of M HC-linked susceptibility to virally induced leukemias, which are generally due to defective immune response of mice with certain MHC haplotypes to murine leukemia virus (MuLV) virions and MuLV antigens on the cell surface. These effects of MHC genes are based on the same molecular mechanisms as the MHC effects on immune response against most viral antigensnamely the formation of complexes of viral proteins, or of their fragments, with the class I or class I1 MHC molecules. These complexes are recognized by the antigen receptors of cytotoxic or helper T lymphocytes, respectively (for discussion see Section V,C). Thus, while most tumor susceptibility genes affect the susceptibility of the cell to the neoplastic process, the MHC genes affecting susceptibility to virally induced tumors operate quite differently. They should be classified as immune-response genes affecting defense against virus infection (MuLV, MTV), rather than as tumor susceptibility genes. This view is supported by the finding that H-2 has very little, if any, influence on susceptibility to carcinogen-induced leukemias (Oomen et al., 1988), in contrast to the large effects of the H-2 genotype on susceptibility to virally induced leukemias. Schematically, three types of genetic effects can be recognized.

1. The MHC immune-response genes, affecting the immunological response, especially antibody production against viral antigens on virions or on virus-infected normal and tumor cells. These genes operate mainly systemically, and the molecular mechanism of their effects is well known; it is the general mechanism of antigen presentation by MHC products, and it is not related to the actual process of neoplastic development. 2. The MHC-linked genes with nonimmunological effects affecting susceptibility to hormonal regulation of cellular functions and expression of oncogenes. In some cases these genes were shown to operate in the tumor cell; in other cases the site of action is not yet known but is likely to reside in the target cell (see Section VI). 3. Non-MHC-linked genes. They form the majority of tumor susceptibility genes. Most of these genes affect the susceptibility of the target cell to the neoplastic process, and their systemic effect is in most instances small, if

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any, although non-MHC genes that affect tumor susceptibility systemically (e.g., through control of immune response or hormone production) may also be demonstrated in the future. The molecular mechanism of action of these genes is generally not known, and their study will likely provide new information about the role of genetic factors in the neoplastic transformation of cells. 111. Biology of Tumor Susceptibility Genes

In order to assess correctly the potential contribution of the study of tumor susceptibility genes to tumor biology, it is necessary (1) to correlate the phenomenon of tumor susceptibility with the two presently investigated classes of genetic factors in tumorigenesis, that is, oncogenes and tumor suppression genes; and (2) to relate the effects of tumor susceptibility genes to the individual stages of the neoplastic process.

A.

TUMOR SUPPRESSION GENES, TUMORSUSCEPTIBILITY

ONCOGENES, AND

Oncogenes are genes involved in the neoplastic process (for review see Varmus, 1987; Bishop, 1987; Klein and Klein, 1986), because they have been found (1) to be present in tumor cells or potentially neoplastic cells in an altered form, as compared with normal cells, or, (2) to be expressed inappropriately in tumor cells, often as a result of an increased number of genes (trisomy, gene amplification) or increased transcription rate caused by alteration in the adjacent DNA by retroviral insertions or chromosomal translocations, and (3) to transform in uitro, upon transfection, suitable indicator cells. Tumor suppression genes (anti-oncogenes, Knudson, 1985) were identified originally in family studies in humans. Certain types of cancer exhibit familial aggregation in rare instances. In such families heterozygosity for a mutation of a certain chromosomal region has often been demonstrated (retinoblastoma, chromosome 13: Knudson, 1985; Wilms’ tumor, chromosome 11: Koufos et al., 1984; colon carcinoma or familial polyposis coli, chromosome 5) either cytologically or by molecular techniques (Solomon et al., 1987; Bodmer et al., 1987), indicating that a gene locus responsible for normal cellular function or differentiation and thus preventing or suppressing the tumorigenesis is located in this segment. In normal tissue in affected members of such families, the mutation has been heterozygous (i.e., one normal chromosome has been present), but in tumor cells a second deletion encompassing the postulated tumor suppression gene locus has been found. The tumor suppression genes are apparently involved in control of the normal differentiation of the cell. Frequent occurrence of deletions of certain

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chromosomal segments in some types of tumors suggests that such segments may also carry specific tumor suppression genes, for example chromosome 22 in neuroma and meningioma (Seizinger et al., 1986), and chromosome 17 in colon carcinoma (Fearon et al. 1987). Genetical and cytological information about localization of tumor suppression genes and availability of cells with homozygous deletions open the way for the molecular isolation of these genes. The retinoblastoma tumor suppression gene (Rb) has been cloned and extensively analyzed (see Section IV,E). The product of the adenovirus oncogene Eal can combine in the cell with the Rb-encoded protein, presumably preventing the Rb product from carrying out its function in the cell (Whyte et al., 1988). Oncogenes and tumor suppression genes offer the possibility of combining molecular and cellular approaches in the analysis of neoplastic transformation. The results of this effort revealed that oncogenes and possibly also tumor susceptibility genes are related to certain classes of genes responsible for regulation of normal functions of the cell: genes encoding growth factors, growth factor receptors, hormone receptors, proteins participating in certain stages of signal transduction, and nuclear proteins, some of which were shown to be DNA-binding proteins (Varmus, 1987; Bishop, 1987; Lee et al., 1987). Expression of these genes during various stages of prenatal and early postnatal development or during cell activation supports the notion that their primary function is regulation of cell differentiation and proliferation. These data are also compatible with the concept of cancer as a caricature of normal tissue renewal (Pierce and Speers, 1988). The pattern of expression of most protooncogenes in different cell lineages and developmental stages does not suggest a highly specific function of these genes in development (Bishop, 1983), the exception possibly being the int-l gene, which was reported to be homologous with the wing2ess developmental gene of Drosophila (Rijsewijk et al., 1987). What is the possible relationship between oncogenes and tumor suppression genes on one hand and tumor susceptibility genes on the other? Some tumor susceptibility genes may be related to the class of tumor suppression genes. An allele of a tumor suppression gene with an altered function or expression of its product would manifest itself as a tumor susceptibility gene (W. F. Bodmer, personal communication). Some tumor susceptibility genes may be related to cellular protooncogenes. The first such example is provided by Ryan et al. (1987), who observed that one of the factors affecting susceptibility to urethane induction of lung tumors is genetically linked or identical to the Kras-2 proto-oncogene in mice. This indicates that the Kras-2 gene may actually be one of the tumor susceptibility genes. There are several possibilities that could account for this observation. Kras-2 mutations are frequent in chemically induced lung tumors of mice (Stowers et al., 1987) and polymorphism of the structural gene might affect the frequency of muta-

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tions either by modifying the access of the mutagen compound to the target DNA site or by affecting locally the efficiency of the DNA repair (Topal, 1988). Alternatively, the Kras-2-linked susceptibility might be due to adjacent sequences or closely linked genes that affect the expression of the Kras-2 gene. Genetic differences between mouse strains may influence the preferential retroviral integration sites found in tumors. Spontaneous lymphomas in the high lymphoma strain AKR are induced by mink cell focus-forming (MCF) virus, which is produced by recombination between ecotropic and xenotropic MuLV sequences in AKR mice. The recombinant inbred strains (RIS) produced between the AKR strain and the low-lymphoma strain DBAI2 (AK x D strains) exhibit strain-specific prevalence for certain types of leukopoietic tumors. In some strains, T lymphomas induced by MCF virus were prevalent, in other strains mainly B lymphomas caused by ecotropic virus were seen, and in one strain myeloid leukemias rather than lymphomas appeared (Mucenski et al., 1986, 1987). In the latter strain, the myeloid leukemias shared a common novel integration site, Eui-l, which has been found sporadically also in B-cell and pre-B-cell lymphomas in some, but not all AK x D RIS (Mucenski et al., 1988). Mucenski and colleagues suggest that the segregation of the genes between the RIS, including endogenous proviral loci, might affect the nature of the MuLV formed by recombination with available genomic sequences. This would in turn determine the type of tumor produced. In that case, the specific “tumor susceptibility loci” would be linked with the endogenous retroviral genes or with specific integration sites. Strain-specific differences in the variants of the produced ecotropic MuLV might also be responsible. However, still other genetic factors need to be postulated that determine the strain-related prevalence of myeloid or lymphoid tumors in strains producing ecotropic virus and exhibiting insertions at the Eui-l site in their tumors. Myeloid tumors in another strain, BxH-2, have a different ecotropic virus integration site, Eui-2 (Buchberg et al., 1987). An example of influence of a ‘normal’ cellular gene on the tumorigenic effects of an oncogene has been obtained in transgenic mice carrying c-myc and IgM heavy chain genes. The expression of gene coding for membrane-bound but not for secreted genes form of IgM suppressed the leukemogenic effect of the c-myc gene (Nussenzweig et al., 1988). This suppression correlates with the alteration of B-cell development by the immunoglobulin transgene. B. TUMORSUSCEPTIBILITY GENESAND THE MULTISTAGE PROCESSOF NEOPLASTIC DEVELOPMENT

The development of tumors is a multistep process. In several species the chemical induction of skin tumors can be divided into at least two stages:

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initiation and promotion (reviewed in Slaga, 1983). Further evidence stems from the observations that tumor development often proceeds in discrete stepwise alterations from an incipient benign preneoplastic lesion toward a fully malignant phenotype (Foulds, 1969; Nowell, 1976; Farber and Cameron, 1980), and that malignant transformation by oncogenes in transfected cells or in transgenic mice appears to require multiple genetic changes in the cell (for reviews see, e.g., Klein and Klein, 1986; Groner et al., 1987), in the prospective oncogene (Duesberg, 1987; Temin, 1988), or in both. It appears that different tumor susceptibility genes affect different stages of this process. There are presently not many experimental models that allow precise staging of the effect of tumor susceptibility genes. However, numerous experiments with induction of skin tumors either by a completecarcinogenesis schedule or by a two-stage (initiation and promotion) procedure provided convincing evidence for stage-specific effects of tumor susceptibility genes. The relative susceptibility of inbred mouse strains to the complete-carcinogenesis protocol (tumor induction by a tumorigenic dose of a chemical carcinogen) or to two-stage carcinogenesis (subthreshold dose of a carcinogen, followed by repeated doses of a noncarcinogenic “promoting” agent) is quite different (Slaga and Fischer, 1983), indicating that the tumor susceptibility genes in these strains affect differently the various pathways of tumor induction. In several strains the susceptibility to complete carcinogenesis or response to different promoters in two-stage carcinogenesis on one hand were compared with the activation of promutagen-carcinogen, its binding to DNA, and DNA adduct formation on the other hand. Generally, with exception of the effect of the Ah (aromatic hydrocarbon responsiveness) locus (reviewed in Nebert and Gonzalez, 1987), there is no correlation between tumor susceptibility and carcinogen activation or DNA modifications by carcinogen. The strain SENCAR is much more susceptible to two-stage skin carcinogenesis, using dimethylbenzanthracene (DMBA) with 12-O-tetradecanoylphorbol-13-acetate (TPA)promotion, than both BALB/c (Hennings et al., 1981)and C57BL/6; however, with the DMBA complete-carcinogenesis protocol C57BL/6 mice are more susceptible than SENCAR mice (Reiners et al., 1984). These differences in tumor susceptibility do not correlate with the capacity of the keratinocytes to activate the carcinogen metabolically, nor with carcinogen-DNA binding or adduct formation (Reiners et a l . , 1984; Morse et al., 1987). The genetic difference in response to TPA treatment probably affects a step after binding of TPA to its membrane receptor, since the number and affinity of TPA receptors in different strains do not correlate with their genetic susceptibility to TPA promotion (Wheldrake et al., 1982). The response of inbred strains to different promoting agents may also vary (Naito et al., 1987). The course of the selection for heritable high susceptibil-

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ity to TPA promotion (Fischer et al., 1987) suggests that several genes are involved, some of them affecting the susceptibility of skin to papilloma formation. Analogous to these data are the results with lung tumor induction by urethane and the promoting agent butylated hydroxytoluene (BHT). The promoting effect of BHT is strain-dependent and the genes responsible for the susceptibility to BHT promotion were shown to segregate in RIS (Malkinson and Beer, 1984). Induction of hepatocellular adenomas and carcinomas by treatment of newborn mice N-ethyl-N-nitrosourea (ENU) results in a high number of tumors in C3H strain and a low number of tumors in C57BL/6 strain; however, there is no difference between the two strains in the ethylation of hepatic DNA or in specific adduct formation (Drinkwater and Ginsler, 1986). Collectively, these data demonstrate stage-specific influence of tumor susceptibility genes and suggest that the genetic differences in tumor susceptibility generally affect the postinitiation stage of tumorigenesis. It has not been tested to what extent these genetic factors operate systemically. IV. Genetic Definition of Tumor Susceptibility Genes

Besides the MHC-linked genes, which are discussed in Sections V and VI, a large number of other genes also affecting tumor susceptibility exist. They are located on different chromosomes, and in many instances their effects are larger or at least as large as those of the MHC-linked genes. These genes remain largely unknown, and in studies concerned primarily with the MHC they are usually classified under the humble collective name “non-MHC” tumor susceptibility genes. They are of considerable interest, because they affect mainly the susceptibility of target cell to tumorigenesis. Their effect is revealed by interstrain differences in development and type of spontaneous or induced tumors. Tumorigenesis differs between inbred strains also qualitatively, rather than only quantitatively. Strains may differ in the type of tumors that arise in response to a certain carcinogenic agent. Also, tumors of the same tissue origin may differ between the strains in the degree of their differentiation or in the stage of progression toward malignancy. Strain-specific preference for certain retroviral integration sites in tumor DNA, as well as differences in responsiveness to various promoting agents and in location of the tumors, are other examples of “qualitative” effects of tumor susceptibility genes. Therefore, this “qualitative” aspect of tumor susceptibility genes must be kept in mind in the methodological discussion that follows, because for practical purposes the genetics of tumor susceptibility must be treated as a quantitative trait, by transforming, where necessary, qualitative data into

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quantitative data. However, the aim and the perspective of the genetic studies is to define not only the genes that af€ect the quantitative aspects of the neoplastic process, but also those that determine the various qualitative features of tumors. In most instances of strain differences in tumor susceptibility several genes are involved. This makes the definition and mapping of these genes very difficult and in most cases virtually impossible. Therefore these genes have remained largely unidentified. In the following passages we will discuss the nature of the difficulties of analysis of non-MHC tumor susceptibility genes and propose a possible solution.

A. MULTIGENIC DETERMINATION OF TUMOR SUSCEPTIBILITY Tumor susceptibility must be analyzed as a quantitative genetic trait, because it is expressed as one or more of the quantitative parameters of tumor development: proportion of animals developing certain tumors (tumor incidence), number of tumors per animal (multiplicity), time of appearance of tumors, age at death caused by the tumor, tumor size, growth rate, number or size of metastases, and so on. In uitro assays of tumorigenesis also describe properties of cell populations in quantitative terms-number of foci of transformed cells, number of cells showing anchorage-independent growth, population-doubling time, and so forth. Such in uiuo or in uitro quantitative phenotypes are the outcome of an unknown number of steps or processes, each of them influenced by one or more genes. A difference between two strains or individuals with respect to such phenotypes can be caused by difference in one or more of these genes. In cases with multigenic differences, the values observed in segregation tests (F, or backcross mice) fail to form a small number of clearly defined phenotypic classes that are seen when difference in a single gene is involved. Therefore, various statistical methods for analysis of multigenically controlled quantitative characteristics in segregating populations were developed (reviewed in Roderick and Schlager, 1966; Falconer, 1963), and selection procedures for isolating genes controlling quantitative traits were proposed (Thoday, 1961). The disadvantage of these methods is that ad hocproduced genetically heterogeneous groups (F2, backcross, etc.) are used, in which each animal has a different genotype. Because a quantitative phenotype cannot be established reliably in a single mouse and because it is not possible to characterize such a population for a large number of segregating genetic markers, the establishment of a relationship between genotypes and phenotype is very difficult. Therefore, a genetic tool was needed that would provide genetically characterized homogeneous strains of mice carrying genes of two inbred strains segregated according to a regular pattern.

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B. RECOMBINANT INBREDSTRAINS Bailey (1965, 1971) recognized the need for such a genetic tool and devised the recombinant inbred strains (RIS). Each RIS is the result of consecutive generations of brother-sister matings starting with an F, hybrid male and a female from a cross between two inbred strains. Using a number of pairs of F, mice, a series of RIS is produced (Fig. 1A). Each RIS received approximately half of its genes from each parental inbred strain, and the set of genes inherited from each parental strain is different in each RIS. The use of the RIS revolutionized mouse genetics, because it provided two essential advantages when compared to previously available methods. First, the individual RIS have been genotyped in order to establish which alleles of a particular gene were received from one parental strain and which from the other. As the RIS are maintained permanently in virtually homozygous state, the results of the typing of alleles of different genes become accumulated in time and provide eventually an equivalent of an extensively typed segregating population. This has a great economic advantage for linkage studies, because the established strain distribution pattern of a newly studied gene can be compared with that of all previously typed genes directly, without any further testing. Second, a quantitative phenotype, for example incidence of tumors or plasma level of a hormone, can be established with the same certainty as in an inbred strain, because all mice of an RIS are genotypically identical. The quantitative phenotypes observed in different RIS can then be compared with the strain distribution patterns of previously typed genes in order to find evidence for linkage. Gene mapping with the help of RIS has been very fruitful (Bailey, 1981; Taylor, 1978, 1980) in establishing linkage of various genes. It has been widely hoped that application of this efficient tool will also contribute to the identification of genes involved in tumor susceptibility. This unfortunately generally did not turn out to be the case. In most studies the quantitative phenotypes measured in different RIS (tumor incidence or tumor multiplicity) turned out to form a continuous range of values, rather than clear-cut phenotypic classes (Demant and Hart, 1986). Consequently, no exact information about the genes involved and their linkage relationship could be obtained. In general, therefore, the RIS are a less efficient tool in analysis of quantitative traits like tumor susceptibility. This shortcoming of the RIS applies to analysis of all multigenically controlled quantitative phenotypes, and it is primarily due to additive and nonadditive interactions between individual components of the multigenic system. The additive action of two or more susceptibility genes results in a quantitative phenotypic effect equal to the sum of the effects of individual genes. The correlation between the

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A

A

B

X

BROTHER X SISTER MATING I I I I I

I I I

I

3

RECOMBINANT I N B R E D S T R A I N S

I I I I I I I I ETC

I

AXB-A AXB-B AXB-C AXB-D

AXB-E

AXB-F

ETC.

FIG. 1. The scheme for production of (A) recornbinant inbred strains and (B) recombinant congenic strains.

quantitative phenotypes of RIS and their genotypes, which is essential for any genetic interpretation of the results, is distorted or destroyed by additive interactions, because very similar phenotypes may be caused by quite different genotypes. The negative effects of additive gene interaction on the resolution power of the RIS are illustrated in Tables I and 11, using a hypothetical example of analysis by a series of 16 RIS of tumor susceptibility controlled by three nonlinked loci. Very similar phenotypes are exhibited by genetically quite different RIS (Table 11, strains E, F versus 0, P, and A, B versus K, L). As a corollary of the disruption of the correlation between the phenotype and the genotype in the RIS, the linkage actually indicated by the

129

GENETICS OF TUMOR SUSCEPTIBILITY IN THE MOUSE

B

BACKCROSS

X

B

X

F,

x

Bc2(N,)

BACKCROSS

BROTHER

7

Bc2 (N,)

SISTER MATING

I

I

7 I I

I

I

ETC,

I

I RECOMBINANT

CONGENIC

STRAINS

ACB-A

ACB-B

ACB-C

ACB-D

ACB-E

ACB-F

ETC,

FIG. 1.(B)

RIS for the studied phenotype is frequently spurious and misleading. A gene locus N in Tables I and 11, which is not involved in tumor susceptibility, exhibits the best correlation with it. Effects of nonadditive gene interactions, where the combined phenotypic effect of two or more genes is not equal to the sum of their individual effects, results in an even greater disruption of association between phenotype and genotype in the RIS. In addition, there is no complete representation of all possible genotypes in a series of RIS. Even with a small number of genes involved, the number of possible genotypes is considerable (2n, n = number of loci). They are not likely to be all represented in a series of RIS. For example, for n = 3 the

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TABLE I A QUANTITATIVE TRAITSTUDIEDWITH RIS AND RCS-A

MODEL".^

Strain designation A

RIS Locus 1 Locus 2 Locus 3 Locus N Phenotype RCS Locus 1 Locus 2 Locus 3 Locus N Phenotype

x x x x

100

u u u u

0

B

C

D

E

F

G

H

I

J

x x x x

x x u x

x x u x

x u x x

x u x x

u x x x

u x x x

x u u u

x u u u

u x u u

u u u u

u x u u

x u u u

u u u u

u u u u

100 90

0 4 0

K

L

M

N

O

P

u x u u

u u x u

u u x u

u u u u

u u u u

u u u u

u u u u

u u u u

90

80

80

70

70 50 50 40

40

30 30

u u u x

x u u u

u u u u

u u u x

u u u u

u u x u

u u u u

0 5 0

0

0

0 5 0

0

0 3 0

0

0

0

0

0

0

Modified from Demant and Hart (1986), with permission. Phenotype is given as a sum of percentages with parental strain phenotype: X = 100%and U = 0%. Three nonlinked genes: locus 1, X = 50%. U = 0%; locus 2, X = 40%. U = 0%; locus 3, X = 30%. U = 0%. Irrelevant gene: locus N, X = 0%. U = 0%. a

TABLE II A QUANTITATIVE TRAITSTUDIEDWITH RIS AND RCS-A

MODEL~JJ

Strain designation

RIS Locus I Locus 2 Locus 3 LocusN Phenotype RCS Locus 1 Locus 2 Locus 3 LocusN Phentotype

A

B

x x x

x x x

X

80

X 80

x u u u u u

U 70

U 30

C

D

x x u

E

F

U 80 ~

H

I

J

K

L

M

N

O

P

x x x u u x x u u u u u u x u u x x u u x x u u u u u x x x x u u u u x x u u

X X U U 100 100 30 30

u x u

G

U U 40 40

X 70

X 70

X X 80 80

U 0

U 0

U

U

30

30

u u u u u u u u u u u u u u u u u u u u u u x u u u u x u u u u u u u x u u u X 30

U 0

U 30

X U 30 30

U 30

U 30

U U 30 30

U 40

U 30

U

U

30 30

~~

Modified from Demant and Hart (1986), with permission, Phenotype is given as a sum of percentages with parental strain phenotype: X = 80%and U = 30%. Three nonlinked genes: locus 1, X = 40%. U = 0%;locus 2, X = 0%.U = -50%, locus 3, X = 406,U = 80%. Irrelevant gene: locus N, X = 06,U = 0%. a

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probability that all 8 possible genotypes are represented in a series of 16 RIS is 0.31, and for n = 4 it is almost zero (Demant and Hart, 1986). The incomplete representation of all possible genotypic combinations in a series of RIS makes the assessment of the nature of genetic control of the studied trait difficult.

C. RECOMBINANTCONGENIC STRAINS

The multigenic nature of the control of quantitative phenotype is the principal obstacle in its genetic analysis using the RIS. Therefore, a different genetic tool is needed, one that would retain the advantageous characteristics of the RIS, but that would transform the multigenic effect into a set of single-gene effects, which could be subsequently mapped and analyzed separately. To this end, we devised a new analytical system, the recombinant congenic strains (RCS) (Demant and Hart, 1986). While each RIS received a unique ‘mixture’ of genes originating in approximately equal proportions from each of the two parental strains, in the RCS the genes of one parental inbred strain (the “donor” strain) are randomly ‘dispersed’ in small proportions in the genetic background of the second parental inbred strain (the “background” strain), to achieve the separation of the genes-components of a multigenic system. This is done by backcrossing repeatedly the donor strain to the background strain, and subsequently using pairs of backcross mice to produce a series of new strains by consecutive brother-sister mating (Fig. 1B). In this way, a series of R C S is produced, each R C S containing a different, relatively small set of genes originating from the donor strain. As a result, each of several genes affecting tumor susceptibility in the donor strain will very likely be transferred into a different RCS. Those R C S that received such genes from the donor strain will differ from the background strain in tumor susceptibility, while the majority of the R C S will be phenotypically identical to the background strain. The segregation of the genes relevant for tumor susceptibility in RIS and RCS and the resulting phenotypes are compared, using model situations with three unlinked loci, in Tables I and II. The R C S share with the RIS their two principal advantages. First, as the R C S become genotyped, the information about the distribution of the alleles from the two parental strains among individual R C S accumulates, and the strain distribution of each new traitlgene can be compared with that of the previously typed genes to obtain indication of linkage. Second, the quantitative phenotype of mice of each RCS can be determined reliably by testing the necessary number of animals, which are all identical genetically. Three series of R C S are being presently prepared and analyzed at our laboratory:

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1. The HcB series with the background strain C3H/HeSnA, and the donor strain C57BL/lOScSnA; 2. The OcB series with the background strain 020/A, and the parental strain BlO.O20/Dem (this is an H-2 congenic strain carrying the H-2P" haplotype of 020/A on the C57BL/ lOScSnA background; the OcB series therefore contains non-H-2 genes of the C578Ll105c5nA strain spread on the 020/A genome); 3. The CcS series with the background strain BALB/cHeA, the donor strain STS/A.

These strains may be used to study the genetics of susceptibility to a variety of tumors, because the inbred strains which were used as their parents differ considerably both quantitatively and qualitatively in tumorigenesis in various organs. The main features of the tumor susceptibility resistance of these parental strains are summarized in Table 111.

D. QUANTITATIVE A N D STATISTICAL CONSIDERATIONS The rationale for the production procedure of RCS and the relevant computations have been given in detail by Demant and Hart (1986). The most useful scheme for the construction of the RCS is to produce the second backcross generation and to proceed then with continuous brother-sister mating. In that way, each RCS will receive 12.5% of genes from the donor strain. The sets of donor strain genes that are present in different RCS will overlap. Therefore, there is an effective upper limit to the total portion of the donor strain genes, which can be transferred into a set of RCS. A set of 20 RCS, each containing 12.5% of the donor strain genes, will contain 293% of the donor strain genome; a set of 16 RCS will contain 88% of the donor strain genome. In contrast to the RIS, for which the analytical power is barely affected by possible unintentional selection during the period of their establishment, more caution is required with the RCS. As discussed in detail elsewhere (Demant et a l . , 1988), an unintentional selection during the breeding of the RCS could result in deviation from the expected segregation of the donor strain's genes. As a result, a larger or a smaller proportion than expected might become transferred into the set of RCS, or certain parts of the genome might become overrepresented or underrepresented in the process. If these deviations were of a considerable magnitude, they could decrease or destroy the analytical power of the RCS. It is therefore advisable, at an intermediate stage of RCS production, to test the segregation of several nonlinked genetic markers. Tests of electrophoretical polymorphism of four unlinked enzymes in a set of RCS at an intermediate stage of inbreeding showed no significant deviation from the expected pattern (Demant et al., 1988).

*

TABLE 111 FEATURES OF TUMORSUSCEFTIBILITY RESISTANCE' HcB series

Lung tumors Intestinal tumors Mammary tumors Colon tumors Gross MuLV leukemia Liver tumors Myeloma Exocrine pancreatic nodules

OcB series

CcS series

C3H/HeASn

C57BL/lOScSnA

020/A

B10.020/Dem

BALB/cHeA

STS/A

n.d.6 n.d. susc. n.d. susc. susc. n.d. n.d.

Rest susc. Res. n.d. Res. Res . n.d. n.d.

SUSC.~ Res. Susc. n.d. n.d. n.d. n.d. Res.

Res. Susc. Res. n.d. n.d. n.d. n.d. n.d.

susc. Res. susc. Res. n.d. n.d. susc. susc.

susc. Res. Res. susc. n.d. n.d. Res. n.d.

a The data stem from our preliminary screening (M.A. van der Valk, L.C.J.M. Oomen et al., in preparation) with the exception of colon tumors and myeloma (E. Skamene, personal communication). The lung, intestinal, liver, and mammary tumors were induced by N-ethyl-N-nitrosourea treatment, accompanied, in the case of mammary tumors, with hormonal stimulation. The mammary tumors in the C3H-C57BL/lOScSn combination were induced by C3H-MTV (Miihlbock and Dux, 1981). The data on MuLV-induced leukemia are from literature (for references see Lilly and Mayer, 1980). The preneoplastic pancreatic nodules of exocrine cells in mice treated with ENU and hypophyseal isografts were observed by Dr. M.A. van der Valk at the Netherlands Cancer Institute (unpublished). n.d., not determined. Res., resistant. d Susc., susceptible.

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What is the resolution power of the RCS? The possibility to resolve with the help of RCS the individual components of a multigenic system affecting a quantitative trait depends on the proportion of donor strain genes in the RCS, on the total number of RCS in the set, and on the number of genes responsible for the quantitative difference between the two parental strains. These factors determine the probability that each gene of the donor strain that affects the analyzed trait is represented at least once in a set of RCS, and that no more than one such gene will be present in individual RCS. With 12.5% of donor strain’s genes in each RCS, a rational possibility exists to resolve traits controlled by up to five or six genes (Demant and Hart, 1986). With a higher number of genes involved, the analysis becomes more difK cult, but at least some of the genes involved may become readily identified. Establishment of linkage of the genes isolated in individual RCS by comparing their strain distribution pattern with that of previously typed polymorphic markers should be possible. However, the variance of the linkage estimated with RCS is higher than with RIS (Demant and Hart, 1986). Therefore, each linkage indicated by the RCS has to be confirmed by independent segregation test. This procedure is no more laborious than that using the RIS, since Bailey (1981) pointed out that every linkage indicated by the RIS has to be confirmed by an independent test too. The RCS complement other genetic systems available for analysis of interstrain differences. The basic characteristics of the available genetic systems are listed in Table IV. The special appeal of the RCS for study of tumor susceptibility is based on the fact that tumor susceptibility is often determined by several genes-a situation where the RCS are readily applicable. The choice of the optimal analytical system for any trait has preferably to be established in advance by a preliminary segregation test. A specific comment is required about the relative merits of the use of RCS (or RIS) on one hand, and transgenic mice on the other hand. Transgenic mice have turned out to be a very useful tool in analysis of biological effects of specific genes that were cloned and in many instances also coupled with a

APPROPRIATE

TABLE IV GENETICSYSTEMS FOR GENETICANALYSIS

OF INTERSTRAIN DIFFERENCES

Number of genes involved in the difference

1 2-6 37

Genetic test system

RIS RCS Congenic strains

Proportion of donor strain genome

0.5 0.125 so.01

Detection of linkage Possible Possible Not likely

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desired promoter-enhancer element, or otherwise mutated. The transgenes become transferred through germ-line cells to following generations. By definition each such strain of transgenic mice is a specific instrument for the study of the introduced gene. Therefore, the transgenic mice represent a unigenic analytical system. In contrast, the RCS and RIS represent coordinate or correlational analytical systems, because they use the available information about the distribution of alleles of the genes of the two parental strains among the set RCS or RIS as a kind of set of coordinates, with which one can correlate any new genetic trait. The unigenic systems and coordinate systems differ in economy of scale, in kinds of genes that can be analyzed, and in the possible depth of analysis. Much like a set of numbers along the abscissa and ordinate can define a virtually unlimited number of points in a plane, with the coordinate-correlational system a virtually unlimited number of genes can be analyzed by a single set of 20-25 RIS or RCS, provided that the genes to be analyzed are present in different allelic forms in the two parental strains. With a unigenic system, on the other hand, for each gene a separate strain of transgenic mice is needed. The RIS and RCS can be used to study well-defined genes as well as to define the map genes that are as yet unknown. The transgenic mice can be used only with genes that are already defined and cloned. On the other hand, the possibilities of genetic manipulation of the studied gene offered by transgenic mice cannot be obtained by the RCS or RIS. Obviously, the two types of systems are mutually complementary. The genes defined by the RCS or RIS can be cloned and subsequently used for transgenesis. The congenic strains, which carry only a very small proportion of genetic material of the “donor” parental strain, are in some logistical aspects more similar to transgenic mice than to RCS or RIS.

E. FROMGENETICMAPPINGTO M O L E C U ~ R ISOLATIONOF GENES In order to use the tumor susceptibility genes as an effective tool for analysis of the neoplastic process, it is necessary to clone them. Although for a long time the information on map position of genes had little impact on their study with methods of molecular genetics, for the first time genes have been physically isolated on the basis of their map location. The retinoblastoma gene (Rb) located in the 414 band of the human chromosome 13 has been physically identified (Field et al., 1986) by the standard chromosomal walking procedure with cosmids derived from this chromosomal region serving as the starting points (Fung et al., 1987; Lee et al., 1987). Another locus associated with a disease syndrome, Duchenne muscular dystrophy, lo-

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calized on the X chromosome, has been identified by locus-specific probes (Kunkel et al., 1985) by utilizing the DNA of a male patient with a deletion at this locus in a phenol-emulsion reassociation technique (Kohne et al., 1977). Several technical developments make the task of identifying a gene on the basis of its chromosomal gene map location a realistic undertaking. 1. The use of interspecies somatic-cell hybrids, which carry a limited number (preferably only one) of the chromosomes of the species studied. This approach was originally used by Gusella et al. (1980) to isolate clones of human DNA from chromosome 11, using human HeLa cells-Chinese hamster ovary hybrid cell lines with defined chromosomal constitution. The clones containing human DNA were detected by hybridization with labeled DNA of the human HeLa cell line. An analogous approach has been used by Kasahara et al. (1987) to obtain a DNA library from the mouse chromosome 17. In this case, a cosmid library from a mouse-Chinese hamster line that contained mouse chromosomes 17 and 18 has been screened for cosmids containing mouse DNA with a murine highly repetitive sequence probe. Repetitive sequence-free fragments of such clones were used to identify those clones that carry the genetic material from chromosome 17. 2. Increase in the number of informative markers on mouse chromosomes. In addition to the almost 1200 genes with known chromosomal assignment, 660 different DNA probes or clones with known chromosomal assignment are contained in the listing in the Mouse News Letter for February 1988 (pp. 120-146), a large increase from 287 listed in the previous year (Mouse News Letter, February 1987, pp. 80-90). This trend is likely to continue for several years, and it will result in availability of suitable DNA markers spread with considerable density along the chromosomal DNA. In addition, a rapid expansion of the physically mapped segments of mouse genome can be expected. 3. Introduction of pulse field gradient electrophoresis (PFGE) (Schwartz and Cantor, 1984) and its modifications (Carle and Olson, 1984; Chu et al., 1986; Herrmann et al., 1987; Carle et al., 1986). The PFGE separates electrophoretically DNA fragments of size up to 9 megabase pairs-corresponding to k 5 centimorgans (cM) of the mouse gene map. The efficacy of this method in establishing order and orientation of genes and restriction sites on chromosomes has been demonstrated (e.g., Miller et al., 1987; Brown and Bird, 1986; Herrmann et al., 1987). 4. Another development that will facilitate the creation of physical maps for large segments of chromosomes is the technology of “chromosome jumping” (Poustka and Lehrach, 1986; Poustka et al., 1987). In this technique large fragments of DNA, obtained by digestion with low-frequency-cutting restriction endonucleases, are circularized using a suitable plasmid. The

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circularized DNA is cut and the plasmid segments carrying the two ends of the long DNA fragments are cloned. The resulting clones provided probes defining two ends of a continuous DNA segment demarcated by specific restriction sites. The availability of large cloned fragments of DNA will enhance considerably the effectiveness of isolation and cloning of the genes that occupy a known map position. The ultimate resolution power of meiotic mapping is L O . 1-0.2 cM; this corresponds to *200-400 kb in mouse, and 100-200 kb in humans. The techniques available until recently did not allow cloning of segments >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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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

Glucocorticoid

-

Epithelial Interactions

-

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

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mean number of tumors

4 3.5 3

2.5 2 1.5 1

BlO.A(PR)

BIO.A

BlO.A(BR)

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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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