Relationship Between myc Oncogene Activation and MHC Class I Expression

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

I. Introduction

11. MHC Class I Expression and Cancer

111.

IV. V.

VI.

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

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

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

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

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

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

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

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

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

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

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

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

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

1 . MHC Class I Downmodulation in Experimental Tumors

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

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

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

Tumor Acute lymphoblastic leukemia

HLA Class I downmodulateda (% of tumors)

Allele downmodulatedb

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

A,B A B

1/1 (100)f

AAC

Burkitt lymphomaf

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

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

Cervical carcinoma

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

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

Choriocarcinomaf Colorectal carcinoma

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

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

Basal cell carcinoma B-cell lymphoma Bladder carcinoma

Breast carcinoma

$2-m

AAC AAC Bw6

Correlation with progressionc

Correlation with myc activationd 1 23 4 5,6 6

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

7 8

$2-m

8 7 , B44 A,B,C

Ref.e

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

N o (Ref. 28)

9-15

c-mycg c-mychzi

16 17,18 19

c-myci

20 21 22

c-mycg

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

TABLE I (Continued) ~

Tumor

Eccrine procarcinoma Endometrial carcinoma Gastric carcinoma w

‘9 t~

Laryngeal carcinoma Lung carcinoma (non-small cell)

Lung carcinoma (small cell)

HLAClass I downmodulatedo (% of tumors)

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

B, Bw4 A3

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

$2-m

3/3 (100)

11/12 (92)f Melanoma

Allele downmodulatedb

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

(looy

A3,C AB,C B Ab,C A

Correlation with progression.

~~

~

~~~~~~~

Correlation with myc activationd

c-mycgth

&f.C

27,29,30,32 27 34

35 21 32.36 32 37

Yes

B

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

Yes (Ref. 42,43)

No (c-myc)

38

c-, N-, Lmyc

39 40 40 39 41-44 45

c-, N-, Lmyc c-myc

46 47

Neuroblastoma

Ovarian cancer Renal cell carcinomaf

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

319

0 03

-

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

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

48 49

50 51 52 21

53

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

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

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

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

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

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

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

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

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

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

AND

rus

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

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

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

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

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

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

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D. myc 1. myc Activation

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

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

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

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

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

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

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

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

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

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

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

enhancer B

enhancer A

CAAT

IRE

-200

-

- 160

- 100

TATA

-60

0 bv

bindlng rite for regulatory protelnr: TQQQQATTCCCCA

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

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

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

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

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

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

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

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

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

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

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

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

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6. Role for Methylation

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

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

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

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

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

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

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

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

Plasmid pfosCAT pMHCfosCAT pMoCAT

Number of experiments 4 4

3

70 CAT activity IGR39D

I .6 82.9 53.8

IGR-myc 1.2 40.1 50.3

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

myc

-2.0

ONCOGENE ACTIVATION

- 1.5

- 1.0

AND MHC CLASS I EXPRESSION

-0.5

221

0 kb

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

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

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

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

A. MHC

A N D PROGRESSION

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

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

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

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

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

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

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r

EXPRESSION

227

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

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PETER I . SCHRIER AND LUCY T . C. PELTENBURG

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

myc

229

ONCOGENE ACTIVATION AND MHC CLASS I EXPRESSION

I

I 50 40

.v) v)

30

Y

x

z

20

s

10

IGR39D

myc

gpt-c5

B27-c7 B27-c3 827-c4

cell line fluorescence

%

N K lysis

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

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

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

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

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

VI. Concluding Remarks

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

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A c-rnyc activation early event

___*

activation o f c-myc

-

activation

normal cel l

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

escape from NK and T c e ll surveillance

B c-myc activation late event

___,

oncogene activation

normal cel l

cl : M H C Class I

activation o f c-myc

tumor c e l l killed by T cells

:

NKTS

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

:

tumor peptide

escape from NK and T c e ll surveillance

L ,

:

accessory molecule

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

myc ONCOGENE ACTIVATION

AND MHC CLASS I EXPRESSION

233

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

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