Driving adenovirus type 12-transformed BALB/c mouse cells to express high levels of class I major histocompatibility complex proteins enhances, rather than abrogates, their tumorigenicity.

The tumorigenicity of adenovirus type 12 (Ad12)-transformed cells has been attributed to the low levels of class I major histocompatibility complex (MHC) protein expression by these cells. These levels of class I proteins are thought to be below the threshold critical for cytotoxic T-lymphocyte recognition, a process that may be involved in tumor cell immunosurveillance. We have used gene transfer experiments to investigate the role played by class I protein expression in the tumorigenicity of Ad12-transformed BALB/c mouse cells in naive, syngeneic adult mice. Our Ad12-transformed mouse cells were tumorigenic in adult mice and were similar to other Ad12-transformed mammalian cells in that they expressed low levels of class I MHC mRNA and cell surface proteins. Despite these low levels of expression, the cells were highly immunogenic in syngeneic mice and were rejected as allografts by allogeneic mice. Transfection of genomic H-2Dd or H-2Ld fragments into these cells produced a variety of cell clones that expressed increased levels of cell surface class I proteins. These cells expressing high levels of class I protein were up to 16-fold more tumorigenic than the parental cells in syngeneic adult mice. Thus, by quantitative assays, the tumorigenicity of Ad12-transformed BALB/c mouse cells is not functionally related to the low levels of class I MHC proteins they express. The increased tumorigenicity expressed by H-2Dd- and H-2Ld-transfected cells was not detected in BALB/c nu/nu mice, suggesting that a thymus-dependent mechanism that is not mediated by evasion of cytotoxic T-lymphocyte recognition could contribute to the difference in tumorigenicity of Ad12-transformed BALB/c mouse cells that express low and high levels of class I MHC proteins.     

Viral gene inhibition of class I major histocompatibility antigen expression: not a general mechanism governing the tumorigenicity of adenovirus type 2-, adenovirus type 12-, and simian virus 40-transformed Syrian hamster cells

The association between the level of class I major histocompatibility (MHC) antigen expression and the tumorigenic phenotype was determined for cells from a series of 15 lines of adenovirus type 2 (Ad2)-, Ad12-, and simian virus 40 (SV40)-transformed hamster cells and 16 lines of cells established from hamster tumors induced by SV40 mutants. These cells range from nontumorigenic to highly tumorigenic in both syngeneic and allogeneic adult hamsters. The Ad2-transformed cells--cells that were nontumorigenic in syngeneic adult hamsters--expressed either high levels or low levels of class I MHC antigens. The SV40-transformed cells--cells transformed in vitro that produced tumors with equal efficiency in both syngeneic and allogeneic adult hamsters--or cells derived from SV40-induced tumors expressed very high levels of class I MHC antigens. The Ad12-transformed cells uniformly expressed low levels of class I MHC antigens; these cells produced tumors 200- to 1,000-fold less efficiently in allogeneic adult hamsters than in syngeneic adult hamsters and produced tumors with about the same efficiency in immunoimmature newborns and immunocompetent syngeneic adult hamsters. We conclude that the expression of either high levels or low levels of class I MHC antigens is, at most, a minor factor in the differences observed among these adenovirus- and SV40-transformed cells in their tumor-inducing capacity in naive, immunocompetent hamsters.     

Changes in NF-kappa B and ISGF3 DNA binding activities are responsible for differences in MHC and beta-IFN gene expression in Ad5- versus Ad12-transformed cells.

Changes in MHC class I expression are frequently observed in tumors, which represents at least one mechanism by which tumor cells escape immune surveillance. MHC class I expression is often suppressed in type 12 adenovirus (Ad12)-transformed rodent cells, but is highly induced in Ad5-transformed cells. This difference helps to explain why Ad12 but not Ad5 can induce tumors in immunocompetent syngeneic rats. In this report we demonstrate that only Ad5- but not Ad12-transformed rodent fibroblasts constitutively express beta-IFN which results in ISGF3 factor induction, and stimulation of MHC class I expression. Furthermore, we demonstrate that in contrast to Ad12-transformed cells, Ad5-transformed cells show constitutive levels of nuclear NF-kappa B-like DNA binding activity. This is of particular interest since both the beta-IFN and the MHC class I promoters contain an NF-kappa B DNA binding site. Thus, high levels of MHC class I expression in Ad5-transformed cells are due to a combinatorial stimulation of two cis-regulatory sequences of the MHC class I promoter: the NF-kappa B binding site and the interferon stimulated response element (ISRE), which binds the ISGF3 factor complex. The failure of Ad12-transformed cells to activate this pathway explains their low levels of MHC class I expression and their greater oncogenicity.     

Adenovirus E1A-mediated regulation of class I MHC expression.

Expression of class I MHC transplantation antigens has been shown to be reduced in baby rat kidney (BRK) cells transformed by highly oncogenic adenovirus type 12 (Ad12), as compared with untransformed cells and cells transformed by non-oncogenic Ad5. Here we show that this reduction of class I expression also occurs in a variety of other primary cell cultures transformed by Ad12, and that reduction of class I gene expression occurs for all class I loci. Transfection of Ad5E1 into class I-negative Ad12-transformed BRK cells leads to complete restoration of class I expression. Introduction of Ad12E1 into most class I-positive established cell lines does not result in suppression of class I expression. However, transfection of the Ad12E1A region into a class I-positive cell line which was immortalized by a mutant Ad12E1A region resulted in suppression of class I gene expression, implying that the suppression of class I activity in Ad12-transformed cells is due to an active switching-off process.     

Downregulation of MHC class I expression due to interference with p105-NF kappa B1 processing by Ad12E1A.

We have previously demonstrated that expression of major histocompatibility complex (MHC) class I genes is repressed in baby rat kidney cells transformed by early region 1 of oncogenic adenovirus type 12 (Ad12E1). Reduced expression of MHC class I antigens contributes to the escape of Ad12-transformed cells from T-cell-mediated immune surveillance and to tumour induction. In this study, we show that repression of MHC class I expression by Ad12E1A is mediated via the H2TF1 element of the MHC class I promoter. This element binds NF kappa B and KBF1, two factors which play a major role in the regulation of MHC class I expression in vivo. In extracts from Ad12E1-transformed cells, binding of KBF1 and NF kappa B to the H2TF1 element is decreased. This is caused by reduced production of p50-NF kappa B1, the 50 kDa subunit shared by KBF1 and NF kappa B, due to interference with p105-NF kappa B1 processing by Ad12-13S-E1A protein. Overexpression of the p105-NF kappa B1 cDNA, or of a truncated p105-NF kappa B1 cDNA that codes for p50-NF kappa B1, restores MHC class I expression in Ad12E1-transformed cells. These data demonstrate that downregulation of MHC class I expression in Ad12E1-transformed cells is due to interference with processing of p105-NF kappa B1 by the Ad12-13S-E1A protein.     

Rejection of B16 melanoma induced by expression of a transfected major histocompatibility complex class I gene.

Transfection of a functional major histocompatibility complex class I gene into certain tumor cells, induced by oncogenic viruses or chemical carcinogens, can effectively abrogate their tumorigenic activity. Since experimentally induced tumors possess strong tumor-specific transplantation antigens, expression of cell surface class I antigens may present the tumor cells to appropriate immune effector cells. Most spontaneously arising tumors do not possess tumor-specific transplantation antigens, and their tumorigenicity may not be affected by the expression of a transfected class I gene. We demonstrate that the poorly immunogenic B16-BL6 melanoma can be rendered nontumorigenic in syngeneic mice by the expression of the class I H-2K antigen but not the class II I-A antigen. Furthermore, the poorly tumorigenic, class I-expressing B16-BL6-transfected cells can effectively immunize syngeneic C57BL/6 mice against the highly tumorigenic, class I-deficient B16-BL6 parental cells. Our success in experimentally manipulating the tumorigenicity of a spontaneously derived neoplasm offers hope for a potential modality for the effective treatment of human cancer.     

The level of expression of adenovirus type 2 transforming genes governs sensitivity to nonspecific immune cytolysis and other phenotypic properties of adenovirus 2-simian virus 40-transformed cell hybrids.

Syrian hamster embryo cells transformed by adenovirus type 2 (Ad2) or simian virus 40 (SV40) differ markedly in morphology, tumorigenicity, and susceptibility to in vitro lysis by nonspecific cytotoxic cells. Hybrid cells formed by fusing Ad2- and SV40-transformed Syrian hamster embryo cells may express only SV40 T antigens or both SV40 and Ad2 T antigens. Hybrids that express only SV40 T antigens are indistinguishable from the nonhybrid SV40-transformed phenotype, whereas hybrid cells that express T antigens from both viruses closely resemble the nonhybrid parental Ad2-transformed phenotype. Because these hybrid cells have been useful in the study of neoplastic transformation, we determined the amount of viral antigens that they accumulate in an attempt to correlate the level of expression of the transforming viral genes with some of their phenotypic properties. Hybrid cells that expressed proteins from both viruses showed reduced levels of SV40 T antigens compared with those of hybrid cells that did not express Ad2 T antigens. We also found that the production of several cellular proteins that influence cytomorphology was inhibited in hybrid and nonhybrid cells that expressed Ad2 T antigens, and the repression of these cellular proteins correlated with a change in cytomorphology from fibroblastic to spherical. Finally, we showed that the susceptibility of our hybrid cells to in vitro lysis by natural killer cells and activated macrophages, two putative host-effector cells involved in defense against neoplasia, correlated closely with the level of expression of a 58,000-dalton Ad2 protein. The results reported here, together with the results of previous studies, indicate that the oncogenic potential of hybrid cells that express both Ad2 and SV40 antigens is extremely sensitive to Ad2 expression, whereas other phenotypic properties depend on Ad2 expression in a dose-dependent manner.     

Tumorigenicity and H-2 expression of papillomavirus-transformed mouse cell lines

Summary Tumorigenicity in immunocompetent syngeneic mice and H-2 class I antigen expression of BPV1-transformed mouse cell lines had no correlation. H-2 expression was examined using monoclonal anti-(H-2K^b) and anti-(H-2D^b) antibodies in immunofluorescence staining for flow cytometry analysis and by determining the sensitivity of the cells to cytolysis by allostimulated spleen cells. Nontumorigenic cell lines were as resistant as tumorigenic cell lines to natural killer activity. The results indicate that in our model defence by natural killer cells is not a decisive factor. The results also show that instead of or in addition to H-2 class I antigens other factors (e. g. the presence or absence of virus-specific antigens) are important in determining the tumorigenicity of BPV1-transformed cell lines.     

The loss of class II MHC antigen expression byras-transformed murine fibroblasts passaged as tumours correlates with increased tumorigenicity but is not mediated by T cells

Summary In several murine tumour systems, expression of class II major histocompatibility complex (MHC) antigens by tumour cells, either constitutive or inducible, correlates with reduced tumorigenicity as compared with equivalent class-II-negative cells, and CD4 phenotype T cells together with interferon (which induces the expression of class II) may be involved in the control of the proliferation of class-II-expressing tumours. This implies a potential T-cell-mediated selection pressure against class II expression. To test this possibility, we have repeatedly passaged as tumours in euthymic, syngeneic miceras-transformed murine fibroblast lines, which are class-II-inducible, to determine whether class-II-non-inducible variants are selected. We examined the expression of both class I and class II antigen in tumour cells re-established in vitro. It was found that the inducibility of class II, but not class I, expression rapidly diminished, correlating with augmented tumorigenicity. However, this loss of class II inducibility occurred in athymic as well as euthymic mice. Therefore, despite the fact that the tumorigenicity of these lines is augmented in euthymic mice depleted of CD4 T cells or interferon , we found no evidence of T-cell-mediated selection against class II expression. The loss of class II expression observed must be due to mechanisms other than immune selection. The possibility that this might result from other soluble factors modulating the response to interferon in vivo is discussed.     

Autocrine induction of major histocompatibility complex class I antigen expression results from induction of beta interferon in oncogene-transformed BALB/c-3T3 cells.

By varying growth conditions, we identified a novel mechanism of autocrine regulation of major histocompatibility complex (MHC) class I gene expression by induction of beta interferon gene expression in transformed BALB/c-3T3 cells. Low-serum conditions enhanced MHC class I antigen expression in v-rasKi- and v-mos-transformed BALB/c-3T3 cells but not in untransformed BALB/c-3T3 cells. Transformed and untransformed cells grown under standard serum conditions (10% bovine calf serum) expressed similar cell surface levels of MHC class I antigens. However, low-serum conditions (0.5% bovine calf serum) induced four- to ninefold increases in cell surface levels of MHC class I antigens in both v-rasKi- and v-mos-transformed cells but not in untransformed cells. These increases in MHC class I gene expression were seen at both the mRNA and cell surface protein levels and involved not only the heavy-chain component of the class I antigens but also beta 2 microglobulin. Beta 1 interferon mRNA and beta interferon-inducible 2',5'-oligoadenylate synthetase mRNA were induced by growth under low-serum conditions in transformed BALB/c-3T3 cells, and antibodies to beta interferon blocked the induction of MHC class I antigen expression by serum deprivation in these cells. These results demonstrate that growth under low-serum conditions leads to induction of beta interferon expression in oncogene-transformed cells which then directly mediates autocrine enhancement of MHC class I gene expression.     

In vivo tumorigenicity and in vitro sensitivity to tumor-necrosis-factorα mediated killing of c-Ha-ras-transformed cells

Summary Cellular subclones of high and low tumorigenicity obtained from a mouse c-Ha-ras-transformed clone, were examined for their sensitivity to tumornecrosis-factor (TNF)-mediated cytotoxicity. Cells of the highly tumorigenic subclones showed a significantly enhanced resistance to the cytotoxic effect of TNF plus cyclohexamide (CHI) as compared to cells of the lowtumorigenic subclones. The enhanced resistance to TNF+CHI was not due to a lower expression of TNF receptors on the cells. The c-Ha-ras-transfected cells were transformed and maintained in culture only (C cells). In vivo passage of cells of the initially low-tumorigenic c-Ha-ras subclones through the mouse significantly enhanced the tumorigenic potential of these CTC cells (culture/tumor/culture). In correlation with their enhanced tumorigenicity, the CTC cells were highly resistant to TNF-mediated cytotoxicity as compared to C cells of the same subclone. Furthermore, the involvement of TNF in determining the tumorigenic phenotype of the c-Ha-ras-transformed cells was demonstrated in a more direct manner. Cells of a c-Ha-ras-transformed low-tumorigenic, highly TNF-sensitive subclone were selected by repeated cycles of in vitro exposure to TNF. As a result, a stable, highly TNF-resistant population of cells emerged. These TNF-resistant cells caused more tumors in mice as compared to their original TNF-sensitive cells. These results show that the resistance to the cytotoxic effect of TNF plus cyclohexamide may be involved, at least partially, in the tumorigenic potential of c-Ha-ras-transformed cells and suggest a possible role for TNF in the enhancement of the tumorigenic potential of these cells in mice.     

Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs.

The studies described here demonstrate that the expression of many early adenovirus mRNAs is dependent upon the activity of a pre-early viral product. This viral gene product is defective in adenovirus 5 host range (Ad hr) group I mutants. Adenovirus 5 host range mutants were previously isolated by their ability to replicate in the adenovirus 5-transformed human embryonic cell line 293 and by their inability to replicate efficiently in HeLa cells (Harrison, Graham and Williams, 1977). The group I complementation class of host range mutants has been mapped by marker rescue between 0 and 4.4 units (Frost and Williams, 1978). We have used the S1 nuclease gel technique to examine the expression of early mRNA after infection of HeLa cells with Ad5 hr group I and II mutants. The Ad5 hr group II mutants stimulate the synthesis of a wild-type pattern of early mRNAs. In contrast, infection of HeLa cells with Ad5 hr group I mutants gives rise to only two early mRNAs. These mRNAs map from 1.5–4.4 units, or in the same region as the Ad5 hr group I mutations. Since infection of HeLa cells with Ad5 hr group I mutants was defective for synthesis of cytoplasmic mRNAs complementary to three early regions in the right half of the genome and to the early region 4.5–11.0 units, we also analyzed nuclear RNA from these cells by the S1 nuclease gel technique for the presence of precursor RNA chains. Nuclear precursors were not detected in Ad5 hr group I-infected HeLa cells, suggesting that the gene product defective in these mutants is required for synthesis of stable nuclear RNA from the three early regions in the right half of the genome and from the early region 4.5–11.0 units.