The effect of H-ras expression on tumorigenicity and immunogenicity of Balb/c 3T3 fibroblasts.

In an attempt to define immunological parameters affected by the H-ras oncogene, we have used Balb/c 3T3 cells transfected with either H-ras (98/6), H-ras+v-myc (98/4v) or plasmid only (98/1). We found that while control and oncogene-transfected Balb/c 3T3 cells exhibit similar low sensitivity to lysis by natural killer (NK) cells, H-ras+v-myc-transfected cells could immunize syngeneic Balb/c mice and induce cytotoxic T cells (CTL) with broad specificity, that lysed all types of Balb/c 3T3 cells tested. Immunization of Balb/c mice with 98/4v cells prevented homologous tumor formation and partially inhibited the formation of tumors derived from H-ras-transfected cells. 98/6 cells were not immunogenic in vivo and did not protect the animals from a challenge of 98/6 cells. The results suggested that CTLs but not NK effector cells were important for eliciting in vivo tumor rejection of H-ras+v-myc-transfected cells. In contrast, antigens eliciting the cytotoxic T-cell response, and possibly also the in vivo tumor cell rejection response, were expressed on all cell types tested but were immunogenic only on the surface of 98/4v cells. We further determined major histocompatibility complex (MHC) class-I molecule expression on the outer cell surface and found that H-2K was down-regulated in H-ras-transfected cells. The results support the observation that oncogenes can down-regulate specific MHC antigens, thereby preventing presentation of tumor antigens and allowing tumor escape from immune recognition.

EGF receptor activity and mitogenic response of Balb/3T3 cells expressing Ras and Myc oncogenes. EGF receptor activity in oncogene transformed cells.

EGF receptors are found on the surface of most cells, usually with high and low binding affinities. To investigate functional relationships between EGF (EGF-like growth factors) and oncogenes we have characterized the expression of the epidermal growth factor receptor (EGFr) in H-Ras, v-Myc, and H-Ras-v-Myc transformed Balb/3T3 cells. H-Ras cells show a marked decrease in the number of EGFr molecules per cell compared to parental cells. v-Myc and H-Ras-v-Myc transformed cells express an intermediate level of receptors. The majority of the EGF receptors on the parental and oncogene transformed cells bind EGF with low affinity and this low affinity receptor is down-regulated by oncogene transformation. v-Myc expression, in the H-Ras-v-Myc transformed cells, abrogates the receptor down-regulation seen with H-Ras transformation. The mechanism of abrogation is not a result of a change in the p21-Ras concentration in the H-Ras-v-Myc transformed cells. In addition, the mitogenic response to EGF was examined. H-Ras and H-Ras-v-Myc transformed cells do not respond to EGF mitogenically. In contrast, EGF stimulates DNA synthesis in parental cells and v-Myc transfected cells; this result suggests that growth promoting signals from the EGF receptor may not be required in H-Ras transformed cells.

The H-ras oncogene regulates expression of 70- and 45-kDa cell-surface molecules whose expression correlates with tumor-cell immunogenicity.

The effects of the H-ras oncogene on fibroblast cell tumorigenicity and immunogenicity was studied in transfectants of the BALB/c 3T3 clone A31 fibroblastoid cell-line. Cells that were transfected with MC29-LTR-H-ras (98/6) or MC29-LTR-v-myc + H-ras (98/4v) and were inoculated into syngeneic BALB/c mice were tumorigenic in 100% and 60% of animals respectively. By contrast, transfectants containing the pSV2neo plasmid alone (98/1) displayed normal characteristics both in vitro and in vivo. Inoculation of mice with mitomycin-C-treated 98/1 or 98/4v cells induced an effective protective immunity to a challenge of live 98/4v cells, and a partial immunity against 98/6 cells. Mitomycin-C-treated 98/6 cells failed to render immunity against a challenge of either 98/6 or 98/4v cells. To correlate immunogenicity and tumorigenicity of the different cell types with cell-surface-antigen expression, we prepared MAbs against 98/4v cells in syngeneic mice. Immunohistochemical and immunoblot analysis revealed that MAbs 102 and 104 recognized 2 protein band of 70 and 45 kDa respectively, which were expressed predominantly in 98/1 and 98/4v cells. A third immunoreactive protein band of 44 kDa that reacted with MAb 6 was expressed at a similar cell-surface density on all cell types. Cell-differentiation-inducing agents, such as DMSO, retinoic acid or sodium butyrate, were all found to induce 98/6 cell flattening and morphological changes toward a normal phenotype that were followed by up-regulation of the 70- and 45-kDa antigens. The results suggest that regulation of expression of the 70- and 45-kDa molecules is affected by H-ras, and that expression of these cell-surface molecules may be relevant to tumor cell immunogenicity.