We recently developed a unique, infinite life span human fibroblast cell strain, MSU-1.0, that is diploid, phenotypically normal, and nontumorigenic. From this cell strain, we isolated a spontaneously variant strain, designated (MSU-1.1), that has a stable karyotype (45 chromosomes), which includes two unique chromosomes. From the MSU-1.1 cell strain we isolated a spontaneous variant that has the same stable karyotype, but proliferates rapidly in medium without exogenous growth factors. This strain is designated MSU-1.2. None of these strains are tumorigenic. The fes oncogene was transfected into MSU-1.2 cells, and several clonal transfectant cell strains were isolated. All of these strains were highly malignant with a short latency. Transfection of the fes oncogene into MSSU-1.1 cells did not give rise to tumor cells directly. However, MSU-1.1 cells expressing a transfected H-ras oncogene or the v-sis oncogene were converted to malignant cells by transfection of the fes gene. MSU-1.1 cells were also converted to malignant cells by high expression of transfected H-ra or N-ras oncogenes. MSU-1.1 and MSU-1.2 cells can also be converted to tumor cells by carcinogen treatment and appropriate selection. MSU-1.0 cells have never been neoplastically transformed by any procedure. These results clearly indicate that some critical change for carcinogenesis occurred when the MSU-1.1 cells were generated from the MSU-1.0 cells. Furthermore, they indicate that carcinogen treatment causes MSU-1.1 and MSU-1.2 cells to undergo some change critical to convert them to tumor cells. This proposal seeks to determine what genetic change occurred at these two steps in transforming these human fibroblast-derived cells. Cell fusions will be carried out to determine whether the critical difference between cell strains is the loss of suppressor gene activity or activation of an oncogene. In the case where evidence points to the loss of genetic information as the causal event, we will return the lost chromosome fragment to the cells. We will examine the status of suppressor genes when the evidence suggests that such genes have lost expression. In cases where it appears a dominant oncogene has been activated, we will examine the most obvious candidate genes (e.g., ras, and sis). If one or the other of these efforts does not succeed, we will clone the gene(s) involved by making an appropriate cDNA library in an EBV vector and transfer these cDNA's to suitable recipient cells in the lineage to identify phenotypically modified cells. Plasmids will be isolated from such cells, enriched, retested to see if they reproducibly cause the phenotypic change being tested. If so, the cDNA involved will be sequenced and compared with known sequences.
