Cooperation of oncogenes in the multistep transformation of established fibroblasts in culture.

The multistage nature of carcinogenesis observed in a variety of systems has been linked experimentally to the sequential activation and subsequent cooperation of oncogene proteins. Cellular transformation resulting from cooperative interactions between activated oncogenes has been described previously for cultured primary cells and certain established cell lines. Our laboratory is using the mouse embryonic cell line C3H10T1/2 to investigate cooperative transformation mediated by the activated human H-ras gene and several nuclear oncogenes from both viral and cellular sources. Oncogene cooperation in C3H10T1/2 is marked by an increase in focus number and an alteration in focus morphology. Although ras cooperates with myc and fos oncogenes to transform C3H10T1/2 fibroblasts, cooperative transformation is not observed in cells similarly co-transfected with the H-ras and E1A oncogenes. This result appears to be due to a growth inhibitory effect of the EIA gene product on C3H10T1/2 cells. The observed cooperation between the H-ras and MC29 viral gag-myc oncogenes has been used along with site-directed mutagenesis of the gag-myc gene to identify two structural regions within the gag-myc protein that mediate ras/myc cooperativity. In addition, the gag-myc mutants generated for these cooperation studies have been used to map the nuclear localization signal of the gag-myc protein and relate the proper cellular location of the protein to its activity in several transformation assays.