The role of Ha-ras oncogenes in growth factor independence in rat mammary carcinoma cells.

To determine if activation of the c-Ha-ras-1 gene is involved in the acquisition of growth factor independence in 7,12-dimethylbenz[a]anthracene (DMBA)--and N-nitrosomethylurea (NMU)--induced rat mammary carcinomas, three strategies were used. First, Ha-ras DNA from growth factor-independent DMBA-induced rat mammary tumor cells was amplified using the polymerase chain reaction and examined for the presence of mutations in the first and second exons of Ha-ras-1 by restriction fragment length polymorphism analysis, allele-specific oligonucleotide hybridization, and direct sequencing. No mutations were found in the codon 12/13 or codon 61 regions of the Ha-ras-1 gene. Second, a similar analysis of an NMU-induced mammary carcinoma showed that it harbored an activating mutation in codon 12 of Ha-ras-1. When analyzed for growth factor requirements, these cells were found to express limited growth potential in all media tested, in contrast to growth factor-independent cells, which proliferated extensively in the presence or absence of exogenous growth factors. Third, growth factor-dependent rat mammary tumor cells and spontaneously immortalized rat normal mammary epithelial cells were transfected with an activated form of the Ha-ras-1 (T24) gene, and the growth factor requirements of the transfected cells were examined. The ras-transfected cells retained the growth factor requirements of the normal cells. In addition, ras-transfected cells were transplanted into syngeneic rats and nude mice, and no tumors developed after 6 mo in vivo. These results indicate that, in rat mammary tumor cells, neither growth factor independence in vitro nor transplantability are directly mediated by Ha-ras oncogenes. The results also suggest that ras activation and growth factor independence may be associated with independent pathways to malignancy in rat mammary tumorigenesis.