COOPERATIVITY OF SV40 LARGE T ANTIGEN AND RAS IN PROGRESSIVE STAGES OF TRANSFORMATION OF HUMAN FIBROBLASTS

Abstract

Human diploid fibroblasts (HDF) are more resistant to transformation by viral or chemical oncogenic agents than primary or established rodent cells. HDF immortalized by SV40 T antigen provide an experimental system for studying the progression and synergism in transformation by secondary oncogenes. The human fibroblast line HAL, which was immortalized with an origin-defective SV40 genome encoding a temperature-sensitive T antigen, has been used in this study to analyze the cooperativity between SV40 T antigen and the ras oncogene in the progression of transformation. This study demonstrates that HAL cells possess characteristic growth patterns requiring 10% serum, are anchorage dependent and express a temperature sensitive T antigen. HAL cells rely on the normal functioning of T antigen for continual growth, and therefore, do not proliferate at 39°C, the restrictive temperature. Three new derivatives of the HAL cell line were generated by microinjection of the Harvey rat sarcoma, ras, oncogene. The cell line v-ras-HAL was derived by microinjection of HAL cells with v-Ha-ras DNA. The cell lines c-rasSVneo-HAL and c-rasLTRhygro-HAL were established by microinjection of HAL cells with the plasmids pSV2neoT24 or fpHVT24, respectively. In these two cell lines, the ras gene is transcriptionally regulated by the cellular promoter and either the SV40 enhancer or an upstream LTR enhancer, respectively. The three ras containing cell lines grow in reduced serum concentrations (0 to 5%), are anchorage independent and express both T antigen and ras p21 as detected by Western immunoblotting and indirect immunofluorescence. The v-ras-HAL and c-rasSVneo-HAL cell lines are still dependent upon the normal functioning of T antigen for continual growth at 39°C, however the c-rasLTRhygro-HAL cell line proliferates at 39°C in 10% serum-containing medium. Therefore, neither v-Ha-ras nor c-ras can replace T antigen at 39°C, rather T antigen and ras cooperate in progressive stages of transformation of human fibroblasts (White, J.A., et al., 1992).