Cooperativity of SV40 T antigen and ras in progressive stages of transformation of human fibroblasts.
Human diploid fibroblasts immortalized by SV40 T antigen provide an experimental system for studying the progression and synergism in transformation by secondary oncogenes. We have utilized the human fibroblast line HAL, which was immortalized with an orgin-defective SV40 genome encoding a temperature-sensitive T antigen, to study 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 degrees C. Three new derivatives of the HAL cell line were generated by microinjection of the 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, wherein the ras gene is transcriptionally regulated by the cellular promoter and driven by either the SV40 enhancer or an upstream LTR enhancer. 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. 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 degrees C, however the c-rasLTRhygro-HAL cell line does proliferate at 39 degrees C in 10% serum-containing medium. Therefore, we propose that neither v-Ha-ras nor c-ras can replace T antigen at 39 degrees C; rather T antigen and ras cooperate in progressive stages of transformation of human fibroblasts.
White
J A
JA
Biology Department, Hood College, Frederick, Maryland 21701.
Carter
S G
SG
Ozer
H L
HL
Boyd
A L
AL
D000952:Antigens, Polyomavirus Transforming / Q000032:analysis / Q000235:genetics*; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002999:Clone Cells; D005347:Fibroblasts; D015870:Gene Expression; D011905:Genes, ras; D006801:Humans; D007700:Kinetics; D015689:Oncogene Protein p21(ras) / Q000032:analysis / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000032:analysis / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics*; D013696:Temperature; D013997:Time Factors
D000952:Antigens, Polyomavirus Transforming; C501009:HRAS protein, human; D015689:Oncogene Protein p21(ras); D016283:Proto-Oncogene Proteins p21(ras)