Transformation and radiosensitivity of human diploid skin fibroblasts transfected with SV40 T-antigen mutants defective in RB and P53 binding domains.
A series of human diploid fibroblast cell clones were developed by DNA transfection with either wild-type SV40 T-antigen (SV40 T) or T-antigen mutants defective in its various functional domains. Cell clones expressing the wild-type SV40 T were significantly radioresistant as compared with clones transfected with the neo gene only (D0 = 192 +/- 13 vs 127 +/- 19). This radioresistance persisted in post-crisis, immortalized cell lines. A series of mutants with point or deletion mutations within each functionally active domain of SV40 T were also examined for their ability to alter radiosensitivity and induce morphological transformation. Cell clones transfected with T-antigen mutants defective in nuclear localization or origin binding showed increased radioresistance similar to clones transfected with wild-type T-antigen, and expressed morphological changes characteristic of SV40 T-transfected cells. A retinoblastoma susceptibility gene (RB) binding defective mutant showed moderately increased radioresistance (D0 = 174 +/- 10). However, cell clones transfected with three different p53 binding defective mutants showed no change in radiosensitivity (D0 = 132 +/- 5) as compared with neo gene transfected controls. Transfection with T-antigen mutants defective in either the RB or p53 binding domain yielded no morphological alterations characteristic of transformation. These data suggest that the SV40 T/p53 complex may be of importance in the radioresistance phenotype.
Laboratory of Radiobiology, Harvard School of Public Health, Boston, Massachusetts 02115.
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D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry / Q000235:genetics*; D002448:Cell Adhesion; D002460:Cell Line; D002470:Cell Survival; D002472:Cell Transformation, Viral / Q000235:genetics* / Q000528:radiation effects; D003114:Colony-Forming Units Assay; D004171:Diploidy; D005347:Fibroblasts; D016161:Genes, Retinoblastoma; D016158:Genes, p53; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D009154:Mutation; D011836:Radiation Tolerance; D012867:Skin / Q000166:cytology; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming