Transformation of diploid human fibroblasts by DNA transfection with the v-sis oncogene.
The simian sarcoma virus (SSV) oncogene (v-sis) has a high degree of homology to the cellular gene coding for the B peptide of human platelet-derived growth factor (PDGF), a potent fibroblast mitogen. The cellular homolog of v-sis is activated in some mesenchymal human tumors and cell lines derived from them. To determine the phenotype produced by v-sis in diploid human fibroblasts, we constructed plasmids containing the SSV provirus and drug-resistance markers and transfected them into early-passage human cells. Fibroblasts that had integrated the plasmid were selected for drug resistance and shown to contain and express the v-sis oncogene by DNA and RNA hybridization. The v-sis-expressing cells grew to higher saturation densities than control cells transfected with the vector plasmid alone and formed large, well defined foci. This allowed selection of transfectants directly for focus formation. The v-sis transformed cells continued to grow well in the absence of serum, whereas age-matched, vector-transfected control cells ceased replicating under these conditions so that the final difference in density between the two populations was tenfold. Incorporation of thymidine in serum-free medium by the v-sis-transformed cells was independent of exogenous PDGF. In contrast, PDGF increased thymidine incorporation in such medium by the control cells to the level found in the v-sis-transformed cells with or without added PDGF. These results suggest that expression of the v-sis oncogene in diploid human fibroblasts causes sufficient endogenous synthesis of the B chain of PDGF to allow transformants to grow to abnormally high cell densities. When individual v-sis-transformed cells were grown on a background of normal cells, this higher cell density at confluence could be visualized as a focus.
D002452:Cell Count; D002455:Cell Division; D002471:Cell Transformation, Neoplastic; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D005347:Fibroblasts; D006801:Humans; D009857:Oncogenes; D010982:Platelet-Derived Growth Factor / Q000096:biosynthesis / Q000494:pharmacology; D012190:Retroviridae / Q000235:genetics*; D012842:Sarcoma Virus, Woolly Monkey / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D010982:Platelet-Derived Growth Factor; D004247:DNA