Study of the role of an oncogene in the formation of tumours
The aim of this study was to examine the claim that a single, mutant oncogene can transform NIH 3T3 mouse fibroblasts into transformed, tumorigenic cells, acting in a genetically dominant fashion. A c.Ha-ras 1 oncogene, cloned from the EJ human bladder carcinoma cell line, was inserted into a shuttle vector carrying the selectable marker gene gpt, which encodes the enzyme XPRT (xanthine-guanine phosphoribosyl transferase). This construct, pSV2gptEJ, was transfected into NIH 3T3 cells by the calcium phosphate precipitation method and cells which had incorporated the plasmid were selected by growth in mycophenolic acidcontaining medium, to which gpt confers resistance. A number of clonal lines were established and their tumorigenicity tested. Tumour cell lines derived from these transfectants were back-selected using 2-thioxanthine, a cytotoxic analogue of the xanthine-guanine phosphoribosyl transferase substrate, to isolate clones which no longer contained functional pSV2gptEJ sequences. Six sub-clones which did not express detectable levels of the ras oncogene product, p21H.ras , were obtained. All were judged to be less transformed than the transfected parent cells: they appeared morphologically normal, were more serum-sensitive, showed clear saturation densities and were more anchorage-dependent. Three of these sub-clones were found to be tumorigenic at all sites tested. Cytological examination of the NIH 3T3 transfectants revealed that significant perturbation of their chromosome complement accompanied transfection. The transfection process, in the absence of DNA or with pSV2gpt alone, was found to be capable of transforming NIH 3T3 cells. Finally, a brief investigation of the effect of a "functional EJ-ras gene upon the differentiated phenotypes of these cell lines was attempted by comparing their ability to produce an extracellular matrix.