N-methyl-N-nitrosourea-induced transformation of rat urothelial cells in vitro is not mediated by activation of ras oncogenes

Adult rat urothelial cells were transformed in vitro following treatment with a single dose of N-methyl-N-nitrosourea (MNU) or MNU treatment followed by promotion with sodium saccharin. This in vitro transformation process involves multiple steps: slow-growing 'pre-neoplastic' epithelial foci are induced 70-100 days after MNU treatment and from such foci rapidly proliferating immortal cell lines were established, some of which became tumorigenic after a further latent period. A series of epithelial cell lines and a single fibroblast cell line established in this way were analysed for the presence of transforming genes by DNA transfection into NIH3T3 cells. None of the epithelial cell lines induced foci in a focus formation assay. The single non-epithelial line induced foci and was found to contain an activated c-Ki-ras gene with a G----A transition in codon 12. To assay for the possible presence of transforming genes which were not active in a focus formation assay, two of the epithelial lines were analysed further by co-transfection with a dominant selectable marker, followed by selection and inoculation into nude mice. No tumours were induced within the latent period for tumour production by control cells transfected with NIH3T3 cell DNA (40-60 days). These results suggest that there is cell type specificity for oncogene activation during in vitro rat bladder transformation initiated by a single carcinogen and that ras gene activation is not a necessary step in urothelial transformation in vitro.