A p53 mutation is required for stable transformation of REF52 cells by themyc and ras oncogenes

It is known that neoplastic transformation of rodent primary embryonic fibr oblasts cultured in vitro requires coexpression at least of two cooperating oncogenes. In the case of transduction into cells of oncogenes ras and myc , the cell transformation is poorly effective. To study some additional fac tors necessary for such transformation, c-myc and N-ras(Asp12) were consecu tively introduced into REF52 cells by retroviral infection, and the cell cu ltures obtained were analyzed. Expression of myc broke the regulation of th e cell cycle, in particular, canceled the G1 phase arrest for cells with da maged DNA, despite the normal function of protein p53 and induction of the p53-responsive gene p21(Waf1) in, these cells. The subsequent transduction of ras led to morphological transformation of cells and an increase of the p53 level. However, reversion of the transformed phenotype to normal morpho logy took place after less than five passages. On this background, rare clo nes generated the stable transformed cell lines characterized by accelerate d proliferation and having a mutation in the p53 gene. Attempts to obtain s table transformed cell lines by transduction of ras into REF52 cells not ex pressing exogenous myc were unsuccessful. Analysis of the stable transforme d clones revealed a mutation at codon 271 of the p53 gene, a hot spot of mu tations, which led to the replacement of arginine by cysteine. In these clo nes, p53 is accumulated owing to the increased life time, and has a flexibl e conformation, being able to interact with monoclonal PAb1620 and PAb240 a ntibodies recognizing alternative protein conformations. The results obtain ed suggest that p53 participates in negative regulation of the cell cycle u nder conditions of oncogenic stimulation, and its inactivation is necessary for full transformation of cells by cooperating oncogenes myc and ras.