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

It is known that neoplastic transformation of rodent primary embryonic fibroblasts 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 factors necessary for such transformation, c-myc and N-rasAsp12 were consecutively introduced into REF52 cells by retroviral infection, and the cell cultures obtained were analyzed. Expression of myc broke the regulation of the cell cycle, in particular, canceled the G1 phase arrest for cells with damaged DNA, despite the normal function of protein p53 and induction of the p53-responsive gene p21Waf1 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 morphology took place after less than five passages. On this background, rare clones generated the stable transformed cell lines characterized by accelerated proliferation and having a mutation in the p53 gene. Attempts to obtain stable transformed cell lines by transduction of ras into REF52 cells not expressing exogenous myc were unsuccessful. Analysis of the stable transformed clones revealed a mutation at codon 271 of the p53 gene, a hot spot of mutations, which led to the replacement of arginine by cysteine. In these clones, p53 is accumulated owing to the increased life time, and has a flexible conformation, being able to interact with monoclonal PAb1620 and PAb240 antibodies recognizing alternative protein conformations. The results obtained suggest that p53 participates in negative regulation of the cell cycle under conditions of oncogenic stimulation, and its inactivation is necessary for full transformation of cells by cooperating oncogenes myc and ras.