Regulation of the level of the oncoprotein p53 in non-transformed and transformed cells

Abstract

In order to contribute to the understanding of the activation of the oncoprotein p53 we determined the metabolic stability of p53 in a variety of non-transformed, immortalized and SV40- and non-SV40-transformed cell lines. In addition, we analyzed the metabolic stability of the SV40 large T antigen in SV40 transformed cell lines. Pulse-chase experiments revealed a low stability (t1/2 = 20 min) of p53 in non-transformed cells and in cells immortalized by the p53 construct pLTRp53cG9. In cells transformed by an activated ras oncogene and pLTRp53cG9 and in methylcholanthrene induced mouse sarcoma cells p53 proved to be progressively more stable with half-lives ranging from 5.5 h to 7 h. Sequential immunoprecipitation with p53- or T antigen specific monoclonal antibodies allowed us to separate T-p53 complexes, uncomplexed p53 and free T antigen in cell extracts from cells transformed by SV40 and pLTRp53cG9. In these transformed cells uncomplexed p53 showed an increased stability (t1/2 = 2.8 h) when compared to p53 from non-transformed cells. Complex formation with T antigen resulted in an additional stabilization of p53 (t1/2 = 13.3 h). Furthermore, T-p53 complex formation also seems to increase the stability of T antigen nearly sixfold. In transformed cells two immunological variants of p53, a PAb246 precipitable and a non-precipitable form showed distinctly different stabilities, indicating a correlation between the ability of p53 subclasses to bind hsc70 protein and their metabolic stability. Moreover, binding to hsc70 correlated with the stabilization of T antigen in CTM cells also where the mutant T antigen is localized exclusively in the cytoplasm. In abortively infected cells p53, even in complex with T antigen, exhibited a relatively low stability (t1/2 = 87 min) indicating that complex formation per se is not sufficient for fully stabilizing p53.