DNA clones of the wild-type p53 proto-oncogene inhibit the ability of E1A plus ras or mutant p53 plus ras-activated oncogenes to transform primary rat embryo fibroblasts. The rare clones of transformed foci that result from E1A plus ras plus wild-type p53 triple transfections all contain the p53 DNA in their genome, but the great majority fail to express the p53 protein. The three cell lines derived from such foci that express p53 all produce mutant p53 proteins with properties similar or identical to transformation-activated p53 proteins. The p53 mutants selected in this fashion (transformation in vitro) resemble the p53 mutants selected in tumors (in vivo). These results suggest that the p53 proto-oncogene can act negatively to block transformation.

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The 11-4 p53 cDNA clone failed to transform primary rat fibroblasts when cotransfected with the ras oncogene. Two linker insertion mutations at amino acid 158 or 215 (of 390 amino acids) activated this p53 cDNA for transformation with ras. These mutant cDNAs produced a p53 protein that lacked an epitope, recognized by monoclonal antibody PAb246 (localized at amino acids 88 to 110 in the protein) and preferentially bound to a heat shock protein, hsc70. In rat cells transformed by a genomic p53 clone plus ras, two populations of p53 proteins were detected, PAb246+ and PAb246-, which did or did not bind to this monoclonal antibody, respectively. The PAb246-p53 preferentially associated with hsc70, and this protein had a half-life 4-to 20-fold longer than free p53 (PAb246+). These data suggest a possible functional role for hsc70 in the transformation process. cDNAs for p53 derived from methylcholanthrene-transformed cells transform rat cells in cooperation with the ras oncogene and produce a protein that bound with the heat shock proteins. Recombinant clones produced between a Meth A cDNA and 11-4 were tested for the ability to transform rat cells. A single amino acid substitution at residue 132 was sufficient to activate the 11-4 p53 cDNA for transformation. These studies have identified a region between amino acids 132 and 215 in the p53 protein which, when mutated, can activate the p53 cDNA. These results also call into question what the correct p53 wild-type sequence is and whether a wild-type p53 gene can transform cells in culture.The nuclear oncogene p53 is often expressed at elevated levels in tumor-derived as well as virally and chemically transformed cell lines (4,6,17). The levels of p53 in nontransformed cells are quite low, and the half-life of the protein is short (6 to 30 min) (19,25,26). In simian virus 40 (SV40)-and adenovirus-transformed cells, p53 is found in an oligomeric protein complex with the SV40 large T antigen (16,17) or the adenovirus Elb 55,000-Mr (55K) protein, respectively (29). In these virus-transformed cell lines, the p53 levels are as much as 100-fold higher than in nontransformed counterparts, and the half-life of the protein is correspondingly extended (19,25). It has been suggested that the elevation of p53 levels is involved in the process of viral transformation and that the increased amounts result from the stabilization of p53 in these oligomeric protein complexes. Increased levels of p53 have also been implicated in alterations in the growth control of primary rodent cells. Elevated levels of p53 resulted in the immortalization of primary cells (14) or, when assayed in conjunction with an activated ras gene, in the full transformation of rat embryo fibroblasts (7, 21). Pinhasi-Kimhi et al. (24) and Hinds et al. (13) recently demonstrated that in p53-plus-ras-transformed cells, p53 is found in oligomeric protein complexes with a member(s) of the mammalian 70K heat shock protein (hsp70) family. Similar p53-p70 complexes have been observed in several transformed cell lines e...