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...

Mutant forms of the p53 cellular tumor antigen elicit neoplastic transformation in vitro. Recent evidence indicated that loss of normal p53 expression is a frequent event in certain types of tumors, raising the possibility that such loss provides transformed cells with a selective growth advantage. Thus, it was conceivable that the mutants might contribute to transformation by abrogating normal p53 function. We therefore studied the effect of plasmids encoding wild-type (wt) p53 on the ability of primary rat embryo fibroblasts to be transformed by a combination of mutant p53 and ras. It was found that wt p53 plasmids indeed caused a marked reduction in the number of transformed foci. Furthermore, wt p53 plasmids also suppressed the induction of transformed foci by combinations of bona fide oncogenes, such as myc plus ras or adenovirus EIA plus ras. On the other hand, plasmids carrying mutations in the p53 coding region totally failed to inhibit oncogene-mediated focus induction and often even slightly stimulated it. Hence, such mutations completely abolished the activity of wt p53 that is responsible for the "suppressor" effect. The latter fact is of special interest, since similar mutations in p53 are often observed in human and rodent tumors. The inhibitory effect of wt p53 was most pronounced when early-passage cells were used as targets, whereas established cell lines were less sensitive. These data support the notions that wt p53 expression may be restrictive to neoplastic progression and that p53 inactivation may play a crucial role in tumorigenesis.

The cellular tumour antigen p53 is found at elevated levels in a wide variety of transformed cells (for reviews see refs 1, 2). Very little is yet known about the precise relationship of p53 to malignant transformation. Although the increase in p53 levels could be a secondary by-product of the transformed state, it is equally possible that p53 is actively involved in altering cellular growth properties, especially as it has been implicated in the regulation of normal cell proliferation. We sought to test whether p53 could behave in a manner similar to known genes in a biological test system, and we demonstrate here that p53 can cooperate with the activated Ha-ras oncogene to transform normal embryonic cells. The resultant foci contain cells of a markedly altered morphology which produce high levels of p53. Cell lines established from such foci elicit tumours in syngeneic animals.

The p53 cellular tumour antigen, long known to be overproduced in a variety of neoplastically transformed cells, was recently shown to be directly involved in transformation. Thus, p53 can complement activated Ha-ras in transforming secondary rat embryo fibroblasts into grossly altered, tumorigenic cells. Moreover, p53 can also be shown to possess immortalizing activity. Our previous results indicated, however, that the contribution of p53 to the transformation was not synonymous with immortalization, suggesting that the two activities of the protein are probably separable. We demonstrate here that this is indeed the case, as overproduction of p53 in an established cell line, while not causing gross morphological changes, endows these cells with an overt tumorigenic potential. Furthermore, the tumorigenic efficiency of such cell lines may be correlated with the extent of p53 over-production.

The possible involvement of p53 overproduction in simian virus 40 (SV40)mediated transformation was studied by using the rat embryo fibroblast focus formation assay. Transformation by wild-type SV40 was enhanced two- to threefold by cotransfection of a plasmid overexpressing mouse p53. More significantly, such a plasmid could partially complement a transformation-defective deletion mutant of SV40. Hence, the ability of SV40 T antigen to induce high p53 levels may indeed be directly relevant to the viral transforming potential.