Different tumor-derived p53 mutants exhibit distinct biological activities.

In its wild-type form, the protein p53 can interfere with neoplastic processes. Tumor-derived cells often express mutant p53. Full-length mutant forms of p53 isolated so far from transformed mouse cells exhibit three common properties in vitro: loss of transformation-suppressing activity, gain of pronounced transforming potential, and ability to bind the heat shock protein cognate hsc70. A tumor-derived mouse p53 variant is now described, whose site of mutation corresponds to a hot spot for p53 in human tumors. While absolutely nonsuppressing, it is only weakly transforming and exhibits no detectable hsc70 binding. The data suggest that the ability of a p53 mutant to bind endogenous p53 is not the sole determinant of its oncogenic potential. The data also support the existence of gain-of-function p53 mutants.

Overproduction of protein p53 contributes to simian virus 40-mediated transformation.

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.

Induction of growth arrest by a temperature-sensitive p53 mutant is correlated with increased nuclear localization and decreased stability of the protein.

A temperature-sensitive mutant of p53, p53Val-135, was found to be able to arrest cell proliferation when overexpressed at 32.5 degrees C. While much of the protein was cytoplasmic in cells proliferating at 37.5 degrees C, it became predominantly nuclear at 32.5 degrees C. Concomitantly, p53Val-135 became destabilized, although not to the extent seen in primary fibroblasts.

p53 mutations: gains or losses?

Although the case for p53 as a tumor suppressor gene appears very strong, one should still keep an open eye for the possibility that mutations in p53 do not necessarily imply a mere loss of "suppressor" activity. It is still possible that the presence of a p53 mutation in a tumor contributes, in a dominant positive manner, to tumorigenesis. In other words, certain p53 mutants may well be oncogenic in their own right, and carry distinct activities that promote growth deregulation and malignant progression. Elucidating this issue also has practical implications, since the nature of the resident mutations may greatly dictate the consequences of attempts to reintroduce wild-type (wt) p53 into particular types of tumor cells. There are two major obstacles along the road to meaningful answers: the limitations of the experimental systems used for evaluating the biological activities of wt and mutant p53 and a fundamental lack of knowledge about the relevant biochemistry of the p53 protein. These two aspects constitute primary experimental challenges for investigators in the field.

Conditional inhibition of transformation and of cell proliferation by a temperature-sensitive mutant of p53.

Mutant p53 can contribute to transformation, while wild-type (wt) p53 is not oncogenic and actually inhibits transformation. Furthermore, wt p53 may act as a suppressor gene in human carcinogenesis. We now describe the temperature-sensitive behavior of a particular mutant, p53val135. Like other p53 mutants, it can elicit transformation at 37.5 degrees C. However, at 32.5 degrees C it suppresses transformation, behaving like authentic wt p53. Moreover, the proliferation of transformed cells expressing p53val135 is dramatically inhibited at the permissive temperature. Significantly, the inhibition of both transformation and proliferation is reversible upon temperature upshift. These data demonstrate that the ability of wt p53 to suppress transformation is not due to a general lethal effect, but rather to a reversible growth arrest. p53val135 may prove instrumental for gaining insight into the cellular and molecular properties of wt p53.

Overproduction of p53 antigen makes established cells highly tumorigenic.

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.

Wild-type p53 can inhibit oncogene-mediated focus formation.

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 E1A 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 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.

Specific interaction between the p53 cellular tumour antigen and major heat shock proteins.

The protein p53 is capable of participating in neoplastic transformation and can form specific complexes with the large-T antigen of simian virus 40 (SV40). This interaction probably results in the stabilization of p53 (refs 7,8) and may contribute to SV40-mediated transformation. Several non-SV40-transformed cells also exhibit a stabilized p53 which is present in elevated levels. Recently, this stabilization was shown to coincide with the ability to precipitate a polypeptide (p68) of relative molecular mass (Mr) 68,000-70,000 by anti-p53 monoclonal antibodies. We now report that this co-precipitation indeed represents a specific complex between the two proteins; the complex sediments on a sucrose gradient as a relatively broad peak of 10-14S and can be dissociated in vitro. Furthermore, p68 is the HSP70 heat shock protein cognate, found in elevated levels in a p53-overproducing cell line. On heat-shock treatment of such overproducers, p53 also forms a complex with the related highly inducible HSP68.

Activated Ha-ras can cooperate with defective simian virus 40 in the transformation of nonestablished rat embryo fibroblasts.

We studied the ability of activated Ha-ras to cooperate with simian virus 40 (SV40) in the transformation of nonestablished rat embryo fibroblasts. Cotransfection with Ha-ras greatly accelerated the rate of focus induction by wild-type SV40. Moreover, a series of transformation-defective SV40 mutants could be partially complemented by Ha-ras. This was true not only for mutants retaining an intact N-terminal immortalization-competent domain, but also for a nonkaryophilic SV40 mutant. In the latter case, all detectable T antigen was cytoplasmic, indicating that efficient transformation can be achieved through the interaction of two nonnuclear proteins. By employing cell lines derived with various SV40 mutants, it was determined that the ability to complex with p53 depends on the integrity of a relatively large region in the C-terminal half of large T. Finally, we report that nonkaryophilic SV40 large T forms a complex with the major heat shock protein HSP70, and we discuss its possible implications.

Simian virus 40 can overcome the antiproliferative effect of wild-type p53 in the absence of stable large T antigen-p53 binding.

In simian virus 40 (SV40)-transformed cells, a tight complex is formed between the viral large T antigen (large T) and p53. It has been proposed that this complex interferes with the antiproliferative activity of p53. This notion was tested in primary rat fibroblasts by assessing the ability of SV40-mediated transformation to be spared from the inhibitory effect of wild-type (wt) p53. The data indicate that relative to transformation induced by myc plus ras, SV40-plus-ras-mediated focus formation was indeed much less suppressed by p53 plasmids. A majority of the resultant cell lines made a p53 protein with properties characteristic of a wt conformation. Furthermore, cell lines expressing stably both SV40 large T and a temperature-sensitive p53 mutant continued to proliferate at a temperature at which this p53 assumes wt-like properties and normally causes a growth arrest. Surprisingly, at least partial resistance to the growth-inhibitory effect of wt p53 was also evident when transformation was mediated by an SV40 deletion mutant, encoding a large T which does not bind p53 detectably. In addition to supporting the idea that SV40 can overcome the growth-restrictive activity of wt p53, these findings strongly suggest that at least part of this effect does not require a stable association between p53 and large T.