Mutant p53 DNA clones from human colon carcinomas cooperate with ras in transforming primary rat cells: a comparison of the "hot spot" mutant phenotypes

The majority of the p53 genes derived from human colorectal carcinomas contain point mutations. A significant number of these mutations occur in or around amino acids 143, 175, 273, or 281. Experiments presented here demonstrate for the first time that p53 DNA clones containing any one of these mutations cooperate with the activated ras oncogene to transform primary rat embryo cells in culture. These transformed cells produce elevated levels of the human p53 protein, which has extended half-lives (1.5-7 h), as compared to the wild-type human p53 protein (20-30 min). The p53 mutant with an alteration at residue 175 (p53-175H) binds tightly to the cellular heat shock protein, hsc70. In contrast, the p53 mutants possessing mutations at either residue 273 or 281 (p53-273H/281G) do not bind detectably to this heat shock protein and generally are less efficient at forming transformed foci in culture. The transformed cell lines are tumorigenic in nude mice. Thus, two classes of p53 mutant proteins can be distinguished: p53-175H, which cooperates with ras efficiently and binds to hsc70, and p53-273H/281G, which has a reduced efficiency of transformed foci formation and does not bind hsc70. This demonstrates that complex formation between mutant p53 and hsc70 is not required for p53-mediated transformation, but rather it facilitates this function, perhaps by ensuring sequestration of the endogenous wild-type p53 protein. The positive effect on cell proliferation by these mutant p53 proteins is consistent with a role for activated p53 mutants in the genesis of colorectal carcinomas.     

Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines.

A rabbit antiserum was prepared against the C-terminal peptide of 21 amino acids from the human heat shock protein hsp70. These antibodies were shown to be specific for this highly inducible heat shock protein (72 kilodaltons [kDa] in rat cells), and for a moderately inducible, constitutively expressed heat shock protein, hsc70 (74 kDa). In six independently derived rat cell lines transformed by a murine cDNA-genomic hybrid clone of p53 plus an activated Ha-ras gene, elevated levels of p53 were detected by immunoprecipitation by using murine-specific anti-p53 monoclonal antibodies. In all cases, the hsc70, but not the hsp70, protein was coimmunoprecipitated with the murine p53 protein. Similarly, antiserum to heat shock protein coimmunoprecipitated p53. Western blot (immunoblot) analysis demonstrated that the hsc70 and p53 proteins did not share detectable antigenic epitopes. The results provide clear immunological evidence for the specific association of a single heat shock protein, hsc70, with p53 in p53-plus-ras-transformed cell lines. A p53 cDNA clone, p11-4, failed to produce clonable cell lines from foci of primary rat cells transfected with p11-4 plus Ha-ras. A mutant p53 cDNA clone derived from p11-4, SVKH215, yielded a 2- to 35-fold increase in the number of foci produced after transfection of rat cells with SVKH215 plus Ha-ras. When cloned, 87.5% of these foci produced transformed cell lines. SVKH215 encodes a mutant p53 protein that binds preferentially to the heat shock proteins of 70 kDa compared with binding by the parental p11-4 p53 gene product. These data suggest that the p53-hsc70 protein complex could have functional significance in these transformed cells.     

Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form.

Point mutations in the p53 gene are the most frequently identified genetic change in human cancer. They convert murine p53 from a tumour suppressor gene into a dominant transforming oncogene able to immortalize primary cells and bring about full transformation in combination with an activated ras gene. In both the human and murine systems the mutations lie in regions of p53 conserved from man to Xenopus. We have developed a monoclonal antibody to p53 designated PAb240 which does not immunoprecipitate wild type p53. A series of different p53 mutants all react more strongly with PAb240 than with PAb246. The PAb240 reactive form of p53 cannot bind to SV40 large T antigen but does bind to HSP70. In contrast, the PAb246 form binds to T antigen but not to HSP70. PAb240 recognizes all forms of p53 when they are denatured. It reacts with all mammalian p53 and chicken p53 in immunoblots. We propose that immunoprecipitation of p53 by PAb240 is diagnostic of mutation in both murine and human systems and suggest that the different point mutations which convert p53 from a recessive to a dominant oncogene exert a common conformational effect on the protein. This conformational change abolishes T antigen binding and promotes self-oligomerization. These results are consistent with a dominant negative model where mutant p53 protein binds to and neutralizes the activity of p53 in the wild type conformation.     

Mutation is required to activate the p53 gene for cooperation with the ras oncogene and transformation.

Previous experiments have brought into question which amino acid sequence of the p53 oncogene product should be considered wild type and whether the normal protein is capable of cooperating with the ras oncogene to transform cells in culture. To address these questions, a series of p53 cDNA-genomic hybrid clones have been compared for the ability to cooperate with the ras oncogene in transformation assays. From these experiments, it has become clear that the amino acid alanine at position 135, in either the genomic clone or the cDNA clone, failed to produce a p53 protein that cooperated with the ras oncogene and transformed cells. Replacing alanine with valine at this position in either the genomic or the cDNA clone activated for transformation in this assay. Using restriction enzyme polymorphisms in the p53 gene, it was shown that normal mouse DNA encodes alanine at position 135 in the p53 protein. Thus, mutation is required to activate the p53 protein for cooperation with the ras oncogene. After cotransfection with the activated ras gene, the genomic p53 DNA clone always produced more transformed cell foci (1.7-fold) than similar cDNA clones and these foci were more readily cloned (3.6-fold) into permanent cell lines. A series of deletion mutants of the genomic p53 clone were employed to show that the presence of intron 4 in the p53 gene was sufficient to provide much enhanced clonability of transformed foci from culture dishes. The presence of introns in the p53 gene constructions also resulted in elevated levels of p53 protein in the p53-plus-ras-transformed cell lines. Thus, qualitative changes in the p53 protein are required to activate p53 for transformation with the oncogene ras. Quantitative improvements of transformation frequencies are associated with the higher expression levels of altered p53 protein that are provided by having one of the p53 introns in the transforming plasmid.     

Identification of the p53 protein domain involved in formation of the simian virus 40 large T-antigen-p53 protein complex.

An expression vector utilizing the enhancer and promoter region of the simian virus 40 (SV40) DNA regulating a murine p53 cDNA clone was constructed. The vector produced murine p53 protein in monkey cells identified by five different monoclonal antibodies, three of which were specific for the murine form of p53. The murine p53 produced in monkey cells formed an oligomeric protein complex with the SV40 large tumor antigen. A large number of deletion mutations, in-frame linker insertion mutations, and linker insertion mutations resulting in a frameshift mutation were constructed in the cDNA coding portion of the p53 protein expression vector. The wild-type and mutant p53 cDNA vectors were expressed in monkey cells producing the SV40 large T antigen. The conformation and levels of p53 protein and its ability to form protein complexes with the SV40 T antigen were determined by using five different monoclonal antibodies with quite distinct epitope recognition sites. Insertion mutations between amino acid residues 123 and 215 (of a total of 390 amino acids) eliminated the ability of murine p53 to bind to the SV40 large T antigen. Deletion (at amino acids 11 through 33) and insertion mutations (amino acids 222 through 344) located on either side of this T-antigen-binding protein domain produced a murine p53 protein that bound to the SV40 large T antigen. The same five insertion mutations that failed to bind with the SV40 large T antigen also failed to react with a specific monoclonal antibody, PAb246. In contrast, six additional deletion and insertion mutations that produced p53 protein that did bind with T antigen were each recognized by PAb246. The proposed epitope for PAb246 has been mapped adjacent (amino acids 88 through 109) to the T-antigen-binding domain (amino acids 123 through 215) localized by the mutations mapped in this study. Finally, some insertion mutations that produced a protein that failed to bind to the SV40 T antigen appeared to have an enhanced ability to complex with a 68-kilodalton cellular protein in monkey cells.     

The gene for the rat heat-shock cognate, hsc70, can suppress oncogene-mediated transformation.

In cells transformed by mutant mouse p53 plus ras, the former protein is found to be complexed with the heat-shock protein cognate hsc70. To determine whether hsc70 can directly affect neoplastic transformation, nonestablished rat embryo fibroblasts (REF) were transfected with rat genomic hsc70 DNA in conjunction with various oncogenes. We report here that the hsc70 gene could efficiently suppress focus induction by mutant p53 plus ras, as well as by myc plus ras. No inhibitory effect of hsc70 was detectable in assays monitoring the ability of REF to be immortalized by mutant p53, arguing against a nonspecific deleterious effect of the hsc70 genomic clone on REF survival and proliferation. Lines generated in the presence of the hsc70 plasmid produced augmented levels of hsc70. Plasmids encoding only short NH2-terminal fragments of hsc70 could also, in some cases, partially reduce oncogene-mediated focus formation. However, a maximal inhibitory effect required the production of a functional hsc70 protein. The data presented here raise the possibility that hsc70 may be directly involved in the modulation of oncogene-mediated transformation.     

Association between the cellular p53 and the adenovirus 5 E1B-55kd proteins reduces the oncogenicity of Ad-transformed cells.

The association of the cellular p53 protein with the E1B-55kd protein of adenovirus 5 (Ad5) is thought to result in inactivation of the p53 recessive oncogene product. Here we show that Ad5 E1-transformed 3Y1 rat cells which express low levels of the 55 kd E1B protein do not contain the p53-E1B 55kd complex. These cells have nuclearly located p53 and are highly oncogenic in nude mice. In 3Y1 cells expressing the E1B protein at a sufficiently high level, association between p53 and E1B-55kd occurs, resulting in an almost complete trapping of p53 into a discrete cytoplasmic body. These cells only form tumors after a very long latency period and in the tumors that eventually appear selection has occurred in favor of cells lacking the complex and containing free nuclear p53. Comparable results were found when highly oncogenic Ad12-transformed cells were supertransfected with the Ad5 E1B region. In none of the Ad-transformed mouse, rat and human cell lines examined, could we detect p53 of abnormal molecular weight or association with hsc70, neither could we immunoprecipitate p53 by the mutant specific antibody PAb240. These data suggest that a high level of nuclear p53 with a wild-type conformation contributes to the oncogenicity of Ad transformed cells.     

Purification of complexes of nuclear oncogene p53 with rat and Escherichia coli heat shock proteins: in vitro dissociation of hsc70 and dnaK from murine p53 by ATP.

Oligomeric protein complexes containing the nuclear oncogene p53 and the simian virus 40 large tumor antigen (D. I. H. Linzer and A. J. Levine, Cell 17:43-51, 1979), the adenovirus E1B 55-kilodalton (kDa) tumor antigen, and the heat shock protein hsc70 (P. Hinds, C. Finlay, A. Frey, and A. J. Levine, Mol. Cell. Biol. 7:2863-2869, 1987) have all been previously described. To begin isolating, purifying, and testing these complexes for functional activities, we have developed a rapid immunoaffinity column purification. p53-protein complexes are eluted from the immunoaffinity column by using a molar excess of a peptide comprising the epitope recognized by the p53 monoclonal antibody. This mild and specific elution condition allows p53-protein interactions to be maintained. The hsc70-p53 complex from rat cells is heterogeneous in size, with some forms of this complex associated with a 110-kDa protein. The maximum apparent molecular mass of such complexes is 660,000 daltons. Incubation with micromolar levels of ATP dissociates this complex in vitro into p53 and hsc70 110-kDa components. Nonhydrolyzable substrates of ATP fail to promote this dissociation of the complex. Murine p53 synthesized in Escherichia coli has been purified 660-fold on the same antibody affinity column and was found to be associated with an E. coli protein of 70 kDa. Immunoblot analysis with specific antisera demonstrated that this E. coli protein was the heat shock protein dnaK, which has extensive sequence homology with the rat hsc70 protein. Incubation of the immunopurified p53-dnaK complex with ATP resulted in the dissociation of the p53-dnaK complex as it did with the p53-hsc70 complex. This remarkable conservation of p53-heat shock protein interactions and the specificity of dissociation reactions suggest a functionally important role for heat shock proteins in their interactions with oncogene proteins.     

Mutant p53 tumor suppressor alleles release ras-induced cell cycle growth arrest.

Overexpression of an activated ras gene in the rat embryo fibroblast line REF52 results in growth arrest at either the G1/S or G2/M boundary of the cell cycle. Both the DNA tumor virus proteins simian virus 40 large T antigen and adenovirus 5 E1a are able to rescue ras induced lethality and cooperate with ras to fully transform REF52 cells. In this report, we present evidence that the wild-type activity of the tumor suppressor gene p53 is involved in the negative growth regulation of this model system. p53 genes encoding either a p53Val-135 or p53Pro-193 mutation express a highly stable p53 protein with a conformation-dependent loss of wild-type activity and the ability to eliminate any endogenous wild-type p53 activity in a dominant negative manner. In cotransfection assays, these mutant p53 genes are able to rescue REF52 cells from ras-induced growth arrest, resulting in established cell lines which express elevated levels of the ras oncoprotein and show morphological transformation. Full transformation, as assayed by tumor formation in nude mice, is found only in the p53Pro-193-plus-ras transfectants. These cells express higher levels of the ras protein than do the p53Val-135-plus-ras-transfected cells. Transfection of REF52 cells with ras alone or a full-length genomic wild-type p53 plus ras results in growth arrest and lethality. Therefore, the selective event for p53 inactivation or loss during tumor progression may be to overcome a cell cycle restriction induced by oncogene overexpression (ras). These results suggest that a normal function of p53 may be to mediate negative growth regulation in response to ras or other proliferative inducing signals.     

Monoclonal antibody analysis of p53 expression in normal and transformed cells.

The cellular phosphoprotein p53 binds tightly and specifically to simian virus 40 T antigen and the 58,000-molecular-weight adenovirus E1b protein. Many human and murine tumor cell lines contain elevated levels of the p53 protein even in the absence of these associated viral proteins. Recently the cloned p53 gene, linked to strong viral promoters, has been shown to complement activated ras genes in transformation of primary rodent cell cultures. Overexpression of the p53 gene alone rescues some primary rodent cell cultures from senescence. We isolated three new monoclonal antibodies to the p53 protein, designated PAb242, PAb246, and PAb248, and mapped the epitopes they recognized on p53 in comparison with other previously isolated antibodies. At least five sterically separate epitopes were defined on murine p53. One of the antibodies, PAb246, recognizes an epitope on p53 that is unstable in the absence of bound simian virus 40 T antigen. This effect is demonstrable in vivo and in newly developed in vitro assays of T-p53 complex formation. Using the panel of anti-p53 antibodies and sensitive immunocytochemical methods, we found that p53 has a predominantly nuclear location in established but not transformed cells as well as in the vast majority of transformed cell lines. Several monoclonal antibodies to p53 showed cross-reactions with non-p53 components in immunocytochemical staining.