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1.
The formation of complexes between oncoproteins of DNA tumor viruses and the cellular protein p53 is thought to result in inactivation of the growth suppressor function of p53. In cells transformed by nononcogenic human adenovirus type 5 (Ad5), the 55-kDa protein encoded by E1B forms a stable complex with p53 and sequesters it in the cytoplasm. However, the homologous 54-kDa protein of highly oncogenic Ad12 does not detectably associate with p53. Yet in Ad12-transformed cells, p53 is metabolically stable, is present at high levels in the nucleus, and contributes to the oncogenicity of the cells. Such properties have previously been described for mutant forms of p53. Here, we show that stable p53 in Ad12-transformed cells is wild type rather than mutant and that stabilization of p53 is a direct consequence of the expression of the Ad12 E1B protein. We also compared the effects of the E1B proteins on transformation of rodent cells by different combinations of oncogenes. A synergistic interaction was observed for the gene encoding the 54-kDa E1B protein of Ad12 with myc plus ras oncogenes, resembling the effect of mutant p53 on myc plus ras. In contrast, the Ad5 55-kDa E1B protein strongly inhibited transformation by myc plus ras but stimulated transformation by E1A plus ras. The data are explained in terms of different interactions of the two E1B proteins with endogenous p53. The results suggest that in cultured rat cells, endogenous wild-type p53 plays an essential role in cell proliferation, even in the presence of myc plus ras. The dependence on p53 is lost, however, when the adenovirus E1A oncogene is present.  相似文献   

2.
Expression of a p53-associated protein, Mdm-2 (murine double minute-2), can inhibit p53-mediated transactivation. In this study, overexpression of the Mdm-2 protein was found to result in the immortalization of primary rat embryo fibroblasts (REFs) and, in conjunction with an activated ras gene, in the transformation of REFs. The effect of wild-type p53 on the transforming properties of mdm-2 was determined by transfecting REFs with ras, mdm-2, and normal p53 genes. Transfection with ras plus mdm-2 plus wild-type p53 resulted in a 50% reduction in the number of transformed foci (relative to the level for ras plus mdm-2); however, more than half (9 of 17) of the cell lines derived from these foci expressed low levels of a murine p53 protein with the characteristics of a wild-type p53. These results are in contrast to previous studies which demonstrated that even minimal levels of wild-type p53 are not tolerated in cells transformed by ras plus myc, E1A, or mutant p53. The mdm-2 oncogene can overcome the previously demonstrated growth-suppressive properties of p53.  相似文献   

3.
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.  相似文献   

4.
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.  相似文献   

5.
Simian virus 40 (SV40) T antigen binds to the tumor suppressor p53 protein, and this association may contribute to oncogenic transformation by the virus. We investigated the importance of this binding on transformation by examining three replication-competent mutants of SV40 (402DE, 402DN, and 402DH). These mutants express T antigens defective in binding to human and monkey p53s but retain some binding with mouse p53. All showed significant reduction in their ability to induce transformed cell foci of two normal human cell lines as well as a slight reduction with mouse embryo cells. Other comparable mutants which express T antigens retaining the ability to complex with p53 were able to induce foci at wild-type levels in both human and mouse cells. Further studies were performed with five T-antigen-positive clones isolated from the few human cell foci that appeared after transfection with 402 mutant DNAs. All five clones reached senescence at about the same point as did the parental untransformed cells. However, six other human cell clones obtained after transfection with DNA from nondefective mutants or wild-type virus were still growing well at more than 10 passages beyond their expected life span. These results suggest that the ability of T antigen to form stable complexes with p53 is necessary for SV40 to extend the life span and partially transform human cells in culture.  相似文献   

6.
Rat embryo fibroblasts transformed with the HPV-16 E7 gene and the activated c-H-ras gene fall into two distinct phenotypic classes. At high cell density, clones of one class form colonies in methylcellulose supplemented with low serum; at low cell density, these cells display responsiveness to mitogenic factors present in serum-free conditioned medium from rat embryo fibroblasts. In contrast, clones of the second class exhibit an absolute dependency on growth factors present in serum at all cell densities in the methylcellulose colony assay and fail to respond to conditioned medium. We find that the status of the endogenous p53 gene is tightly correlated with these two classes of clones. Clones of the first class contain missense mutations in the p53 gene and have lost the wild-type allele. Clones of the second class express wild-type p53 protein. The importance of mutant p53 expression in reducing the growth factor dependency of transformed clones was confirmed in a separate series of experiments in which rat embryo fibroblasts were transformed with three genes, E7 + ras + mutant p53. The growth behaviour of these triply transfected clones was similar to that of the E7 + ras clones expressing endogenous mutant p53. We demonstrate that the enhanced proliferation of E7 + ras clones expressing mutant p53 protein involves an autocrine mechanism.  相似文献   

7.
8.
The 289-residue (289R) and 243R early region 1A (E1A) proteins of human adenovirus type 5 induce cell transformation in cooperation with either E1B or activated ras. Here we report that Ser-132 in both E1A products is a site of phosphorylation in vivo and is the only site phosphorylated in vitro by purified casein kinase II. Ser-132 is located in conserved region 2 near the primary binding site for the pRB tumor suppressor and, in 289R, just upstream of the conserved region 3 transactivation domain involved in regulation of early viral gene expression. Mutants containing alanine or glycine in place of Ser-132 interacted with pRB-related proteins at somewhat reduced efficiency; however, all Ser-132 mutants transformed primary rat cells in cooperation with E1B as well as or better than the wild type when both major E1A proteins were expressed. Such was not the case with mutants expressing only 289R. In cooperation with E1B, the Asp-132 and Gly-132 mutants yielded reduced numbers of smaller transformed foci. With activated ras, all Ser-132 mutants were significantly defective for transformation and the rare foci produced were small and contained extensive areas populated by low densities of flat cells. In the absence of E1B, all Ser-132 mutants induced p53-independent cell death more readily than virus expressing wild-type 289R. These results suggested that phosphorylation at Ser-132 may enhance the binding of pRB and related proteins and also reduce the toxicity of E1A 289R, thus increasing transforming activity.  相似文献   

9.
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.  相似文献   

10.
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.  相似文献   

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