Chromosomal assignment of the murine gene encoding the transformation-related protein p53.

p53 is a transformation-related protein that is encoded by the cellular genome and is synthesized at elevated levels in a wide range of different cell line types and in primary tumors of various species. By using several independently established anti-p53 monoclonal antibodies, it was possible to distinguish between p53 of mouse origin and p53 of Chinese hamster origin. By analysis of a series of mouse X Chinese hamster hybrid cell lines containing various mouse chromosomes, we mapped the p53 gene product to mouse chromosome 11.     

Monoclonal antibodies displaying a novel species specificity for the primate transformation-related protein, p53

SV40 large T antigen associates with a cellular phosphoprotein, p53, in virus-transformed cells. We have raised three new monoclonal antibodies, PAb1101, PAb1102 and PAb1103, to this cellular protein, derived from SV40-transformed human fibroblasts. These define at least two non-overlapping determinants on human p53 that are in different areas of the molecule from those recognised by previously available antibodies. Unlike those antibodies, PAb1102 and PAb1103 do not react with rodent p53. PAb1101 reacts far more weakly with rodent p53 than with primate p53. All three antibodies show a preference for binding to the large T-associated form of p53, an effect that is particularly marked with PAb1102. The novel specificity of these antibodies allows further probing of the nature and function of the large T/p53 complex in human cells.     

Variation in antigenic determinants of p53 transformation-related protein obtained from various species

p53 is a cellular-encoded transformation-related protein. It is synthesized at elevated levels in tumor cells but has also been detected at low concentrations in several types of nontransformed cells. The p53 of tumor cells is immunogenic and elicits specific antibody production. The antigenic determinants of the p53 protein were studied by specific binding to anti-p53 monoclonal antibodies obtained from the RA3-2C2, PAb122, and PAb421 established hybridoma cell lines, and their conservation was followed in various animal species. We found that whereas mouse p53 efficiently immunoprecipitated with all three anti-p53 monoclonal antibodies, human and rat p53 bound PAb122 and PAb421 but lacked a determinant binding RA3-2C2. The hamster p53 molecule represented a third category, which immunoprecipitated with polyclonal anti-p53 antibodies but failed to bind all three monoclonal antibodies analyzed here. Using these monoclonal antibodies, we detected no variations between p53 found in transformed and p53 found in nontransformed cells, within a given species. The results also showed that RA3-2C2, which recognizes a mouse-specific determinant, binds a site located at a proteolytic digestion fragment of the p53 molecule that differs from that containing PAb122 and PAb421 recognition site(s). p53 is a single protein that can be immunoprecipitated through different antigenic determinants that vary between species.     

Stabilization of the p53 transformation-related protein in mouse fibrosarcoma cell lines: effects of protein sequence and intracellular environment.

The transformation-related protein p53 is normally very labile. The stability of p53 is significantly increased in a number of fibrosarcoma cell lines derived from mouse tumors induced by treatment with physical or chemical agents. In many instances, p53 stabilization is correlated with the ability to form a stable complex with the heat shock protein cognate hsc70. We describe a line in which p53 is very stable yet has no detectable interaction with hsc70. The inability to form such a complex probably resides in the primary structure of the endogenous p53, since introduction of other p53 variants into those cells resulted in the appearance of a p53-hsc70 complex. The factors affecting p53 stability were investigated by stable transfection experiments. The results indicated that the primary structure of the p53 protein is a major determinant of its turnover rate; different p53 variants were degraded at distinct and characteristic rates in a number of transformed cell types. However, at least one p53 variant was degraded differently in nontransformed BALB/c-3T3 than in transformed fibrosarcoma cells, demonstrating that the specific cellular environment can also affect the stability of p53.     

Intranuclear distribution of SV40 large T-antigen and transformation-related protein p53 in abortively infected cells

The intranuclear localization of SV40 T-antigen (T-Ag) and the cellular protein p53 was studied in SV40 abortively infected baby mouse kidney cells using two complementary methods of ultrastructural immunocytochemistry in combination with preferential staining of nuclear RNP components and electron microscope autoradiography. Both proteins were revealed in association with peri- and interchromatin RNP fibrils containing the newly synthesized hnRNA. In addition, T-Ag and p53 remained bound, at least in part, to the residual internal nuclear matrix following nuclease and salt extractions of infected cells. The localization of T-Ag was different in SV40 lytically infected monkey kidney cells since, in addition to hnRNP fibrils, the viral protein was also associated with cellular chromatin. However, when lytic infection was performed in conditions of blocked viral DNA replication, T-Ag was no longer associated with the cellular chromatin but remained bound to the hnRNP fibrils. We conclude that the transforming and lytic functions of T-Ag can be distinguished by different subnuclear distributions. The significance of the association of T-Ag and p53 with hnRNP fibrils and the internal nuclear matrix is discussed in relation to the role of these structures in the control of cellular mRNA biogenesis.     

Inactivation of the cellular p53 gene is a common feature of Friend virus-induced erythroleukemia: relationship of inactivation to dominant transforming alleles.

The Friend erythroleukemia virus complex contains no cell-derived oncogene. Transformation by this virus may therefore involve mutations affecting cellular gene expression. We provide evidence that inactivating mutations of the cellular p53 gene are a common feature in Friend virus-induced malignancy, consistent with an antioncogene role for p53 in this disease. We have shown that frequent rearrangements of the p53 gene cause loss of expression or synthesis of truncated proteins, whereas overexpression of p53 protein is seen in other Friend cell lines. We now demonstrate that p53 expression in the latter cells is also abnormal, as a result of missense mutations in regions encoding highly conserved amino acids. Three of these aberrant alleles obtained from cells from different mice were cloned and found to function as dominant oncogenes in gene transfer assays, supporting the view that certain naturally occurring missense mutations in p53 confer a dominant negative phenotype on the encoded protein.     

Expression of p185 and p53 in benign and malignant colorectal lesions

The c-erbB2 gene has been found to be amplified in a number of human adenocarcinomas, leading to elevated levels of expression of its encoded product, p185. Mutations in the p53 gene are also common in colorectal carcinomas, brain tumours, leukaemia and lymphomas.In this study, p185 and p53 overexpression was analyzed in colorectal adenomas (22 tubular adenomas and 2 tubulo-villous adenomas) and moderately differentiated adenocarcinomas (n = 22) in order to determine whether there was a relationship between these two proteins. The proteins are encoded by two genes located in the same chromosome. p185 and p53 expression was determined on tissue sections by immunohistochemical staining procedure.Expression of p185 was significantly higher (p < 0.01) in preneoplastic lesions (95.8% of cases) than colorectal cancer (63.6% of cases). p53 showed an inverse pattern to p185, being expressed in 58.3% of benign lesions and 72.7% of adenocarcinomas.These results confirm that p185 overexpression is associated with the early stages of colorectal cancer, whereas p53 is associated with more advanced stages. Although there was no correlation between p185 and p53 expression in premalignant lesions and adenocarcinomas, these two proteins have an important role in the adenoma–carcinoma sequence.     

Poly(ADP-ribose) polymerase-1 regulates the stability of the wild-type p53 protein.

We investigated the interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the product of the tumor suppressor gene p53 using two different approaches. In the first approach, we used primary and immortalized cells derived from wt and PARP-1 -/- mice. We examined whether PARP-1 deficiency would affect the expression of the wild-type (wt) p53 protein. The inactivation of the PARP-1 gene markedly affected the constitutive expression of the wt p53 protein. Interestingly, only the regularly spliced form of wt p53 was reduced to a barely detectable level in consequence to an approximately 8-fold shortening of its half-life, whereas the level of alternatively spliced p53 remained unchanged. Moreover, reconstitution of cells lacking the PARP-1 gene with the human counterpart restored the normal stability of the regularly spliced p53 protein. In the second approach, we performed experiments with c-Ha-ras transformed primary rat cells overexpressing the p53135val mutant alone or in combination with PARP-1. The advantage of this temperature sensitive p53135val mutant is its oncogenic character at 37 degrees C, connected with cytoplasmic localization of p53, and its tumor suppressor activity at 32 degrees C, accompanied by p53 translocation into the nucleus. No noticeable differences in proliferation and G1 accumulationwere observed between cells expressing p53135val with or without PARP-1. On the other hand, a comparison of the recovery of G1 arrested cells after a shift up to 37 degrees C for both cell lines showed dramatic differences in the kinetics. While cells expressing p53135val rapidly reached the characteristic S-phase level after a shift up to basal temperature, cells additionally expressing PARP-1 rested in G1 despite the temperature elevation. This coincided with exclusively cytoplasmic p53 protein in cells expressing p53135val and predominantly nuclear localization of p53 in p53135val +PARP-1 cells, as evidenced by immunostaining. Determination of the p53 level during the maintenance of cells at 32 degrees C revealed a marked decrease in the level of p53 in cells expressing p53135val alone, whereas in cells coexpressing PARP-1, the level of p53 remained largely unaffected. This indicates that the stability of wild-type p53 greatly differed between both cell lines. Furthermore, the inhibition of PARP-1 activity in G1 arrested cells by 3-aminobenzamide abolished its stabilizing effect on the wild-type p53 protein. Taken together, our results indicate that PARP-1 regulates the stability of the wt p53 protein and that its enzymatic activity is necessary for this stabilizing action.     

Subgenomic fragment of molecular cloned Friend murine leukemia virus DNA contains the gene(s) responsible for Friend murine leukemia virus-induced disease.

Friend murine leukemia virus (G-MuLV) is a helper-independent, type C retrovirus isolated from stocks of Friend virus complex (spleen focus-forming virus plus MuLV). In cell culture, F-MuLV has an ecotropic and NB-tropic host range and causes XC cells to fuse. When injected into newborn NIH Swiss mice, F-MuLV produces hepatosplenomegaly, severe anemia, and numerous circulating hematopoietic precursors in the peripheral blood with normal thymus and lymph nodes after 3 to 6 weeks. Recently, we molecularly cloned an 8.5-kilobase pair (kbp) form of F-MuLV DNA from which we could recover the pathogenic F-MuLV virus by DNA transfection of NIH 3T3 cells. From this molecularly cloned F-MuLV DNA, we have now subcloned in pBR322 a 4.1-kbp HindIII fragment which contains in continuity 3.0 kbp from the 3' terminus (env and c region), 0.6 kbp of the terminal repeat sequences, and 0.5 kbp from the 5'terminus of the viral RNA (genome). NIH 3T3 fibroblasts were transfected with this DNA fragment an then infected with the wild mouse amphotropic retrovirus (cl 1504-A). In cell culture, 1504-A is a helper-independent type C virus which has an N-tropic host range and does not cause fusion of XC cells. When injected into newborn NIH Swiss mice, 1504-A does not produce splenomegaly or thymic enlargement in mice held for up to 8 months. The transfection with the F-MuLV fragment and the infection with 1504-A consistently yielded virus preparations that were XC positive. From such virus stocks we were able to isolate both helper-independent and replication-defective XC-positive viruses. The helper-independent virus was shown to be a recombinant virus since it contains a gp70 molecule derived at least in part from F-MuLV and a specific gag precursor derived from 1504-A as determined by radioactive immune precipitation assays. When injected into newborn Swiss mice, the recombinant helper-independent virus caused hepatosplenomegaly in approximately 50% of the mice in 6 to 8 weeks. The histology of the diseased splenic tissue was indistinguishable from that seen in the disease caused by the whole F-MuLV. The replication-defective virus could be pseudotyped with new 1504-A virus, and this viral complex also caused the F-MuLV disease picture when the complex was injected into newborn Swiss mice. We conclude that the genetic information responsible for the pathogenicity of F-MuLV is contained within the 4.1-kbp DNA fragment, which includes env gene sequences, the terminal repeat sequences, and the c region sequences of the F-MuLV genome.     

p53蛋白多肽片段在大肠杆菌中的表达

在所有研究过的人体肿瘤组织或细胞中,ｐ５３似乎是突变频率最高的一个基因,研究和检测ｐ５３基因及其编码产物的变化将具有重要的意义。我们将野生型ｐ５３基因编码区３’端７０３ｂｐ的ｃＤ－ＮＡ片段插入到大肠杆菌表达载体ｐＢＶ２２０中,得到了一个重组体表达质粒ｐＲＲ３３,经热诱导表达,用ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ印迹法证实ｐ５３蛋白多肽在大肠杆菌中得到了表达,它不仅可用于抗ｐ５３蛋白抗体的制备,而且也可用于对ｐ５３蛋白羧基端多肽功能的研究。     

Expression of p53 during mouse embryogenesis.

By in situ hybridisation we have examined the expression of p53 during mouse embryogenesis from day 8.5 to day 18.5 post coitum (p.c.). High levels of p53 mRNA were detected in all cells of the day 8.5 p.c. and 10.5 p.c. mouse embryo. However, at later stages of development, expression became more pronounced during differentiation of specific tissues e.g. of the brain, liver, lung, thymus, intestine, salivary gland and kidney. In cells undergoing terminal differentiation, the level of p53 mRNA declined strongly. In the brain, hybridisation signals were also observed in postmitotic but not yet terminally differentiated cells. Therefore, gene expression of p53 does not appear to be linked with cellular proliferation in this organ. A proposed role for p53 in cellular differentiation is discussed.     

Enhanced binding of a 95 kDa protein to p53 in cells undergoing p53-mediated growth arrest.

To explore the biochemical functions of p53, we have initiated a search for cellular p53-binding proteins. Coprecipitation of three polypeptides was observed when cell lines overexpressing a temperature-sensitive (ts) p53 mutant were maintained at 32.5 degrees C (wild-type p53 activity, leading to growth arrest) but not at 37.5 degrees C (mutant p53 activity). One of these three proteins, designated p95 on the basis of its apparent molecular mass, was highly abundant in p53 immune complexes. We demonstrate herein that p95 is a p53-binding protein, which exhibits poor p53-binding in cells overproducing several distinct mutant p53 proteins. Yet, p95 associates equally well with both the wild-type (wt) and the mutant conformations of the ts p53 in transformed cells growth-arrested at 32.5 degrees C. On the basis of our findings we suggest that wt p53 activity increases p53-p95 complex formation and that such interaction may play a central role in p53 mediated tumour suppression.     

Effects of oncogenic mutations and DNA response elements on the binding of p53 to p53-binding protein 2 (53BP2)

The tumor suppressor p53 is frequently mutated in human cancers. Upon activation it can induce cell cycle arrest or apoptosis. ASPP2 can specifically stimulate the apoptotic function of p53 but not cell cycle arrest, but the mechanism of enhancing the activation of pro-apoptotic genes over cell cycle arrest genes remains unknown. In this study, we analyzed the binding of 53BP2 (p53-binding protein 2, the C-terminal domain of ASPP2) to p53 core domain and various mutants using biophysical techniques. We found that several p53 core domain mutations (R181E, G245S, R249S, R273H) have different effects on the binding of DNA response elements and 53BP2. Further, we investigated the existence of a ternary complex consisting of 53BP2, p53, and DNA response elements to gain insight into the specific pro-apoptotic activation of p53. We found that binding of 53BP2 and DNA to p53 is mutually exclusive in the case of GADD45, p21, Bax, and PIG3. Both pro-apoptotic and non-apoptotic response elements were competed off p53 by 53BP2 with no indication of a ternary complex.