Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P04637 (p53)
 77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Several species of alternatively spliced mRNAs are transcribed from the E6 gene region of human papillomavirus (HPV) 16. These have the coding capacity for either the full length E6 of 151 amino acids (aa) or four truncated variants, E6I-E6IV, of 43-64 aa. As the first step to identify the putative E6 variants and their functions, we generated cDNAs corresponding to the various E6 open reading frames (ORF) and examined their expression employing in vitro transcription/translation systems and the bacterial pET system. In wheat germ extract, in vitro translation resulted in the production of all five proteins, E6 and E6I-E6IV. These proteins were also expressed as stable fusion proteins from the pET16b and pET17 x b vectors in Escherichia coli. Mobilites of the E6 variant proteins on SDS-acrylamide gels were consistent with their predicted sizes. The authenticity of the synthesized proteins was confirmed by immunoprecipitation with specific antibodies directed against epitopes in the N-terminal portion of E6 as well as antibodies raised against the individual variant proteins produced in E. coli. In rabbit reticulocyte lysate, however, only the full length E6 and the E6IV variant were synthesized. This could be due to inefficient translation as well as lower stability of the short variants. E6I-III, in reticulocyte lysate (RTL). The ability of the E6 variants to associate with p53 and target its proteolytic degradation in vitro, was examined in coimmunoprecipitation assays, using in vitro synthesized proteins and monoclonal antibodies to p53. Results of these assays indicated that only the full length E6 efficiently binds to and promotes the degradation of p53. The E6 variants E6I-E6IV, although able to associate with p53 at a low efficiency, were unable to target its degradation.
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PMID:The E6 variant proteins E6I-E6IV of human papillomavirus 16: expression in cell free systems and bacteria and study of their interaction with p53. 880 76

Previous studies have identified a 180-kDa mouse cardiomyocyte phosphoprotein with limited epitopic homology to p53. In this study, the protein was purified and partially sequenced. Oligonucleotide probes based on the available amino acid sequence data were used to isolate cDNA clones. Sequence analyses revealed that the clones encoded a protein with regional homology to the yeast RAD50 gene product. Expression of the mouse cDNA rescued the methyl methanesulfonate-sensitive phenotype in rad50 mutant yeast, indicating that the cardiomyocyte phosphoprotein is the mammalian homologue of the yeast RAD50 gene product. Fluorescence in situ hybridization analyses localized the mouse RAD50 gene to the A5-B1 region of chromosome 11. Northern blot analyses demonstrated a complex pattern of RAD50 expression during mouse development which was further complicated by the presence of several alternatively spliced transcripts. High levels of RAD50 expression was evident in the adult myocardium, a somewhat surprising observation given the absence of DNA synthesis in adult cardiomyocytes.
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PMID:Mouse RAD50 has limited epitopic homology to p53 and is expressed in the adult myocardium. 891 May 85

The onset of p53-dependent apoptosis results from the accumulation of damaged DNA. Recently, it was shown that the C' terminus of the p53 protein plays a central role in sensing damaged DNA. In our present study, we examined the role of the C' terminus in the induction of apoptosis. A temperature-sensitive (ts) mutant of the alternatively spliced form of p53 (p53AS-ts) and the ts mutant of the regularly spliced form (p53RS-ts) were used to generate series of stable clones with increasing amounts of p53 protein. Apoptotic patterns induced by either the regularly spliced p53 product (p53RS) or a C'-terminally alternatively spliced p53 product (p53AS) were compared. We found that although both forms of p53 induced apoptosis following expression of the wild-type protein conformation, the kinetics were different. Apoptosis induced by the p53AS protein was attenuated compared to that induced by p53RS. The delay in the manifestation of the apoptotic features following p53AS expression was in agreement with a delay in the regulation of the expression of apoptosis-related genes. The observation that p53 with an altered C' terminus is still capable of inducing apoptosis suggests that the actual onset of the apoptotic process most probably involves structural domains other than the C' terminus of the p53 molecule. However, the fact that the apoptotic activity mediated by the p53AS product was slower than that mediated by the p53RS product suggests that the C' terminus indeed exerts a certain control on the apoptotic activity of the p53 molecule.
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PMID:The murine C'-terminally alternatively spliced form of p53 induces attenuated apoptosis in myeloid cells. 900 Dec 25

In the present study, we investigated the role of p53 in the differentiation of epidermal keratinocyte cells. The interrelationship between p53 expression and the various stages of epidermal differentiation and the role of the COOH terminus of the p53 molecule in this process were determined by comparing the expression of the regularly spliced p53 (RSp53) molecule and that of the COOH-terminal alternatively spliced (ASp53) form. p53 mRNA distribution was studied by in situ analysis of frozen skin sections and by reverse transcription-PCR analysis of the various wild-type p53 forms expressed in neonatal skin cell fractions separated by Percoll gradient. p53 protein levels were measured by fluorescence-activated cell sorting analysis and immunohistochemistry, using antibodies that recognize either the COOH terminus of RSp53 or ASp53. The results show that although less mature keratinocyte cells predominantly express the RSp53 form, the more mature cells preferentially express the ASp53 form. Therefore, it is possible that the two p53 forms are associated with different functions required at the various stages of keratinocyte differentiation. The results suggest that the COOH-terminal domain of the p53 molecule is important for its activity in the process of keratinocyte differentiation.
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PMID:Differential expression of the regularly spliced wild-type p53 and its COOH-terminal alternatively spliced form during epidermal differentiation. 926 94

Upregulation of the p53 protein was shown to induce cell cycle arrest at the G1/S border and in some cases at the G2/M border. Furthermore, it was suggested that p53 is associated with the induction of the various DNA repair pathways. Previously, we demonstrated that cells co-expressing endogenous wild type p53 protein, together with dominant negative mutant p53, exhibit deregulation of apoptosis, G1 arrest and delay in G2 following gamma-irradiation. In the present study, we investigated the role of p53 protein in the DNA damage response at the G2 phase. Using p53-null, wild type p53 and mutant p53-producer cell lines, we found that the two C-terminally spliced p53 forms could prevent gamma-irradiation induced mutagenesis prior to mitosis, at the G2/M checkpoint. We found that at the G2 phase, p53 may facilitate repair of DNA breaks giving rise to micronuclei, and regulate the exit from the G2 checkpoint. At the G1 phase, only the regularly spliced form of p53 caused growth arrest. In contrast, both the regularly and the alternatively spliced p53 forms directed postmitotic micronucleated cells towards apoptosis. These results provide a functional explanation for the cell cycle-independent expression of p53 in normal cycling cells, as well as in cells where p53 is up-regulated, following DNA damage.
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PMID:Role of wild type p53 in the G2 phase: regulation of the gamma-irradiation-induced delay and DNA repair. 939 47

The DNA binding activity of wild type p53 is central to its activity. The "central" part of the molecule, where most mutations appear in primary human tumors, is the actual DNA binding domain. The C-terminal part was shown to exert a negative effect on the DNA binding activity. In the present study we show that while anti-p53 antibodies recognizing the C terminus of the wild type p53 facilitate DNA binding activity, blocking of the wild type specific epitope by specific anti-p53 antibodies, inhibited the DNA binding activity of the wild type p53 protein. An alternatively spliced p53 protein exhibits an augmented DNA binding activity. The fact that most p53 mutants have lost the wild type p53 conformation specific epitope, coupled with the observation that blocking of this site by binding specific antibodies, prevents the interaction of wild type p53 with DNA, suggests that maintaining the correct structural conformation of this site is central for DNA binding activity. Still, the internal structure of the p53 target and particularly the length of the sequence between the two tandem inverted repeats, is critical for protein-DNA interaction behavior.
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PMID:DNA-binding activity of wild-type p53 protein is mediated by the central part of the molecule and controlled by its C terminus. 946 43

The mdm2 oncogene encodes a 90-kDa nuclear phosphoprotein that binds and inhibits the function of the p53 tumor suppressor protein. It was recently reported that the expression of alternatively spliced variants of mdm2 correlated with malignancy in ovarian tumors and bladder carcinomas. We analyzed the presence of alternatively spliced mdm2 variants and studied their correlation to p53 status in a total of 66 human astrocytic tumors, including 32 glioblastomas multiforme, 17 anaplastic astrocytomas, 12 astrocytomas, and 5 pilocytic astrocytomas, using a specific nested reverse transcription-PCR technique. The full-length mdm2 transcript was demonstrated in all of the cases. Multiple-sized PCR products were found in 29 cases. Two of 5 pilocytic astrocytomas (40%), none of 12 astrocytomas, and 5 of 17 anaplastic astrocytomas (29%) showed alternative splice variants. In contrast, 22 of 32 glioblastomas (69%) showed the presence of splice variants, demonstrating a significantly higher frequency than in lower-grade astrocytomas (P < 0.0003). A majority of the splice variants were 707 base-type (mdm2-b), which was confirmed by sequence analysis. There was no apparent correlation of the presence of mdm2 splice variants with p53 gene status. These results suggest a new role for mdm2, independent of p53 gene status, as an oncogene in the development of malignant astrocytic tumors.
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PMID:Short alternative splice transcripts of the mdm2 oncogene correlate to malignancy in human astrocytic neoplasms. 948 8

The C-terminal of p53 (amino-acids 368-383) represses the DNA binding activity of p53. In vitro, phosphorylation of this region by Protein Kinase C (PKC) is associated with increased DNA binding activity. However, whether PKC can directly modulate p53 function in vivo is not known. Here, we demonstrate that cotransfection of p53 with either PKC alpha or PKC zeta increases p53's transcriptional activity. Mutagenesis of p53 indicates that serine 371 is the major site for phosphorylation by PKC alpha in vitro. Mutation of serine 371 caused a small decline in p53 activation by PKC alpha and PKC zeta. However, the alternatively spliced murine p53, which lacks the PKC phosphorylation sites, still demonstrated increased transcriptional activation when cotransfected with either PKC alpha or PKC zeta. The results indicate that phosphorylation of p53 by PKC in vitro does not correlate with the ability of PKC to upregulate p53's transcriptional activity in vivo.
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PMID:Regulation of the p53 protein by protein kinase C alpha and protein kinase C zeta. 957 Nov 86

Most solid tumors are unable to grow in the ascites form, unless selected by prolonged serial transfer of peritoneal fluid (Klein, 1955). Established ascites tumor cells grow under highly crowded, virtually anoxic conditions (Warburg and Hiepler, 1953). Hypoxia was recently identified as a powerful inducer of p53 dependent apoptosis (Graeber et al., 1996). We wished to examine whether the conversion of relatively well-vascularized solid mouse tumors into freely growing ascitic cell variants favors cell with mutated or deleted p53. We have sequenced exons 4-9 of p53 cDNA from two serially transplanted methylcholanthrene induced sarcomas (MCIM and MSWBS) that were available in the original solid and the gradually converted ascites form. We have also examined five additional solid tumors, four carcinomas and one sarcoma and six additional ascites tumors, five carcinomas and one sarcoma. Sequence analysis showed that all solid tumors carried exclusively wild type p53. Among the eight ascites tumors, five carried mutant p53 and three had only the wild type gene. In one of the two isogenic pairs, the original solid tumor line had only wild type, whereas the derived ascites line had only mutant p53. In the second pair, the solid tumor was wild type whereas the ascitic variant was heterozygous. The naturally occurring alternatively spliced p53 (p53as) mRNA was detected in all solid tumors, but not in five of the eight ascites tumors. Our findings indicate that conversion of solid into ascites tumors favors the selection of cell variants with mutated p53 and of cells that lack the alternatively spliced form of p53.
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PMID:Is conversion of solid into more anoxic ascites tumors associated with p53 inactivation? 981 64

The interaction between poly(ADP-ribose) polymerase (PARP) and the product of the tumor suppressor gene p53 has been described previously. Here, we have investigated whether PARP deficiency may affect the expression and regulation of wild-type (wt) p53. For this purpose, we have used immortalized cells derived from wt and PARP knockout mice. We have found a clearly reduced basal level of PAb421 immunoreactive wt p53 protein in PARP-deficient cells. The monoclonal antibody PAb421 is known to recognize an epitope in the COOH terminus of normally spliced p53 protein. Under indirect immunofluorescence, this antibody stained nuclei in normal but not in PARP-deficient cells. Despite marked reduction of wt p53 protein in PARP knockout cells, no significant difference of the p53 transcription rate was observed between wt and PARP-deficient cells. Interestingly, in both cell types, an additional p53 transcript representing the alternatively spliced (AS) p53 form was detected. Because of its reactivity with different specific anti-p53 antibodies, we have determined that the p53 protein present in PARP knockout mouse cells possesses characteristic features of AS p53. Our results clearly show that PARP-deficient cells constitutively express the AS form of wt p53 and indicate that the regularly spliced p53 is extremely unstable in the absence of PARP. Moreover, PARP-/- cells fail to transactivate p53-responsive genes. Treatment of PARP-/- cells with genotoxic agents primarily leads to the activation of AS p53 protein.
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PMID:Reduced stability of regularly spliced but not alternatively spliced p53 protein in PARP-deficient mouse fibroblasts. 989 79


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