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)

Wild-type p53 is a short-lived protein which turns over very rapidly via selective proteolysis in the ubiquitin-proteasome pathway. Most p53 mutations, however, encode for protein products which display markedly increased intracellular levels and are associated with positive tumor-promoting activity. The mechanism by which mutation leads to impairment of ubiquitination and proteasome-mediated degradation is unknown, but it has been noted that many transforming p53 mutants are found in stable physical association with molecular chaperones of the hsp70 class. To explore a possible role for aberrant chaperone interactions in mediating the altered function of mutant p53 and its intracellular accumulation, we examined the chaperone proteins which physically associate with a temperature-sensitive murine p53 mutant. In lysate prepared from A1-5 cells grown under mutant temperature conditions, hsp70 coprecipitated with p53Val135 as previously reported by others, but in addition, other well-recognized elements of the cellular chaperone machinery, including hsp90, cyclophilin 40, and p23, were detected. Under temperature conditions favoring wild-type p53 conformation, the coprecipitation of chaperone proteins with p53 was lost in conjunction with the restoration of its transcriptional activating activity. Chaperone interactions similar to those demonstrated in A1-5 cells under mutant conditions were also detected in human breast cancer cells expressing two different hot-spot mutations. To examine the effect of directly disrupting chaperone interactions with mutant p53, we made use of geldanamycin (GA), a selective hsp90-binding agent which has been shown to alter the chaperone associations regulating the function of unliganded steroid receptors. GA treatment of cells altered heteroprotein complex formation with several different mutant p53 species. It increased p53 turnover and resulted in nuclear translocation of the protein in A1-5 cells. GA did not, however, appear to restore wild-type transcriptional activating activity to mutant p53 proteins in either A1-5 cells or human breast cancer cell lines.
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PMID:The physical association of multiple molecular chaperone proteins with mutant p53 is altered by geldanamycin, an hsp90-binding agent. 948 68

Pyrrolidine dithiocarbamate (PDTC) is a thiol compound widely used to study the activation of redox-sensitive transcription factors. Although normally used as an antioxidant, PDTC has been shown to exert pro-oxidant activity on proteins both in vitro and in vivo. Because p53 redox status has been shown to alter its DNA binding capability, we decided to test the effect of PDTC on p53 activation. In this communication, we report that PDTC inhibits the activation of temperature-sensitive murine p53(Val-135) (TSp53) in the transformed rat embryo fibroblast line, A1-5, as well as wild-type human p53 in the normal diploid fibroblast line, WS1neo. In A1-5 cells, PDTC abrogated UV- and temperature shift-induced TSp53 nuclear translocation and p53-mediated transactivation of MDM2. PDTC also blocked UV-induced accumulation of wild-type p53 in WS1neo cells. Continual presence of PDTC was required for its effect as both UV-induced nuclear translocation and accumulation resumed after PDTC removal. We next investigated whether PDTC treatment altered the p53 redox state. We found that PDTC increased p53 cysteine residue oxidation in vivo. This represents the first direct evidence showing that the p53 redox state can be altered in vivo and that increased oxidation correlates with its inability to perform its downstream functions.
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PMID:Pyrrolidine dithiocarbamate prevents p53 activation and promotes p53 cysteine residue oxidation. 966 66

A possible role of p53-dependent transcription in the induction of DNA repair was explored by transfecting a UV-irradiated chloramphenicol acetyl transferase (CAT) reporter plasmid (pRGC.FOS.CAT), containing a minimal FOS promoter driven by a consensus p53 binding site, into a p53 negative-mouse cell line [(10)1]. When a p53-expressing plasmid (pSV.p53) was cotransfected into these cells, CAT expression levels persisted even after prolonged UV irradiation. In comparison, CAT expression from pSV2.CAT, which lacks a p53-responsive element in its SV40 promoter, dropped off much more precipitously after UV irradiation in the absence or presence of WT p53 expression. A similar sharp drop was observed with three other constructs when the reporter gene was under the control of the ras, beta-actin or fos promoter. Mouse cells (A1-5) that constitutively express a temperature-sensitive mutant (135 AV) of mouse p53 also generated, at 32 degrees C, higher levels of enzyme expressed from UV-irradiated pRGC.FOS.CAT than from UV-irradiated pSV2.CAT. The frequency of cyclobutane pyrimidine dimers in UV-irradiated pRGC.FOS.CAT was determined with T4 endo V, and the probability of having an undamaged CAT coding strand was calculated by the Poisson distribution for various times of UV-irradiation. The observed relative CAT expression levels from irradiated pSV2.CAT and pRGC.FOS.CAT in the absence of p53 were consistent with those numbers. These results show that WT p53-mediated transcription directs a resistance of the transcribed DNA to UV inactivation and reactivates the reporter gene. Furthermore, some single point substitution mutants of p53 that maintain a near normal ability to activate transcription had lost their ability to extend CAT gene expression after UV irradiation. Conversely, other mutants with reduced transcriptional activity retained this ability. This indicates that although resistance to UV inactivation is transcriptionally-dependent, these two activities are genetically distinct. These data, taken together, suggest that the transcription of UV-damaged DNA by a p53-dependent process promotes its repair.
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PMID:p53-mediated transcription induces resistance of DNA to UV inactivation. 969 32

Exposure of mammalian cells to ultraviolet (UV) light and other DNA-damaging agents triggers the UV response which is characterized by induction of a large number of genes including c-fos, c-jun, and the genes for DNA repair enzymes and cell-cycle regulatory proteins such as p21 WAF1 and p53. Upon DNA damage, the p53 tumor suppressor protein transmits signals to restrict cell-cycle progression, thereby allowing time for DNA repair to occur. Cells also respond to genotoxic stress by activation of the jun N-terminal kinase (JNK)/stress-activated protein kinase pathway. In this report we investigated the effects of modulation of the level of wild-type and mutant p53 protein on basal and UV-inducible JNK activity. We used the A1-5 rat fibroblast cell line, which contains a p53 gene coding for a temperature-sensitive p53 protein, which allows us to regulate the relative level of wild-type and mutant p53 protein produced in a cell. We measured the relative levels of JNK activity in sham-irradiated and UV-irradiated cells by using the immune complex kinase assay and then computed the fold induction of JNK after UV exposure. We demonstrated that cells expressing p53 protein in the wild-type conformation (when grown at 32 degrees C) exhibited a very low level of JNK activity that was induced 14- to 16-fold by UVC irradiation. When cells were grown at 37 degrees C or 39 degrees C to express predominantly mutant p53 protein, basal JNK activity was significantly higher than at 32 degrees C. UVC irradiation of cells expressing mutant p53 protein resulted in JNK activation, although the overall fold-induction was only two-fold because JNK1 activity was already high in the sham-treated controls. UVB irradiation also induced JNK1 activity, although we again observed a relatively high level of basal JNK activity in sham-irradiated cells expressing mutant p53 protein compared with cells expressing wild-type p53. Control experiments confirmed that JNK1 basal activity was not affected by temperature alone. Western blot analysis of cell extracts indicated that expression of p21 WAF protein was significantly higher in cells expressing wild-type p53 protein and was associated with low basal levels of JNK1 activity. In contrast, cells expressing mutant p53 protein and very low levels of p21 WAF1 protein were found to have a higher level of basal JNK1 activity. We also observed a reduced ability to induce JNK1 after UV irradiation of several other cell lines with p53-mutant or p53-null genotypes. Our results provide evidence for a novel connection between p53 status and the basal level of JNK1, a critical enzyme in the stress-activated protein kinase family. In addition, these studies suggest that the presence of mutant p53 protein in a cell not only affects basal activity of JNK1 but also affects the ability of a cell to respond to UV-induced stress by transmitting signals via induction or activation of the JNK1 cascade.
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PMID:Mutational status of the p53 gene modulates the basal level of jun N-terminal kinase and its inducibility by ultraviolet irradiation in A1-5 rat fibroblasts. 1044 33

P53 is inactivated in tumors by mechanisms other than mutations in the p53 gene itself. To gain insight into the mechanisms by which this inactivation occurs, we chemically mutagenized A1-5 cells expressing high levels of temperature sensitive p53val135 (tsp53) and selected for clones that were capable of growth at the permissive temperature for p53 activation. We expanded 22 clones (ALTR cells for A1-5 Low Temperature Resistant) that could grow at the permissive temperature. Most exhibited cytoplasmic sequestration as the mechanism by which p53 was inactivated. We show here that this cytoplasmically sequestered tsp53 protein is maintained in a mutant conformation. Only in clones with nuclear localized p53 is it also expressed in the wild-type conformation suggesting that subcellular localization of tsp53 is important in determining the conformation of the protein. Consistent with this, we show that the changes in conformation of p53 in A1-5 and SK-N-SH cells induced by ionizing radiation also correlate with nuclear translocation of p53. We suggest that nuclear translocation of p53 can result in a change in the conformation from mutant to wild-type but that these may be two separable events. Oncogene (2000) 19, 4042 - 4049.
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PMID:Conformational phenotype of p53 is linked to nuclear translocation. 1096 61

Manipulation of checkpoint response to DNA damage can be developed as a means for protecting astronauts from the adverse effects of unexpected, or background exposures to ionizing radiation. To achieve this goal reagents need to be developed that protect cells from radiation injury by prolonging checkpoint response, thus promoting repair. We present evidence for a low molecular weight substance excreted by cells that dramatically increases the duration of the G2-delay. This compound is termed G2-Arrest Modulating Activity (GAMA). A rat cell line (A1-5) generated by transforming rat embryo fibroblasts with a temperature sensitive form of p53 plus H-ras demonstrates a dramatic increase in radiation resistance after exposure to low LET radiation that is not associated with an increase in the efficiency of rejoining of DNA double strand breaks. Radioresistance in this cell line correlates with a dramatic increase in the duration of the G2 arrest that is modulated by a GAMA produced by actively growing cells. The properties of GAMA suggest that it is a low molecular weight heat-stable peptide. Further characterization of this substance and elucidation of its mechanism of action may allow the development of a biological response modifier with potential applications as a radioprotector. GAMA may be useful for protecting astronauts from radiation injury as preliminary evidence suggests that it is able to modulate the response of cells exposed to heavy ion radiation, similar to that encountered in outer space.
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PMID:Evidence for factors modulating radiation-induced G2-delay: potential application as radioprotectors. 1177 63

Exposure of mammalian cells to genotoxic stress results in activation of the c-jun amino-terminal kinase (JNK)-stress-activated protein kinase (SAPK) pathway and induction of DNA repair enzymes and cell cycle-regulatory proteins such as p53 and p21waf1. The p53 tumor suppressor protein transmits signals that activate p21waf1 gene expression. The p21waf1 protein then restricts cell-cycle progression, thereby allowing time for DNA repair to occur. In this study, we investigated the effects of modulation of the level of wild-type and mutant p53 protein on basal JNK1 activity in the A1-5 rat fibroblast cell line. This cell line contains a p53 gene coding for a temperature-sensitive p53 protein, which allows us to regulate the relative level of wild-type and mutant p53 protein produced in cells. Using the immune complex kinase assay to measure JNK1 activity, we demonstrated that cells expressing the wild-type-conformation p53 protein (when grown at 32.5 degrees C) exhibited a very low level of JNK1 activity. When cells were grown at 37 degrees C or 39 degrees C to express predominantly mutant p53 protein, basal level of JNK1 activity was significantly higher than at 32.5 degrees C. We also demonstrated protein-protein interactions between the p53, p21waf1, and JNK1 proteins in this cell line. Both wild-type p53 protein (expressed at 32.5 degrees C) and mutant p53(val135) protein (expressed at 37 degrees C and 39 degrees C) were present in immunocomplexes of JNK1 protein. Under conditions where wild-type p53 protein was present to induce p21waf1 expression (at 32.5 degrees C), a higher level of p21waf1 protein was also detected in the JNK1 immunocomplexes than in those at 37 degrees C and 39 degrees C. We next investigated the effect that co-association of p53 protein and p21waf1 protein would have on JNK1 activity. We measured basal levels of JNK1 activity in cells expressing wild-type p53 and p21waf1, or in p21waf1-null cells, and demonstrated that cells expressing both p53 and p21waf1 proteins exhibited an approximately threefold lower basal level of JNK1 activity when compared with p21waf1-null cells. To confirm that p21waf1 protein expression in cells resulted in reduced JNK1 activity, we transfected p21waf1-/- cells with a p21waf1 expression vector. We observed that JNK1 activity was inhibited after exogenous p21waf1 protein was expressed in these cells. Our results provide evidence for modulation of the JNK1 pathway by p53 and p21waf1 proteins and support the hypothesis that modulation of JNK1 activity occurred through protein-protein interactions between JNK1, p53, and p21waf1 proteins.
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PMID:Association of JNK1 with p21waf1 and p53: modulation of JNK1 activity. 1250 78

P53 is a tumor suppressor gene that plays a crucial role in suppressing tumorigenesis by inducing either cell cycle arrest or apoptosis in cells with DNA damage. In more than 50% of tumors p53 is inactivated by gene mutations. However, there have also been reports of tumor cells in which p53 remains wild type and is present in elevated concentrations. Here we utilized a set of mutant cell lines, which, unlike the parental A1-5 cell line, which expresses a mouse tsp53 and becomes growth arrested at 32 degrees C, are capable of growth at this same incubation temperature. We found that the tsp53 in the two cell lines, ALTR-17 and ALTR-24, was identical to the parental A1-5s and concentrated in the nucleus at 32 degrees C. Examination of both lines revealed that p21 was induced at 32 degrees C, although to a lesser extent than in parental cells and that the p21 genes were not mutated. Interestingly, evaluation of the conformation of tsp53 using conformation-specific antibodies showed that the protein existed in different forms, which were found to bind DNA using chromatin immunoprecipitation assays and which we showed could induce expression of a p21 reporter construct. We conclude that the tsp53 may exist in various forms capable of binding DNA.
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PMID:DNA binding and selective gene induction by different forms of the p53 protein. 1713 Aug 40

Although the p53 tumor suppressor is most frequently inactivated by genetic mutations, exclusion from the nucleus is also seen in human tumors. We have begun to examine p53 nuclear importation by isolating a series of mutant cells in which the temperature-sensitive murine p53(Val135) mutant is sequestered in the cytoplasm. We previously showed that that three of them (ALTR12, ALTR19, and ALTR25) constituted a single complementation group. Here, we found that ALTR12 cells are more sensitive to heat stress than either ALTR19 or ALTR25 and that there was a complete lack of induction of Hsp70 in response to heat shock. Western blot analysis showed no expression of the Hsf1 transcription factor, and neither heat shock nor azetidine could induce p53 nuclear localization in ALTR12 cells but did in parental A1-5 cells. Suppression of Hsf1 in A1-5 cells with quercetin or an Hsf1 siRNA reduced p53 nuclear importation and inhibited p53-mediated activation of a p21 reporter. Most convincingly, p53 nuclear importation could be restored in ALTR12 cells by introducing an exogenous Hsf1 gene. Collectively, our result suggests that Hsf1 is required for p53 nuclear importation and activation and implies that heat shock factors play a role in the regulation of p53.
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PMID:Hsf1 is required for the nuclear translocation of p53 tumor suppressor. 1881 48