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)

A camptothecin (CPT)-resistant cell line (MCF-7/C4) was established from MCF-7 cells by mutagenic treatment with methylmethanesulfonate and selection with CPT. MCF-7/C4 is 30-fold resistant to CPT and is cross-resistant to UV and cis-dichlorodiammineplatinum(II) but not to VP-16 or ionizing radiation. Topoisomerase I (top1)-mediated cleavable complexes in the presence of CPT, measured by oligonucleotide assay and by alkaline elution, were similar in both cell lines. Other top1 parameters such as top1 protein, RNA levels, and DNA relaxation were also similar in both cell lines. Thus, CPT resistance is not due to alterations in top1 activity but is caused by changes in the downstream pathways from the top1-induced damage. Both cell lines had similar doubling time (22 hr), but MCF-7/C4 cells showed reduced S-phase fraction in the absence of CPT and reduced G2 delay after CPT treatment. p53, GADD45, and p21WAF1/CIP1 were induced similarly by CPT in both cell lines. The overall repair capacity estimated by the ability of cells to reactivate UV-damaged pSV-CAT plasmid was increased in MCF-7/C4 cells. These observations suggest that enhanced DNA repair is one of the factors involved in CPT resistance.
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PMID:Acquired camptothecin resistance of human breast cancer MCF-7/C4 cells with normal topoisomerase I and elevated DNA repair. 896 67

Alterations of the tumor suppressor gene p53 are uncommon in differentiated thyroid neoplasia but are detected at high frequency in anaplastic thyroid carcinoma suggesting that impaired p53 function may contribute to the undifferentiated and highly aggressive phenotype of these tumors. Effects of wild type p53 (wt-p53) re-expression were investigated in a human anaplastic thyroid carcinoma cell line (ARO) expressing a mutated p53. ARO cells were stably transfected with the temperature-sensitive p53 Val135 gene (ts-p53) which exhibits wild type-like activity at 32 degrees C. Exogenous wt-p53 function in ARO-tsp53 clones was assessed by evaluating its transcriptional activity on a CAT reporter vector containing p53 binding sites. At 32 degrees C, a significant reduction in the proliferation rate (approximately or equal to 50%) was observed, with accumulation of cells in the G0/G1 phase of the cell cycle. This effect was accompanied by induction of the expression of the growth inhibitor p21/Waf1 gene. At 32 degrees C, ARO-tsp53 clones also showed a marked impairment of their tumorigenic potential. Furthermore, transfected clones re-acquired the ability to respond to thyrotropin (TSH) stimulation showing an increased expression of thyroid-specific genes (thyroglobulin, thyroperoxidase and TSH receptor). In conclusion, re-expression of wt-p53 activity in ARO cells, inhibits cell proliferation and restores responsiveness to physiological stimuli.
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PMID:p53 re-expression inhibits proliferation and restores differentiation of human thyroid anaplastic carcinoma cells. 903 81

sgk is a novel member of the serine/threonine protein kinase family that is transcriptionally regulated by serum and glucocorticoids in Rat2 fibroblasts and in mammary epithelial cells. 5'-Deletion analysis of the sgk promoter, using a series of sgk-CAT. (chloramphenicol acetyltransferase) chimeric reporter gene plasmids, defined a glucocorticoid-responsive region that contains a glucocorticoid response element (sgkGRE) between -1000 and -975 bp. The sgkGRE is specifically bound by glucocorticoid receptors and is sufficient to confer glucocorticoid responsiveness to a heterologous promoter in several cell lines. Strikingly, cotransfection of either the murine or human wild type p53, but not a mutant p53, repressed the dexamethasone-stimulated transactivation of reporter plasmids containing either the sgkGRE or a consensus GRE. Gel shift analysis revealed that in vitro synthesized p53 prevented binding of the glucocorticoid receptor both to the sgkGRE as well as to a consensus GRE. The p53-mediated repression of dexamethasone-induced sgkGRE activity required both the DNA binding and transactivation functions of the p53 protein. Activation of endogenous p53, by exposure to UV light, repressed the glucocorticoid receptor transactivation of a consensus GRE-CAT reporter plasmid in transfected cells. Conversely, activated glucocorticoid receptors suppressed the transactivation function of p53, while transrepression by p53 was largely unaffected. The presented data demonstrate that sgk is a primary glucocorticoid-responsive protein kinase gene that implicates a new pathway of cross-talk between steroid receptor signaling and cellular phosphorylation cascades. In addition, our study provides the first evidence of mutual interference of transactivation functions of p53 and the glucocorticoid receptor, possibly through their direct interaction.
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PMID:Repression of glucocorticoid receptor transactivation and DNA binding of a glucocorticoid response element within the serum/glucocorticoid-inducible protein kinase (sgk) gene promoter by the p53 tumor suppressor protein. 905 78

Redox modulation of wild-type p53 plays a role in sequence-specific DNA binding in vitro . Reduction produces a DNA-binding form of the protein while oxidation produces a non-DNA-binding form. Primer extension analysis reveals that increasing concentrations of reduced p53 result in enhanced protection of the consensus sequence, while increasing concentrations of oxidized p53 confer minimal protection of the consensus sequence. DNA binding by oxidized p53 is, therefore, not sequence-specific. In contrast, there is no observable difference in the binding of oxidized p53 and reduced p53 to double-stranded non-specific or mismatched DNA in gel mobility shift assays. Both forms of p53 bind equally well, suggesting that redox modulation of p53 does not play a role in its binding to non-specific or mismatched DNA. In view of the in vitro evidence that redox state influences the sequence-specific DNA-binding of p53, we have examined the effect of oxidative stress on the in vivo ability of p53 to bind to and transactivate PG13-CAT, a reporter construct containing multiple copies of the p53 consensus binding site linked to the chloramphenicol acetyltransferase gene. Hydrogen peroxide treatment of cells cotransfected with p53 results in a marked decrease in CAT activity, suggesting that oxidation of p53 decreases the ability of the protein to bind to consensus DNA and transactivate target genes in vivo.
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PMID:Redox state regulates binding of p53 to sequence-specific DNA, but not to non-specific or mismatched DNA. 909 41

The role of the tumor suppressor p53 in repair of ultraviolet light (UV)-induced DNA damage was evaluated using a host-cell reactivation (HCR) assay. HCR determines a cell's ability to repair UV-damaged DNA through reactivation of a transfected CAT reported plasmid. Most UV damage is removed through nucleotide excision repair (NER). Primary murine keratinocytes isolated from p53-deficient and wild-type p53 mice were used in the HCR assay. The NER was reduced in p53-/- keratinocytes as compared with p53+/+ keratinocytes. The reduced DNA repair in p53-/- mice was confirmed with a radioimmunoassay comparing cyclobutane dimers (CPDs) and (6-4) photoproducts in p53+/+ and p53-/- keratinocytes after the cells were exposed to UV irradiation. Our results demonstrate that wildtype p53 plays a significant role in regulating NER. Furthermore, as there is evidence that p53 protein levels decrease after keratinocytes become differentiated, we sought to determine whether p53 plays a role in NER in differentiated keratinocytes. Differentiation of the keratinocytes by increasing the Ca2+ concentration in the culture media resulted in a marked reduction in NER equally in both p53+/+ and p53-/- groups. This finding suggests that reduced DNA repair after differentiation is p53 independent. A similar reduction in HCR was confirmed in differentiated human keratinocytes. These data, taken together, indicate that p53 or p53-regulated proteins enhance NER in basal undifferentiated keratinocytes but not in differentiated cells. As nonmelanoma skin cancers originate from the basal keratinocytes, our findings suggest that loss of p53 may contribute to the pathogenesis of this common skin cancer.
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PMID:Differentiation-dependent p53 regulation of nucleotide excision repair in keratinocytes. 909

The crystallographic structure of the p53 core domain showed that most of the p53 mutations found in human tumors are located in conserved regions of the p53 DNA-binding domain. The aim of our study was to investigate the effect on DNA-binding and transactivation of three p53 mutations frequently found in hepatocellular carcinomas (HCC). Two of these mutations are located near the DNA-binding surface and are induced by aflatoxin B1 (249ser) and oxiradicals (249met). In contrast, mutation 220cys is not associated with a specific carcinogen in HCCs and is located outside the DNA binding structures of p53. Cotransfection experiments in two HCC cell lines, with mutated or deleted P53 genes, showed that all three mutations did not enhance reporter gene activity (RGC-CAT), in contrast to wt p53. However, in hepatoma cell lines all three mutations did suppress the p53 wildtype (wt) transactivation in a dose-dependent fashion. DNA-binding was monitored by gel shift assays using the consensus-, Waf-, and RGC-p53 binding sites. All three p53 mutations did decrease DNA-binding versus all binding sites included. Interestingly although all mutations showed the same DNA-binding and transactivation properties, differences in the ectopic expression in different hepatoma cells were observed. Therefore our results indicate that p53 mutations in HCC found in the DNA-binding domain and outside the conserved DNA-binding structures modulate target gene expression by decreasing sequence specific DNA-binding in a dominant negative fashion. The cellular environment may contribute to an additional selection advantage of some mutations.
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PMID:Target gene modulation in hepatocellular carcinomas by decreased DNA-binding of p53 mutations. 909 90

Using HeLa cells stably transfected with an HIV-LTR-CAT construct, we demonstrated a peak in CAT induction that occurs in viable (but not necessarily cell-division-competent) cells 24 h following exposure to some cell-killing agents. gamma rays were the only cell-killing agent which did not induce HIV transcription; this can be attributed to the fact that gamma-ray-induced apoptotic death requires functional p53, which is not present in HeLa cells. For all other agents, HIV-LTR induction was dose-dependent and correlated with the amount of cell killing that occurred in the culture. Doses which caused over 99% cell killing induced HIV-LTR transcription maximally, demonstrating that cells that will go on to die by 14 days are the cells expressing HIV-LTR-CAT.
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PMID:HIV expression is induced in dying cells. 911 23

We have previously shown that p53 disruption sensitizes certain cancer cell types to cisplatin (CDDP) (Fan et al., 1995). In the present study we investigated the role of the p53 downstream effector, p21CIP1/WAF1 (p21), in this sensitization. Studies were performed in human colon cancer HCT-116 cells and murine embryonic fibroblasts (MEF) with intact versus disrupted p21 genes. For comparison, HCT-116 cells lacking p53 function were also prepared through stable transfection with the human papillomavirus type-16 E6 gene. HCT-116/E6 cells were found to be more sensitive than control transfectants to CDDP and another DNA crosslinking agent, nitrogen mustard (HN2). HCT-116 cells with disrupted p21 genes also exhibited greater CDDP and HN2-sensitivity than parental HCT-116 cells. In contrast, the clonogenic survival of HCT-116 cells exposed to ionizing radiation, adriamycin, taxol or vincristine was not affected by p53 or p21 disruption. Sensitization of HCT-116/p21-/- cells to CDDP and HN2 was not limited to the HCT-116 cell background since MEF from p21 knockout mice were also more sensitive to these DNA crosslinking agents. Investigations into a possible cause of this enhanced sensitivity revealed that HCT-116 cells lacking p53 or p21 function exhibited a reduced ability to repair cisplatin-damaged CAT-reporter plasmids transfected into the cells. In addition, we found that HCT-116/p21-/- cells were much more susceptible to HN2-induced cell cycle delay than parental cells. Our results suggest that p21 disruption preferentially sensitizes at least some cell types to DNA crosslinking agents.
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PMID:Cells lacking CIP1/WAF1 genes exhibit preferential sensitivity to cisplatin and nitrogen mustard. 917 48

Transcriptional activation of the human c-myc gene by SV40 large T antigen was examined using HepG2 cells by co-transfecting a T antigen expression plasmid with a myc-CAT construct containing the 2.3-kb upstream region from the P1 promoter and the P2 promoter region fused to the CAT gene. T antigen increased the basal activity of the P2 promoter region containing the E2F binding site, but both the P2 promoter region and the upstream region from the P1 promoter were important for overall activation by T antigen. CAT assay using mutated T antigen lacking p53 or the RB binding site indicated that p53 or RB was not mainly involved in transcriptional activation of the c-myc gene. It appears that activation of the c-myc gene by T antigen is probably dependent upon E2F and a cellular factor through a mechanism which is independent of binding of T antigen to p53 and RB.
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PMID:Transcriptional activation of the human c-myc gene by simian virus 40 large T antigen without binding to p53 and RB proteins in the transient expression system. 919 53

Genotoxic stress results in transcriptional activation of the p53 promoter. To gain more detailed information on genotoxic induction of the p53 promoter at a uniform genomic locus, we have developed an efficient strategy for replacing a defined genomic segment in mouse NIH 3T3 cells with exogenous transfected DNA using a 'double lox' targeting strategy mediated by Cre DNA recombinase. The strategy utilizes a pair of heterospecific lox sites engineered both into the genome and onto the targeting DNA. This allows direct replacement of genomic DNA by a Cre-catalyzed double crossover event. p53-CAT reporter constructs were site-specifically placed into the genomic target 20-fold more efficiently by double lox recombination than by Cre-mediated single crossover insertional recombination, and the absolute frequency of site-specific double lox targeting exceeded the frequency of transformation due to random illegitimate recombination of transfected DNA into the genome. Resulting targeted single-copy integrants of the p53-CAT reporter show strong genotoxic induction by mitomycin C, and a dynamic range of induction that exceeds that seen in transient transfection assays. The double lox strategy is generally applicable to Cre-mediated genomic targeting in any cell and should be of particular utility in the site-specific targeting of DNA into embryonic stem (ES) cells for the production of gene-modified mice.
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PMID:Segmental genomic replacement by Cre-mediated recombination: genotoxic stress activation of the p53 promoter in single-copy transformants. 920 31


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