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Query: UNIPROT:P04637 (p53)
 77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The hepatitis B virus X protein (HBx) is implicated in liver cancer development, and this presumably involves its ability to bind and functionally inactivate the p53 tumour suppressor. For example expression of HBx in cultured cells has been shown to inhibit global nucleotide excision repair, a p53-dependent subpathway of nucleotide excision repair (NER) which eliminates helix-distorting DNA adducts, e.g., UV-induced cyclobutane pyrimidine dimers (CPDs), from the genome overall. However it remains undetermined whether HBx also interferes with transcription-coupled NER (TCNER), another NER subpathway which removes DNA adducts uniquely from the transcribed strand (TS) of active genes. To address this, we employed the model human lymphoblastoid strain TK6 and its isogenic p53-null counterpart NH32, in conjunction with derivatives of these strains constitutively expressing HBx (TK6-HBx and NH32-HBx). Relative to TK6, following exposure to either UVB (290-320 nm) or UVC (254 nm), TK6-HBx, NH32 and NH32-HBx manifested significantly reduced apoptotic capacity to varying degrees, although no striking differences in clonogenic survival between the four strains were observed. As previously documented in our laboratory [Proc. Natl. Acad. Sci. 100 (2003) 7219-7224], ligation-mediated PCR analysis revealed NH32 to be deficient compared with TK6 in CPD removal along the TS strand of the chromosomal c-jun locus following UVB exposure, but to be proficient in this respect following UVC exposure, i.e., the requirement for p53 in TCNER exhibits wavelength dependence in human cells. Remarkably however, in contrast to the situation for NH32, TK6-HBx and NH32-HBx manifested defective repair along the TS of c-jun after irradiation with either UVB or UVC. The data demonstrate that HBx expression can reduce the efficiency of TCNER in addition to GNER in human cells via p53-independent as well as p53-dependent pathways.
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PMID:Expression of hepatitis B virus X oncoprotein inhibits transcription-coupled nucleotide excision repair in human cells. 1545 Apr 28

Despite significant evidence of a role of hypoxia in cellular resistance to ionizing radiation-induced toxicity, the underlying molecular mechanisms remain unclear. This study focused on the influence of hypoxia on radiation-induced signals in TK6 human lymphoblastoid cells. Hypoxic (<10 ppm oxygen) and aerobic cells were exposed to equilethal doses of ionizing radiation, radiation dose ratio, 3:1 (hypoxia:air). Hypoxia alone or radiation treatment under aerobic or hypoxic conditions led to increased levels of phospho-p44/42 mitogen-activated protein kinase. Levels of phospho-p38 mitogen-activated protein kinase did not change as a result of either hypoxia or irradiation. Hypoxia alone had no effect on expression of phospho-stress-activated protein kinase (SAPK), wild-type p53, or cleaved caspase 3. Irradiation under aerobic conditions resulted in an increase in the phospho-SAPK signal, whereas hypoxia suppressed the irradiation-induced increase in the level of phospho-SAPK. Both hypoxic and aerobic cells showed increases in p53 levels in response to radiation. Hypoxia blocked radiation-induced cleavage of caspase 3 and poly-ADP-ribose polymerase. Irradiation of aerobic and hypoxic TK6 cells using 6 and 18 Gy, respectively, resulted in a similar and significant increase in fraction of apoptotic cells within 24 hours postirradiation. In contrast, basal levels of apoptosis were observed at 24 hours postirradiation in aerobic and hypoxic NH32 cells, a p53 null derivative of TK6 cells. These results suggest that radiation-induced apoptosis under hypoxia occurs independent of phospho-SAPK and caspase 3, and the p53 response is an obligatory apoptotic signal in TK6 cells.
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PMID:Effects of hypoxia on radiation-responsive stress-activated protein kinase, p53, and caspase 3 signals in TK6 human lymphoblastoid cells. 1569 2

Gene expression responses of human cell lines exposed to a diverse set of stress agents were compared by cDNA microarray hybridization. The B-lymphoblastoid cell line TK6 (p53 wild-type) and its p53-null derivative, NH32, were treated in parallel to facilitate investigation of p53-dependent responses. RNA was extracted 4 h after the beginning of treatment when no notable decrease in cell viability was evident in the cultures. Gene expression signatures were defined that discriminated between four broad general mechanisms of stress agents: Non-DNA-damaging stresses (heat shock, osmotic shock, and 12-O-tetradecanoylphorbol 13-acetate), agents causing mainly oxidative stress (arsenite and hydrogen peroxide), ionizing radiations (neutron and gamma-ray exposures), and other DNA-damaging agents (ultraviolet radiation, methyl methanesulfonate, adriamycin, camptothecin, and cis-Platinum(II)diammine dichloride (cisplatin)). Within this data set, non-DNA-damaging stresses could be discriminated from all DNA-damaging stresses, and profiles for individual agents were also defined. While DNA-damaging stresses showed a strong p53-dependent element in their responses, no discernible p53-dependent responses were triggered by the non-DNA-damaging stresses. A set of 16 genes did exhibit a robust p53-dependent pattern of induction in response to all nine DNA-damaging agents, however.
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PMID:Stress-specific signatures: expression profiling of p53 wild-type and -null human cells. 1582 34

Structural chromosomal rearrangements are commonly observed in tumor karyotypes and in radiation-induced genomic instability. Here we report the effects of TP53 deficiency on karyotypic stability before and after irradiation using related cells and clones differing in cellular TP53 status. The parental cell line, TK6, is a TP53 wild-type human B-lymphoblastoid line with a highly stable karyotype. In the two TK6 derivatives used here, TP53 has been inactivated by biochemical means (expression of HPV16 E6; TK6-5E) or genetic means (allelic inactivation; NH32). Biochemical inactivation of TP53 (TK6-5E) had little effect on the spontaneous karyotype, whereas allelic inactivation of TP53 (NH32) resulted in a modest increase in spontaneous karyotypic instability. After 2 Gy gamma irradiation, the number of unstable clones derived from TP53-deficient cells was significantly elevated compared to the TP53 wild-type counterpart. Extensively destabilized clones were common after irradiation in the set of clones derived from NH32 cells, and one was observed in the set of TK6-5E clones; however, they were never observed in TK6-derived clones. In two of the irradiated NH32 clones, whole chromosomes or chromosome bands were preferentially involved in alterations. These results suggest that genomic instability may differ both quantitatively and qualitatively as a consequence of altered TP53 expression. Some of the results showing repeated and preferential chromosome involvement in aberrations support a model in which instability may be driven by cis mechanisms.
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PMID:Spontaneous and radiation-induced genomic instability in human cell lines differing in cellular TP53 status. 1618 38

The p53 protein has been implicated in multiple cellular responses related to DNA damage. Alterations in any of these cellular responses could be related to increased genomic instability. Our previous study has shown that mutations in p53 lead to hypermutability to ionizing radiation. To investigate further how p53 is involved in regulating mutational processes, we used 8K cDNA microarrays to compare the patterns of gene expression among three closely related human cell lines with different p53 status including TK6 (wild-type p53), NH32 (p53-null), and WTK1 (mutant p53). Total RNA samples were collected at 1, 3, 6, 9, and 24 h after 10 Gy gamma-irradiation. Template-based clustering analysis of the gene expression over the time course showed that 464 genes are either up or downregulated by at least twofold following radiation treatment. In addition, cluster analyses of gene expression profiles among these three cell lines revealed distinct patterns. In TK6, 165 genes were upregulated, while 36 genes were downregulated. In contrast, in WTK1 75 genes were upregulated and 12 genes were downregulated. In NH32, only 54 genes were upregulated. Furthermore, we found several genes associated with DNA repair namely p53R2, DDB2, XPC, PCNA, BTG2, and MSH2 that were highly induced in TK6 compared to WTK1 and NH32. p53R2, which is regulated by the tumor suppressor p53, is a small subunit of ribonucleotide reductase. To determine whether it is involved in radiation-induced mutagenesis, p53R2 protein was inhibited by siRNA in TK6 cells and followed by 2 Gy radiation. The background mutation frequencies at the TK locus of siRNA-transfected TK6 cells were about three times higher than those seen in TK6 cells. The mutation frequencies of siRNA-transfected TK6 cells after 2 Gy radiation were significantly higher than the irradiated TK6 cells without p53R2 knock down. These results indicate that p53R2 was induced by p53 protein and is involved in protecting against radiation-induced mutagenesis.
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PMID:Transcriptional responses to ionizing radiation reveal that p53R2 protects against radiation-induced mutagenesis in human lymphoblastoid cells. 1624 78

Glutathione (GSH) plays an important role in cellular defense response in many in vitro and in vivo models. Here we investigated its role in NO()-induced toxicity in cell culture and mouse models. Wild-type (TK6) and p53-null (NH32) human lymphoblastoid cells were treated with NO(.) at a steady-state concentration of 0.6 muM, similar to the level estimated to occur in inflamed tissues. In both cell types, GSH was depleted by this exposure in a dose- and time-dependent manner. Contrary to expectations, prior depletion of GSH by treatment with l-buthionine-SR-sulfoximine did not potentiate NO(.)-induced cell killing or DNA deamination in TK6 cells. In activated RAW264.7 murine macrophages producing NO(.), intracellular GSH content did not change, although gamma-glutamate-cysteine ligase was upregulated. NO(.) overproduction in RcsX lymphoma-bearing SJL mice resulted in significantly elevated GSH levels in various organs. Administration of the NO(.) synthase inhibitor N-methylarginine abolished the increase in GSH in these animals. Collectively, these data indicate a multifaceted and complex involvement of GSH in responses of cells and tissues to toxic levels of NO(.). NO(.) treatment effectively depleted GSH levels in human lymphoblastoid cells, but this alteration was not a critical initiating factor for NO(.)-mediated toxicity. Murine macrophages maintained GSH homeostasis when exposed to endogenously produced NO(.). In RcsX lymphoma-bearing mice, upregulation of de novo synthesis of GSH appeared to be a response to the toxic effects of NO(.).
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PMID:Biological role of glutathione in nitric oxide-induced toxicity in cell culture and animal models. 1627 84

Toxicity induced by nitric oxide (NO(*)) has been extensively investigated in many in vitro and in vivo experimental models. Recently, our laboratories found that both concentration and cumulative total dose are critical determinants of cell death caused by NO(*). Here, we report results of studies designed to define total dose thresholds and threshold effects for several NO(*)-induced toxicity and cellular responses and to determine impacts of p53 on them. We exposed human lymphoblastoid TK6 cells harboring wild-type p53 and isogenic p53-null NH32 cells to NO(*) delivered by a membrane delivery system. Cells were exposed at a steady state concentration of 0.6 microM for varying lengths of time to deliver increasing cumulative doses (expressed in units of microM min), and several end points of cytotoxicity and mutagenesis were quantified. Threshold doses for NO(*)-induced cytotoxicity were 150 microM min in TK6 cells and 300 microM min in NH32 cells, respectively. Threshold doses for NO(*)-induced apoptosis were identical to those for cytotoxicity, but mitochondrial depolarization thresholds were lower than those for cytotoxicity and apoptosis in both cell types. To gain insight into underlying mechanisms, cells of both types were exposed to sublethal (33% of cytotoxicity threshold), cytotoxicity threshold, or toxic (twice the cytotoxicity threshold) doses of NO(*). In TK6 cells (p53), the sublethal threshold dose induced DNA double-strand breaks, but nucleobase deamination products (xanthine, hypoxanthine, and uracil) in DNA were increased only modestly (<50%) by toxic doses. Increased mutant fraction at the thymidine kinase gene (TK1) locus was observed only at the toxic dose of NO(*). Treatment of NH32 cells with NO(*) at the threshold or toxic dose elevated mutagenesis of the TK1 gene, but did not cause detectable levels of DNA double-strand breaks. At similar levels of cell viability, the frequency of DNA recombinational repair was higher in p53-null NH32 cells than in wild-type TK6 cells. NO(*) treatment induced p53-independent cell cycle arrest predominately at the S phase. Akt signaling pathway and antioxidant proteins were involved in the modulation of toxic responses of NO(*). These findings indicate that exposure to doses of NO(*) at or above the cytotoxicity threshold dose induces DNA double-strand breaks, mutagenesis, and protective cellular responses to NO(*) damage. Furthermore, recombinational repair of DNA may contribute to resistance to NO(*) toxicity and potentially increase the risk of mutagenesis. The p53 plays a central role in these responses in human lymphoblastoid cells.
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PMID:Threshold effects of nitric oxide-induced toxicity and cellular responses in wild-type and p53-null human lymphoblastoid cells. 1654 44

In response to DNA damage by genotoxic agents, histone H2AX is phosphorylated on Ser-139. However, during the cell cycle, predominantly in S and G(2)M phase, histone H2AX is also phosphorylated in untreated normal and tumour cells. This constitutive H2AX phosphorylation is markedly reduced by exposure of cells to the reactive oxygen species scavenger N-acetyl-L-cysteine. Therefore, it appears likely that constitutive H2AX phosphorylation reflects the ongoing oxidative DNA damage induced by the reactive oxygen species during progression through the cell cycle. Because the tumour suppressor p53 (tumour protein p53) is known to induce transcription of genes associated with cell response to oxidative stress, we have compared the intensity of constitutive H2AX phosphorylation, and the effect of N-acetyl-L-cysteine on it, in cells with different tumour protein p53 status. These were human lymphoblastoid cell lines derived from WIL2 cells: TK6, a p53 wt line, NH32, a tumour protein p53 knock-out derived from TK6, and WTK1, a WIL2-derived line that expresses a homozygous mutant of tumour protein p53. Also tested were the tumour protein p53-null promyelocytic HL-60 cells. The degree of constitutive H2AX phosphorylation was distinctly lower in NH32, WTK1 and HL-60 compared to TK6 cells in all phases of the cell cycle. Also, the degree of attenuation of constitutive H2AX phosphorylation by N-acetyl-L-cysteine was less pronounced in NH32, WTK1, and HL-60, compared to TK6 cells. However, the level of reactive oxygen species detected by the cells' ability to oxidize carboxyl-dichlorodihydrofluorescein diacetate was not significantly different in the cell lines studied, which would suggest that regardless of tumour protein p53 status, the level of oxidative DNA damage was similar. The observed higher level of constitutive H2AX phosphorylation in cells harbouring wt tumour protein p53 may thus indicate that tumour protein p53 plays a role in facilitating histone H2AX phosphorylation, an important step in the mobilization of the DNA repair machinery at the site of DNA double-strand breaks.
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PMID:Extent of constitutive histone H2AX phosphorylation on Ser-139 varies in cells with different TP53 status. 1687 65

Mutations of NBS1 are responsible for the human hereditary disease Nijmegen breakage syndrome (NBS), which is characterized by an extremely high cancer rate. In this study, we investigated the influence of NBS1 on ionizing radiation (IR) induced apoptosis. Using small interfering RNA (siRNA) transfection, we knocked down NBS1 protein in three closely related human lymphoblastoid cell lines differing in p53 status: TK6 with a wild-type p53, NH32 with a null mutation of p53, and WTK1 with a mutant p53. We found that up to 48h after 5Gy IR, all three lines showed an obvious induction of apoptosis regardless of the p53 status. The magnitude of apoptosis induction was TK6>NH32>WTK1. This suggested that although p53 is an important modulator of IR-induced apoptosis, other p53-independent apoptosis pathway also exists. Moreover, NBS1 knockdown led to reduction of IR-induced apoptosis in all three lines and both NBS1/ATM/p53/BAX and NBS1/ATM/CHK2/E2F1 apoptosis pathways were partially inactivated. Our results suggest that NBS1 plays an important role in IR-induced apoptosis via both p53-dependent and p53-independent mechanisms. The impaired apoptosis response to DNA damage in NBS1 deficient cells might be one of the important mechanisms of cancer predisposition in NBS patients.
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PMID:The effects of NBS1 knockdown by small interfering RNA on the ionizing radiation-induced apoptosis in human lymphoblastoid cells with different p53 status. 1753 95

Many studies have shown that an alteration of p53 affects various cellular responses to DNA damage after treatment with ionizing radiation. The human lymphoblast cell WTK1, which contains a mutant p53 (ile237), is 10-fold hypermutable at the thymidine kinase (tk) locus compared with TK6 cells, which are from the same donor but contain wild-type p53. These results implied that the specific p53 mutation found in WTK1 may actively contribute to mutagenesis in a gain of function manner. To further investigate this, the present experiments involved transfecting WTK1 cells with a wild-type p53 vector; this restored p53 activity in WTK1 cells, as evidenced by radiation-induced expression of p21. We compared radiosensitivity, as measured both by clonogenic survival and the induction of apoptosis, as well as mutant fractions (MFs) at the tk locus. WTK1 cells expressing wild-type p53 were more sensitive to gamma-ray-induced toxicity as measured by either clonogenic survival or apoptosis. The mutation assays revealed that both the spontaneous and gamma-ray-induced MFs were significantly decreased in WTK1 cells expressing wild-type p53; the MFs were similar to those observed in p53-null NH32 cells, also derived from the same donor. These results indicate that wild-type p53 can reduce the apparent gain-of-function hypermutable effects of a particular p53 gene mutation and thereby help maintain genomic stability.
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PMID:Wild-type p53 reduces radiation hypermutability in p53-mutated human lymphoblast cells. 1756 29


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