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)

Doxorubicin (DOX) is a common anticancer drug. The mechanisms of DOX induced apoptosis and the involvement of reactive oxygen species (ROS) in apoptotic signaling were investigated in p53-null human osteosarcoma Saos-2 cells. Accumulation of pre-G1 phase cells and induction of DNA laddering, which are the hallmarks of apoptosis, were detected in cells at 48 h upon DOX treatment. Furthermore, DOX increased the intracellular hydrogen peroxide and superoxide levels, followed by mitochondrial membrane depolarization, cytochrome c release, caspase-3 activation, prior to DNA laddering in Saos-2 cells. In addition, DOX treatment also upregulated Bax and downregulated Bcl-2 levels in the cells. The role of ROS in DOX induced cell death was confirmed by the suppression effect of catalase on DOX induced ROS formation, mitochondrial cytochrome c release, procaspase-3 cleavage, and apoptosis in Saos-2 cells. The catalase treatment however only suppressed DOX induced Bax upregulation but had no effect on Bcl-2 downregulation. Results from the present study suggested that ROS might act as the signal molecules for DOX induced cell death and the process is still functional even in the absence of p53.
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PMID:Reactive oxygen species mediate doxorubicin induced p53-independent apoptosis. 1289 28

Double autoimmunostaining by a sequential twice-repeated enzyme-labeled polymer method was examined on archival paraffin sections of formalin-fixed human tissue using an autoimmunostaining apparatus to determine optimal conditions for glycine treatment, to select the best combination of dyes for the horseradish peroxidase-hydrogen peroxide reaction, and to investigate mounting methods for preparing permanent specimens. The optimal glycine treatment determined by changing the incubation time in 0.1 M glycine hydrochloride buffer, pH 2.2, was glycine buffer washing three times for 1 min each, with suppression of nonspecific binding of the primary antibody by protein blocking. Combinations of DAB and AEC, SG and AEC with Ultramount, and DAB and VIP or NovaRED and SG with the VectaMount were found usable for the double autoimmunostaining, based on color analysis of the dyes. Pairs of primary antibodies, CD68 and anti-fascin antibodies CD3 and CD79a, and anti-Ki-67 antigen and anti-p53 antibodies were applicable in double autoimmunostaining with appropriate antigen retrieval for each pair of primary antibodies. Consequently, good sequential double autoimmunostaining should include masking the nonspecific binding of primary antibodies, optimal glycine treatment, and selection of adequate dyes and mounting methods.
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PMID:Double autoimmunostaining with glycine treatment. 1292 42

We have recently demonstrated that the redox reactant pyruvate prevents hydrogen peroxide (H2O2)-induced endothelial apoptosis and that its anti-apoptotic feature is mediated partially through the mitochondrial compartment. However, little is known about molecular signal pathways that mediate the anti-apoptotic feature of pyruvate. A biochemical approach to elucidate such signal pathways was attempted in human umbilical vein endothelial cells (HUVECs). Effects of antioxidant pyruvate were compared with those of cytosolic reductant L-lactate, redox-neutral acetate, and malate-aspartate shuttle blocker aminooxyacetate. Various indices of endothelial apoptosis were correlated with cell viability. Submillimolar H2O2 caused >50% cell killing, as manifested by its oxidant insult. The massive cell death induced by H2O2 was inhibited by pyruvate but not by L-lactate or aminooxyacetate, suggesting a role of cytosolic NADH reducing equivalents, possibly via stimulated oxidant generation. The induction and nuclear translocation of p53 by H2O2 was blocked by pyruvate and appeared to be somewhat enhanced by L-lactate or aminooxyacetate in association with oxidant generation. Nuclear translocation of p53 accompanied the transactivation of bax and downregulation of bcl-2. The pyruvate-related redox manipulation inhibited the H2O2-induced p53 activation, restored the downregulated bcl-2 and the upregulated bax, and hence enhanced the bcl-2/bax expression ratio. In contrast, L-lactate, acetate, or aminooxyacetate had no such effect. These results indicate that pyruvate could modulate key regulatory signal pathways in cytosol and mitochondrial matrix, thereby inactivating endothelial death pathways. Furthermore, it is suggested that stabilizing the expression of bcl-2 and bax genes by metabolic antioxidants may be an effective strategy for endothelial protection against oxidative stress.
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PMID:Mechanisms of pyruvate inhibition of oxidant-induced apoptosis in human endothelial cells. 1293 67

Defects in DNA mismatch repair (MMR) are common in human cancers, confer tolerance to certain types of chemotherapeutic agents, and lead to genomic instability. In addition to their mismatch-correcting roles during DNA replication, MMR proteins can bind to certain DNA lesions and signal p53 and apoptosis by an unknown mechanism. To further study the mechanism by which the MMR protein MLH1 is involved in the induction of p53 and apoptosis, we exposed the colon carcinoma cell line HCT116 (MLH1-deficient) and mlh1-corrected HCT116 sublines to alkylating agents or hydrogen peroxide (H2O2). It was found that while alkylating agents induced both apoptosis and phosphorylation of the Ser-15 site of p53 in a MLH1-dependent manner, induction of apoptosis, but not p53 phosphorylation, was MLH1 dependent following treatment with H2O2. The MLH1-dependent induction of p53 phosphorylation by alkylating agents did not appear to be cell cycle dependent, arguing against a futile repair mechanism operating during S phase as the sole mechanism for the MLH1-dependent DNA damage signaling. Importantly, we found that both alkylating agents and H2O2 caused significant inhibition of mRNA synthesis in MLH1-expressing but not in MLH1-deficient cells. These findings suggest a novel mechanism of MLH1 in the induction p53 and apoptosis by inhibiting RNA polymerase II-dependent transcription on damaged DNA templates.
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PMID:Potential role of MLH1 in the induction of p53 and apoptosis by blocking transcription on damaged DNA templates. 1293

We had earlier shown that higher concentration of hydrogen peroxide (H(2)O(2)) induced p53-dependent apoptosis in glioma cell line with wild type p53 but had minimal effect on cells with mutated p53. Here we show a potentiating effect of hydroxylamine (HA), an inhibitor of catalase, on a nontoxic dose of H(2)O(2) in glioma cells. HA sensitized both p53 wild type and mutated glioma cells to 0.25 mM H(2)O(2). Potentiating effect of HA was independent of p53. Higher levels of reactive oxygen species (ROS) generation were observed in cells treated with HA+H(2)O(2) as compared to cells treated with each component alone in both the cell lines. Dimethyl sulfoxide (DMSO) protected cells. Cytosolic cytochrome c and activated caspase 3 were detected at 4h. The results suggest that higher levels of intracellular ROS, generated by HA+H(2)O(2) act as a molecular switch in activating a rapidly acting p53-independent mitochondrial apoptotic pathway.
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PMID:Hydroxylamine potentiates the effect of low dose hydrogen peroxide in glioma cells independent of p53. 1296 3

Most of the cancer-associated mutations in the tumor suppressor p53 map to its DNA-binding core domain. Many of them inactivate p53 by decreasing its thermodynamic stability. We have previously designed the superstable quadruple mutant M133L/V203A/N239Y/N268D containing the second-site suppressor mutations N239Y and N268D, which specifically restore activity and stability in several oncogenic mutants. Here we present the x-ray structure of this quadruple mutant at 1.9 A resolution, which was solved in a new crystal form in the absence of DNA. This structure reveals that the four point mutations cause only small local structural changes, whereas the overall structure of the central beta-sandwich and the DNA-binding surface is conserved. The suppressor mutation N268D results in an altered hydrogen bond pattern connecting strands S1 and S10, thus bridging the two sheets of the beta-sandwich scaffold in an energetically more favorable way. The second suppressor mutation N239Y, which is located in close proximity to the DNA-binding surface in loop L3, seems to reduce the plasticity of the structure in large parts of loop L3 as indicated by decreased crystallographic temperature factors. The same is observed for residues in the vicinity of the N268D substitution. This increase in rigidity provides the structural basis for the increase in thermostability and an understanding how N268D and N239Y rescue some of the common cancer mutants.
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PMID:Crystal structure of a superstable mutant of human p53 core domain. Insights into the mechanism of rescuing oncogenic mutations. 1453 97

The p53 tumor suppressor protein is involved in apoptosis and cell cycle checkpoints. We have shown recently that p53 also facilitates base excision repair (BER). To further examine p53 involvement in the regulation of BER we chose to focus on 3-methyladenine DNA glycosylase (3-MeAde DNA glycosylase), the first enzyme acting in the BER pathway. 3-MeAde DNA glycosylase activity was found to be modulated by the p53 protein. This modulation was dependent on the type of genotoxic stress used. Gamma-irradiation damage resulted in activation of glycosylase, which was enhanced by p53. Doxorubicin and hydrogen peroxide (H2O2) treatment, although inducing p53 stabilization, did not cause the activation of glycosylase. Nitric oxide (NO) resulted in activation of 3-MeAde DNA glycosylase. Surprisingly this activation was down regulated by wild-type p53. The down regulation of 3-MeAde DNA glycosylase activity was due to trans repression of glycosylase mRNA by p53. Furthermore, we found that AP endonuclease (APE) activity was not altered by NO. Our study provides evidence for a possible antimutagenic role for p53 following exposure of cells to NO species. In the absence of p53, NO exposure results in elevation of 3-MeAde DNA glycosylase activity that results in elevation in the number of AP sites in DNA. At the same time, APE activity does not rise and removal of the AP sites is not further processed resulting in a mutator phenotype. When p53 is present, it down regulates the transcription of 3-MeAde DNA glycosylase. This provides a new model by which p53 prevents the creation of a mutator phenotype.
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PMID:The role of p53 in base excision repair following genotoxic stress. 1455 12

To clarify the apoptotic and survival signal transduction pathways in activated vascular endothelial cells exposed to oxidative stress, the effects of inhibitors of signal transduction on hydrogen peroxide (H(2)O(2))-induced apoptosis in bovine aortic vascular endothelial cells (BAEC) were examined. Treatment of BAEC with 1 mM H(2)O(2) caused increases of DNA fragmentation, p53 expression, Bax/Bcl-2 ratio, and the activities of caspases 3 and 9. The increases of DNA fragmentation, Bax/Bcl-2 ratio, and caspase activities were abrogated by BAPTA-AM (an intracellular Ca(2+) chelator) and N-acetyl-L-cysteine (an antioxidant), and augmented by wortmannin [a phosphatidylinositol 3-kinase (PI3K) inhibitor]. The increase of the intracellular Ca(2+) concentration ([Ca(2+)](i)) observed in H(2)O(2)-stimulated cells was unaffected by wortmannin, suggesting that the potentiating effect of wortmannin on the apoptosis was not due to an alteration of [Ca(2+)](i). H(2)O(2) increased the levels of PI3K activity and Akt phosphorylation. Both were attenuated by wortmannin and, to a lesser extent, by genistein (a tyrosine kinase inhibitor) and suramin (a growth factor receptor inhibitor), but not affected by BAPTA-AM. These results suggest that H(2)O(2) induces Ca(2+)-dependent apoptosis and Ca(2+)-independent survival signals such as redox-regulated activation of PI3K/Akt, which is partly mediated by the activation of growth factor receptors in BAEC.
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PMID:Redox regulation of PI3K/Akt and p53 in bovine aortic endothelial cells exposed to hydrogen peroxide. 1458 44

Differentiated melanocytic cells produce melanin, through several redox reactions including tyrosinase-catalyzed DOPA oxidation to DOPA quinone. We now developed a method based on DOPA oxidase in-gel detection and Sypro Ruby fluorometric normalization to investigate induction of specific DOPA oxidase isoforms in response to hydrogen peroxide-mediated stress, and to ask whether this is associated with p53-dependent adaptive responses. This report shows that hydrogen peroxide leads to comparable induction of 60 and 55 kDa DOPA oxidases in poorly pigmented B16 melanoma, in contrast to sole induction of a major 55 kDa DOPA oxidase in their highly pigmented counterparts. In the latter cells, this response also increases p53 concomitant with joint induction of p53-activated proteins like the cell-cycle inhibitor p21WAF1 and pro-apoptotic bax, with no comparable effect on expression of anti-apoptotic bcl-2. Together, these data suggest that response to hydrogen peroxide involves p53-mediated growth-restrictive signaling and unequal induction of specific DOPA oxidases in melanocytic cells with unequal basal pigmentation.
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PMID:Hydrogen peroxide increases a 55-kDa tyrosinase concomitantly with induction of p53-dependent p21 waf1 expression and a greater Bax/Bcl-2 ratio in pigmented melanoma. 1463 45

The restriction site mutation (RSM) method has been developed over the past 13 years as a sensitive mutation test which can detect mutations in restriction sites in any gene. Due to the fact that 5/8 of the main mutation hotspots in the TP53 gene fall within restriction sites, RSM can analyse them for the presence of rare mutations (1 mutation in 10000 non-mutated copies). After validating the usefulness of RSM in detecting mutagen-induced mutations, we recently turned our attention to looking for TP53 mutations in pre-malignant tissue. We show here that RSM can detect early TP53 mutations in pre-malignant tissue of the oesophagus, stomach, colon and bladder. We can also use these clinical mutation data to speculate as to the causative mutagens involved in these cancer conditions. We here use an example of mutations detected in gastric tissue and those induced in vitro by hydrogen peroxide.
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PMID:The restriction site mutation (RSM) method: clinical applications. 1468 8


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