UNIPROT:P16104 - FACTA Search

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Query: UNIPROT:P16104 (H2AX)
3,930 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

HDAC inhibitors induce histone hyperacetylation by a relative increase of histone acetyltransferase activity. Histone hyperacetylation may affect chromatin structure and susceptibility to DNA-damaging stress, such as IR. We here investigate whether these inhibitors can radiosensitize human gastric MKN45 and colorectal DLD1 adenocarcinoma cells. In both cells, FK228 pretreatment at minimally toxic concentrations clearly augmented IR-induced cell death, DNA fragmentation and caspase-3/-8 activation. In contrast, 5-FU did not clearly augment IR-induced cell death and caspase-3 activation. FK228 increased expression of proapoptotic BH3-only Bim proteins, and gene transfer-mediated overexpression of Bimalpha radiosensitized DLD1 cells. These data suggest that the FK228-mediated increase of Bim expression may at least partially contribute to its augmentation of radiation-induced apoptosis. However, FK228 did not distinctly affect IR-induced phosphorylation of H2AX, which is an initial event followed by DNA damage. FK228 strongly augmented IR-induced growth suppression of MKN45 tumor xenografts. In addition, other HDAC inhibitors, MS275 and CBHA, similarly augmented IR-induced cell death in both cell types. Our results suggest that these HDAC inhibitors may enhance the efficacy of radiation therapy in gastrointestinal cancer cells.
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PMID:Histone deacetylase inhibitors FK228, N-(2-aminophenyl)-4-[N-(pyridin-3-yl-methoxycarbonyl)amino- methyl]benzamide and m-carboxycinnamic acid bis-hydroxamide augment radiation-induced cell death in gastrointestinal adenocarcinoma cells. 1506 98

DNA double strand breaks (DSBs) are potentially carcinogenic lesions. The induction of DSBs triggers phosphorylation of histone H2AX. Phosphorylated H2AX, denoted p-H2AX, may be detected immunocytochemically and the intensity of p-H2AX immunofluorescence (IF) reveals the frequency of DSBs. Using this assay we tested whether the exposure of A549 human pulmonary adenocarcinoma cells to tobacco smoke, and normal human bronchial epithelial cells (NHBE) to tobacco smoke condensate, induces DSBs. Cellular p-H2AX IF and DAPI fluorescence of individual cells were measured by laser scanning cytometry (LSC). Exposure of A549 cells to tobacco smoke and NHBE cells to smoke condensate led to H2AX phosphorylation in both a time and dose dependent manner. The maximal rate of H2AX phosphorylation was seen during the initial 4h of cell treatment. At high doses (50 microg/ml of smoke condensate), H2AX phosphorylation continued to increase for up to 24h. No differences in the level of H2AX phosphorylation were apparent between cells in G(1) vs S vs G(2)/M phase of the cell cycle in response to treatment with smoke condensate. The data provide strong evidence that exposure of A549 cells to tobacco smoke or NHBE cells to smoke condensate rapidly induces DSBs in these cells. The present assay to detect and measure DSBs induced by tobacco products complements other mutagenicity assays and may be applied to test potential carcinogens in other products.
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PMID:Induction of H2AX phosphorylation in pulmonary cells by tobacco smoke: a new assay for carcinogens. 1525 92

DNA double-strand breaks (DSBs) are potentially mutagenic/carcinogenic lesions. Induction of DSBs triggers phosphorylation of histone H2AX on Ser-139. Phosphorylated H2AX (gammaH2AX) can be detected immunocytochemically, and the intensity of gammaH2AX immunofluorescence (IF), reflecting the number of gammaH2AX-IF foci per nucleus, reveals the frequency of DSBs. Using multiparameter cytometric analysis of gammaH2AX-IF, we previously observed that DSBs are induced in normal human bronchial epithelial (NHBE) and A549 pulmonary adenocarcinoma cells following exposure to cigarette smoke (CS) or smoke condensate. In the present study, we show that N-acetyl L-cysteine (NAC) and glutathione, both effective scavengers of free radicals, prevented induction of DSBs by CS in these cells. In contrast, the glutathione synthesis inhibitor, DL-Buthionine-[S,R]-sulfoximine (BSO), enhanced the induction of DSBs by CS. The observed reduction of DSBs by NAC correlated with protection of the reproductive capability (clonogenicity) of A549 cells treated with CS. The data implicate formation of free radicals by CS as factors generating DSBs and affecting cell survival. Interestingly, at the conditions of exposure to CS when clonogenicity was only moderately affected, S-phase cells showed significantly higher sensitivity in terms of induction of DSBs compared with G1 or G2M cells. In light of the evidence that CS increases oxidative stress and induces cell proliferation in the lungs of smokers, the high propensity of S-phase cells to develop DSBs upon exposure to CS has to be considered as a potentially pathogenic event in smoke-induced tumor development. This is the first report to reveal cell cycle-phase specificity in both the induction of DSBs by CS and their prevention by free radical scavengers. The detection of gammaH2AX to assess the induction of CS-induced DSBs and their relationship to cell cycle phase provides a convenient tool to explore approaches to protect cells from this type of genotoxic damage.
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PMID:Induction of DNA double-strand breaks in A549 and normal human pulmonary epithelial cells by cigarette smoke is mediated by free radicals. 1668 50

For patients with solid tumors, the tolerance of surrounding tissues often limits the dose of radiation that can be delivered. Thus, agents that preferentially increase the cytotoxic effects of radiation toward tumor cells would significantly alter the therapeutic ratio and improve patient survival. Using a high-throughput, unbiased screening approach, we have identified 4'-bromo-3'-nitropropiophenone (NS-123) as a radiosensitizer of human glioma cells in vitro and in vivo. NS-123 radiosensitized U251 glioma cells in a dose-dependent and time-dependent manner, with dose enhancement ratios ranging from 1.3 to 2.0. HT-29 colorectal carcinoma and A549 lung adenocarcinoma cells were also radiosensitized by NS-123 in vitro, whereas NS-123 did not increase the radiation sensitivity of normal human astrocytes or developmental abnormalities or lethality of irradiated Zebrafish embryos. In a novel xenograft model of U251 cells implanted into Zebrafish embryos, NS-123 enhanced the tumor growth-inhibitory effects of ionizing radiation (IR) with no apparent effect on embryo development. Similar results were obtained using a mouse tumor xenograft model in which NS-123 sensitized U251 tumors to IR while exhibiting no overt toxicity. In vitro pretreatment with NS-123 resulted in accumulation of unrepaired IR-induced DNA strand breaks and prolonged phosphorylation of the surrogate markers of DNA damage H2AX, ataxia telangiectasia mutated protein, DNA-dependent protein kinase, and CHK2 after IR, suggesting that NS-123 inhibits a critical step in the DNA repair pathway. These results show the potential of this cell-based, high-throughput screening method to identify novel radiosensitizers and suggest that NS-123 and similar nitrophenol compounds may be effective in antiglioma modalities.
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PMID:Identification and biological evaluation of a novel and potent small molecule radiation sensitizer via an unbiased screen of a chemical library. 1787 20

As demonstrated recently, ionizing radiation (IR) can mediate phosphorylation of DNA-PKcs in human tumor cells through stimulation of the PI3K/Akt pathway. It is also known that DNA-PKcs directly interacts the X-ray repair cross-complementing group 1 protein (XRCC1) involved in base excision repair (BER). Therefore, in the present study we investigated the role of PI3K/Akt activity and DNA-PKcs on XRCC1 expression/stabilization. In contrast to the DNA-PKcs-deficient glioblastoma cell line MO59J, the DNA-PKcs-proficient counterpart MO59K as well as human lung adenocarcinoma A549 cells presented a high basal level of XRCC1 expression. Radiation doses of 3-12Gy did not stimulate a further enhanced expression of XRCC1 in DNA-PKcs-proficient cells (MO59K and A549) within 180min post-irradiation. However, a marked induction of XRCC1 expression was apparent in DNA-PKcs-deficient MO59J cells. Targeting of DNA-PKcs as well as PI3K/Akt pathway by specific kinase inhibitors and/or siRNA reduced basal XRCC1 expression in un-irradiated DNA-PKcs-proficient cells to the level observed in DNA-PKcs-deficient cells. Reduction of basal expression of XRCC1 by XRCC1-siRNA, AKT-siRNA as well as DNA-PKcs inhibitor facilitated IR-induced XRCC1 expression. XRCC1 expression induced by irradiation, however, was independent of PI3K/Akt signaling, but dependent of MAPK-ERK1/2. By immuno-precipitation experiments and confocal microscopy a complex formation of XRCC1 and DNA-PKcs was shown. Applying gamma-H2AX foci analysis it was shown that basal expression of XRCC1 is important for the repair of IR-induced DNA-double strand breaks (DNA-DSBs). These data indicate that IR-induced XRCC1 expression is dependent on the expression level of DNA-PKcs and basal activity status of PI3K/Akt signaling. Likewise, potential of IR-induced XRCC1 expression depends on its basal expression level.
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PMID:PI3K-Akt signaling regulates basal, but MAP-kinase signaling regulates radiation-induced XRCC1 expression in human tumor cells in vitro. 1867 86

The DNA topoisomerase I (topo1) inhibitor topotecan (TPT) and topo2 inhibitor mitoxantrone (MXT) damage DNA inducing formation of DNA double-strand breaks (DSBs). We have recently examined the kinetics of ATM and Chk2 activation as well as histone H2AX phosphorylation, the reporters of DNA damage, in individual human lung adenocarcinoma A549 cells treated with these drugs. Using a phospho-specific Ab to tumor suppressor protein p53 phosphorylated on Ser15 (p53-Ser15(P)) combined with an Ab that detects p53 regardless of the phosphorylation status and multiparameter cytometry we correlated the TPT- and MXT-induced p53-Ser15(P) with ATM and Chk2 activation as well as with H2AX phosphorylation in relation to the cell cycle phase. In untreated interphase cells, p53-Ser15(P) had "patchy" localization throughout the nucleoplasm while mitotic cells showed strong p53-Ser15(P) cytoplasmic immunofluorescence (IF). The intense phosphorylation of p53-Ser15, combined with activation of ATM and Chk2 (involving centrioles) as well as phosphorylation of H2AX seen in the untreated mitotic cells, suggest mobilization of the DNA damage detection/repair machinery in controlling cytokinesis. In the nuclei of cells treated with TPT or MXT, the expression of p53-Ser15(P) appeared as closely packed foci of intense IF. Following TPT treatment, the induction of p53-Ser15(P) was most pronounced in S-phase cells while no significant cell cycle phase differences were seen in cells treated with MXT. The maximal increase in p53-Ser15(P) expression, rising up to 2.5-fold above the level of its constitutive expression, was observed in cells treated with TPT or MXT for 4-6 h. This maximum expression of p53-Ser15(P) coincided in time with the peak of Chk2 activation but not with ATM activation and H2AX phosphorylation, both of which crested 1-2 h after the treatment with TPT or MXT. The respective kinetics of p53-Ser15 phosphorylation versus ATM and Chk2 activation suggest that in response to DNA damage by TPT or MXT, Chk2 rather than ATM mediates p53 phosphorylation.
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PMID:Phosphorylation of p53 on Ser15 during cell cycle caused by Topo I and Topo II inhibitors in relation to ATM and Chk2 activation. 1880 8

Resveratrol decreases cancer risk and improves health of laboratory animals. However, it can also promote genomic instability. Part of the beneficial activity of resveratrol may result from the activation of SIRT1 deacetylase. We examined how resveratrol influenced the growth of human cancer cell lines of different origin: osteosarcoma (U-2 OS) and lung adenocarcinoma (A549) and how it modulated the expression as well as the localization of key proteins, involved in DNA repair and cell cycle regulation. Resveratrol-induced growth arrest was associated with signs of stress-induced senescence. Differential expression of BRCA1, cyclin B1, pRb and p21 in U-2 OS and A549 cells indicates that resveratrol can engage various molecular mechanisms to arrest cell cycle progression. In subset of U-2 OS cells, the upregulated BRCA1 formed foci closely associated with WRN and the telomeric protein (TRF1). Moreover, resveratrol induced telomeric instability in U-2 OS cells and the activation of DNA damage signaling in both cell lines, manifested as the phosphorylation of histone H2AX at serine 139 and of p53 at serines 15 and 37. Our data are consistent with the hypothesis that resveratrol inhibits cell growth and induces senescence by altering DNA metabolism.
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PMID:Resveratrol induces senescence-like growth inhibition of U-2 OS cells associated with the instability of telomeric DNA and upregulation of BRCA1. 1955 22

We have investigated the role of reactive oxygen species and thiol-oxidizing agents in the induction of cell death and have shown that adenocarcinoma gastric (AGS) cells respond differently to the oxidative challenge according to the signaling pathways activated. In particular, apoptosis in AGS cells is induced via the mitochondrial pathway upon treatment with thiol-oxidizing agents, such as diamide. Apoptosis is associated with persistent oxidative damage, as evidenced by the increase in carbonylated proteins and the expression/activation of DNA damage-sensitive proteins histone H2A.X and DNA-dependent protein kinase. Resistance to hydrogen peroxide is instead associated with Keap1 oxidation and rapid translocation of Nrf2 into the nucleus. Sensitivity to diamide and resistance to hydrogen peroxide are correlated with GSH redox changes, with diamide severely increasing GSSG, and hydrogen peroxide transiently inducing protein-GSH mixed disulfides. We show that p53 is activated in response to diamide treatment by the oxidative induction of the Trx1/p38(MAPK) signaling pathway. Similar results were obtained with another carcinoma cell line, CaCo2, indicating that these findings are not limited to AGS cells. Our data suggest that thiol-oxidizing agents could be exploited as inducers of apoptosis in tumor histotypes resistant to ROS-producing chemotherapeutics.
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PMID:Redox mechanisms involved in the selective activation of Nrf2-mediated resistance versus p53-dependent apoptosis in adenocarcinoma cells. 1964 29

The aim of the current study was to determine the signaling differences between gamma- and proton beam-irradiations. A549 lung adenocarcinoma cells were irradiated with 2 Gy proton beam or gamma-radiation. Proton beam was found to be more cytotoxic than gamma-radiation. Proton beam-irradiated cells showed phosphorylation of H2AX, ATM, Chk2, and p53. The mechanism of excessive cell killing in proton beam-irradiated cells was found to be upregulation of Bax and downregulation of Bcl-2. The noteworthy finding of this study is the biphasic activation of the sensor proteins, ATM, and DNA-PK and no activation of ATR by proton irradiation.
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PMID:Low energy proton beam induces efficient cell killing in A549 lung adenocarcinoma cells. 2021 May 20

Phosphorylation of histone H2AX (gammaH2AX) is a sensitive marker of DNA damage, particularly induction of DNA double-strand breaks. Using multiparameter cytometry we explored the effects of doxorubicin (DOX), cisplatin (CDDP) and 5-fluorouracil (5-FU) on four types of endometrioid adenocarcinoma cell lines (HEC-1A, HEC-1B, Ishikawa, KLE) correlating the drug-induced increases in phosphorylated H2AX (gammaH2AX) with cell cycle phase, induction of apoptosis and induction of cell senescence, the latter detected by analysis of beta-galactosidase. The study revealed significant differences among the cell lines in the effects of DNA damage vis-a-vis cell cycle phase specificity, induction of apoptosis or senescence following drug treatment. DOX treatment showed little cell cycle specificity in terms of induction of gammaH2AX, and its mechanism, which is similar to another anthracycline DNA topoisomerase II inhibitor mitoxantrone, may involve oxidative DNA damage modulated by other factors. Treatment with CDDP and 5-FU led to phosphorylation of H2AX preferentially in S-phase cells, consistent with the induction of replication stress. The response of Ishikawa cells expressing wt p53 was different compared to other cell lines. The data suggest that the treatment of endometrioid adenocarcinoma with these drugs may have to be customized to individual patients. The flow cytometric bivariate analysis of gammaH2AX and DNA content is a useful technique for better understanding the effects of antitumor agents and may contribute to customized patient treatments.
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PMID:DNA damage detected with gammaH2AX in endometrioid adenocarcinoma cell lines. 2037 80

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