Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P16104 (H2AX)
 3,930 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA in live cells undergoes continuous oxidative damage caused by metabolically generated endogenous as well as external oxidants and oxidant-inducers. The cumulative oxidative DNA damage is considered the key factor in aging and senescence while the effectiveness of anti-aging agents is often assessed by their ability to reduce such damage. Oxidative DNA damage also preconditions cells to neoplastic transformation. Sensitive reporters of DNA damage, particularly the induction of DNA double-strand breaks (DSBs), are activation of ATM, through its phosphorylation on Ser 1981, and phosphorylation of histone H2AX on Ser 139; the phosphorylated form of H2AX has been named gammaH2AX. We review the observations that constitutive ATM activation (CAA) and H2AX phosphorylation (CHP) take place in normal cells as well in the cells of tumor lines untreated by exogenous genotoxic agents. We postulate that CAA and CHP, which have been measured by multiparameter cytometry in relation to the cell cycle phase, are triggered by oxidative DNA damage. This review also presents the findings on differences in CAA and CHP in various cell lines as well as on the effects of several agents and growth conditions that modulate the extent of these histone and ATM modifications. Specifically, described are effects of the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC), and the glutathione synthetase inhibitor buthionine sulfoximine (BSO) as well as suppression of cell metabolism by growth at higher cell density or in the presence of the glucose antimetabolite 2-deoxy-D-glucose. Collectively, the reviewed data indicate that multiparameter cytometric measurement of the level of CHP and/or CAA allows one to estimate the extent of ongoing oxidative DNA damage and to measure the DNA protective-effects of antioxidants or agents that reduce or amplify generation of endogenous ROS.
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PMID:Constitutive histone H2AX phosphorylation and ATM activation, the reporters of DNA damage by endogenous oxidants. 1694 Jul 54

We applied a flow cytometric method to quantify IR-induced histone H2AX phosphorylation at serine 139 (gammaH2AX) and compared those values to those obtained using a standard microscopy based foci counting method. After PFA fixation, methanol permeabilization was suitable for both FITC- or Alexa647-gammaH2AX. In contrast, Alexa647-gammaH2AX was not suitable for ethanol permeabilization. Antibody concentrations at 1-2 microg/ml yielded the highest gammaH2AX positive percentage for both antibodies. Without DAPI staining, gammaH2AX formation can be measured as a relative fold increase. Values determined by bivariant flow cytometric analysis and those obtained using microscopic foci formation exhibited a good quantitative correlation. Values obtained by both methods could vary according to the gating or threshold setting used. gammaH2AX positive cells increased as a function of radiation dose (2-16 Gy) followed by a dose-dependent decay. The free radical scavenger N-acetyl-L-cysteine (NAC), if administered at a concentration of 4 mM 30 min before IR, was effective in reducing IR-induced gammaH2AX formation in all phases of the cell cycle. We have developed a simplified and quantitative flow cytometry based method to measure IR-induced gammaH2AX in cells and demonstrated strong correlation to values obtained by a standard automated digital microscopic foci analysis along with NIH ImageJ custom macro software.
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PMID:Flow cytometric analysis of phosphorylated histone H2AX following exposure to ionizing radiation in human microvascular endothelial cells. 1696 Mar 36

Etoposide (VP-16) belongs to the family of DNA topoisomerase II (topo2) inhibitors, drugs widely used in cancer chemotherapy. Their presumed mode of action is stabilization of "cleavable complexes" between topo2 and DNA; collisions of DNA replication forks with these complexes convert them into DNA double-strand breaks (DSBs), potentially lethal lesions that may trigger apoptosis. Immunocytochemical detection of activation of ATM (ATM-S1981P) and histone H2AX phosphorylation (gammaH2AX) provides a sensitive probe of the induction of DSBs in individual cells. Using multiparameter cytometry we measured the expression of ATM-S1981P and gammaH2AX as well as initiation of apoptosis (caspase-3 activation) in relation to the cell cycle phase in etoposide-treated human lymphoblastoid TK6 cells. The induction of ATM-S1981P and gammaH2AX was seen in all phases of the cell cycle. The G(1)-phase cells, however, preferentially underwent apoptosis. The extent of etoposide-induced H2AX phosphorylation was partially reduced by N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS). The maximal reduction of H2AX phosphorylation by NAC, seen in G(1)-phase cells, was nearly 50%. NAC also protected a fraction of G(1) cells from etoposide-induced apoptosis, but had no such effect on S or G(2)M cells. However, no significant rise in the intracellular level of ROS upon treatment with etoposide was detected. The effects of etoposide were compared with the previously investigated effects of another topo2 inhibitor, mitoxantrone. The latter was seen to induce a maximal level of ATM-S1981P and gammaH2AX (partially abrogated by NAC) in G(1)-phase cells, but unlike etoposide, triggered apoptosis exclusively of S-phase cells. The data suggest that in addition to the generally accepted mechanism involving collisions of replication forks with the "cleavable complexes", other mechanisms which appear to be different for etoposide vs. mitoxantrone, may contribute to formation of DSBs and to triggering of apoptosis.
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PMID:Induction of ATM activation, histone H2AX phosphorylation and apoptosis by etoposide: relation to cell cycle phase. 1729 10

Human microvascular endothelial cells (HMEC) were exposed to ionizing radiation at doses ranging from 0 to 16 Gy in either the presence or absence of the active thiol forms of amifostine (WR1065), phosphonol (WR255591), N-acetyl-l-cysteine (NAC), captopril or mesna. Each of these clinically relevant thiols, administered to HMEC at a dose of 4 mM for 30 min prior to irradiation, is known to exhibit antioxidant properties. The purpose of this investigation was to determine the relationship(s), if any, between the frequency of radiation-induced histone H2AX phosphorylation at serine 139 (gamma-H2AX) in cells and subsequent survival, as assessed by colony-forming ability, in exposed cell populations as a function of the presence or absence of each of the five thiol compounds during irradiation. gamma-H2AX formation in irradiated cells, as a function of relative DNA content, was quantified by bivariant flow cytometry analysis with FITC-conjugated gamma-H2AX antibody and nuclear DAPI staining. gamma-H2AX formation in cells was measured as the relative fold increase as a function of the treatment conditions. The frequency of gamma-H2AX-positive cells increased with increasing dose of radiation followed by a dose- and time-dependent decay. The most robust response for gamma-H2AX formation occurred 1 h after irradiation with their relative frequencies decreasing as a function of time 4 and 24 h later. To assess the effects of the various thiols on gamma-H2AX formation, all measurements were made 1 h after irradiation. WR1065 was not only effective in protecting HMEC against gamma-H2AX formation across the entire dose range of radiation exposures used, but it was also significantly more cytoprotective than either its prodrug (WR2721) or disulfide (WR33278) analogue. WR1065 had no significant effect on gamma-H2AX formation when administered immediately or up to 30 min after radiation exposure. An inhibitory effect against gamma-H2AX formation induced by 8 Gy of radiation was expressed by each of the thiols tested. NAC, captopril and mesna were equally effective in reducing the frequency of gamma-H2AX formation, with both WR1065 and WR255591 exhibiting a slightly more robust protective effect. Each of the five thiols was effective in reducing the frequency of gamma-H2AX-positive cells across all phases of the cell cycle. In contrast to the relative ability of each of these thiols to inhibit gamma-H2AX formation after irradiation, NAC, captopril and mesna afforded no protection to HMEC as determined using a colony-forming survival assay. Only WR1065 and WR255591 were effective in reducing the frequencies of radiation-induced gamma-H2AX-positive cells as well as protecting against cell death. These results suggest that the use of gamma-H2AX as a biomarker for screening the efficacy of novel antioxidant radioprotective compounds is highly problematic since their formation and disappearance may be linked to processes beyond simply the formation and repair of radiation-induced DSBs.
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PMID:Relationship between phosphorylated histone H2AX formation and cell survival in human microvascular endothelial cells (HMEC) as a function of ionizing radiation exposure in the presence or absence of thiol-containing drugs. 1772 2

Aminoflavone (5-amino-2-(4-amino-3-fluorophenyl)-6,8-difluoro-7-methylchromen-4-one; AF; NSC 686288), a novel anticancer candidate agent, is undergoing clinical evaluation. AF induces DNA-protein cross-links (DPCs), Gamma-H2AX phosphorylation, aryl hydrocarbon receptor (AhR) signaling, apoptosis and its own metabolism via cytochrome P4501A1 and 1A2 (CYP1A1/1A2) activation in sensitive estrogen receptor positive (ER+) MCF7 breast cancer cells. Estrogen receptor negative (ER-) breast cancer is typically more aggressive with a poorer prognosis. In this investigation, we evaluated the ability of AF to induce reactive oxygen species (ROS) formation, oxidative DNA damage and apoptosis in ER- MDA-MB-468 breast cancer cells. The antioxidant, N-acetyl-L-cysteine (NAC), attenuated the cytotoxic effects of AF in MDA-MB-468 cells; an effect is also observed in ER+ T47D breast cancer cells. Nonmalignant MCF10A breast epithelial cells were resistant to the cytotoxic effects of AF. AF increased intracellular ROS, an effect blocked by NAC and the CYP1A1/1A2 inhibitor, alpha-Naphthoflavone (alpha-NF). AF induced oxidative DNA damage as evidenced by increased 8-oxo-7,8-dihydroguanine (8-oxodG) levels and DPC formation in these cells. AF caused S-phase arrest corresponding to an increase in p21((waf1/cip1)) protein expression. AF induced caspase 3, 8 and 9 activation, caspase-dependent apoptotic body formation and poly [ADP-ribose] polymerase (PARP) cleavage. Pretreatment with the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp(OMe)-fluoromethylketone inhibited apoptosis and partially inhibited ROS formation and oxidative DNA damage. Pretreatment with NAC attenuated AF-induced apoptotic body formation and caspase 3 activation. These studies suggest AF inhibits the growth of breast cancer cells in part, by inducing ROS production, oxidative DNA damage and apoptosis and has the potential to treat hormone-independent breast cancer.
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PMID:Aminoflavone induces oxidative DNA damage and reactive oxidative species-mediated apoptosis in breast cancer cells. 1805 23

The fragile FHIT gene is among the first targets of DNA damage in preneoplastic lesions, and recent studies have shown that Fhit protein is involved in surveillance of genome integrity and checkpoint response after genotoxin exposure. We now find that Fhit-deficient hematopoietic cells, exposed to the genotoxin hydroquinone, are resistant to the suppression of stem cell in vitro colony formation observed with wild-type (Wt) hematopoietic cells. In vivo-transplanted, hydroquinone-exposed, Fhit-deficient bone marrow cells also escaped the bone marrow suppression exhibited by Wt-transplanted bone marrow. Comparative immunohistochemical analyses of bone marrow transplants showed relative absence of Bax in Fhit-deficient bone marrow, suggesting insensitivity to apoptosis; assessment of DNA damage showed that occurrence of the oxidized base 8-hydroxyguanosine, a marker of DNA damage, was also reduced in Fhit-deficient bone marrow, as was production of intracellular reactive oxygen species. Treatment with the antioxidant N-acetyl-l-cysteine relieved hydroquinone-induced suppression of colony formation by Wt hematopoietic cells, suggesting that the decreased oxidative damage to Fhit-deficient cells, relative to Wt hematopoietic cells, accounts for the survival advantage of Fhit-deficient bone marrow. Homology-dependent recombination repair predominated in Fhit-deficient cells, although not error-free repair, as indicated by a higher incidence of 6-thioguanine-resistant colonies. Tissues of hydroquinone-exposed Fhit-deficient bone marrow-transplanted mice exhibited preneoplastic alterations, including accumulation of histone H2AX-positive DNA damage. The results indicate that reduced oxidative stress, coupled with efficient but not error-free DNA damage repair, allows unscheduled long-term survival of genotoxin-exposed Fhit-deficient hematopoietic stem cells carrying deleterious mutations.
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PMID:Fhit-deficient hematopoietic stem cells survive hydroquinone exposure carrying precancerous changes. 1848 48

Ionizing radiation (IR) induces DNA strand breaks leading to cell death or deleterious genome rearrangements. In the present study, we examined the role of N-acetyl-L-cysteine (NAC), a clinically proven safe agent, for it's ability to protect against gamma-ray-induced DNA strand breaks and/or DNA deletions in yeast and mammals. In the yeast Saccharomyces cerevisiae, DNA deletions were scored by reversion to histidine prototrophy. Human lymphoblastoid cells were examined for the frequency of gamma-H2AX foci formation, indicative of DNA double strand break formation. DNA strand breaks were also measured in mouse peripheral blood by the alkaline comet assay. In yeast, NAC reduced the frequency of IR-induced DNA deletions. However, NAC did not protect against cell death. NAC also reduced gamma-H2AX foci formation in human lymphoblastoid cells but had no protective effect in the colony survival assay. NAC administration via drinking water fully protected against DNA strand breaks in mice whole-body irradiated with 1Gy but not with 4Gy. NAC treatment in the absence of irradiation was not genotoxic. These data suggest that, given the safety and efficacy of NAC in humans, NAC may be useful in radiation therapy to prevent radiation-mediated genotoxicity, but does not interfere with efficient cancer cell killing.
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PMID:N-acetyl cysteine protects against ionizing radiation-induced DNA damage but not against cell killing in yeast and mammals. 1942 9

Activation of the nuclear transcription factor-kappaB (NF-kappaB) has been implicated in liver tumorigenesis. We evaluated the effects of a novel NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), in two human liver cancer cell lines HA22T/VGH and HuH-6. DHMEQ treatment dose dependently decreased the DNA-binding capacity of the NF-kappaB p65 subunit, inhibited cell growth and proliferation, and increased apoptosis as shown by caspase activation, release of cytochrome c, poly(ADP-ribose) polymerase cleavage, and down-regulation of survivin. DHMEQ also induced a dose-dependent activation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling, and inhibition of this pathway significantly reduced cell growth. It is noteworthy that we observed that DHMEQ stimulated reactive oxygen species (ROS) production in a dose-dependent manner and that pretreatment of the cells with the antioxidant N-acetyl-L-cysteine (NAC) significantly reduced DHMEQ-induced ROS generation. Accordingly, NAC completely reversed the DHMEQ-induced growth inhibition, caspase activation, and cell death. DHMEQ-treated cells exhibited DNA damage, as evaluated by accumulation in nuclear foci of phospho-H2AX, which was completely reversed by NAC. Moreover, DHMEQ induced the expression of genes involved in the endoplasmic reticulum stress response (GRP78, CHOP, TRB3) and promoted the splicing of XBP1 mRNA in a dose-dependent fashion in both cell lines, which was reversed in the presence of NAC. Knockdown of TRB3 mRNA expression by small interference RNA significantly decreased DHMEQ-induced cell growth inhibition. These data suggest that DHMEQ antitumor effects are primarily mediated through ROS generation. Thereby, considering that cancer cells are under increased ER stress and oxidative stress conditions, DHMEQ may greatly improve various anticancer strategies.
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PMID:Antitumor effects of dehydroxymethylepoxyquinomicin, a novel nuclear factor-kappaB inhibitor, in human liver cancer cells are mediated through a reactive oxygen species-dependent mechanism. 1946 Oct 54

The topoisomerase-I (topo-I) inhibitor topotecan, derivative of camptothecin, is the only registered drug for relapsed small cell lung cancer (SCLC). The histone deacetylase inhibitor vorinostat has shown preclinical and clinical antitumor activities in hematologic malignancies and solid tumors, including SCLC, and has recently been approved for the treatment of cutaneous T-cell lymphomas. In this study, we analyzed the antitumor effect of vorinostat combined with topotecan or camptothecin in topo-I inhibitor-sensitive H209 and inhibitor-resistant H526 SCLC cells. Simultaneous or sequential exposure (24 h delay) to either agent resulted in strong synergistic cytotoxic effect in both cell lines, as shown by calculating combination index, and confirmed by growth in soft agar. Combination treatments increased S-phase cell cycle arrest paralleled by apoptosis as measured by hypodiploid peak formation, Annexin V binding, DNA fragmentation, and mitochondria destruction. The apoptotic process was triggered by a caspase-dependent mechanism and can be ascribed to the phosphorylation of H2AX, a reporter of DNA double-strand breaks. These effects were paralleled by an increase of topo-I/DNA covalent complexes induced by combination treatment and suggest a potentiation by vorinostat of topotecan-induced DNA damage. Finally, oxidative injury played a significant functional role in the observed enhanced lethality because coadministration of the antioxidant N-acetyl-l-cysteine blocked reactive oxygen species generation, apoptosis, and mitochondria destruction induced by the vorinostat/topotecan combination. To our knowledge, this is the first demonstration of a synergistic antitumor effect between topotecan and vorinostat in SCLC. Because no well-established treatment is available for recurrent SCLC patients, our results indicate that this drug combination should be explored clinically.
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PMID:Synergistic antitumor effect between vorinostat and topotecan in small cell lung cancer cells is mediated by generation of reactive oxygen species and DNA damage-induced apoptosis. 1988 47

Cells that sustain double-strand breaks (DSB) can develop genomic instability, which contributes to carcinogenesis, and agents that cause DSBs are considered potential carcinogens. We looked for evidence of acid-induced DNA damage, including DSBs, in benign Barrett's epithelial (BAR-T) cell lines in vitro and in patients with Barrett's esophagus in vivo. In BAR-T cells, we also explored the mechanisms underlying acid-induced DNA damage. We exposed BAR-T cells to acid in the presence of a fluorescent probe for reactive oxygen species (ROS) and in the presence or absence of disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (which prevents intracellular acidification) and N-acety-l-cysteine (a scavenger of ROS). DSBs were detected by Western blotting and immunofluorescence for histone H2AX phosphorylation and by CometAssay. During endoscopy in patients with Barrett's esophagus, we took biopsy specimens from the metaplastic mucosa before and after esophageal perfusion with 0.1 N HCl for 3 min and sought DSBs by Western blotting for histone H2AX phosphorylation. In BAR-T cells, acid exposure resulted in ROS production and caused a time-dependent increase in levels of phospho-H2AX that continued for at least 48 h. Pretreatment with disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate or N-acety-l-cysteine prevented the acid-induced increase in phospho-H2AX levels. DSBs also were detected in biopsy specimens of Barrett's metaplasia following esophageal acid perfusion in all of 6 patients with Barrett's esophagus. Acid exposure causes DSBs in Barrett's epithelial cells through ROS produced as a consequence of intracellular acidification. These findings suggest that acid can be considered a carcinogen in Barrett's esophagus.
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PMID:In benign Barrett's epithelial cells, acid exposure generates reactive oxygen species that cause DNA double-strand breaks. 1992 Jan 91


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