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)

Phosphorylation of histone H2AX on serine 139 (gammaH2AX) occurs at sites flanking DNA double-strand breaks and can provide a measure of both number and location of these breaks within the nucleus. Because double-strand breaks are often lethal and are produced by several chemotherapeutic agents, we examined the possibility that expression of gammaH2AX after treatment might be useful as a surrogate indicator of clonogenic cell kill. Chinese hamster V79 cells were exposed for 30 min to drugs known to produce DNA double-stand breaks with different efficiencies: bleomycin, tirapazamine, doxorubicin, etoposide, 4-nitro-quinoline-N-oxide, and hydrogen peroxide. Cells were then allowed 1 h to develop foci before fixation or were plated to measure colony formation ability. Anti-gammaH2AX antibody staining was measured using flow cytometry. Flow histograms were analyzed for the percentage of cells that showed gammaH2AX levels greater than untreated cells, and this percentage was compared with the clonogenic surviving fraction. H2AX expression measured 1 h after treatment predicted cell killing for all of the drugs examined over two logs of cell kill. Moreover, predictive ability was largely independent of drug type in this cell line, and gammaH2AX levels five times background resulted in 50-90% cell kill. This method seems to provide a useful indicator of clonogenic response to treatment with selected chemotherapeutic drugs.
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PMID:Expression of phosphorylated histone H2AX as a surrogate of cell killing by drugs that create DNA double-strand breaks. 1290 3

Decay of an Auger-electron-emitting radioisotope can knock out a targeted gene by producing DNA strand breaks within its sequence. For delivery of Auger emitters to genomic targets we used triplex-forming oligonucleotides (TFOs) that bind specifically to their target sequences by forming hydrogen bonds within the major groove of the target duplex. We named this approach antigene radiotherapy. In our previous studies, we demonstrated that (125)I-labeled TFOs targeted against the human MDR1 gene produced sequence-specific double strand breaks (DSBs) within this gene in live cultured cells. We also found that conjugation of TFO with nuclear localization signal peptide significantly increased the efficiency of targeting. To screen the wide variety of possible TFO modifications a sensitive and robust assay of DNA damage produced by such (125)I-TFOs would be highly desirable. Recently we showed a direct correspondence between the number of decays of (125)I incorporated into DNA as (125)I-UdR and the number of histone gamma-H2AX foci per cell revealed by staining with gamma-H2AX antibodies. The technique is 100-fold more sensitive than other DSB-detection methods, thus it is possible to detect as few as an average of 0.5 DSBs per cell in a population of cultured cells. Here we applied this method to evaluate the intracellular DNA damage produced by two (125)I-TFOs, the first targeted to the single-copy HPRT gene ((125)I-TFO-HPRT) and second to a multicopy repeated sequence (GA)(n) that occurs almost 7000 times in the human genome ((125)I-TFO-GA). DNA damage produced by (125)I-TFO was assessed by staining the cells with gamma-H2AX antibody followed by either direct counting gamma-H2AX foci or by measuring the gamma-H2AX signal using flow cytometry. Both methods produced quantitatively close results; (125)I-TFO-GA with multiple nuclear targets produced on average 1.93 times more gamma-H2AX foci per cell and generated 1.96 times increase in gamma-H2AX antibody staining signal than (125)I-TFO-HPRT with a single target. The gamma-H2AX-based assay requires considerably less time and effort than the direct measurement of DSB by Southern hybridization applied previously. Therefore, we believe that gamma-H2AX-based measurement of DNA damage could be useful for evaluation and cellular DNA accessibility by (125)I-labeled DNA targeting agents.
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PMID:DNA damage produced by 125I-triplex-forming oligonucleotides as a measure of their successful delivery into cell nuclei. 1639 33

Excessive activation of ionotropic glutamate receptors increases oxidative stress, contributing to the neuronal death observed following neurological insults such as ischemia and seizures. Post-translational histone modifications may be key mediators in the detection and repair of damage resulting from oxidative stress, including DNA damage, and may thus affect neuronal survival in the aftermath of insults characterized by excessive glutamate release. In non-neuronal cells, phosphorylation of histone variant H2A.X (termed gamma-H2AX) occurs rapidly following DNA double-strand breaks. We investigated gamma-H2AX formation in rat cortical neurons (days in vitro 14) following activation of N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate glutamate receptors using fluorescent immunohistochemical techniques. Moreover, we evaluated the co-localization of gamma-H2AX 'foci' with Mre11, a double-strand break repair protein, to provide further evidence for the activation of this DNA damage response pathway. Here we show that minimally cytotoxic stimulation of ionotropic glutamate receptors was sufficient to evoke gamma-H2AX in neurons, and that NMDA-induced gamma-H2AX foci formation was attenuated by pretreatment with the antioxidant, Vitamin E, and the intracellular calcium chelator, BAPTA-AM. Moreover, a subset of gamma-H2AX foci co-localized with Mre11, indicating that at least a portion of gamma-H2AX foci is damage dependent. The extent of gamma-H2AX induction following glutamate receptor activation corresponded to the increases we observed following conventional DNA damaging agents [i.e. non-lethal doses of gamma-radiation (1 Gy) and hydrogen peroxide (10 microm)]. These data suggest that insults not necessarily resulting in neuronal death induce the DNA damage-evoked chromatin modification, gamma-H2AX, and implicate a role for histone alterations in determining neuronal vulnerability following neurological insults.
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PMID:Rapid phosphorylation of histone H2A.X following ionotropic glutamate receptor activation. 1670 43

After genotoxic stress poly(ADP-ribose) polymerase-1 (PARP-1) can be hyperactivated, causing (ADP-ribosyl)ation of nuclear proteins (including itself), resulting in NAD(+) and ATP depletion and cell death. Mechanisms of PARP-1-mediated cell death and downstream proteolysis remain enigmatic. beta-lapachone (beta-lap) is the first chemotherapeutic agent to elicit a Ca(2+)-mediated cell death by PARP-1 hyperactivation at clinically relevant doses in cancer cells expressing elevated NAD(P)H:quinone oxidoreductase 1 (NQO1) levels. Beta-lap induces the generation of NQO1-dependent reactive oxygen species (ROS), DNA breaks, and triggers Ca(2+)-dependent gamma-H2AX formation and PARP-1 hyperactivation. Subsequent NAD(+) and ATP losses suppress DNA repair and cause cell death. Reduction of PARP-1 activity or Ca(2+) chelation protects cells. Interestingly, Ca(2+) chelation abrogates hydrogen peroxide (H(2)O(2)), but not N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced PARP-1 hyperactivation and cell death. Thus, Ca(2+) appears to be an important co-factor in PARP-1 hyperactivation after ROS-induced DNA damage, which alters cellular metabolism and DNA repair.
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PMID:Calcium-dependent modulation of poly(ADP-ribose) polymerase-1 alters cellular metabolism and DNA repair. 1692 Jul 18

Despite rapid advances in the field of DNA repair, little is known about the repair of protein-DNA adducts. Previous studies have demonstrated that topoisomerase II (TopII)-DNA adducts (TopII-DNA covalent complexes) are rapidly degraded by the proteasome. It has been hypothesized that proteasomal degradation of TopII-DNA covalent adducts exposes TopII-concealed DNA double-strand breaks (DSBs) for repair. To test this hypothesis, the anticancer drug, VP-16 (etoposide), was employed to induce TopII-DNA covalent complexes in mammalian cells, and the involvement of proteasome in processing TopII-DNA covalent complexes into DSBs was investigated. Consistent with the hypothesis, VP-16-induced DSBs as monitored by neutral comet assay, as well as DNA damage signals (e.g. gamma-H2AX) were significantly reduced in the presence of the proteasome inhibitor, MG132. Using both top2beta knock-out mouse embryonic fibroblasts and Top2beta small interfering RNA knockdown PC12 cells, as well as postmitotic neurons in which TopIIalpha was absent, we showed that VP-16-induced DNA damage signals were attenuated upon proteasome inhibition, suggesting the involvement of proteasome in the repair/processing of both TopIIalpha-DNA and TopIIbeta-DNA adducts. By contrast, hydrogen peroxide-induced gamma-H2AX was unaffected upon proteasome inhibition, suggesting a specific requirement of the proteasome pathway in the processing of TopII-DNA covalent complexes into DNA damage.
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PMID:A protease pathway for the repair of topoisomerase II-DNA covalent complexes. 1697 21

Doxorubicin is among the most effective and widely used anticancer drugs in the clinic. However, cardiotoxicity is one of the life-threatening side effects of doxorubicin-based therapy. Dexrazoxane (Zinecard, also known as ICRF-187) has been used in the clinic as a cardioprotectant against doxorubicin cardiotoxicity. The molecular basis for doxorubicin cardiotoxicity and the cardioprotective effect of dexrazoxane, however, is not fully understood. In the present study, we showed that dexrazoxane specifically abolished the DNA damage signal gamma-H2AX induced by doxorubicin, but not camptothecin or hydrogen peroxide, in H9C2 cardiomyocytes. Doxorubicin-induced DNA damage was also specifically abolished by the proteasome inhibitors bortezomib and MG132 and much reduced in top2beta(-/-) mouse embryonic fibroblasts (MEF) compared with TOP2beta(+/+) MEFs, suggesting the involvement of proteasome and DNA topoisomerase IIbeta (Top2beta). Furthermore, in addition to antagonizing Top2 cleavage complex formation, dexrazoxane also induced rapid degradation of Top2beta, which paralleled the reduction of doxorubicin-induced DNA damage. Together, our results suggest that dexrazoxane antagonizes doxorubicin-induced DNA damage through its interference with Top2beta, which could implicate Top2beta in doxorubicin cardiotoxicity. The specific involvement of proteasome and Top2beta in doxorubicin-induced DNA damage is consistent with a model in which proteasomal processing of doxorubicin-induced Top2beta-DNA covalent complexes exposes the Top2beta-concealed DNA double-strand breaks.
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PMID:Topoisomerase IIbeta mediated DNA double-strand breaks: implications in doxorubicin cardiotoxicity and prevention by dexrazoxane. 1787 25

The differentiation of skeletal myoblasts is characterized by permanent withdrawal from the cell cycle and fusion into multinucleated myotubes. Muscle cell survival is critically dependent on the ability of cells to respond to oxidative stress. Base excision repair (BER) is the main repair mechanism of oxidative DNA damage. In this study, we compared the levels of endogenous oxidative DNA damage and BER capacity of mouse proliferating myoblasts and their differentiated counterpart, the myotubes. Changes in the expression of oxidative stress marker genes during differentiation, together with an increase in 8-hydroxyguanine DNA levels in terminally differentiated cells, suggested that reactive oxygen species are produced during this process. The repair of 2-deoxyribonolactone, which is exclusively processed by long-patch BER, was impaired in cell extracts from myotubes. The repair of a natural abasic site (a preferred substrate for short-patch BER) also was delayed. The defect in BER of terminally differentiated muscle cells was ascribed to the nearly complete lack of DNA ligase I and to the strong down-regulation of XRCC1 with subsequent destabilization of DNA ligase IIIalpha. The attenuation of BER in myotubes was associated with significant accumulation of DNA damage as detected by increased DNA single-strand breaks and phosphorylated H2AX nuclear foci upon exposure to hydrogen peroxide. We propose that in skeletal muscle exacerbated by free radical injury, the accumulation of DNA repair intermediates, due to attenuated BER, might contribute to myofiber degeneration as seen in sarcopenia and many muscle disorders.
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PMID:Terminally differentiated muscle cells are defective in base excision DNA repair and hypersensitive to oxygen injury. 1794 40

Phosphorylated histone H3 at serine 10 and serine 28 (H3Ser10 and H3Ser28) have been recognized as cell cycle markers to evaluate the late-G(2)/M status of cell and tissue samples. I report about the reduction phenomena of H3Ser10 and Ser28 phosphorylation (H3Ser10P and 28P) at the late-G(2) through M cell cycle phases in association with DNA damage caused by hydrogen peroxide (H(2)O(2)). The levels of H3Ser10P and Ser28P decreased between 15 and 60 min after H(2)O(2) addition in an inverse correlation manner with H2AX Ser139 phosphorylation (gammaH2AX). Experiments using wortmannin suggested that the reduction events of H3Ser10P/28P are under the control of phosphatidylinositol 3-kinase-like kinases. Fluorescence microscopic observation showed that H3Ser10 and Ser28 on telomeric portions of condensed M-phase chromosomes retained their strongly phosphorylated status even after 60 min of H(2)O(2) treatment. In addition, these chromosome parts were poorly gammaH2AX positive showing mutually exclusive distribution patterns between H3Ser10P/28P and gammaH2AX. Considering these data, I hypothesize that the reduction of the H3Ser10P/28P is a part of the DNA damage response processes. It is advisable to pay careful attention to these phenomena at the time of designing cell cycle assay protocols with H3Ser10P or Ser28P mitosis markers when DNA damaging process is expected to occur.
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PMID:Reduction of phosphorylated histone H3 serine 10 and serine 28 cell cycle marker intensities after DNA damage. 1839 32

Minutes after DNA damage, the variant histone H2AX is phosphorylated by protein kinases of the phosphoinositide kinase family, including ATM, ATR or DNA-PK. Phosphorylated (gamma)-H2AX-which recruits molecules that sense or signal the presence of DNA breaks, activating the response that leads to repair-is the earliest known marker of chromosomal DNA breakage. Here we identify a dynamic change in chromatin that promotes H2AX phosphorylation in mammalian cells. DNA breaks swiftly mobilize heterochromatin protein 1 (HP1)-beta (also called CBX1), a chromatin factor bound to histone H3 methylated on lysine 9 (H3K9me). Local changes in histone-tail modifications are not apparent. Instead, phosphorylation of HP1-beta on amino acid Thr 51 accompanies mobilization, releasing HP1-beta from chromatin by disrupting hydrogen bonds that fold its chromodomain around H3K9me. Inhibition of casein kinase 2 (CK2), an enzyme implicated in DNA damage sensing and repair, suppresses Thr 51 phosphorylation and HP1-beta mobilization in living cells. CK2 inhibition, or a constitutively chromatin-bound HP1-beta mutant, diminishes H2AX phosphorylation. Our findings reveal an unrecognized signalling cascade that helps to initiate the DNA damage response, altering chromatin by modifying a histone-code mediator protein, HP1, but not the code itself.
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PMID:HP1-beta mobilization promotes chromatin changes that initiate the DNA damage response. 1843 99

Trabectedin (Yondelis; ET-743) is a potent anticancer drug that binds to DNA by forming a covalent bond with a guanine in one strand and one or more hydrogen bonds with the opposite strand. Using a fluorescence-based melting assay, we show that one single trabectedin-DNA adduct increases the thermal stability of the double helix by >20 degrees C. As deduced from the analysis of phosphorylated H2AX and Rad51 foci, we observed that clinically relevant doses of trabectedin induce the formation of DNA double-strand breaks in human cells and activate homologous recombination repair in a manner similar to that evoked by the DNA interstrand cross-linking agent mitomycin C (MMC). Because one important characteristic of this drug is its marked cytotoxicity on cells lacking a functional Fanconi anemia (FA) pathway, we compared the response of different subtypes of FA cells to MMC and trabectedin. Our data clearly show that human cells with mutations in FANCA, FANCC, FANCF, FANCG, or FANCD1 genes are highly sensitive to both MMC and trabectedin. However, in marked contrast to MMC, trabectedin does not induce any significant accumulation of FA cells in G2-M. The critical relevance of FA proteins in the response of human cells to trabectedin reported herein, together with observations showing the role of the FA pathway in cancer suppression, strongly suggest that screening for mutations in FA genes may facilitate the identification of tumors displaying enhanced sensitivity to this novel anticancer drug.
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PMID:Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin. 1848 18


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