Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Farnesyltransferase inhibitors (FTIs) possess antitumor activity. Based on recent findings, we hypothesized that FTIs induce reactive oxygen species (ROS) that damage DNA, leading to DNA damage responses. To test this hypothesis, we investigated the effects of FTIs on the generation of ROS, DNA double-strand breaks (DSB), DNA damage responses, and RhoB, and the effects of quenching ROS on these FTI effects. We evaluated four FTIs in human cancer cell lines of different tissue origins. We found that FTIs induced ROS and DSBs. Suppressing expression of the beta-subunit of farnesyltransferase with siRNA did not induce ROS, but slightly attenuated the ROS induced by FTIs. N-acetyl-L-cysteine (NAC), but not caspase inhibitors, blocked FTI-induced DSBs, suggesting that the DSBs were caused by ROS and did not result from apoptosis. The DSBs led to DNA damage responses. H2AX became phosphorylated and formed nuclear foci. The DNA-damage-sensing molecules involved were probably ataxia-telangiectasia mutated protein (ATM) and DNA-dependent protein kinase (DNA-PK) but not ATM- and Rad3-related protein (ATR). Key components of the homologous recombination and nonhomologous end joining repair pathways (DNA-PK, BRCA1, and NBS1) underwent phosphorylation and formed nuclear foci. RhoB, a mediator of the antineoplastic effect of FTIs and a protein inducible by DNA damage, was increased by FTIs. This increase was blocked by NAC. We concluded that FTIs induced oxidative DNA damage by inducing ROS and initiated DNA damage responses, including RhoB induction, and there was a complex relationship among FTIs, farnesyltransferase, ROS, and RhoB. Our data also imply that inhibitors of DNA repair may accentuate the clinical efficacy of FTIs.
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PMID:Farnesyltransferase inhibitors induce DNA damage via reactive oxygen species in human cancer cells. 1586 62

SPO11 introduces double-strand breaks (DSBs) that trigger the phosphorylation of H2AX during meiotic prophase. In mice, SPO11 is strictly required for initiation of meiotic recombination and synapsis, yet SPO11 is still considered to be dispensable for sex-body formation in mouse spermatocytes. We provide conclusive evidence showing that functional SPO11, and consequently recombination and synapsis, are required for phosphorylation of H2AX in the X-Y chromatin and for sex-body formation in mouse spermatocytes. We investigated the role in meiosis of the three kinases [ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia- and Rad-3-related) and DNA-PKcs (DNA-dependent-protein-kinase catalytic subunit)] known to phosphorylate H2AX in mitotic cells. We found that DNA-PKcs can be ruled out as an essential kinase in this process, whereas ATM is strictly required for the chromatin-wide phosphorylation of H2AX occurring in leptotene spermatocytes in response to DSBs. Remarkably, we discovered that Spo11 heterozygosity can rescue the prophase-I-arrest characteristic of ATM-deficient spermatocytes. Characterization of the rescued Atm-/- Spo11+/- mutant indicates that ATM is dispensable for sex-body formation and phosphorylation of H2AX in this subnuclear domain. The co-localization of ATR, phosphorylated H2AX and the sex chromatin observed in the Atm-/- Spo11+/- mutant, along with ATR transcription kinetics during the first wave of spermatogenesis, confirm and expand recent findings indicating that ATR is the kinase involved in H2AX phosphorylation in the sex body.
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PMID:SPO11 is required for sex-body formation, and Spo11 heterozygosity rescues the prophase arrest of Atm-/- spermatocytes. 1599 65

A variant of histone H2A, H2AX, is phosphorylated on Ser139 in response to DNA double-strand breaks (DSBs), and clusters of the phosphorylated form of H2AX (gamma-H2AX) in nuclei of DSB-induced cells show foci at breakage sites. Here, we show phosphorylation of H2AX in a cell cycle-dependent manner without any detectable DNA damage response. Western blot and immunocytochemical analyses with the anti-gamma-H2AX antibody revealed that H2AX is phosphorylated at M phase in HeLa cells. In ataxia-telangiectasia cells lacking ATM kinase activity, gamma-H2AX was scarcely detectable in the mitotic chromosomes, suggesting involvement of ATM in M-phase phosphorylation of H2AX. Single-cell gel electrophoresis assay and Western blot analysis with the anti-phospho-p53 (Ser15) antibody indicated that H2AX in human M-phase cells is phosphorylated independently of DSB and DNA damage signaling. Even in the absence of DNA damage, phosphorylation of H2AX in normal cell cycle progression may contribute to maintenance of genomic integrity.
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PMID:Phosphorylation of histone H2AX at M phase in human cells without DNA damage response. 1615 2

Topoisomerase I-associated DNA single-strand breaks selectively trapped by camptothecins are lethal after being converted to double-strand breaks by replication fork collisions. BLM (Bloom's syndrome protein), a RecQ DNA helicase, and topoisomerase IIIalpha (Top3alpha) appear essential for the resolution of stalled replication forks (Holliday junctions). We investigated the involvement of BLM in the signaling response to Top1-mediated replication DNA damage. In BLM-complemented cells, BLM colocalized with promyelocytic leukemia protein (PML) nuclear bodies and Top3alpha. Fibroblasts without BLM showed an increased sensitivity to camptothecin, enhanced formation of Top1-DNA complexes, and delayed histone H2AX phosphorylation (gamma-H2AX). Camptothecin also induced nuclear relocalization of BLM, Top3alpha, and PML protein and replication-dependent phosphorylation of BLM on threonine 99 (T99p-BLM). T99p-BLM was also observed following replication stress induced by hydroxyurea. Ataxia telangiectasia mutated (ATM) protein and AT- and Rad9-related protein kinases, but not DNA-dependent protein kinase, appeared to play a redundant role in phosphorylating BLM. Following camptothecin treatment, T99p-BLM colocalized with gamma-H2AX but not with Top3alpha or PML. Thus, BLM appears to dissociate from Top3alpha and PML following its phosphorylation and facilitates H2AX phosphorylation in response to replication double-strand breaks induced by Top1. A defect in gamma-H2AX signaling in response to unrepaired replication-mediated double-strand breaks might, at least in part, explain the camptothecin-sensitivity of BLM-deficient cells.
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PMID:Phosphorylation of BLM, dissociation from topoisomerase IIIalpha, and colocalization with gamma-H2AX after topoisomerase I-induced replication damage. 1619 71

The human immunodeficiency virus type 1 (HIV-1) protein Vpr (viral protein R) arrests cells in the G2 phase of the cell cycle, a process that requires activation of the ATR (ataxia-telangiectasia and Rad3-related) pathway. In this study we demonstrate that the expression of Vpr does not cause DNA double-strand breaks but rather induces ATR activation, as indicated by induction of Chk1 phosphorylation and the formation of gamma-H2AX and 53BP1 nuclear foci. We define a C-terminal domain containing repeated H(F/S)RIG sequences required for Vpr-induced activation of ATR. Further investigation of the mechanism by which Vpr activates the ATR pathway reveals an increase in chromatin binding of replication protein A (RPA) upon Vpr expression. Immunostaining shows that RPA localizes to nuclear foci in Vpr-expressing cells. Furthermore, we demonstrate direct binding of Vpr to chromatin in vivo, whereas Vpr C-terminal domain mutants lose this chromatin-binding activity. These data support a mechanism whereby HIV-1 Vpr induces ATR activation by targeting the host cell DNA and probably interfering with normal DNA replication.
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PMID:Activation of the ATR pathway by human immunodeficiency virus type 1 Vpr involves its direct binding to chromatin in vivo. 1630 15

Despite a clear link between ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using G0-G1 confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser15 (p53Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologically distinct subpool of p53Ser15 is ATM dependent and resistant to 26S-proteasomal degradation. p53Ser15 colocalizes and coimmunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear microbeam irradiation studies confirm p53Ser15 is recruited to sites of DNA damage containing gamma-H2AX, ATM(Ser1981), and DNA-PKcs(Thr2609) in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased gamma-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis.
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PMID:Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo. 1632 27

Various stresses including ionizing radiation give normal human fibroblasts a phenotype of senescence-like growth arrest (SLGA), manifested by p53-dependent irreversible G1 arrest. To determine the mechanism of persistent activation of p53, we examined phosphorylated Ataxia telangiectasia mutated (ATM) and phosphorylated histone H2AX foci formation after X-irradiation. Although the multiple tiny foci, detected soon after (<30 min) irradiation, gradually disappeared, some of these foci changed to large foci and persisted for 5 days. Large foci containing phosphorylated ATM and gamma-H2AX co-localized and foci with p53 phosphorylated at serine 15 also showed the same distribution. Interestingly, the signals obtained by telomere fluorescence in situ hybridization (FISH) assay did not co-localize with 90% of the large foci. Our results indicate that chromatin alteration in interstitial chromosomal regions is the most likely cause of continuous activation of p53, which results in the induction of SLGA by ionizing radiation.
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PMID:Interstitial chromatin alteration causes persistent p53 activation involved in the radiation-induced senescence-like growth arrest. 1636 Jan 20

Double strand DNA breaks in the genome lead to the activation of the ataxia-telangiectasia mutated (ATM) kinase in a process that requires ATM autophosphorylation at serine-1981. ATM autophosphorylation only occurs if ATM is previously acetylated by Tip60. The activated ATM kinase phosphorylates proteins involved in arresting the cell cycle, including p53, and in repairing the DNA breaks. Chloroquine treatment and other manipulations that produce chromatin defects in the absence of detectable double strand breaks also trigger ATM phosphorylation and the phosphorylation of p53 in primary human fibroblasts, while other downstream substrates of ATM that are involved in the repair of DNA double strand breaks remain unphosphorylated. This raises the issue of whether ATM is constitutively activated in patients with genetic diseases that display chromatin defects. We examined lymphoblastoid cell lines (LCLs) generated from patients with different types of chromatin disorders: Immunodeficiency, Centromeric instability, Facial anomalies (ICF) syndrome, Coffin Lowry syndrome, Rubinstein Taybi syndrome and Fascioscapulohumeral Muscular Dystrophy. We show that ATM is phosphorylated on serine-1981 in LCLs derived from ICF patients but not from the other syndromes. The phosphorylated ATM in ICF cells did not phosphorylate the downstream targets NBS1, SMC1 and H2AX, all of which require the presence of double strand breaks. We demonstrate that ICF cells respond normally to ionizing radiation, ruling out the possibility that genetic deficiency in ICF cells renders activated ATM incapable of phosphorylating its downstream substrates. Surprisingly, p53 was also not phosphorylated in ICF cells or in chloroquine-treated wild type LCLs. In this regard the response to chromatin-altering agents differs between primary fibroblasts and LCLs. Our findings indicate that although phosphorylation at serine-1981 is essential in the activation of the ATM kinase, serine-1981 phosphorylation is insufficient to render ATM an active kinase towards downstream substrates, including p53.
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PMID:Constitutive phosphorylation of ATM in lymphoblastoid cell lines from patients with ICF syndrome without downstream kinase activity. 1642 3

Bcl-2 can both promote and attenuate tumorigenesis. Although the former function is relatively well characterized, the mechanism of the latter remains elusive. We report here that enforced Bcl-2 expression in MCF7 cells stabilizes p53, induces phosphorylation of p53 serine 15 (p53pSer15) and inhibits MCF7 cell growth. Consistent with p53 Ser15 being a target of ataxia telangiectasia mutated protein(ATM)/ATR (ATM- and rad3-related) in the DNA damage response, Bcl-2 activates ATM by inducing ATM Ser1981 phosphorylation, which is accompanied with the phosphorylaton of two additional ATM substrates, Chk2 Thr68 and H2AX Ser139. Downregulation of ATM using a specific small interference RNA fragment (ATMRNAi) abolished Bcl-2-induced p53pSer15 and Bcl-2-mediated growth inhibition of MCF7 cells. Ectopic expression of a dominant-negative p53 mutant, p53175H, partially rescued this growth inhibition. Taken together, these observations demonstrate the contribution of ATM-p53 function to Bcl-2-mediated inhibition of MCF7 cell growth, indicating an ATM-mediated surveillance system for regulating Bcl-2 overexpression. Consistent with this concept, we found that MCF7 cells express Bcl-2 heterogeneously with 34.5% of cells being Bcl-2 negative. In general, Bcl-2-positive MCF7 cells proliferate slower than those of Bcl-2 negative. Thus, we provide evidence suggesting that activation of ATM suppresses Bcl-2-induced tumorigenesis, and that attenuation of ATM function may be an important event in breast cancer progression.
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PMID:Identification of an ataxia telangiectasia-mutated protein mediated surveillance system to regulate Bcl-2 overexpression. 1663 71

ATM (ataxia-telangiectasia mutated) is activated by a variety of noxious agent, including oxidative stress, and ATM deficiency results in an anomalous cellular response to oxidative stress. However, the mechanisms for ATM activation by oxidative stress remain to be established. Furthermore, it is not clear whether ATM responds to oxidative DNA damage or to a change in the intracellular redox state, independent of DNA damage. We found that ATM is activated by N-methyl-N'-nitro-nitrosoguanidine (MNNG) and 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), in NBS1- or MSH6-deficient cells. We further found that ATM is activated by treating chromatin-free immunoprecipitated ATM with MNNG or 15d-PGJ(2), which modifies free sulfhydryl (SH) groups, and that 15d-PGJ(2) binds covalently to ATM. Interestingly, 15d-PGJ(2)-induced ATM activation leads to p53 activation and apoptosis, but not to Chk2 or H2AX phosphorylation. These results indicate that ATM is activated through the direct modification of its SH groups, independent of DNA damage, and this activation leads, downstream, to apoptosis.
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PMID:ATM activation by a sulfhydryl-reactive inflammatory cyclopentenone prostaglandin. 1682 97


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