ATM activation and histone H2AX phosphorylation as indicators of DNA damage by DNA topoisomerase I inhibitor topotecan and during apoptosis

Damage that engenders DNA double-strand breaks (DSBs) activates ataxia telangiectasia mutated (ATM) kinase through its auto- or trans-phosphorylation on Ser1981 and activated ATM is one of the mediators of histone H2AX phosphorylation on Ser139. The present study was designed to explore: (i) whether measurement of ATM activation combined with H2AX phosphorylation provides a more sensitive indicator of DSBs than each of these events alone, and (ii) to reveal possible involvement of ATM activation in H2AX phosphorylation during apoptosis. Activation of ATM and/or H2AX phosphorylation in HL-60 or Jurkat cells treated with topotecan (Tpt) was detected immunocytochemically in relation to cell cycle phase, by multiparameter cytometry. Exposure to Tpt led to concurrent phosphorylation of ATM and H2AX in S-phase cells, whereas G1 cells were unaffected. Immunofluorescence (IF) of the S-phase cells immunostained for ATM-S1981P and gammaH2AX combined was distinctly stronger compared to that of the cells stained for each of these proteins alone. However, because of the relatively high ATM-S1981P IF of G1 cells, the ratio of IF of S to G1 cells, that is, the factor that determines competence of the assay in distinction of cells with DSBs, was 2- to 3-fold lower for ATM-S1981P alone, or for ATM-S1981P and gammaH2AX IF combined, than for gammaH2AX alone. ATM activation concurrent with H2AX phosphorylation, likely triggered by induction of DSBs during DNA fragmentation, occurred during apoptosis. The data suggest that frequency of activated ATM and phosphorylated H2AX molecules, per apoptotic cell, is comparable.     

Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis

Reviewed are the methods aimed to detect DNA damage in individual cells, estimate its extent and relate it to cell cycle phase and induction of apoptosis. They include the assays that reveal DNA fragmentation during apoptosis, as well as DNA damage induced by genotoxic agents. DNA fragmentation that occurs in the course of apoptosis is detected by selective extraction of degraded DNA. DNA in chromatin of apoptotic cells shows also increased propensity to undergo denaturation. The most common assay of DNA fragmentation relies on labelling DNA strand breaks with fluorochrome-tagged deoxynucleotides. The induction of double-strand DNA breaks (DSBs) by genotoxic agents provides a signal for histone H2AX phosphorylation on Ser139; the phosphorylated H2AX is named gammaH2AX. Also, ATM-kinase is activated through its autophosphorylation on Ser1981. Immunocytochemical detection of gammaH2AX and/or ATM-Ser1981(P) are sensitive probes to reveal induction of DSBs. When used concurrently with analysis of cellular DNA content and caspase-3 activation, they allow one to correlate the extent of DNA damage with the cell cycle phase and with activation of the apoptotic pathway. The presented data reveal cell cycle phase-specific patterns of H2AX phosphorylation and ATM autophosphorylation in response to induction of DSBs by ionizing radiation, topoisomerase I and II inhibitors and carcinogens. Detection of DNA damage in tumour cells during radio- or chemotherapy may provide an early marker predictive of response to treatment.     

Phosphorylation of histone H2AX at M phase in human cells without DNA damage response

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.     

Monoubiquitination of H2AX protein regulates DNA damage response signaling

Double strand breaks (DSBs) are the most deleterious of the DNA lesions that initiate genomic instability and promote tumorigenesis. Cells have evolved a complex protein network to detect, signal, and repair DSBs. In mammalian cells, a key component in this network is H2AX, which becomes rapidly phosphorylated at Ser(139) (γ-H2AX) at DSBs. Here we show that monoubiquitination of H2AX mediated by the RNF2-BMI1 complex is critical for the efficient formation of γ-H2AX and functions as a proximal regulator in DDR (DNA damage response). RNF2-BMI1 interacts with H2AX in a DNA damage-dependent manner and is required for monoubiquitination of H2AX at Lys(119)/Lys(120). As a functional consequence, we show that the H2AX K120R mutant abolishes H2AX monoubiquitination, impairs the recruitment of p-ATM (Ser(1981)) to DSBs, and thereby reduces the formation of γ-H2AX and the recruitment of MDC1 to DNA damage sites. These data suggest that monoubiquitination of H2AX plays a critical role in initiating DNA damage signaling. Consistent with these observations, impairment of RNF2-BMI1 function by siRNA knockdown or overexpression of the ligase-dead RNF2 mutant all leads to significant defects both in accumulation of γ-H2AX, p-ATM, and MDC1 at DSBs and in activation of NBS1 and CHK2. Additionally, the regulatory effect of RNF2-BMI1 on γ-H2AX formation is dependent on ATM. Lacking their ability to properly activate the DNA damage signaling pathway, RNF2-BMI1 complex-depleted cells exhibit impaired DNA repair and increased sensitivity to ionizing radiation. Together, our findings demonstrate a distinct monoubiquitination-dependent mechanism that is required for H2AX phosphorylation and the initiation of DDR.     

Nitrogen Oxide-Releasing Aspirin Induces Histone H2AX Phosphorylation,ATM Activation and Apoptosis Preferentially in S-Phase Cells: Involvement of Reactive Oxygen Species

Nitric oxide-releasing acetylsalicylic acid (NO-ASA; NO-aspirin) developed as an anti-inflammatory agent that was expected to avoid some of the adverse effects of aspirin (ASA), was recently shown to be cytotoxic to cells of different tumor lines. The cytotoxic properties and potency of NO-ASA are different than those of ASA which implies that the intracellular targets for NO-ASA and ASA, and their mechanism of action, are different. The aim of the present study was to reveal whether the cytotoxicity induced by NO-ASA is mediated by damage to DNA. We observed that even brief (1 h) treatment of human B-lymphoblastoid TK6 cells with ? 5 μM NO-ASA led to DNA damage revealed by the alkaline and neutral comet assays, histone H2AX phosphorylation on Ser 139, and ATM phosphorylation on Ser 1981, a marker of activation of this kinase. The induction of H2AX phosphorylation was preferential to S-phase cells. Exposure to ? 5 μM NO-ASA for over 3 h led to apoptosis, also preferentially of S-phase cells. Apoptosis was atypical; while chromatin was highly condensed there was no evidence of nuclear fragmentation nor were the cells positive in the TUNEL assay though they did express activated caspase-3. The induction of phosphorylation of H2AX on Ser 139 by NO-ASA was markedly attenuated in the presence of N-acetyl-L-cysteine, a scavenger of reactive oxygen species (ROS). The data imply that the NO-ASA induces DNA damage through oxidative stress; the oxidation-generated lesions provide a signal for induction of H2AX phosphorylation during DNA replication, perhaps when the progressing replication forks collide with the primary lesions converting them to DNA double-strand breaks. Because neither induction of H2AX phosphorylation nor apoptosis were observed at equimolar concentrations of ASA, the NO moiety attached to ASA appeared to mediate these effects.     

Deficiency in DNA damage response,a new characteristic of cells infected with latent HIV-1

Viruses can interact with host cell molecules responsible for the recognition and repair of DNA lesions, resulting in dysfunctional DNA damage response (DDR). Cells with inefficient DDR are more vulnerable to therapeutic approaches that target DDR, thereby raising DNA damage to a threshold that triggers apoptosis. Here, we demonstrate that 2 Jurkat-derived cell lines with incorporated silent HIV-1 provirus show increases in DDR signaling that responds to formation of double strand DNA breaks (DSBs). We found that phosphorylation of histone H2AX on Ser139 (gamma-H2AX), a biomarker of DSBs, and phosphorylation of ATM at Ser1981, Chk2 at Thr68, and p53 at Ser15, part of signaling pathways associated with DSBs, are elevated in these cells. These results indicate a DDR defect even though the virus is latent. DDR-inducing agents, specifically high doses of nucleoside RT inhibitors (NRTIs), caused greater increases in gamma-H2AX levels in latently infected cells. Additionally, latently infected cells are more susceptible to long-term exposure to G-quadruplex stabilizing agents, and this effect is enhanced when the agent is combined with an inhibitor targeting DNA-PK, which is crucial for DSB repair and telomere maintenance. Moreover, exposing these cells to the cancer drug etoposide resulted in formation of DSBs at a higher rate than in un-infected cells. Similar effects of etoposide were also observed in population of primary memory T cells infected with latent HIV-1. Sensitivity to these agents highlights a unique vulnerability of latently infected cells, a new feature that could potentially be used in developing therapies to eliminate HIV-1 reservoirs.     

DNA damage response as a biomarker in treatment of leukemias

Early assessment of cancer response to the treatment is of great importance in clinical oncology. Most antitumor drugs, among them DNA topoisomerase (topo) inhibitors, target nuclear DNA. The aim of the present study was to explore feasibility of the assessment of DNA damage response (DDR) as potential biomarker, eventually related to the clinical response, during treatment of human leukemias. We have measured DDR as reported by activation of ATM through its phosphorylation on Ser 1981 (ATM-S1981P) concurrent with histone H2AX phosphorylation on Ser139 (γH2AX) in leukemic blast cells from the blood of twenty patients, 16 children/adolescents and 4 adults, diagnosed with acute leukemias and treated with topo2 inhibitors doxorubicin, daunomycin, mitoxantrone or idarubicin. Phosphorylation of H2AX and ATM was detected using phospho-specific Abs and measured in individual cells by flow cytometry. The increase in the level of ATM-S1981P and γH2AX, varying in extent between the patients, was observed in blasts from the blood collected one hour after completion of the drug infusion with respect to the pre-treatment level. A modest degree of correlation was observed between the induction of ATM activation and H2AX phosphorylation in blasts of individual patients. The number of the studied patients (20) and the number of the clinically non-responding ones (2) was too low to draw a conclusion whether the assessment of DDR can be clinically prognostic. The present findings, however, demonstrate the feasibility of assessment of DDR during the treatment of leukemias with drugs targeting DNA.     

AIF-mediated caspase-independent necroptosis requires ATM and DNA-PK-induced histone H2AX Ser139 phosphorylation

The alkylating DNA-damage agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces a form of caspase-independent necroptosis implicating the mitochondrial flavoprotein apoptosis-inducing factor (AIF). Following the activation of PARP-1 (poly(ADP-ribose) polymerase-1), calpains, BID (BH3 interacting domain death agonist), and BAX (Bcl-2-associated X protein), the apoptogenic form of AIF (tAIF) is translocated to the nucleus where, associated with Ser139-phosphorylated histone H2AX (γH2AX), it creates a DNA-degrading complex that provokes chromatinolysis and cell death by necroptosis. The generation of γH2AX is crucial for this form of cell death, as mutation of H2AX Ser139 to Ala or genetic ablation of H2AX abolish both chromatinolysis and necroptosis. On the contrary, reintroduction of H2AX-wt or the phosphomimetic H2AX mutant (H2AX-S139E) into H2AX^−/− cells resensitizes to MNNG-triggered necroptosis. Employing a pharmacological approach and gene knockout cells, we also demonstrate in this paper that the phosphatidylinositol-3-OH kinase-related kinases (PIKKs) ATM (ataxia telangiectasia mutated) and DNA-dependent protein kinase (DNA-PK) mediate γH2AX generation and, consequently, MNNG-induced necroptosis. By contrast, H2AX phosphorylation is not regulated by ATR or other H2AX-related kinases, such as JNK. Interestingly, ATM and DNA-PK phosphorylate H2AX at Ser139 in a synergistic manner with different kinetics of activation. Early after MNNG treatment, ATM generates γH2AX. Further, DNA-PK contributes to H2AX Ser139 phosphorylation. In revealing the pivotal role of PIKKs in MNNG-induced cell death, our data uncover a milestone in the mechanisms regulating AIF-mediated caspase-independent necroptosis.     

Phosphorylation of p53 on Ser15 during cell cycle and caused by Topo I and Topo II inhibitors in relation to ATM and Chk2 activation

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-Ser15P) combined with an Ab that detects p53 regardless of the phosphorylation status and multiparameter cytometry we correlated the TPT- and MXT- induced p53-Ser15P with ATM and Chk2 activation as well as with H2AX phosphorylation in relation to the cell cycle phase. In untreated interphase cells, p53-Ser15P had "patchy" localization throughout the nucleoplasm while mitotic cells showed strong p53-Ser15P 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-Ser15P appeared as closely packed foci of intense IF. Following TPT treatment, the induction of p53-Ser15P 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-Ser15P 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-Ser15P 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.     

Induction of ATM Activation,Histone H2AX Phosphorylation and Apoptosis by Etoposide: Relation to Cell Cycle Phase

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 (γH2AX) provides a sensitive probe of the induction of DSBs in individual cells. Using multiparameter cytometry we measured the expression of ATM-S1981P and γH2AX 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 γH2AX was seen in all phases of the cell cycle. The G1-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 G1-phase cells, was nearly 50%. NAC also protected a fraction of G1 cells from etoposide-induced apoptosis, but had no such effect on S or G2M 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 γH2AX (partially abrogated by NAC) in G1-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.     

Collaborative roles of gammaH2AX and the Rad51 paralog Xrcc3 in homologous recombinational repair

One of the earliest events in the signal transduction cascade that initiates a DNA damage checkpoint is the phosphorylation on serine 139 of histone H2AX (gammaH2AX) at DNA double-strand breaks (DSBs). However, the role of gammaH2AX in DNA repair is poorly understood. To address this question, we generated chicken DT40 cells carrying a serine to alanine mutation at position 139 of H2AX (H2AX(-/S139A)) and examined their DNA repair capacity. H2AX(-/S139A) cells exhibited defective homologous recombinational repair (HR) as manifested by delayed Rad51 focus formation following ionizing radiation (IR) and hypersensitivity to the topoisomerase I inhibitor, camptothecin (CPT), which causes DSBs at replication blockage. Deletion of the Rad51 paralog gene, XRCC3, also delays Rad51 focus formation. To test the interaction of Xrcc3 and gammaH2AX, we disrupted XRCC3 in H2AX(-/S139A) cells. XRCC3(-/-)/H2AX(-/S139A) mutants were not viable, although this synthetic lethality was reversed by inserting a transgene that conditionally expresses wild-type H2AX. Upon repression of the wild-type H2AX transgene, XRCC3(-/-)/H2AX(-/S139A) cells failed to form Rad51 foci and exhibited markedly increased levels of chromosomal aberrations after CPT treatment. These results indicate that H2AX and XRCC3 act in separate arms of a branched pathway to facilitate Rad51 assembly.     

Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation

Elevated level of oxygen (hyperoxia) is widely used in critical care units and in respiratory insufficiencies. In addition, hyperoxia has been implicated in many diseases such as bronchopulmonary dysplasia or acute respiratory distress syndrome. Although hyperoxia is known to cause DNA base modifications and strand breaks, the DNA damage response has not been adequately investigated. We have investigated the effect of hyperoxia on DNA damage signaling and show that hyperoxia is a unique stress that activates the ataxia telangiectasia mutant (ATM)- and Rad3-related protein kinase (ATR)-dependent p53 phosphorylations (Ser6, -15, -37, and -392), phosphorylation of histone H2AX (Ser139), and phosphorylation of checkpoint kinase 1 (Chk1). In addition, we show that phosphorylation of p53 (Ser6) and histone H2AX (Ser139) depend on both ATM and ATR. We demonstrate that ATR activation precedes ATM activation in hyperoxia. Finally, we show that ATR is required for ATM activation in hyperoxia. Taken together, we report that ATR is the major DNA damage signal transducer in hyperoxia that activates ATM.     

Tissue-specific DNA-PK-dependent H2AX phosphorylation and gamma-H2AX elimination after X-irradiation in vivo

Histone H2AX rapidly undergoes phosphorylation at Ser139 (γ-H2AX) in response to DNA double-strand breaks. Although ATM kinase and DNA-PK phosphorylate Ser139 of H2AX in culture cells, the regulatory mechanism of γ-H2AX level remains unclear in vivo. Here, we detected the phosphorylation of H2AX and the elimination of γ-H2AX in the mouse skin after X-irradiation. Furthermore, following X-irradiation, the level of γ-H2AX also increased in mice lacking either ATM or DNA-PK. Although the elimination after X-irradiation was detected in the skin of these mutant mice, the elimination in DNA-PK-deficient mice was slower than that in C3H and ATM knockout mice, suggesting that a fraction of γ-H2AX in the skin is eliminated in a DNA-PK-dependent manner. Although the DNA-PK-dependent elimination of γ-H2AX was also detected in the liver, kidney, and spleen, the DNA-PK-dependent phosphorylation of H2AX was detected in the spleen only. These results suggest that the regulatory mechanism of γ-H2AX level is tissue-specific.     

Phosphorylation of Histone H2AX on Ser 139 and Activation of ATM During Oxidative Burst in Phorbol Ester-Treated Human Leukocytes

Oxidative burst is a defense mechanism used by specialized phagocytes such as granulocytes or monocytes to kill the invading microorganisms through generation of superoxide anions. Oxidative burst also results in DNA damage of the phagocytes. Phagocytes are terminally differentiated and some of very short life-span cells. We could find no reports whether oxidative burst-mediated DNA damage triggers in such cells histone H2AX-Ser139 phosphorylation and activation of Ataxia Telangiectasia Mutated (ATM), the signals otherwise used to activate DNA repair and checkpoint pathways in proliferating cells. We now present the evidence that induction of oxidative stress in human peripheral blood leukocytes by phorbol myristate acetate (PMA) was associated with intense phosphorylation of histone H2AX and with ATM activation, seen already 60 min after exposure to PMA. The modifications of H2AX and ATM in individual granulocytes, monocytes and lymphocytes were detected prior to caspases activation and thus were unrelated to induction of apoptosis. A large intercellular variation in response was observed, and only a fraction of cells in these subpopulations showed H2AX and ATM modifications. The data are compatible with the earlier observations of DNA damage during oxidative burst and suggest that even in terminally differentiated cells that have a short life-span, DNA damage triggers recruitment of the DNA repair machinery. The observed H2AX phosphorylation in lymphocytes may reflect their DNA damage by the superoxide ions propagating from the neighboring granulocytes and/or monocytes.     

Cytometry of ATM activation and histone H2AX phosphorylation to estimate extent of DNA damage induced by exogenous agents.

This review covers the topic of cytometric assessment of activation of Ataxia telangiectasia mutated (ATM) protein kinase and histone H2AX phosphorylation on Ser139 in response to DNA damage, particularly the damage that involves formation of DNA double-strand breaks. Briefly described are molecular mechanisms associated with activation of ATM and the downstream events that lead to recruitment of DNA repair machinery, engagement of cell cycle checkpoints, and activation of apoptotic pathway. Examples of multiparameter analysis of ATM activation and H2AX phosphorylation vis-a-vis cell cycle phase position and induction of apoptosis that employ flow- and laser scanning-cytometry are provided. They include cells treated with a variety of exogenous genotoxic agents, such as ionizing and UV radiation, DNA topoisomerase I (topotecan) and II (mitoxantrone, etoposide) inhibitors, nitric oxide-releasing aspirin, DNA replication inhibitors (aphidicolin, hydroxyurea, thymidine), and complex environmental carcinogens such as present in tobacco smoke. Also presented is an approach to identify DNA replicating (BrdU incorporating) cells based on selective photolysis of DNA that triggers H2AX phosphorylation. Listed are strategies to distinguish ATM activation and H2AX phosphorylation induced by primary DNA damage by genotoxic agents from those effects triggered by DNA fragmentation that takes place during apoptosis. While we review most published data, recent new findings also are included. Examples of multivariate analysis of ATM activation and H2AX phosphorylation presented in this review illustrate the advantages of cytometric flow- and image-analysis of these events in terms of offering a sensitive and valuable tool in studies of factors that induce DNA damage and/or affect DNA repair and allow one to explore the linkage between DNA damage, cell cycle checkpoints and initiation of apoptosis.     

Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA-PK in human cells exposed to ionizing radiation and treated with kinase inhibitors

In eukaryotic cells, DNA double strand breaks (DSBs) cause the prompt phosphorylation of serine 139 at the carboxy terminus of histone H2AX to generate gamma-H2AX, detectable by Western blotting or immunofluorescence. The consensus sequence at the phosphorylation site implicates the phosphatidylinositol 3-like family of protein kinases in H2AX phosphorylation. It remains open whether ATM (ataxia telangiectasia mutated) is the major H2AX kinase, or whether other members of the family, such as DNA-PK (DNA dependent protein kinase) or ATR (ATM and Rad3 related), contribute in a functionally complementary manner. To address this question, we measured global H2AX phosphorylation in cell lysates and foci formation in individual cells of either wild type or mutant (ATM or DNA-PK) genetic background. Normal global phosphorylation kinetics is observed after irradiation in cells defective either in ATM or DNA-PK alone, suggesting a complementary contribution to H2AX phosphorylation. This is further supported by the observation that initial H2AX phosphorylation is delayed when both kinases are inhibited by wortmannin, as well as when ATM is inhibited by caffeine in DNA-PK deficient cells. However, robust residual global phosphorylation is detectable under all conditions of genetic or chemical inhibition suggesting the function of additional kinases, such as ATR. Treatment with wortmannin, caffeine, or UCN-01 produces a strong DNA-PK dependent late global hyperphosphorylation of H2AX, uncoupled from DNA DSB rejoining and compatible with an inhibition of late steps in DNA DSB processing. Evaluation of gamma-H2AX foci formation confirms the major conclusions made on the basis of global H2AX phosphorylation, but also points to differences particularly several hours after exposure to IR. The results in aggregate implicate DNA-PK, ATM and possibly other kinases in H2AX phosphorylation. The functional significance and the mechanisms of coordination in space and time of these multiple inputs require further investigation.     

Constitutive Histone H2AX Phosphorylation and ATM Activation,the Reporters of DNA Damage by Endogenous Oxidants

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 γH2AX. 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.     

Sequential phosphorylation of Ser-10 on histone H3 and ser-139 on histone H2AX and ATM activation during premature chromosome condensation: relationship to cell-cycle phase and apoptosis.

BACKGROUND: Histone H1 and H3 phosphorylation associated with chromatin condensation during mitosis has been studied extensively. Less is known on histone modifications that occur during premature chromosome condensation (PCC). The aim of the present study was to reveal the status of histone H3 and H2AX phosphorylation on Ser-10 and Ser-139, respectively, as well as ATM activation through phosphorylation on Ser-1981, during PCC, and relate these events to cell-cycle phase and to initiation of apoptosis. MATERIALS AND METHODS: To induce PCC, A549 and HL-60 cells were exposed to the phosphatase inhibitor calyculin A (Cal A). Phosphorylation of histone H3 and H2AX as well as ATM activation were detected immunocytochemically concurrent with analysis of cellular DNA content and activation of caspase-3, a marker of apoptosis. The intensity of cellular fluorescence was measured by flow- or laser scanning cytometry. RESULTS: Induction of PCC led to rapid histone H3 phosphorylation, followed by activation of ATM and then H2AX phosphorylation in both, HL-60 and A549 cells. All these events occurred sequentially, prior to caspase-3 activation, and affected cells in all phases of the cell cycle. ATM activation and H2AX phosphorylation was seen during mitosis of A549 but not HL-60 cells. CONCLUSIONS: Because the Cal A-induced phosphorylation of histone H3 and H2AX, and of ATM, precede caspase-3 activation these modifications are pertinent to PCC and not to apoptosis-associated chromatin condensation. The sequence of histone H3 and H2AX phosphorylation and ATM activation during PCC is compatible with a role of ATM in mediating phosphorylation of H2AX but not H3. Mitosis in some cell types may proceed without ATM activation and H2AX phosphorylation.     

Dynamic change of histone H2AX phosphorylation independent of ATM and DNA-PK in mouse skin in situ

Histone H2AX undergoes phosphorylation on Ser 139 (γ-H2AX) rapidly in response to DNA double-strand breaks induced by exogenous stimuli, such as ionizing radiation. However, the endogenous phosphorylation pattern and modifier of H2AX remain unclear. Here we show that H2AX is regulated physically at the level of phosphorylation at Ser139 during a hair cycle in the mouse skin. In anagen hair follicles, γ-H2AX-positive cells were observed in the outer root sheath (ORS) and hair bulb in a cycling inferior region but not in a permanent superficial region. In telogen hair follicles, γ-H2AX-positive cells were only detected around the germ cell cap. In contrast, following X-irradiation, γ-H2AX was observed in various cell types including the ORS cells in the permanent superficial region. Furthermore, γ-H2AX-positive cells were detected in the skin of mice lacking either ATM or DNA-PK, suggesting that these kinases are not essential for phosphorylation in vivo.