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.     

Induction of DNA damage signaling by oxidative stress in relation to DNA replication as detected using "click chemistry"

Induction of DNA damage by oxidants such as H(2) O(2) activates the complex network of DNA damage response (DDR) pathways present in cells to initiate DNA repair, halt cell cycle progression, and prepare an apoptotic reaction. We have previously reported that activation of Ataxia Telangiectasia Mutated protein kinase (ATM) and induction of γH2AX are among the early events of the DDR induced by exposure of cells to H(2) O(2) , and in human pulmonary carcinoma A549 cells, both events were expressed predominantly during S-phase. This study was designed to further explore a correlation between these events and DNA replication. Toward this end, we utilized 5-ethynyl-2'deoxyuridine (EdU) and the "click chemistry" approach to label DNA during replication, followed by exposure of A549 cells to H(2) O(2) . Multiparameter laser scanning cytometric analysis of these cells made it possible to identify DNA replicating cells and directly correlate H(2) O(2) -induced ATM activation and induction of γH2AX with DNA replication on a cell by cell basis. After pulse-labeling with EdU and exposure to H(2) O(2) , confocal microscopy was also used to examine the localization of DNA replication sites ("replication factories") versus the H2AX phosphorylation sites (γH2AX foci) in nuclear chromatin in an attempt to observe the absence or presence of colocalization. The data indicate a close association between DNA replication and H2AX phosphorylation in A549 cells, suggesting that these DNA damage response events may be triggered by stalled replication forks and perhaps also by induction of DNA double-strand breaks at the primary DNA lesions induced by H(2) O(2) .     

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.     

Effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on Ser 1981 and histone H2AX on Ser 139 in relation to cell cycle phase and induction of apoptosis.

BACKGROUND: DNA replication stress often induces DNA damage. The antitumor drug hydroxyurea (HU), a potent inhibitor of ribonucleotide reductase that halts DNA replication through its effects on cellular deoxynucleotide pools, was shown to damage DNA inducing double-strand breaks (DSBs). Aphidicolin (APH), an inhibitor of alpha-like DNA polymerases, was also reported to cause DNA damage, but the evidence for induction of DSBs by APH is not straightforward. Histone H2AX is phosphorylated on Ser 139 in response to DSBs and one of the protein kinases that phosphorylate H2AX is ataxia telangiectasia mutated (ATM); activation of ATM is through its phosphorylation of Ser 1981. The present study was undertaken to reveal whether H2AX is phosphorylated in cells exposed to HU or APH and whether its phosphorylation is mediated by ATM. MATERIALS AND METHODS: HL-60 cells were treated in cultures with 0.1-5.0 mM HU or 1-4 muM APH for up to 5 h. Activation of ATM and H2AX phosphorylation was detected immunocytochemically using Ab specific to Ser1981-ATM or Ser 139-H2AX epitopes, respectively, concurrent with measurement of cellular DNA content. RESULTS: While exposure of cells to HU led to H2AX phosphorylation selectively during S phase and the cells progressing through the early portion of S (DI = 1.1-1.4) were more affected than late-S phase (DI = 1.6-1.9) cells, ATM was not activated by HU. In fact, the level of constitutive ("programmed") ATM phosphorylation was distinctly suppressed, in all phases of the cell cycle, at 0.1-5.0 mM HU. Cells' exposure to APH also resulted in H2AX phosphorylation at Ser139 with no evidence of ATM activation, and as in the case of HU, the early-S cells were more affected than the late-S phase cells. The rise in frequency of apoptotic cells became apparent after 2 h of exposure to HU or APH, and all apoptotic cells had markedly elevated levels of both H2AX-Ser139 and ATM-Ser1981 phosphorylation. CONCLUSIONS: The lack of correlation between H2AX phosphorylation and ATM activation indicates that protein kinase(s) other than ATM (ATR and/or DNA-dependent protein kinase) are activated by DSBs induced by replication stress. Interestingly, HU inhibits the constitutive ("programmed") level of ATM phosphorylation in untreated cells. However, DNA fragmentation during apoptosis activates ATM and dramatically increases level of H2AX phosphorylation.     

Nitric Oxide Induces Ataxia Telangiectasia Mutated (ATM) Protein-dependent γH2AX Protein Formation in Pancreatic β Cells

In this study, the effects of cytokines on the activation of the DNA double strand break repair factors histone H2AX (H2AX) and ataxia telangiectasia mutated (ATM) were examined in pancreatic β cells. We show that cytokines stimulate H2AX phosphorylation (γH2AX formation) in rat islets and insulinoma cells in a nitric oxide- and ATM-dependent manner. In contrast to the well documented role of ATM in DNA repair, ATM does not appear to participate in the repair of nitric oxide-induced DNA damage. Instead, nitric oxide-induced γH2AX formation correlates temporally with the onset of irreversible DNA damage and the induction of apoptosis. Furthermore, inhibition of ATM attenuates cytokine-induced caspase activation. These findings show that the formation of DNA double strand breaks correlates with ATM activation, irreversible DNA damage, and ATM-dependent induction of apoptosis in cytokine-treated β cells.     

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.     

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.     

Gamma-herpesvirus kinase actively initiates a DNA damage response by inducing phosphorylation of H2AX to foster viral replication

DNA virus infection can elicit the DNA damage response in host cells, including ATM kinase activation and H2AX phosphorylation. This is considered to be the host cell response to replicating viral DNA. In contrast, we show that during infection of macrophages murine gamma-herpesvirus 68 (gammaHV68) actively induces H2AX phosphorylation by expressing a viral kinase (orf36). GammaHV68-encoded orf36 kinase and its EBV homolog, BGLF4, induce H2AX phosphorylation independently of other viral genes. The process requires the kinase domain of Orf36 and is enhanced by ATM. Orf36 is important for gammaHV68 replication in infected animals, and orf36, H2AX, and ATM are all critical for efficient gammaHV68 replication in primary macrophages. Thus, activation of proximal components of the DNA damage signaling response is an active viral kinase-driven strategy required for efficient gamma-herpesvirus replication.     

DNA damage response in renal ischemia–reperfusion and ATP-depletion injury of renal tubular cells

Renal ischemia–reperfusion leads to acute kidney injury (AKI) that is characterized pathologically by tubular damage and cell death, followed by tubular repair, atrophy and interstitial fibrosis. Recent work suggested the possible presence of DNA damage response (DDR) in AKI. However, the evidence is sketchy and the role and regulation of DDR in ischemic AKI remain elusive. In this study, we demonstrated the induction of phosphorylation of ATM, H2AX, Chk2 and p53 during renal ischemia–reperfusion in mice, suggesting DDR in kidney tissues. DDR was also induced in vitro during the recovery or “reperfusion” of renal proximal tubular cells (RPTCs) after ATP depletion. DDR in RPTCs was abrogated by supplying glucose to maintain ATP via glycolysis, indicating that the DDR depends on ATP depletion. The DDR was also suppressed by the general caspase inhibitor z-VAD and the overexpression of Bcl-2, supporting a role of apoptosis-associated DNA damage in the DDR. N-acetylcysteine (NAC), an antioxidant, suppressed the phosphorylation of ATM and p53 and, to a less extent, Chk2, but NAC increased the phosphorylation and nuclear foci formation of H2AX. Interestingly, NAC increased apoptosis, which may account for the observed H2AX activation. Ku55933, an ATM inhibitor, blocked ATM phosphorylation and ameliorated the phosphorylation of Chk2 and p53, but it increased H2AX phosphorylation and nuclear foci formation. Ku55933 also increased apoptosis in RPTCs following ATP depletion. The results suggest that DDR occurs during renal ischemia–reperfusion in vivo and ATP-depletion injury in vitro. The DDR is partially induced by apoptosis and oxidative stress-related DNA damage. ATM, as a sensor in the DDR, may play a cytoprotective role against tubular cell injury and death.     

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.     

Lycopene inhibits Helicobacter pylori-induced ATM/ATR-dependent DNA damage response in gastric epithelial AGS cells

Oxidative stress linked to DNA damage is involved in the pathogenesis of Helicobacter pylori-associated gastric diseases. The DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair, and apoptosis through the activation of ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) and their target proteins. However, neither H. pylori-induced DDR nor the effects of antioxidants on the DNA damage have been established. This study aimed to investigate the detailed process of H. pylori-induced DNA damage and to examine whether lycopene, a natural antioxidant, inhibits DNA damage and cellular response of gastric epithelial AGS cells infected with H. pylori. AGS cells were cultured with H. pylori in Korean isolates and treated with or without lycopene. Cell viability, DNA damage indices, levels of 8-OH-dG, and reactive oxygen species (ROS) as well as cell-cycle distributions were determined. The activation of ATM, ATR, Chk1, and Chk2; histone H2AX focus formation; activation and induction of p53; and levels of Bax and Bcl-2 and poly(ADP-ribose) polymerase-1 (PARP-1) were assessed. The results showed that H. pylori induced apoptosis in AGS cells with increased Bax and decreased Bcl-2 expression as well as PARP-1 cleavage. Culture with H. pylori led to increases in intracellular ROS, 8-OH-dG, double-strand DNA breaks (DSBs), and DNA fragmentation. H. pylori induced activation of the ATM/Chk2 and ATR/Chk1 pathways, phosphorylation of H2AX and p53, and a delay in the progression of the cells entering the S phase. Lycopene inhibited H. pylori-induced increases in ROS, apoptosis, alterations in cell-cycle distribution, DSBs, and ATM- and ATR-mediated DDR in AGS cells. In conclusion, lycopene may be beneficial for treatment of H. pylori-induced gastric diseases associated with oxidative DNA damage.     

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.     

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.     

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.     

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.     

MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals

MDC1 functions in checkpoint activation and DNA repair following DNA damage. To address the physiological role of MDC1, we disrupted the MDC1 gene in mice. MDC1-/- mice recapitulated many phenotypes of H2AX-/- mice, including growth retardation, male infertility, immune defects, chromosome instability, DNA repair defects, and radiation sensitivity. At the molecular level, H2AX, MDC1, and ATM form a positive feedback loop, with MDC1 directly mediating the interaction between H2AX and ATM. MDC1 binds phosphorylated H2AX through its BRCT domain and ATM through its FHA domain. Through these interactions, MDC1 accumulates activated ATM flanking the sites of DNA damage, facilitating further ATM-dependent phosphorylation of H2AX and the amplification of DNA damage signals. In the absence of MDC1, many downstream ATM signaling events are defective. These results suggest that MDC1, as a signal amplifier of the ATM pathway, is vital in controlling proper DNA damage response and maintaining genomic stability.     

ATM phosphorylates histone H2AX in response to DNA double-strand breaks

A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated), and ATR (ATM and Rad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. To definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs-/- and Atm-/- cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs-/- cells and severely compromised in Atm-/- cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm-/- fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm-/- fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks.     

Functional interaction of H2AX, NBS1, and p53 in ATM-dependent DNA damage responses and tumor suppression

Ataxia-telangiectasia (A-T) mutated (ATM) kinase signals all three cell cycle checkpoints after DNA double-stranded break (DSB) damage. H2AX, NBS1, and p53 are substrates of ATM kinase and are involved in ATM-dependent DNA damage responses. We show here that H2AX is dispensable for the activation of ATM and p53 responses after DNA DSB damage. Therefore, H2AX functions primarily as a downstream mediator of ATM functions in the parallel pathway of p53. NBS1 appears to function both as an activator of ATM and as an adapter to mediate ATM activities after DNA DSB damage. Phosphorylation of ATM and H2AX induced by DNA DSB damage is normal in NBS1 mutant/mutant (NBS1m/m) mice that express an N-terminally truncated NBS1 at lower levels. Therefore, the pleiotropic A-T-related systemic and cellular defects observed in NBS1m/m mice are due to the disruption of the adapter function of NBS1 in mediating ATM activities. While H2AX is required for the irradiation-induced focus formation of NBS1, our findings indicate that NBS1 and H2AX have distinct roles in DNA damage responses. ATM-dependent phosphorylation of p53 and p53 responses are largely normal in NBS1m/m mice after DNA DSB damage, and p53 deficiency greatly facilitates tumorigenesis in NBS1m/m mice. Therefore, NBS1, H2AX, and p53 play synergistic roles in ATM-dependent DNA damage responses and tumor suppression.     

Oxidative stress induces cell cycle-dependent Mre11 recruitment,ATM and Chk2 activation and histone H2AX phosphorylation

DNA damage response recruits complex molecular machinery involved in DNA repair, arrest of cell cycle progression, and potentially in activation of apoptotic pathway. Among the first responders is the Mre11- (MRN) complex of proteins (Mre11, Rad50, Nbs1), essential for activation of ATM; the latter activates checkpoint kinase 2 (Chk2) and phosphorylates histone H2AX. In the present study the recruitment of Mre11 and phosphorylation of ATM, Chk2 and H2AX (γH2AX) detected immunocytochemically were measured by laser scanning cytometry to assess kinetics of these events in A549 cells treated with H2O2. Recruitment of Mre11 was rapid, peaked at 10 min of exposure to the oxidant, and was of similar extent in all phases of the cell cycle. ATM and Chk2 activation as well as H2AX phosphorylation reached maximum levels after 30 min of treatment with H2O2; the extent of phosphorylation of each was most prominent in S-, less in G₁-, and the least in G₂M- phase cells. A strong correlation between activation of ATM and Chk2, measured in the same cells, was seen in all phases of the cycle. In untreated cells activated Chk2 and Mre11 were distinctly present in centrosomes while in interphase cells they had characteristic punctate nuclear localization. The punctate expression of activated Chk2 both in untreated and H₂O₂ treated cells was accentuated when measured as maximal pixel rather than integrated value of immunofluorescence (IF) per nucleus, and was most pronounced in G₁ cells, likely reflecting the function of Chk2 in activating Cdc25A. Subpopulations of G₁ and G₂M cells with strong maximal pixel of Chk2-Thr68P IF in association with centrosomes were present in untreated cultures. Cytometric multiparameter assessment of the DNA damage response utilizing quantitative image analysis that allows one to measure inhomogeneity of fluorochrome distribution (e.g. maximal pixel) offers unique advantage in studies of the response of different cell constituents in relation to cell cycle position.