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.     

TGFβ1-Induced Activation of ATM and p53 Mediates Apoptosis in a Smad7-Dependent Manner

ATM, a DNA-damage sensitive kinase and p53, are frequently inactivated in a variety of cancers as they together with γH2AX are critical guardians against DNA damage. Here, we report of a functional cross-talk between the cytokine TGFβ and p53, leading to apoptosis of epithelial cells, involving Smad7, a TGF-β target gene, p38 MAP kinase, and ATM. Using ectopic expression of p53, siRNA for Smad7, p38α -/- deficient cells and specific inhibitors, we show that TGF-β induces apoptosis via ATM and p53 in epithelial cells. Intriguingly, Smad7 act as a scaffold protein to promote functional interactions between p38, ATM and p53 upon TGFβ treatment, facilitating their activation. Smad7 colocalizes with γH2AX in DNA damage foci and was required for proper cell cycle checkpoints to prevent genetic instability. Our data imply that Smad7 plays a crucial role upstream of ATM and p53 to protect the genome from insults evoked by extracellular stress.     

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.     

Activation of DNA damage response signaling in mouse embryonic stem cells

Mouse embryonic stem cells (mESC) are characterized by high proliferation activity. mESC are highly sensitive to genotoxic stresses and do not undergo G1/S checkpoint upon DNA-damage. mESC are supposed to develop sensitive mechanisms to maintain genomic integrity provided by either DNA damage repair or elimination of defected cells by apoptosis. The issue of how mESC recognize the damages and execute DNA repair remains to be studied. We analyzed the kinetics of DNA repair foci marked by antibodies to phosphorylated ATM kinase and histone H2AX (γH2AX). We showed that mESC display non-induced DNA single-strand breaks (SSBs), as revealed by comet-assay, and a noticeable background of γH2AX staining. Exposure of mESC to γ-irradiation induced the accumulation of phosphorylated ATM-kinase in the nucleus as well as the formation of additional γH2AX foci, which disappeared thereafter. To decrease the background of γH2AX staining in control non-irradiated cells, we pre-synchronized mESC at the G2/M by low concentration of nocodazol for a short time (6 h). The cells were then irradiated and stained for γH2AX. Irradiation induced the formation of γH2AX foci both in G2-phase and mitotic cells, which evidenced for the active state of DNA-damage signaling at these stages of the cell cycle in mESC. Due to the G1/S checkpoint is compromised in mESCs, we checked, whether wild-type p53, a target for ATM kinase, was phosphorylated in response to γ-irradiation. The p53 was barely phosphorylated in response to irradiation, which correlated with a very low expression of p53-target p21/Waf1 gene. Thus, in spite of the dysfunction of the p53/Waf1 pathway and the lack of cell cycle checkpoints, the mESC are capable of activating ATM and inducing γH2AX foci formation, which are necessary for the activation of DNA damage response.     

Growth of persistent foci of DNA damage checkpoint factors is essential for amplification of G1 checkpoint signaling

Several DNA damage checkpoint factors form nuclear foci in response to ionizing radiation (IR). Although the number of the initial foci decreases concomitantly with DNA double-strand break repair, some fraction of foci persists. To date, the physiological role of the persistent foci has been poorly understood. Here we examined foci of Ser1981-phosphorylated ATM in normal human diploid cells exposed to 1Gy of X-rays. While the initial foci size was approximately 0.6microm, the one or two of persistent focus (foci) grew, whose diameter reached 1.6microm or more in diameter at 24h after IR. All of the grown persistent foci of phosphorylated ATM colocalized with the persistent foci of Ser139-phosphorylated histone H2AX, MDC1, 53BP1, and NBS1, which also grew similarly. When G0-synchronized normal human cells were released immediately after 1Gy of X-rays and incubated for 24h, the grown large phosphorylated ATM foci (> or =1.6microm) were rarely (av. 0.9%) observed in S phase cells, while smaller foci (<1.6microm) were frequently (av. 45.9%) found. We observed significant phosphorylation of p53 at Ser15 in cells with a single grown phosphorylated ATM focus. Furthermore, persistent inhibition of foci growth of phosphorylated ATM by an ATM inhibitor, KU55933, completely abrogated p53 phosphorylation. Defective growth of the persistent IR-induced foci was observed in primary fibroblasts derived from ataxia-telangiectasia (AT) and Nijmegen breakage syndrome (NBS) patients, which were abnormal in IR-induced G1 checkpoint. These results indicate that the growth of the persistent foci of the DNA damage checkpoint factors plays a pivotal role in G1 arrest, which amplifies G1 checkpoint signals sufficiently for phosphorylating p53 in cells with a limited number of remaining foci.     

Deregulation of DNA damage signal transduction by herpesvirus latency-associated M2

Infected cells recognize viral replication as a DNA damage stress and elicit a DNA damage response that ultimately induces apoptosis as part of host immune surveillance. Here, we demonstrate a novel mechanism where the murine gamma herpesvirus 68 (gammaHV68) latency-associated, anti-interferon M2 protein inhibits DNA damage-induced apoptosis by interacting with the DDB1/COP9/cullin repair complex and the ATM DNA damage signal transducer. M2 expression constitutively induced DDB1 nuclear localization and ATM kinase activation in the absence of DNA damage. Activated ATM subsequently induced Chk activation and p53 phosphorylation and stabilization without eliciting H2AX phosphorylation and MRN recruitment to foci upon DNA damage. Consequently, M2 expression inhibited DNA repair, rendered cells resistant to DNA damage-induced apoptosis, and induced a G(1) cell cycle arrest. Our results suggest that gammaHV68 M2 blocks apoptosis-mediated intracellular innate immunity, which might ultimately contribute to its role in latent infection.     

Activation of ATM/ATR signaling in human embryonic stem cells after DNA damage

Embryonic stem cells (ESCs) are the progenitors of all adult cells; consequently, genomic abnormalities in them may be catastrophic for the developing organism. ESCs are characterized by high proliferation activity and do not stop in checkpoints upon DNA-damage executing only G₂/M delay after DNA damage. ATM and ATR kinases are key sensors of double-strand DNA breaks and activate downstream signaling pathways involving checkpoints, DNA repair, and apoptosis. We examined activation of ATM/ATR signaling in human ESCs and revealed that irradiation induced ATM, ATR, and Chk2 phosphorylation, and γH2AX foci formation and their colocalization with 53BP1 and Rad51 proteins. Interestingly, human ESCs exhibit noninduced γH2AX foci colocalized with Rad51 and marking single-strand DNA breaks. Next, we revealed the significant contribution of ATM, Chk1, and Chk2 kinases to G₂/M block after irradiation and ATM-dependent activation (phosphorylation) of p53 in human ESCs. However, p53 activation and subsequent induction of p21 ^Waf1 gene expression after DNA damage do not result in p21^Waf1 protein accumulation due to its proteasomal degradation.     

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.     

Acetylation of Histone H2AX at Lys 5 by the TIP60 Histone Acetyltransferase Complex Is Essential for the Dynamic Binding of NBS1 to Damaged Chromatin

The association and dissociation of DNA damage response (DDR) factors with damaged chromatin occurs dynamically, which is crucial for the activation of DDR signaling in a spatiotemporal manner. We previously showed that the TIP60 histone acetyltransferase complex acetylates histone H2AX, to facilitate H2AX exchange at sites of DNA damage. However, it remained unclear how the acetylation of histone H2AX by TIP60 is related to the DDR signaling. We found that the acetylation but not the phosphorylation of H2AX is essential for the turnover of NBS1 on damaged chromatin. The loss of H2AX acetylation at Lys 5 by TIP60 in cells disturbed the accumulation of NBS1 at sites of DNA damage. Although the phosphorylation of H2AX is also reportedly required for the retention of NBS1 at damage sites, our data indicated that the acetylation-dependent NBS1 turnover by TIP60 on damaged chromatin restricts the dispersal of NBS1 foci from the sites of DNA damage. These findings indicate the importance of the acetylation-dependent dynamic binding of NBS1 to damaged chromatin, created by histone H2AX exchange, for the proper accumulation of NBS1 at DNA damage sites.     

DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1

Activation of the ataxia telangiectasia mutated (ATM) kinase triggers diverse cellular responses to ionizing radiation (IR), including the initiation of cell cycle checkpoints. Histone H2AX, p53 binding-protein 1 (53BP1) and Chk2 are targets of ATM-mediated phosphorylation, but little is known about their roles in signalling the presence of DNA damage. Here, we show that mice lacking either H2AX or 53BP1, but not Chk2, manifest a G2-M checkpoint defect close to that observed in ATM(-/-) cells after exposure to low, but not high, doses of IR. Moreover, H2AX regulates the ability of 53BP1 to efficiently accumulate into IR-induced foci. We propose that at threshold levels of DNA damage, H2AX-mediated concentration of 53BP1 at double-strand breaks is essential for the amplification of signals that might otherwise be insufficient to prevent entry of damaged cells into mitosis.     

Impaired elimination of DNA double-strand break-containing lymphocytes in ataxia telangiectasia and Nijmegen breakage syndrome

The repair of DNA double-strand breaks is critical for genome integrity and tumor suppression. Here we show that following treatment with the DNA-intercalating agent actinomycin D (ActD), normal quiescent T cells accumulate double-strand breaks and die, whereas T cells from ataxia telangiectasia (AT) and Nijmegen breakage syndrome (NBS) patients are resistant to this death pathway despite a comparable amount of DNA damage. We demonstrate that the ActD-induced death pathway in quiescent T lymphocytes follows DNA damage and H2AX phosphorylation, is ATM- and NBS1-dependent and due to p53-mediated cellular apoptosis. In response to genotoxic 2-Gy gamma-irradiation, on the other hand, quiescent T cells from normal donors survive following complete resolution of the damage thus induced. T cells from AT and NBS patients also survive, but retain foci of phosphorylated H2AX due to a subtle double-strand break (DSB) repair defect. A common consequence of these two genetic defects in the DSB response is the apparent tolerance of cells containing DNA breaks. We suggest that this tolerance makes a major contribution to the oncogenic risk of patients with chromosome instability syndromes.     

Enhanced H2AX Phosphorylation,DNA Replication Fork Arrest,and Cell Death in the Absence of Chk1

H2AX phosphorylation at serine 139 (γH2AX) is a sensitive indicator of both DNA damage and DNA replication stress. Here we show that γH2AX formation is greatly enhanced in response to replication inhibitors but not ionizing radiation in HCT116 or SW480 cells depleted of Chk1. Although H2AX phosphorylation precedes the induction of apoptosis in such cells, our results suggest that cells containing γH2AX are not committed to death. γH2AX foci in these cells largely colocalize with RPA foci and their formation is dependent upon the essential replication helicase cofactor Cdc45, suggesting that H2AX phosphorylation occurs at sites of stalled forks. However Chk1-depleted cells released from replication inhibitors retain γH2AX foci and do not appear to resume replicative DNA synthesis. BrdU incorporation only occurs in a minority of Chk1-depleted cells containing γH2AX foci after release from thymidine arrest and, in cells incorporating BrdU, DNA synthesis does not occur at sites of γH2AX foci. Furthermore activated ATM and Chk2 persist in these cells. We propose that the γH2AX foci in Chk1-depleted cells may represent sites of persistent replication fork damage or abandonment that are unable to resume DNA synthesis but do not play a direct role in the Chk1 suppressed death pathway.     

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.     

Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX

53BP1 participates in the cellular response to DNA damage. Like many proteins involved in the DNA damage response, 53BP1 becomes hyperphosphorylated after radiation and colocalizes with phosphorylated H2AX in megabase regions surrounding the sites of DNA strand breaks. However, it is not yet clear whether the phosphorylation status of 53BP1 determines its localization or vice versa. In this study we mapped a region upstream of the 53BP1 C terminus that is required and sufficient for the recruitment of 53BP1 to these DNA break areas. In vitro assays revealed that this region binds to phosphorylated but not unphosphorylated H2AX. Moreover, using H2AX-deficient cells reconstituted with wild-type or a phosphorylation-deficient mutant of H2AX, we have shown that phosphorylation of H2AX at serine 140 is critical for efficient 53BP1 foci formation, implying that a direct interaction between 53BP1 and phosphorylated H2AX is required for the accumulation of 53BP1 at DNA break sites. On the other hand, radiation-induced phosphorylation of the 53BP1 N terminus by the ATM (ataxia-telangiectasia mutated) kinase is not essential for 53BP1 foci formation and takes place independently of 53BP1 redistribution. Thus, these two damage-induced events, hyperphosphorylation and relocalization of 53BP1, occur independently in the cell.     

The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks

The maintenance of genome stability requires efficient DNA double-stranded break (DSB) repair mediated by the phosphorylation of multiple histone H2AX molecules near the break sites. The phosphorylated H2AX (γ-H2AX) molecules form foci covering many megabases of chromatin. The formation of g-H2AX foci is critical for efficient DNA damage response (DDR) and for the maintenance of genome stability, however, the mechanisms of protein organization in foci is largely unknown. To investigate the nature of γ-H2AX foci formation, we analyzed the distribution of γ-H2AX and other DDR proteins at DSB sites using a variety of techniques to visualize, expand and partially disrupt chromatin. We report here that γ-H2AX foci change composition during the cell cycle, with proteins 53BP1, NBS1 and MRE11 dissociating from foci in G2 and mitosis to return at the beginning of the following G1. In contrast, MDC1 remained colocalized with g-H2AX during mitosis. In addition, while γ-H2AX was found to span large domains flanking DSB sites, 53BP1 and NBS1 were more localized and MDC1 colocalized in doublets in foci. H2AX and MDC1 were found to be involved in chromatin relaxation after DSB formation. Our data demonstrates that the DSB repair focus is a heterogeneous and dynamic structure containing internal complexity.     

Cell cycle-dependent DNA damage signaling induced by ICRF-193 involves ATM, ATR, CHK2, and BRCA1

Topoisomerase II is essential for cell proliferation and survival and has been a target of various anticancer drugs. ICRF-193 has long been used as a catalytic inhibitor to study the function of topoisomerase II. Here, we show that ICRF-193 treatment induces DNA damage signaling. Treatment with ICRF-193 induced G2 arrest and DNA damage signaling involving gamma-H2AX foci formation and CHK2 phosphorylation. DNA damage by ICRF-193 was further demonstrated by formation of the nuclear foci of 53BP1, NBS1, BRCA1, MDC1, and FANCD2 and increased comet tail moment. The DNA damage signaling induced by ICRF-193 was mediated by ATM and ATR and was restricted to cells in specific cell cycle stages such as S, G2, and mitosis including late and early G1 phases. Downstream signaling of ATM and ATR involved the phosphorylation of CHK2 and BRCA1. Altogether, our results demonstrate that ICRF-193 induces DNA damage signaling in a cell cycle-dependent manner and suggest that topoisomerase II might be essential for the progression of the cell cycle at several stages including DNA decondensation.     

Differentiation-induced radioresistance in muscle cells

DNA damage induces cell cycle arrest and DNA repair or apoptosis in proliferating cells. Terminally differentiated cells are permanently withdrawn from the cell cycle and partly resistant to apoptosis. To investigate the effects of genotoxic agents in postmitotic cells, we compared DNA damage-activated responses in mouse and human proliferating myoblasts and their differentiated counterparts, the myotubes. DNA double-strand breaks caused by ionizing radiation (IR) induced rapid activating autophosphorylation of ataxia-teleangiectasia-mutated (ATM), phosphorylation of histone H2AX, recruitment of repair-associated proteins MRE11 and Nbs1, and activation of Chk2 in both myoblasts and myotubes. However, IR-activated, ATM-mediated phosphorylation of p53 at serine 15 (human) or 18 (mouse) [Ser15(h)/18(m)], and apoptosis occurred in myoblasts but was impaired in myotubes. This phosphorylation could be enforced in myotubes by the anthracycline derivative doxorubicin, leading to selective activation of proapoptotic genes. Unexpectedly, the abundance of autophosphorylated ATM was indistinguishable after exposure of myotubes to IR (10 Gy) or doxorubicin (1 microM/24 h) despite efficient phosphorylation of p53 Ser15(h)/18(m), and apoptosis occurred only in response to doxorubicin. These results suggest that radioresistance in myotubes might reflect a differentiation-associated, pathway-selective blockade of DNA damage signaling downstream of ATM. This mechanism appears to preserve IR-induced activation of the ATM-H2AX-MRE11/Rad50/Nbs1 lesion processing and repair pathway yet restrain ATM-p53-mediated apoptosis, thereby contributing to life-long maintenance of differentiated muscle tissues.