ATM signaling and genomic stability in response to DNA damage

DNA double strand breaks represent the most threatening lesion to the integrity of the genome in cells exposed to ionizing radiation and radiomimetic chemicals. Those breaks are recognized, signaled to cell cycle checkpoints and repaired by protein complexes. The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) plays a central role in the recognition and signaling of DNA damage. ATM is one of an ever growing number of proteins which when mutated compromise the stability of the genome and predispose to tumour development. Mechanisms for recognising double strand breaks in DNA, maintaining genome stability and minimizing risk of cancer are discussed.     

Viral oncogene-induced DNA damage response is activated in Kaposi sarcoma tumorigenesis

Kaposi sarcoma is a tumor consisting of Kaposi sarcoma herpesvirus (KSHV)-infected tumor cells that express endothelial cell (EC) markers and viral genes like v-cyclin, vFLIP, and LANA. Despite a strong link between KSHV infection and certain neoplasms, de novo virus infection of human primary cells does not readily lead to cellular transformation. We have studied the consequences of expression of v-cyclin in primary and immortalized human dermal microvascular ECs. We show that v-cyclin, which is a homolog of cellular D-type cyclins, induces replicative stress in ECs, which leads to senescence and activation of the DNA damage response. We find that antiproliferative checkpoints are activated upon KSHV infection of ECs, and in early-stage but not late-stage lesions of clinical Kaposi sarcoma specimens. These are some of the first results suggesting that DNA damage checkpoint response also functions as an anticancer barrier in virally induced cancers.     

WRN is required for ATM activation and the S-phase checkpoint in response to interstrand cross-link-induced DNA double-strand breaks

Werner syndrome (WS) is a human genetic disorder characterized by extensive clinical features of premature aging. Ataxia-telengiectasia (A-T) is a multisystem human genomic instability syndrome that includes premature aging in some of the patients. WRN and ATM, the proteins defective in WS and A-T, respectively, play significant roles in the maintenance of genomic stability and are involved in several DNA metabolic pathways. A role for WRN in DNA repair has been proposed; however, this study provides evidence that WRN is also involved in ATM pathway activation and in a S-phase checkpoint in cells exposed to DNA interstrand cross-link–induced double-strand breaks. Depletion of WRN in such cells by RNA interference results in an intra-S checkpoint defect, and interferes with activation of ATM as well as downstream phosphorylation of ATM target proteins. Treatment of cells under replication stress with the ATM kinase inhibitor KU 55933 results in a S-phase checkpoint defect similar to that observed in WRN shRNA cells. Moreover, γH2AX levels are higher in WRN shRNA cells than in control cells 6 and 16 h after exposure to psoralen DNA cross-links. These results suggest that WRN and ATM participate in a replication checkpoint response, in which WRN facilitates ATM activation in cells with psoralen DNA cross-link–induced collapsed replication forks.     

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.     

DNA ligase I is an in vivo substrate of DNA-dependent protein kinase and is activated by phosphorylation in response to DNA double-strand breaks

DNA-dependent protein kinase (DNA-PK) phosphorylates several cellular proteins in vitro, but its cellular function and natural substrate(s) in vivo are not established. We reported activation of DNA ligase in cultured normal human epidermal keratinocytes (NHEK) on exposure to the DNA-damaging compound bis-(2-chloroethyl) sulfide. The activated enzyme was identified as DNA ligase I, and this activation was attributed to phosphorylation of the enzyme. Here, we show that the phosphorylation is mediated by DNA-PK and that DNA ligase I is one of its natural substrates in vivo. DNA ligase I phosphorylation-cum-activation is a response specific to DNA double-strand breaks. We also demonstrate that affinity-purified inactive DNA ligase I is phosphorylated and activated in vitro by HeLa Cell DNA-PK confirming the in vivo observations. The findings specify the roles of DNA-PK and DNA ligase I in mammalian DNA double-strand break repair.     

Nuclear diacylglycerol kinase-theta is activated in response to alpha-thrombin

Currently, there is substantial evidence that nuclear lipid metabolism plays a critical role in a number of signal transduction cascades. Previous work from our laboratory showed that stimulation of quiescent fibroblasts with alpha-thrombin leads to the production of two lipid second messengers in the nucleus: an increase in nuclear diacylglycerol mass and an activation of phospholipase D, which catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid. Diacylglycerol kinase (DGK) catalyzes the conversion of diacylglycerol to phosphatidic acid, making it an attractive candidate for a signal transduction component. There is substantial evidence that this activity is indeed regulated in a number of signaling cascades (reviewed by van Blitterswijk, W. J., and Houssa, B. (1999) Chem. Phys. Lipids 98, 95-108). In this report, we show that the addition of alpha-thrombin to quiescent IIC9 fibroblasts results in an increase in nuclear DGK activity. The examination of nuclei isolated from quiescent IIC9 cells indicates that DGK-theta and DGK-delta are both present. We took advantage of the previous observations that phosphatidylserine inhibits DGK-delta (reviewed by Sakane, F., Imai, S., Kai, M., Wada, I., and Kanoh, H. (1996) J. Biol. Chem. 271, 8394-8401), and constitutively active RhoA inhibits DGK-theta (reviewed by Houssa, B., de Widt, J., Kranenburg, O., Moolenaar, W. H., and van Blitterswijk, W. J. (1999) J. Biol. Chem. 274, 6820-6822) to identify the activity induced by alpha-thrombin. Constitutively active RhoA inhibited the nuclear stimulated activity, whereas phosphatidylserine did not have an inhibitory effect. In addition, a monoclonal anti-DGK-theta antibody inhibited the alpha-thrombin-stimulated nuclear activity in vitro. These results demonstrate that DGK-theta is the isoform responsive to alpha-thrombin stimulation. Western blot and immunofluorescence microscopy analyses showed that alpha-thrombin induced the translocation of DGK-theta to the nucleus, implicating that this translocation is at least partly responsible for the increased nuclear activity. Taken together, these data are the first to demonstrate an agonist-induced activity of nuclear DGK-theta activity and a nuclear localization of DGK-delta.     

Akt is activated in response to an apoptotic signal

Akt is a serine-threonine kinase known to exert antiapoptotic effects through several downstream targets. Akt is cleaved during mitochondrial-mediated apoptosis in a caspase-dependent manner. The reason for this is not clear, however, because Akt has not been demonstrated to be activated in response to mitochondrial apoptotic stimuli. Accordingly, we explored whether the well described mitochondrial apoptotic stimuli staurosporine (STS) and etoposide activate Akt and whether such activation impacts apoptosis. Both STS and etoposide activated Akt in NIH 3T3 cells, maximally at 8 and 2 h, respectively, preceding the onset of apoptosis and poly(ADP-ribose) polymerase cleavage. The overexpression of Akt delayed STS-induced apoptosis with an even more pronounced delay observed with overexpression of constitutively active Akt. Akt activation by proapoptotic stimuli lay upstream of mitochondria, because neither caspase inhibitors nor overexpression of Bcl-2 or Bcl-x(L) could prevent it. Activation depended on phosphatidylinositol 3-kinase activity, however. Conversely, inhibition of phosphatidylinositol 3-kinase with wortmannin sensitized cells to apoptosis initiated by STS. These data demonstrate that mitochondrial apoptotic stimuli also activate Akt and such activation modulates apoptosis in this setting.     

53BP1, an activator of ATM in response to DNA damage

p53 Binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DNA damage checkpoint proteins with C-terminal BRCT domains and is most likely the human ortholog of the budding yeast Rad9 protein, the first cell cycle checkpoint protein to be described. 53BP1 localizes rapidly to sites of DNA double strand breaks (DSBs) and its initial recruitment to these sites has not been shown to be dependent on any other protein. Initially, 53BP1 was thought to be a mediator of DNA DSB signaling, but now it has been shown to function upstream of ataxia-telangiectasia mutated (ATM), in one of at least two parallel pathways leading to ATM activation in response to DNA damage. Currently, only a single tudor and two BRCT domains are recognized in 53BP1; however, their precise functional role is not understood. Elucidating the function of 53BP1 will be critical to understanding how cells recognize DNA DSBs and how ATM is activated.     

Proapoptotic BID is an ATM effector in the DNA-damage response

The "BH3-only" proapoptotic BCL-2 family members are sentinels of intracellular damage. Here, we demonstrated that the BH3-only BID protein partially localizes to the nucleus in healthy cells, is important for apoptosis induced by DNA damage, and is phosphorylated following induction of double-strand breaks in DNA. We also found that BID phosphorylation is mediated by the ATM kinase and occurs in mouse BID on two ATM consensus sites. Interestingly, BID-/- cells failed to accumulate in the S phase of the cell cycle following treatment with the topoisomerase II poison etoposide; reintroducing wild-type BID restored accumulation. In contrast, introducing a nonphosphorylatable BID mutant did not restore accumulation in the S phase and resulted in an increase in cellular sensitivity to etoposide-induced apoptosis. These results implicate BID as an ATM effector and raise the possibility that proapoptotic BID may also play a prosurvival role important for S phase arrest.     

ATR/ATM targets are phosphorylated by ATR in response to hypoxia and ATM in response to reoxygenation

The ATR kinase phosphorylates both p53 and Chk1 in response to extreme hypoxia (oxygen concentrations of less than 0.02%). In contrast to ATR, loss of ATM does not affect the phosphorylation of these or other targets in response to hypoxia. However, hypoxia within tumors is often transient and is inevitably followed by reoxygenation. We hypothesized that ATR activity is induced under hypoxic conditions because of growth arrest and ATM activity increases in response to the oxidative stress of reoxygenation. Using the comet assay to detect DNA damage, we find that reoxygenation induced significant amounts of DNA damage. Two ATR/ATM targets, p53 serine 15 and histone H2AX, were both phosphorylated in response to hypoxia in an ATR-dependent manner. These phosphorylations were then maintained in response to reoxygenation-induced DNA damage in an ATM-dependent manner. The reoxygenation-induced p53 serine 15 phosphorylation was inhibited by the addition of N-acetyl-l-cysteine (NAC), indicating that free radical-induced DNA damage was mediated by reactive oxygen species. Taken together these data implicate both ATR and ATM as critical roles in the response of hypoxia and reperfusion in solid tumors.     

The ATM kinase induces microRNA biogenesis in the DNA damage response

The DNA damage response involves a complex network of processes that detect and repair DNA damage. Here we show that miRNA biogenesis is globally induced upon DNA damage in an ATM-dependent manner. About one-fourth of miRNAs are significantly upregulated after DNA damage, while loss of ATM abolishes their induction. KH-type splicing regulatory protein (KSRP) is a key player that translates DNA damage signaling to miRNA biogenesis. The ATM kinase directly binds to and phosphorylates KSRP, leading to enhanced interaction between KSRP and pri-miRNAs and increased KSRP activity in miRNA processing. Mutations of the ATM phosphorylation sites of KSRP impaired its activity in regulating miRNAs. These findings reveal a mechanism by which DNA damage signaling is linked to miRNA biogenesis.     

Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response

Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition.     

Death-associated protein kinase is activated by dephosphorylation in response to cerebral ischemia

Death-associated protein kinase (DAPK) is a calcium calmodulin-regulated serine/threonine protein kinase involved in ischemic neuronal death. In situ hybridization experiments show that DAPK mRNA expression is up-regulated in brain following a global ischemic insult and down-regulated in ischemic tissues after focal ischemia. DAPK is inactive in normal brain tissues, where it is found in its phosphorylated state and becomes rapidly and persistently dephosphorylated and activated in response to ischemia in vivo. A similar dephosphorylation pattern is detected in primary cortical neurons subjected to oxygen glucose deprivation or N-methyl-D-aspartate (NMDA)-induced toxicity. Both a calcineurin inhibitor, FK506, and a selective NMDA receptor antagonist, MK-801, inhibit the dephosphorylation of DAPK after in vitro ischemia. This indicates that DAPK could be activated by NMDA receptor-mediated calcium flux, activation of calcineurin, and subsequent DAPK dephosphorylation. Moreover, concomitantly to dephosphorylation, DAPK is proteolytically processed by cathepsin after ischemia. Furthermore, a selective DAPK inhibitor is neuroprotective in both in vitro and in vivo ischemic models. These results indicate that DAPK plays a key role in mediating ischemic neuronal injury.     

Artemis is a phosphorylation target of ATM and ATR and is involved in the G2/M DNA damage checkpoint response

Mutations in Artemis in both humans and mice result in severe combined immunodeficiency due to a defect in V(D)J recombination. In addition, Artemis mutants are radiosensitive and chromosomally unstable, which has been attributed to a defect in nonhomologous end joining (NHEJ). We show here, however, that Artemis-depleted cell extracts are not defective in NHEJ and that Artemis-deficient cells have normal repair kinetics of double-strand breaks after exposure to ionizing radiation (IR). Artemis is shown, however, to interact with known cell cycle checkpoint proteins and to be a phosphorylation target of the checkpoint kinase ATM or ATR after exposure of cells to IR or UV irradiation, respectively. Consistent with these findings, our results also show that Artemis is required for the maintenance of a normal DNA damage-induced G2/M cell cycle arrest. Artemis does not appear, however, to act either upstream or downstream of checkpoint kinase Chk1 or Chk2. These results define Artemis as having a checkpoint function and suggest that the radiosensitivity and chromosomal instability of Artemis-deficient cells may be due to defects in cell cycle responses after DNA damage.     

DNA binding to proteolytically activated TLR9 is sequence-independent and enhanced by DNA curvature

Toll-like receptor 9 (TLR9) recognizes microbial DNA in endolysosomal compartments. The ectodomain of TLR9 must be proteolytically cleaved by endosomal proteases to produce the active receptor capable of inducing an innate immune signal. We show that the cleaved TLR9 ectodomain is a monomer in solution and that DNA ligands with phosphodiester backbones induce TLR9 dimerization in a sequence-independent manner. Ligands with phosphorothioate (PS) backbones induce the formation of large TLR9-DNA aggregates, possibly due to the propensity of PS ligands to self-associate. DNA curvature-inducing proteins including high-mobility group box 1 and histones H2A and H2B significantly enhance TLR9 binding, suggesting that TLR9 preferentially recognizes curved DNA backbones. Our work sheds light on the molecular mechanism of TLR9 activation by endogenous protein-nucleic acid complexes, which are associated with autoimmune diseases including systemic lupus erythematosus.     

Differential epithelium DNA damage response to ATM and DNA-PK pathway inhibition in human prostate tissue culture

The ability of cells to respond and repair DNA damage is fundamental for the maintenance of genomic integrity. Ex vivo culturing of surgery-derived human tissues has provided a significant advancement to assess DNA damage response (DDR) in the context of normal cytoarchitecture in a non-proliferating tissue. Here, we assess the dependency of prostate epithelium DDR on ATM and DNA-PKcs, the major kinases responsible for damage detection and repair by nonhomologous end-joining (NHEJ), respectively. DNA damage was caused by ionizing radiation (IR) and cytotoxic drugs, cultured tissues were treated with ATM and DNA-PK inhibitors, and DDR was assessed by phosphorylation of ATM and its targets H2AX and KAP1, a heterochromatin binding protein. Phosphorylation of H2AX and KAP1 was fast, transient and fully dependent on ATM, but these responses were moderate in luminal cells. In contrast, DNA-PKcs was phosphorylated in both luminal and basal cells, suggesting that DNA-PK-dependent repair was also activated in the luminal cells despite the diminished H2AX and KAP1 responses. These results indicate that prostate epithelial cell types have constitutively dissimilar responses to DNA damage. We correlate the altered damage response to the differential chromatin state of the cells. These findings are relevant in understanding how the epithelium senses and responds to DNA damage.Key words: DNA damage, prostate, γH2AX, ATM, DNA-PK     

Differential epithelium DNA damage response to ATM and DNA-PK pathway inhibition in human prostate tissue culture

The ability of cells to respond and repair DNA damage is fundamental for the maintenance of genomic integrity. Ex vivo culturing of surgery-derived human tissues has provided a significant advancement to assess DNA damage response (DDR) in the context of normal cytoarchitecture in a non-proliferating tissue. Here, we assess the dependency of prostate epithelium DDR on ATM and DNA-PKcs, the major kinases responsible for damage detection and repair by nonhomologous end-joining (NHEJ), respectively. DNA damage was caused by ionizing radiation (IR) and cytotoxic drugs, cultured tissues were treated with ATM and DNA-PK inhibitors, and DDR was assessed by phosphorylation of ATM and its targets H2AX and KAP1, a heterochromatin binding protein. Phosphorylation of H2AX and KAP1 was fast, transient and fully dependent on ATM, but these responses were moderate in luminal cells. In contrast, DNA-PKcs was phosphorylated in both luminal and basal cells, suggesting that DNA-PK-dependent repair was also activated in the luminal cells despite the diminished H2AX and KAP1 responses. These results indicate that prostate epithelial cell types have constitutively dissimilar responses to DNA damage. We correlate the altered damage response to the differential chromatin state of the cells. These findings are relevant in understanding how the epithelium senses and responds to DNA damage.