An overactivated ATR/CHK1 pathway is responsible for the prolonged G2 accumulation in irradiated AT cells

Induction of checkpoint responses in G1, S, and G2 phases of the cell cycle after exposure of cells to ionizing radiation (IR) is essential for maintaining genomic integrity. Ataxia telangiectasia mutated (ATM) plays a key role in initiating this response in all three phases of the cell cycle. However, cells lacking functional ATM exhibit a prolonged G2 arrest after IR, suggesting regulation by an ATM-independent checkpoint response. The mechanism for this ataxia telangiectasia (AT)-independent G2-checkpoint response remains unknown. We report here that the G2 checkpoint in irradiated human AT cells derives from an overactivation of the ATR/CHK1 pathway. Chk1 small interfering RNA abolishes the IR-induced prolonged G2 checkpoint and radiosensitizes AT cells to killing. These results link the activation of ATR/CHK1 with the prolonged G2 arrest in AT cells and show that activation of this G2 checkpoint contributes to the survival of AT cells.     

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.     

Dissecting cellular responses to irradiation via targeted disruptions of the ATM-CHK1-PP2A circuit

Exposure of proliferating cells to genotoxic stresses activates a cascade of signaling events termed the DNA damage response (DDR). The DDR preserves genetic stability by detecting DNA lesions, activating cell cycle checkpoints and promoting DNA damage repair. The phosphoinositide 3-kinase-related kinases (PIKKs) ataxia telangiectasia-mutated (ATM), ATM and Rad 3-related kinase (ATR) and DNA-dependent protein kinase (DNA-PK) are crucial for sensing lesions and signal transduction. The checkpoint kinase 1 (CHK1) is a traditional ATR target involved in DDR and normal cell cycle progression and represents a pharmacological target for anticancer regimens. This study employed cell lines stably depleted for CHK1, ATM or both for dissecting cross-talk and compensatory effects on G?/M checkpoint in response to ionizing radiation (IR). We show that a 90% depletion of CHK1 renders cells radiosensitive without abrogating their IR-mediated G?/M checkpoint arrest. ATM phosphorylation is enhanced in CHK1-deficient cells compared with their wild-type counterparts. This correlates with lower nuclear abundance of the PP2A catalytic subunit in CHK1-depleted cells. Stable depletion of CHK1 in an ATM-deficient background showed only a 50% reduction from wild-type CHK1 protein expression levels and resulted in an additive attenuation of the G?/M checkpoint response compared with the individual knockdowns. ATM inhibition and 90% CHK1 depletion abrogated the early G?/M checkpoint and precluded the cells from mounting an efficient compensatory response to IR at later time points. Our data indicates that dual targeting of ATM and CHK1 functionalities disrupts the compensatory response to DNA damage and could be exploited for developing efficient anti-neoplastic treatments.     

Both ERK1 and ERK2 kinases promote G2/M arrest in etoposide-treated MCF7 cells by facilitating ATM activation

The MEK–ERK pathway plays a role in DNA damage response (DDR). This has been thoroughly studied by modulating MEK activation. However, much less has been done to directly examine the contributions of ERK1 and ERK2 kinases to DDR. Etoposide induces G2/M arrest in a variety of cell lines, including MCF7 cells. DNA damage-induced G2/M arrest depends on the activation of the protein kinase ataxia-telangiectasia mutated (ATM). ATM subsequently activates CHK2 by phosphorylating CHK2 threonine 68 (T68) and CHK2 inactivates CDC25C via phosphorylation of its serine 216 (S216), resulting in G2/M arrest. To determine the contribution of ERK1 and ERK2 to etoposide-induced G2/M arrest, we individually knocked-down ERK1 and ERK2 in MCF7 cells using specific small interfering RNA (siRNA). Knockdown of either kinases significantly reduced ATM activation in response to etoposide treatment, and thereby attenuated phosphorylation of the ATM substrates, including the S139 of H2AX (γH2AX), p53 S15, and CHK2 T68. Consistent with these observations, knockdown of either ERK1 or ERK2 reduced etoposide-induced CDC25C S216 phosphorylation and significantly compromised etoposide-induced G2/M arrest in MCF7 cells. Taken together, we demonstrated that both ERK1 and ERK2 kinases play a role in etoposide-induced G2/M arrest by facilitating activation of the ATM pathway. These observations suggest that a cellular threshold level of ERK kinase activity is required for the proper checkpoint activation in MCF7 cells.     

DNA replication defects, spontaneous DNA damage, and ATM-dependent checkpoint activation in replication protein A-deficient cells

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding complex comprised of 70-kDa (RPA1), 32-kDa (RPA2), and 14-kDa (RPA3) subunits that is essential for DNA replication, recombination, and repair in eukaryotes. In addition, recent studies using vertebrate model systems have suggested an important role for RPA in the initiation of cell cycle checkpoints following exposure to DNA replication stress. Specifically, RPA has been implicated in the recruitment and activation of the ATM-Rad3-related protein kinase, ATR, which in conjunction with the related kinase, ATM (ataxia-telangiectasia-mutated), transmits checkpoint signals via the phosphorylation of downstream effectors. In this report, we have explored the effects of RPA insufficiency on DNA replication, cell survival, and ATM/ATR-dependent signal transduction in response to genotoxic stress. RNA interference-mediated suppression of RPA1 caused a slowing of S phase progression, G2/M cell cycle arrest, and apoptosis in HeLa cells. RPA-deficient cells demonstrated high levels of spontaneous DNA damage and constitutive activation of ATM, which was responsible for the terminal G2/M arrest phenotype. Surprisingly, we found that neither RPA1 nor RPA2 were essential for the hydroxyurea- or UV-induced phosphorylation of the ATR substrates CHK1 and CREB (cyclic AMP-response element-binding protein). These findings reveal that RPA is required for genomic stability and suggest that activation of ATR can occur through RPA-independent pathways.     

p14ARF activates a Tip60-dependent and p53-independent ATM/ATR/CHK pathway in response to genotoxic stress

p14ARF is a tumor suppressor that controls a well-described p53/Mdm2-dependent checkpoint in response to oncogenic signals. Here, new insights into the tumor-suppressive function of p14ARF are provided. We previously showed that p14ARF can induce a p53-independent G2 cell cycle arrest. In this study, we demonstrate that the activation of ATM/ATR/CHK signaling pathways contributes to this G2 checkpoint and highlight the interrelated roles of p14ARF and the Tip60 protein in the initiation of this DNA damage-signaling cascade. We show that Tip60 is a new direct p14ARF binding partner and that its expression is upregulated and required for ATM/CHK2 activation in response to p14ARF. Strikingly, both p14ARF and Tip60 products accumulate following a cell treatment with alkylating agents and are absolutely required for ATM/CHK2 activation in this setting. Moreover, and consistent with p14ARF being a determinant of CHK2 phosphorylation in lung carcinogenesis, a strong correlation between p14ARF and phospho-CHK2 (Thr68) protein expression is observed in human lung tumors (P < 0.00006). Overall, these data point to a novel regulatory pathway that mediates the p53-independent negative-cell-growth control of p14ARF. Inactivation of this pathway is likely to contribute to lung carcinogenesis.     

Inactivation of ATM/ATR DNA Damage Checkpoint Promotes Androgen Induced Chromosomal Instability in Prostate Epithelial Cells

The ATM/ATR DNA damage checkpoint functions in the maintenance of genetic stability and some missense variants of the ATM gene have been shown to confer a moderate increased risk of prostate cancer. However, whether inactivation of this checkpoint contributes directly to prostate specific cancer predisposition is still unknown. Here, we show that exposure of non-malignant prostate epithelial cells (HPr-1AR) to androgen led to activation of the ATM/ATR DNA damage response and induction of cellular senescence. Notably, knockdown of the ATM gene expression in HPr-1AR cells can promote androgen-induced TMPRSS2: ERG rearrangement, a prostate-specific chromosome translocation frequently found in prostate cancer cells. Intriguingly, unlike the non-malignant prostate epithelial cells, the ATM/ATR DNA damage checkpoint appears to be defective in prostate cancer cells, since androgen treatment only induced a partial activation of the DNA damage response. This mechanism appears to preserve androgen induced autophosphorylation of ATM and phosphorylation of H2AX, lesion processing and repair pathway yet restrain ATM/CHK1/CHK2 and p53 signaling pathway. Our findings demonstrate that ATM/ATR inactivation is a crucial step in promoting androgen-induced genomic instability and prostate carcinogenesis.     

Distinct roles for DNA-PK,ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress

DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.     

Caffeine inhibits checkpoint responses without inhibiting the ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases

The ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) kinases regulate cell cycle checkpoints by phosphorylating multiple substrates including the CHK1 and -2 protein kinases and p53. Caffeine has been widely used to study ATM and ATR signaling because it inhibits these kinases in vitro and overcomes cell cycle checkpoint responses in vivo. Thus, caffeine has been thought to overcome the checkpoint through its ability to prevent phosphorylation of ATM and ATR substrates. Surprisingly, I have found that multiple ATM-ATR substrates including CHK1 and -2 are hyperphosphorylated in cells treated with caffeine and genotoxic agents such as hydroxyurea or ionizing radiation. ATM autophosphorylation in cells is also increased when caffeine is used in combination with inhibitors of replication suggesting that ATM activity is not inhibited in vivo by caffeine. Furthermore, CHK1 hyperphosphorylation induced by caffeine in combination with hydroxyurea is ATR-dependent suggesting that ATR activity is stimulated by caffeine. Finally, the G2/M checkpoint in response to ionizing radiation or hydroxyurea is abrogated by caffeine treatment without a corresponding decrease in ATM-ATR-dependent signaling. This data suggests that although caffeine is an inhibitor of ATM-ATR kinase activity in vitro, it can block checkpoints without inhibiting ATM-ATR activation in vivo.     

ATP activates ataxia-telangiectasia mutated (ATM) in vitro. Importance of autophosphorylation

Ataxia-telangiectasia Mutated (ATM), mutated in the human disorder ataxia-telangiectasia, is rapidly activated by DNA double strand breaks. The mechanism of activation remains unresolved, and it is uncertain whether autophosphorylation contributes to activation. We describe an in vitro immunoprecipitation system demonstrating activation of ATM kinase from unirradiated extracts by preincubation with ATP. Activation is both time- and ATP concentration-dependent, other nucleotides fail to activate ATM, and DNA is not required. ATP activation is specific for ATM since it is not observed with kinase-dead ATM, it requires Mn2+, and it is inhibited by wortmannin. Exposure of activated ATM to phosphatase abrogates activity, and repeat cycles of ATP and phosphatase treatment reveal a requirement for autophosphorylation in the activation process. Phosphopeptide mapping revealed similarities between the patterns of autophosphorylation for irradiated and ATP-treated ATM. Caffeine inhibited ATM kinase activity for substrates but did not interfere with ATM autophosphorylation. ATP failed to activate either A-T and rad3-related protein (ATR) or DNA-dependent protein kinase under these conditions, supporting the specificity for ATM. These data demonstrate that ATP can specifically induce activation of ATM by a mechanism involving autophosphorylation. The relationship of this activation to DNA damage activation remains unclear but represents a useful model for understanding in vivo activation.     

S phase and G2 arrests induced by topoisomerase I poisons are dependent on ATR kinase function.

ATR, a human phosphatidylinositol 3-kinase-related kinase, is an important component of the cellular response to DNA damage. In the present study, we evaluated the role of ATR in modulating the response of cells to S phase-associated DNA double-stranded breaks induced by topoisomerase poisons. Prolonged exposure to low doses of the topoisomerase I poison topotecan (TPT) resulted in S phase slowing because of diminished DNA synthesis at late-firing replicons. In contrast, brief TPT exposure, as well as prolonged exposure to the topoisomerase II poison etoposide, resulted in subsequent G(2) arrest. These responses were associated with phosphorylation of the checkpoint kinase Chk1. The cell cycle responses and phosphorylation of Chk1 were markedly diminished by forced overexpression of a dominant negative, kinase-inactive allele of ATR. In contrast, deficiency of the related kinase ATM had no effect on these events. The loss of ATR-dependent checkpoint function sensitized GM847 human fibroblasts to the cytotoxic effects of the topoisomerase I poisons TPT and 7-ethyl-10-hydroxycamptothecin, as assessed by inhibition of colony formation, increased trypan blue uptake, and development of apoptotic morphological changes. Expression of kdATR also sensitized GM847 cells to the cytotoxic effects of prolonged low dose etoposide and doxorubicin, albeit to a smaller extent. Collectively, these results not only suggest that ATR is important in responding to the replication-associated DNA damage from topoisomerase poisons, but also support the view that ATM and ATR have unique roles in activating the downstream kinases that participate in cell cycle checkpoints.     

Depletion of ATR selectively sensitizes ATM-deficient human mammary epithelial cells to ionizing radiation and DNA-damaging agents

DNA damage response (DDR) to double strand breaks is coordinated by 3 phosphatidylinositol 3-kinase-related kinase (PIKK) family members: the ataxia-telangiectasia mutated kinase (ATM), the ATM and Rad3-related (ATR) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). ATM and ATR are central players in activating cell cycle checkpoints and function as an active barrier against genome instability and tumorigenesis in replicating cells. Loss of ATM function is frequently reported in various types of tumors, thus placing more reliance on ATR for checkpoint arrest and cell survival following DNA damage. To investigate the role of ATR in the G₂/M checkpoint regulation in response to ionizing radiation (IR), particularly when ATM is deficient, cell lines deficient of ATM, ATR, or both were generated using a doxycycline-inducible lentiviral system. Our data suggests that while depletion of ATR or ATM alone in wild-type human mammary epithelial cell cultures (HME-CCs) has little effect on radiosensitivity or IR-induced G₂/M checkpoint arrest, depletion of ATR in ATM-deficient cells causes synthetic lethality following IR, which correlates with severe G₂/M checkpoint attenuation. ATR depletion also inhibits IR-induced autophagy, regardless of the ATM status, and enhances IR-induced apoptosis particularly when ATM is deficient. Collectively, our results clearly demonstrate that ATR function is required for the IR-induced G₂/M checkpoint activation and subsequent survival of cells with ATM deficiency. The synthetic lethal interaction between ATM and ATR in response to IR supports ATR as a therapeutic target for improved anti-cancer regimens, especially in tumors with a dysfunctional ATM pathway.     

ATR pathway is the primary pathway for activating G2/M checkpoint induction after re-replication

DNA replication is tightly controlled to ensure accurate chromosome duplication and segregation in each cell cycle. Inactivation of Geminin, an inhibitor of origin licensing, leads to re-replication in human tumor cells within the same cell cycle and triggers a G(2)/M checkpoint. We find that the primary pathway to signal that re-replication has been detected is the ATR kinase and the Rad9-Rad1-Hus1 (9-1-1) clamp complex together with Rad17-RFC clamp loader. ATM kinase and the Mre11-Rad50-Nbs1 complex do not appear to play significant roles in the checkpoint. Chk1 activation occurs at early stages, whereas Chk2 activation occurs much later. Overall we conclude that ATR/Chk1 pathway is activated at an early time point after the loss of Geminin and contributes to checkpoint arrest essential for the accumulation of re-replicated cells, whereas activation of the ATM/Chk2 pathway is a by-product of DNA re-replication at a later period.     

The DNA crosslink-induced S-phase checkpoint depends on ATR-CHK1 and ATR-NBS1-FANCD2 pathways

The genetic syndrome Fanconi anemia (FA) is characterized by aplastic anemia, cancer predisposition and hypersensitivity to DNA interstrand crosslinks (ICLs). FA proteins (FANCs) are thought to work in pathway(s) essential for dealing with crosslinked DNA. FANCs interact with other proteins involved in both DNA repair and S-phase checkpoint such as BRCA1, ATM and the RAD50/MRE11/NBS1 (RMN) complex. We deciphered the previously undefined pathway(s) leading to the ICLs-induced S-phase checkpoint and the role of FANCs in this process. We found that ICLs activate a branched pathway downstream of the ATR kinase: one branch depending on CHK1 activity and the other on the FANCs-RMN complex. The transient slow-down of DNA synthesis was abolished in cells lacking ATR, whereas CHK1-siRNA-treated cells, NBS1 or FA cells showed partial S-phase arrest. CHK1 RNAi in NBS1 or FA cells abolished the S-phase checkpoint, suggesting that CHK1 and FANCs/NBS1 proteins work on parallel pathways. Furthermore, we found that ICLs trigger ATR-dependent FANCD2 phosphorylation and FANCD2/ATR colocalization. This study demonstrates a novel relationship between the FA pathway(s) and the ATR kinase.     

The relative contribution of CHK1 and CHK2 to Adriamycin-induced checkpoint

Topoisomerase II poisons like Adriamycin (ADR, doxorubicin) are clinically important chemotherapeutic agents. Adriamycin-induced DNA damage checkpoint activates ATM and ATR, which could in turn inhibit the cell cycle engine through either CHK1 or CHK2. In this study, we characterized whether CHK1 or CHK2 is required for Adriamycin-induced checkpoint. We found that both CHK1 and CHK2 were phosphorylated after Adriamycin treatment. Several lines of evidence from dominant-negative mutants, short hairpin RNA (shRNA), and knockout cells indicated that CHK1, but not CHK2, is critical for Adriamycin-induced cell cycle arrest. Disruption of CHK1 function bypassed the checkpoint, as manifested by the increase in CDC25A, activation of CDC2, increase in histone H3 phosphorylation, and reduction in cell survival after Adriamycin treatment. In contrast, CHK2 is dispensable for Adriamycin-induced responses. Finally, we found that CHK1 was upregulated in primary hepatocellular carcinoma (HCC), albeit as an inactive form. The presence of a stockpile of dormant CHK1 in cancer cells may have important implications for treatments like topoisomerase II poisons. Collectively, the available data underscore the pivotal role of CHK1 in checkpoint responses to a variety of stresses.     

Ku70/80 modulates ATM and ATR signaling pathways in response to DNA double strand breaks

Double strand break (DSB) recognition is the first step in the DSB damage response and involves activation of ataxia telangiectasia-mutated (ATM) and phosphorylation of targets such as p53 to trigger cell cycle arrest, DNA repair, or apoptosis. It was reported that activation of ATM- and Rad3-related (ATR) kinase by DSBs also occurs in an ATM-dependent manner. On the other hand, Ku70/80 is known to participate at a later time point in the DSB response, recruiting DNA-PKcs to facilitate non-homologous end joining. Because Ku70/80 has a high affinity for broken DNA ends and is abundant in nuclei, we examined their possible involvement in other aspects of the DSB damage response, particularly in modulating the activity of ATM and other phosphatidylinositol (PI) 3-related kinases during DSB recognition. We thus analyzed p53(Ser18) phosphorylation in irradiated Ku-deficient cells and observed persistent phosphorylation in these cells relative to wild type cells. ATM or ATR inhibition revealed that this phosphorylation is mainly mediated by ATM-dependent ATR activity at 2 h post-ionizing radiation in wild type cells, whereas in Ku-deficient cells, this occurs mainly through direct ATM activity, with a secondary contribution from ATR via a novel ATM-independent mechanism. Using ATM/Ku70 double-null cell lines, which we generated, we confirmed that ATM-independent ATR activity contributed to persistent phosphorylation of p53(Ser18) in Ku-deficient cells at 12 h post-ionizing radiation. In summary, we discovered a novel role for Ku70/80 in modulating ATM-dependent ATR activation during DSB damage response and demonstrated that these proteins confer a protective effect against ATM-independent ATR activation at later stages of the DSB damage response.     

Two sides of the Myc-induced DNA damage response: from tumor suppression to tumor maintenance

ABSTRACT: Activation of oncogenes is generally associated with the induction of DNA damage response (DDR) signaling, which acts as a barrier to tumor progression. In this review we will present an overview of the DDR associated with oncogenic activation of Myc, with special focus on two opposite and paradoxical aspects of this response: (1) the role of the Myc-induced DDR in tumor suppression; (2) its role in dampening Myc-induced replication stress, thereby protecting the viability of prospective cancer cells. These opposing effects on cancer progression are controlled by two different branches of DDR signaling, respectively ATM/CHK2 and ATR/CHK1. Indeed, while ATM activity constitutes a barrier to malignant transformation, full activation of ATR and CHK1 is essential for tumor maintenance, providing important opportunities for therapeutic intervention. Thus, the Myc-induced DDR acts as a double-edged sword in tumor progression.