DNA Damage Checkpoint Kinase Chk1 Research Articles

Ubiquitination of the DNA-damage checkpoint kinase CHK1 by TRAF4 is required for CHK1 activation

BackgroundAberrant activation of DNA damage response (DDR) is a major cause of chemoresistance in colorectal cancer (CRC). CHK1 is upregulated in CRC and contributes to therapeutic resistance. We investigated the upstream signaling pathways governing CHK1 activation in CRC.MethodsWe identified CHK1-binding proteins by mass spectrometry analysis. We analyzed the biologic consequences of knockout or overexpression of TRAF4 using immunoblotting, immunoprecipitation, and immunofluorescence. CHK1 and TRAF4 ubiquitination was studied in vitro and in vivo. We tested the functions of TRAF4 in CHK1 phosphorylation and CRC chemoresistance by measuring cell viability and proliferation, anchorage-dependent and -independent cell growth, and mouse xenograft tumorigenesis. We analyzed human CRC specimens by immunohistochemistry.ResultsTRAF4 catalyzed the ubiquitination of CHK1 in multiple CRC cell lines. Following DNA damage, ubiquitination of CHK1 at K132 by TRAF4 is required for CHK1 phosphorylation and activation mediated by ATR. Notably, TRAF4 was highly expressed in chemotherapy-resistant CRC specimens and positively correlated with phosphorylated CHK1. Furthermore, depletion of TRAF4 impaired CHK1 activity and sensitized CRC cells to fluorouracil and other chemotherapeutic agents in vitro and in vivo.ConclusionsThese data reveal two novel steps required for CHK1 activation in which TRAF4 serves as a critical intermediary and suggest that inhibition of the ATR–TRAF4–CHK1 signaling may overcome CRC chemoresistance.

Eya2 promotes cell cycle progression by regulating DNA damage response during vertebrate limb regeneration

How salamanders accomplish progenitor cell proliferation while faithfully maintaining genomic integrity and regenerative potential remains elusive. Here we found an innate DNA damage response mechanism that is evident during blastema proliferation (early- to late-bud) and studied its role during tissue regeneration by ablating the function of one of its components, Eyes absent 2. In eya2 mutant axolotls, we found that DNA damage signaling through the H2AX histone variant was deregulated, especially within the proliferating progenitors during limb regeneration. Ultimately, cell cycle progression was impaired at the G1/S and G2/M transitions and regeneration rate was reduced. Similar data were acquired using acute pharmacological inhibition of the Eya2 phosphatase activity and the DNA damage checkpoint kinases Chk1 and Chk2 in wild-type axolotls. Together, our data indicate that highly-regenerative animals employ a robust DNA damage response pathway which involves regulation of H2AX phosphorylation via Eya2 to facilitate proper cell cycle progression upon injury.

Abstract 4298: Targeting the DNA damage checkpoint kinase Chk1 induces multiple pathways of cytotoxicity

Abstract DNA damaging agents are standard therapies for many cancers, but result in limited long-term clinical benefit. DNA damage checkpoints arrest DNA synthesis and provide time for repair and recovery. Using DNA checkpoint inhibitors to sensitize cancer cells to traditional chemotherapeutics is an emerging strategy. However, clinical trials involving Checkpoint Kinase 1 inhibitors (Chk1i) plus DNA-damaging chemotherapies have shown little clinical benefit. This may be due to sub-optimal scheduling of combinations. We have published that delaying Chk1i until 18 h after gemcitabine confers the greatest sensitization of cancer cells. We have now characterized the mechanisms by which the Chk1i, MK-8776, sensitizes human cancer cells to gemcitabine and SN38. SN38, the active metabolite of irinotecan, traps topoisomerase I on DNA resulting in S phase arrest. Chk1i re-activates replication and drives cells into mitosis before the damage from SN38 is repaired. Co-incubation with a CDK1/2 inhibitor surprisingly failed to prevent S phase progression, but did block mitotic entry and prevented further DNA damage. Conversely, the replication-associated kinase, CDC7, is essential for Chk1i-mediated S phase abrogation. CDC7 is presumably required to activate dormant origins of replication. Gemcitabine inhibits ribonucleotide reductase, thus preventing production of deoxyribonucleotides. Addition of Chk1i 18 h following gemcitabine causes aberrant activation of DNA helicases, but without deoxyribonucleotides, this results in excessive single-stranded DNA. Eventually, the level of single-stranded DNA exceeds cellular stores of the protective protein, RPA, which leads to nucleolytic cleavage and double-strand DNA breaks. Chk1i-mediated helicase activation is again dependent on CDC7, but insensitive to a CDK1/2 inhibitor. Concurrent incubation with gemcitabine plus MK8776 does not activate DNA helicases. These data provide a rationale for a delayed administration of Chk1i when patients are receiving gemcitabine. These investigations have detailed how Chk1i elicits distinct mechanisms of cytotoxicity depending on the context of DNA damage. Understanding of these mechanisms may help identify biomarkers to predict patient response. Furthermore, the clinical development of several Chk1i have been terminated due to unfavorable toxicity, but the cause of toxicity remains unknown. The work described here may help the clinical development by determining whether the observed toxicities are due to on- or off-targets. Citation Format: Nicholas Warren, Jennifer Ditano, Alan Eastman. Targeting the DNA damage checkpoint kinase Chk1 induces multiple pathways of cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4298.

The drinking water contaminant dibromoacetonitrile delays G1-S transition and suppresses Chk1 activation at broken replication forks

Chlorination of drinking water protects humans from water-born pathogens, but it also produces low concentrations of dibromoacetonitrile (DBAN), a common disinfectant by-product found in many water supply systems. DBAN is not mutagenic but causes DNA breaks and elevates sister chromatid exchange in mammalian cells. The WHO issued guidelines for DBAN after it was linked with cancer of the liver and stomach in rodents. How this haloacetonitrile promotes malignant cell transformation is unknown. Using fission yeast as a model, we report here that DBAN delays G1-S transition. DBAN does not hinder ongoing DNA replication, but specifically blocks the serine 345 phosphorylation of the DNA damage checkpoint kinase Chk1 by Rad3 (ATR) at broken replication forks. DBAN is particularly damaging for cells with defects in the lagging-strand DNA polymerase delta. This sensitivity can be explained by the dependency of pol delta mutants on Chk1 activation for survival. We conclude that DBAN targets a process or protein that acts at the start of S phase and is required for Chk1 phosphorylation. Taken together, DBAN may precipitate cancer by perturbing S phase and by blocking the Chk1-dependent response to replication fork damage.

Two Distinct Cdc2 Pools Regulate Cell Cycle Progression and the DNA Damage Response in the Fission Yeast S.pombe

The activity of Cdc2 (CDK1) kinase, which coordinates cell cycle progression and DNA break repair, is blocked upon its phosphorylation at tyrosine 15 (Y15) by Wee1 kinase in the presence of DNA damage. How Cdc2 can support DNA repair whilst being inactivated by the DNA damage checkpoint remains to be explained. Human CDK1 is phosphorylated by Myt1 kinase at threonine 14 (T14) close to its ATP binding site before being modified at threonine 161 (T167Sp) in its T-loop by the CDK-activating kinase (CAK). While modification of T161 promotes association with the cyclin partner, phosphorylation of T14 inhibits the CDK1-cyclin complex. This inhibition is further enforced by the modification of Y15 by Wee1 in the presence of DNA lesions. In S.pombe, the dominant inhibition of Cdc2 is provided by the phosphorylation of Y15 and only a small amount of Cdc2 is modified at T14 when cells are in S phase. Unlike human cells, both inhibitory modifications are executed by Wee1. Using the novel IEFPT technology, which combines isoelectric focusing (IEF) with Phos-tag SDS electrophoresis (PT), we report here that S.pombe Cdc2 kinase exists in seven forms. While five forms are phosphorylated, two species are not. Four phospho-forms associate with cyclin B (Cdc13) of which only two are modified at Y15 by Wee1. Interestingly, only one Y15-modified species carries also the T14 modification. The fifth phospho-form has a low affinity for cyclin B and is neither Y15 nor T14 modified. The two unphosphorylated forms may contribute directly to the DNA damage response as only they associate with the DNA damage checkpoint kinase Chk1. Interestingly, cyclin B is also present in the unphosphorylated pool. We also show that the G146D mutation in Cdc2.1w, which renders Cdc2 insensitive to Wee1 inhibition, is aberrantly modified in a Wee1-dependent manner. In conclusion, our work adds support to the idea that two distinct Cdc2 pools regulate cell cycle progression and the response to DNA damage.

Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints

Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as “authentic” bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents’ gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient’s tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers.

Abstract B57: Bosutinib and bosutinib-isomer are novel Chk1 and Wee1 kinase inhibitors that sensitize cells to DNA damaging agent by overriding cell cycle checkpoint arrest.

Abstract Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well tolerated by patients. We took advantage of the promiscuous nature of kinase inhibitors and screened 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells (Panc1) to a sub-lethal concentration of gemcitabine. bosutinib, dovitinib and BEZ-235, were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA approved Src / Abl inhibitor for the treatment of chronic myelogenous leukemia (CML). Other Src-family kinase inhibitors did not exhibit chemosensitization activity. Unbeknownst to us, the bosutinib we purchased was an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as "authentic" bosutinib. The two compounds differed only in the arrangement of the same R groups around the aniline ring. Authentic bosutinib is designated 2, 4 dichloro, 5-methoxy, while Bosutinib isomer is 3, 5 dichloro, 4-methyoxy. Although our screen identified the isomer, we found that authentic bosutinib also had chemosensitization activity In vitro. Both bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1 in vitro, with Bos-I showing greater potency. These inhibitors enhanced sensitivity to genotoxic agents that either arrest cells in S phase (gemcitabine, cisplatin) or G2 (doxorubicin). Live-cell imaging and immunofluorescence staining showed that Bos-I forced drug-treated cells to override the checkpoint arrest and entered an aberrant mitosis. The in vivo efficacy of bosutinib and Bos-I were validated using cells derived directly from a pancreatic cancer patient's tumor. Notably, the xenograft studies showed that the combination of gemcitabine and bosutinib or Bos-I were significantly more effective in suppressing tumor growth than any of the agents used alone. Consistent with the in vitro data that showed Bos-I was more potent checkpoint inhibitor than bosutinib, higher concentrations of bosutinib were needed to suppress tumor growth by gemcitabine. Molecular modeling of bosutinib and Bos-I bound to Chk1 and Wee1 indicate that the gatekeeper residues and the position of the methoxy group in the aniline ring may be critical determinants of drug binding. The methoxy group of bosutinib (position 5) clashes within the binding pocket particularly near the gatekeeper residues Leu and Asn, in Chk1 and Wee1 respectively. The methoxy group in the Bos-I (position 3) is shifted away from the gatekeeper and allows the inhibitor to bind deeper in the binding site for both kinases. The structural predictions are supported by relative binding energy calculations of bosutininb and Bos-I bound to these kinases. In addition, in vitro kinase assays that show that changing the gatekeeper residue in Wee1 reduced its sensitivity to Bos-I but enhanced its sensitivity to the authentic bosutinib. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers. Checkpoint override by bosutinib and its isomer maybe due to ability to dually inhibit Chk1 and Wee1 kinases. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B57. Citation Format: Neil Beeharry, Eugenia Banina, Vladmir Khazak, Sean Deacon, Jeff Peterson, Mark Andrake, Igor Astsaturov, Tim J. Yen. Bosutinib and bosutinib-isomer are novel Chk1 and Wee1 kinase inhibitors that sensitize cells to DNA damaging agent by overriding cell cycle checkpoint arrest. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B57.

Combination Therapy Targeting the Chk1 and Wee1 Kinases Shows Therapeutic Efficacy in Neuroblastoma

Neuroblastoma is uniquely sensitive to single-agent inhibition of the DNA damage checkpoint kinase Chk1, leading us to examine downstream effectors of this pathway and identify mitotic regulator Wee1 as an additional therapeutic target in this disease. Wee1 was overexpressed in both neuroblastoma cell lines and high-risk patient tumors. Genetic or pharmacologic abrogation of Wee1 signaling results in marked cytotoxicity in 10 of 11 neuroblastoma cell lines with a median IC(50) of 300 nmol/L for the Wee1-selective small-molecule inhibitor MK-1775. Murine tumor lines derived from mice that were either heterozygous or homozygous for MycN were particularly sensitive to single-agent inhibition of Wee1 (IC(50)s of 160 and 62 nmol/L, respectively). Simultaneous pharmacologic inhibition of Chk1 and Wee1 acted in a synergistic fashion to further impede neuroblastoma cell growth in vitro, in a manner greater than the individual inhibitors either alone or combined with chemotherapy. Combination Chk1 and Wee1 inhibition also revealed in vivo efficacy in neuroblastoma xenografts. Taken together, our results show that neuroblastoma cells depend on Wee1 activity for growth and that inhibition of this kinase may serve as a therapeutic for patients with neuroblastoma.

Structure-Specific DNA Endonuclease Mus81/Eme1 Generates DNA Damage Caused by Chk1 Inactivation

The DNA-damage checkpoint kinase Chk1 is essential in higher eukaryotes due to its role in maintaining genome stability in proliferating cells. CHK1 gene deletion is embryonically lethal, and Chk1 inhibition in replicating cells causes cell-cycle defects that eventually lead to perturbed replication and replication-fork collapse, thus generating endogenous DNA damage. What is the cause of replication-fork collapse when Chk1 is inactivated, however, remains poorly understood. Here, we show that generation of DNA double-strand breaks at replication forks when Chk1 activity is compromised relies on the DNA endonuclease complex Mus81/Eme1. Importantly, we show that Mus81/Eme1-dependent DNA damage—rather than a global increase in replication-fork stalling—is the cause of incomplete replication in Chk1-deficient cells. Consequently, Mus81/Eme1 depletion alleviates the S-phase progression defects associated with Chk1 deficiency, thereby increasing cell survival. Chk1-mediated protection of replication forks from Mus81/Eme1 even under otherwise unchallenged conditions is therefore vital to prevent uncontrolled fork collapse and ensure proper S-phase progression in human cells.

The DNA Damage Response Signaling Cascade Regulates Proliferation of the Phytopathogenic FungusUstilago maydisin Planta

In the phytopathogenic fungus Ustilago maydis, the dikaryotic state dominates the period of growth occurring during the infectious phase. Dikaryons are cells in which two nuclei, one from each parent cell, share a single cytoplasm for a period of time without undergoing nuclear fusion. In fungal cells, maintenance of the dikaryotic state requires an intricate cell division process that often involves the formation of a structure known as the clamp connection as well as the sorting of one of the nuclei to this structure to ensure that each daughter dikaryon inherits a balance of each parental genome. Here, we describe an atypical role of the DNA damage checkpoint kinases Chk1 and Atr1 during pathogenic growth of U. maydis. We found that Chk1 and Atr1 collaborate to control cell cycle arrest during the induction of the virulence program in U. maydis and that Chk1 and Atr1 work together to control the dikaryon formation. These findings uncover a link between a widely conserved signaling cascade and the virulence program in a phytopathogen. We propose a model in which adjustment of the cell cycle by the Atr1-Chk1 axis controls fidelity in dikaryon formation. Therefore, Chk1 and Atr1 emerge as critical cell type regulators in addition to their roles in the DNA damage response.

Abstract 3872: The role and regulation of C/EBPalpha in the DNA damage-induced G1 checkpoint

Abstract The ability of cells to correctly respond to DNA damage is a vital step in maintaining genome stability and suppressing tumorigenesis. The C/EBP family of transcription factors is involved in the regulation of cell proliferation, growth arrest, differentiation, inflammation, senescence and energy metabolism in a range of cell types. Previously, we have shown that C/EBPα is highly inducible by DNA damage through a p53-dependent mechanism and has a novel role in the DNA damage G1 checkpoint. Furthermore, cells deficient in C/EBPα display an impaired G1 checkpoint and inappropriate entry into S-phase following DNA damage; suggesting loss of C/EBPα could result in genome/checkpoint instability. While it is known that C/EBPα has a critical role in the DNA damage-induced G1 checkpoint, the molecular mechanism through which it regulates the DNA damage G1 checkpoint is unknown. Moreover, it is not known whether C/EBPα itself is regulated by post-translational modifications (PTMs) downstream of DNA damage. We report that C/EBPα undergoes PTMs involving phosphorylation in response to UVB-induced DNA damage. These modifications are in part dependent of the activity of the DNA damage checkpoint kinase Chk1 and occur in both cultured mouse keratinocytes and in vivo mouse epidermis that have been exposed to UVB. Co-immunoprecipitation studies revealed that C/EBPα forms a complex with E2F1 and this interaction is increased following UVB-induced DNA damage. We observed that ectopic expression of C/EBPα in keratinocytes inhibits E2F1 transcriptional activity. These findings indicate that C/EBPα can function downstream of DNA damage to regulate E2F1and induce a cell cycle arrest independent of C/EBPα transcriptional activity. Additionally, we observe that C/EBPα functions in the canonical DNA damage-induced G1 checkpoint response through regulation of the CDK inhibitor p21. Ablation of C/EBPα in primary mouse keratinocytes or in vivo mouse epidermis results in severely impaired induction of p21 in response to DNA damage. Furthermore, we show that the diminished p21 protein levels in C/EBPα deficient mice following exposure to UVB occur without any changes in p21 mRNA levels. We propose that C/EBPα is required to stabilize p21 in response to DNA damage; enabling p21 to accumulate to levels necessary to elicit the DNA damage G1 checkpoint response. In conclusion, we report that C/EBPα undergoes post-translational modifications downstream of DNA damage and has a multi-faceted role in the DNA damage-induced G1 checkpoint response. We propose C/EBPα inhibits cell cycle progression in response to DNA damage through suppression of E2F1 transcriptional activity and stabilization of the CDK inhibitor p21 protein. This study describes a novel mechanism for the function and regulation of C/EBPα in the DNA damage-induced G1 checkpoint. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3872.

A role for the DNA-damage checkpoint kinase Chk1 in the virulence program of the fungusUstilago maydis

During induction of the virulence program in the phytopathogenic fungus Ustilago maydis, the cell cycle is arrested on the plant surface and it is not resumed until the fungus enters the plant. The mechanism of this cell cycle arrest is unknown, but it is thought that it is necessary for the correct implementation of the virulence program. Here, we show that this arrest takes place in the G2 phase, as a result of an increase in the inhibitory phosphorylation of the catalytic subunit of the mitotic cyclin-dependent kinase Cdk1. Sequestration in the cytoplasm of the Cdc25 phosphatase seems to be one of the reasons for the increase in inhibitory phosphorylation. Strikingly, we also report the DNA-damage checkpoint kinase Chk1 appears to be involved in this process. Our results support the emerging idea that checkpoint kinases have roles other than in the DNA-damage response, by virtue of their ability to interact with the cell cycle machinery.

Kinase Drug Discovery by Affinity Selection/Mass Spectrometry (ASMS): Application to DNA Damage Checkpoint Kinase Chk1

Kinase enzymes are involved in a vast array of biological processes associated with human disease; therefore, selective kinase inhibition by small molecules and therapeutic antibodies is an area of intense study. The authors show that drug candidates with immediate value for biological preclinical evaluation can be identified directly through ultra-efficient affinity screening of kinase enzymes and random compound mixtures. The screening process comprises sampling and trapping equilibrium binding between candidate ligands and protein in solution, followed by removal of unbound ligands via 3 rounds of ultrafiltration and direct identification of bound ligands by mass spectrometry. Evaluation of significant peaks is facilitated by automated integration and collation of the mass spectral data and import into custom software for analysis. One Chk1-selective ligand found by using this process is presented in detail. The compound is potent in both enzymatic and Chk1-dependent cellular assays, and specific contacts in the Chk1 active site are shown by X-ray crystallography.

Schlafen-1 Causes a Cell Cycle Arrest by Inhibiting Induction of Cyclin D1

Schlafen-1 (Slfn-1), the prototypic member of the Schlafen family of proteins, was described as an inducer of growth arrest in T-lymphocytes and causes a cell cycle arrest in NIH3T3 fibroblasts prior to the G1/S transition. How Slfn-1 exerts its effects on the cell cycle is not currently known. We report that synchronized murine fibroblasts expressing Slfn-1 do not exit G1 when stimulated with fetal calf serum, platelet-derived growth factor BB (PDGF-BB) or epidermal growth factor (EGF). The induction of cyclin D1 by these stimuli was blocked in the presence of Slfn-1 as were all downstream cell cycle processes. Overexpression of cyclin D1 in growth-arrested, Slfn-1-expressing cells induced an increase in cell growth consistent with this protein being the biological target of Slfn-1. Activation of the mitogen-activated protein kinase pathway by EGF or phorbol 12-myristate 13-acetate was unaffected by Slfn-1 expression. PDGF signaling was, however, almost completely blocked. This was due to a lack of PDGF receptor expression in Slfn-1-expressing cells consistent with Slfn-1 blocking the cell cycle in G1 where PDGF receptor expression is normally down-regulated. Finally, overexpression of Slfn-1 inhibited the activation of the cyclin D1 promoter. Slfn-1 therefore causes a cell cycle arrest during G1 by inhibiting induction of cyclin D1 by mitogens.

A DNA damage checkpoint response in telomere-initiated senescence.

Most human somatic cells can undergo only a limited number of population doublings in vitro. This exhaustion of proliferative potential, called senescence, can be triggered when telomeres--the ends of linear chromosomes-cannot fulfil their normal protective functions. Here we show that senescent human fibroblasts display molecular markers characteristic of cells bearing DNA double-strand breaks. These markers include nuclear foci of phosphorylated histone H2AX and their co-localization with DNA repair and DNA damage checkpoint factors such as 53BP1, MDC1 and NBS1. We also show that senescent cells contain activated forms of the DNA damage checkpoint kinases CHK1 and CHK2. Furthermore, by chromatin immunoprecipitation and whole-genome scanning approaches, we show that the chromosome ends of senescent cells directly contribute to the DNA damage response, and that uncapped telomeres directly associate with many, but not all, DNA damage response proteins. Finally, we show that inactivation of DNA damage checkpoint kinases in senescent cells can restore cell-cycle progression into S phase. Thus, we propose that telomere-initiated senescence reflects a DNA damage checkpoint response that is activated with a direct contribution from dysfunctional telomeres.