Study of the cytolethal distending toxin (CDT)-activated cell cycle checkpoint. Involvement of the CHK2 kinase

The bacterial cytolethal distending toxin (CDT) triggers a G2/M cell cycle arrest in eukaryotic cells by inhibiting the CDC25C phosphatase-dependent CDK1 dephosphorylation and activation. We report that upon CDT treatment CDC25C is fully sequestered in the cytoplasmic compartment, an effect that is reminiscent of DNA damage-dependent checkpoint activation. We show that the checkpoint kinase CHK2, an upstream regulator of CDC25C, is phosphorylated and activated after CDT treatment. In contrast to what is observed with other DNA damaging agents, we demonstrate that the activation of CHK2 can only take place during S-phase. Use of wortmannin and caffeine suggests that this effect is not dependent on ATM but rather on another as yet unidentified PI3 kinase family member. These results confirm that the CDT is therefore responsible for specific genomic injuries that block cell proliferation by activating a cell cycle checkpoint.     

CHK2在肿瘤中作用的研究进展

细胞周期检测点激酶2(cell cycle checkpoint kinase 2,CHK2)是抑癌基因CHK2编码的丝氨酸/苏氨酸激酶,广泛存在于哺乳动物中。它可在DNA双链断裂后参与DNA修复应答,并作为重要的信号转导蛋白,使细胞周期进程发生阻滞,从而促进DNA修复或诱导细胞凋亡。目前在肺癌、乳腺癌、结直肠癌等多种恶性肿瘤均发现CHK2的缺失,研究者发现CHK2是增强DNA损伤治疗在癌症中治疗效果的良好靶标。CHK2基因作为肿瘤治疗的重要研究靶点,为肿瘤的治疗研究提供了新的方向。本文就CHK2目前在肿瘤中的研究进展做简要综述。     

Differential mode of regulation of the checkpoint kinases CHK1 and CHK2 by their regulatory domains

CHK1 and CHK2 are key mediators that link the machineries that monitor DNA integrity to components of the cell cycle engine. Despite the similarity and potential redundancy in their functions, CHK1 and CHK2 are unrelated protein kinases, each having a distinctive regulatory domain. Here we compare how the regulatory domains of human CHK1 and CHK2 modulate the respective kinase activities. Recombinant CHK1 has only low basal activity when expressed in cultured cells. Surprisingly, disruption of the C-terminal regulatory domain activates CHK1 even in the absence of stress. Unlike the full-length protein, C-terminally truncated CHK1 displays autophosphorylation, phosphorylates CDC25C on Ser(216), and delays cell cycle progression. Intriguingly, enzymatic activity decreases when the entire regulatory domain is removed, suggesting that the regulatory domain contains both inhibitory and stimulatory elements. Conversely, the kinase domain suppresses Ser(345) phosphorylation, a major ATM/ATR phosphorylation site in the regulatory domain. In marked contrast, CHK2 expressed in either mammalian cells or in bacteria is already active as a kinase against itself and CDC25C and can delay cell cycle progression. Unlike CHK1, disruption of the regulatory domain of CHK2 abolishes its kinase activity. Moreover, the regulatory domain of CHK2, but not that of CHK1, can oligomerize. Finally, CHK1 but not CHK2 is phosphorylated during the spindle assembly checkpoint, which correlates with the inhibition of the kinase. The mitotic phosphorylation of CHK1 requires the regulatory domain, does not involve Ser(345), and is independent on ATM. Collectively, these data reveal the very different mode of regulation between CHK1 and CHK2.     

CHK1 Affecting Cell Radiosensitivity is Independent of Non-Homologous End Joining

CHK1 is one of the most important checkpoint proteins in mammalian cells for responding toDNA damage. Cells defective in CHK1 are sensitive to ionizing radiation (IR). The mechanismby which CHK1 protects cells from IR-induced killing remains unclear. DNA double strandbreaks (DSBs) induced by IR are critical lesions for cell survival. Two major complementaryDNA DSBs repair pathways exist in mammalian cells, homologous recombination repair (HRR)and non-homologous end joining (NHEJ). By using CHK1 kinase dead human cell linesestablished in our laboratory, we show here that although these human cell lines have differentCHK1 activities with different sensitivities to IR-induced killing and G2 accumulation, all thesecell lines show similar inductions and rejoining rates of DNA DSBs. These results indicate thatthe different radiosensitivities and G2 checkpoint responses in these cell lines are independent ofNHEJ, suggesting that CHK1-regulated checkpoint facilitates HRR and therefore protects cellsfrom IR-induced killing.     

CHK1-Regulated S-phase Checkpoint Response Reduces Camptothecin Cytotoxity

The cytotoxicity of camptothecin (CPT) is S phase specific and is associated with an inhibition of DNA replication. The relationship between CPT-induced inhibition of DNA replication and CPT cytotoxicity remains unclear. We previously reported that the CPT-induced inhibition reflects an activated S-phase (S) checkpoint response and that this response is mainly regulated by ATR/CHK1 pathway. In this study, by comparing A1-5 and B4, the two transformed rat embryo fibroblasts cell lines, we showed that with higher CHK1 expression, A1-5 cells had a stronger S checkpoint response and were more resistant to CPT-treatment. The data suggested that over-activated CHK1 in CPT-treated A1-5 cells regulated the strong S checkpoint response through the CDC25A/CDK2 pathway. When the CHK-1 regulated strong S checkpoint response was abolished, A1-5 cells became much more sensitive to CPT-induced killing. These data indicated that CHK1 regulated S checkpoint response protected cells from CPT-induced killing.

Key Words:

CHK1, S-phase checkpoint, Camptothecin, DNA damage     

CHK1-regulated S-phase checkpoint response reduces camptothecin cytotoxicity

The cytotoxicity of camptothecin (CPT) is S phase specific and is associated with an inhibition of DNA replication. The relationship between CPT-induced inhibition of DNA replication and CPT cytotoxicity remains unclear. We previously reported that the CPT-induced inhibition reflects an activated S-phase (S) checkpoint response and that this response is mainly regulated by ATR/CHK1 pathway. In this study, by comparing A1-5 and B4, the two transformed rat embryo fibroblasts cell lines, we showed that with higher CHK1 expression, A1-5 cells had a stronger S checkpoint response and were more resistant to CPT-treatment. The data suggested that over-activated CHK1 in CPT-treated A1-5 cells regulated the strong S checkpoint response through the CDC25A/CDK2 pathway. When the CHK-1 regulated strong S checkpoint response was abolished, A1-5 cells became much more sensitive to CPT-induced killing. These data indicated that CHK1 regulated S checkpoint response protected cells from CPT-induced killing.     

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.     

Loss of Geminin induces rereplication in the presence of functional p53

Strict regulation of DNA replication is essential to ensure proper duplication and segregation of chromosomes during the cell cycle, as its deregulation can lead to genomic instability and cancer. Thus, eukaryotic organisms have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. Here, we show that inactivation of Geminin, an inhibitor of origin licensing, leads to rereplication in human normal and tumor cells within the same cell cycle. We found a CHK1-dependent checkpoint to be activated in rereplicating cells accompanied by formation of gammaH2AX and RAD51 nuclear foci. Abrogation of the checkpoint leads to abortive mitosis and death of rereplicated cells. In addition, we demonstrate that the induction of rereplication is dependent on the replication initiation factors CDT1 and CDC6, and independent of the functional status of p53. These data show that Geminin is required for maintaining genomic stability in human cells.     

CHK2-dependent phosphorylation of BRCA1 in hypoxia

Hypoxia induces a diverse spectrum of changes in the expression and activity of numerous DNA repair factors within the tumor microenvironment. In particular, we and others have shown that hypoxia induces phosphorylation and activation of the checkpoint kinase, CHK2, in an ATM-dependent manner. One downstream target of CHK2, the BRCA1 protein, plays a critical role in both DNA repair and cell cycle checkpoint regulation in mammalian cells. Here we report that BRCA1 is specifically phosphorylated on Serine 988 in response to hypoxic stress, and phosphorylation at this site is dependent on CHK2 expression. These findings enhance our understanding of ATM-CHK2 pathway activation in hypoxia, and they identify a novel role for BRCA1 in the response to hypoxic stress.     

Inhibition of checkpoint kinase 2 (CHK2) enhances sensitivity of pancreatic adenocarcinoma cells to gemcitabine

Checkpoint kinase 2 (CHK2) plays pivotal function as an effector of cell cycle checkpoint arrest following DNA damage. Recently, we found that co‐treatment of NSC109555 (a potent and selective CHK2 inhibitor) potentiated the cytotoxic effect of gemcitabine (GEM) in pancreatic cancer MIA PaCa‐2 cells. Here, we further examined whether NSC109555 could enhance the antitumour effect of GEM in pancreatic adenocarcinoma cell lines. In this study, the combination treatment of NSC109555 plus GEM demonstrated strong synergistic antitumour effect in four pancreatic cancer cells (MIA PaCa‐2, CFPAC‐1, Panc‐1 and BxPC‐3). In addition, the GEM/NSC109555 combination significantly increased the level of intracellular reactive oxygen species (ROS), accompanied by induction of apoptotic cell death. Inhibition of ROS generation by N‐acetyl cysteine (NAC) significantly reversed the effect of GEM/NSC109555 in apoptosis and cytotoxicity. Furthermore, genetic knockdown of CHK2 by siRNA enhanced GEM‐induced apoptotic cell death. These findings suggest that inhibition of CHK2 would be a beneficial therapeutic approach for pancreatic cancer therapy in clinical treatment.     

TTK/hMps1 participates in the regulation of DNA damage checkpoint response by phosphorylating CHK2 on threonine 68

CHK2/hCds1 plays important roles in the DNA damage-induced cell cycle checkpoint by phosphorylating several important targets, such as Cdc25 and p53. To obtain a better understanding of the CHK2 signaling pathway, we have carried out a yeast two-hybrid screen to search for potential CHK2-interacting proteins. Here, we report the identification of the mitotic checkpoint kinase, TTK/hMps1, as a novel CHK2-interacting protein. TTK/hMps1 directly phosphorylates CHK2 on Thr-68 in vitro. Expression of a TTK kinase-dead mutant, TTK(D647A), interferes with the G(2)/M arrest induced by either ionizing radiation or UV light. Interestingly, induction of CHK2 Thr-68 phosphorylation and of several downstream events, such as cyclin B1 accumulation and Cdc2 Tyr-15 phosphorylation, is also affected. Furthermore, ablation of TTK expression using small interfering RNA results not only in reduced CHK2 Thr-68 phosphorylation, but also in impaired growth arrest. Our results are consistent with a model in which TTK functions upstream from CHK2 in response to DNA damage and suggest possible cross-talk between the spindle assembly checkpoint and the DNA damage checkpoint.     

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.     

Phosphorylation at Threonine 288 by Cell Cycle Checkpoint Kinase 2 (CHK2) Controls Human Monopolar Spindle 1 (Mps1) Kinetochore Localization

Human Mps1 (hMps1) is a mitotic checkpoint kinase responsible for sensing the unattached and tensionless kinetochore. Despite its importance in safeguarding proper chromosome segregation, how hMps1 is recruited to the kinetochore remains incompletely understood. Here, we demonstrate that phosphorylation at Thr-288 by the cell cycle checkpoint kinase CHK2 is involved in this process. We discovered that the phosphorylation-deficient T288A mutant has an impaired ability to localize to the kinetochore and cannot reestablish the mitotic checkpoint in hMps1-depleted cells. In support, we found that nocodazole induced hMps1 phosphorylation at the previously identified CHK2 site Thr-288 and that this could be detected at the kinetochore in a CHK2-dependent manner. Mechanistically, phosphorylation at Thr-288 promoted the interaction with the KMN (KNL1-Mis12-Ndc80 network) protein HEC1. Forced kinetochore localization corrected the defects associated with the T288A mutant. Our results provide evidence of a newly identified hMps1 phosphorylation site that is involved in the mitotic checkpoint and that CHK2 contributes to chromosomal stability through hMps1.     

NEK11−Linking CHK1 and CDC25A in DNA damage checkpoint signaling

The DNA damage induced G₂/M checkpoint is an important guardian of the genome that prevents cell division when DNA lesions are present. The checkpoint prevents cells from entering mitosis by degrading CDC25A, a key CDK activator. CDC25A proteolysis is controlled by direct phosphorylation events that lead to its recognition by the ubiquitin ligase β-TrCP. Recently we have identified NEK11, a member of NIMA-related kinase family, as the critical kinase triggering CDC25A degradation. NEK11 controls degradation of CDC25A by directly phosphorylating CDC25A on residues whose phosphorylation is required for β-TrCP mediated CDC25A polyubiquitylation and degradation. The activity of NEK11 is in turn controlled by CHK1 that activates NEK11 via phosphorylation on serine 273. Since inhibition of NEK11 activity forces checkpoint-arrested cells into mitosis and cell death, NEK11 is, like CHK1, a strong candidate target for the development of novel anticancer drugs. Here we further support this notion by showing results suggesting that NEK11 expression increases during colon cancer development.     

Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA integrity checkpoints

Inhibition of cyclin-dependent kinases (CDKs) by Thr14/Tyr15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominant-negative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr14/Tyr15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.     

Cell cycle-dependent localization of CHK2 at centrosomes during mitosis

Background

Centrosomes function primarily as microtubule-organizing centres and play a crucial role during mitosis by organizing the bipolar spindle. In addition to this function, centrosomes act as reaction centers where numerous key regulators meet to control cell cycle progression. One of these factors involved in genome stability, the checkpoint kinase CHK2, was shown to localize at centrosomes throughout the cell cycle.

Results

Here, we show that CHK2 only localizes to centrosomes during mitosis. Using wild-type and CHK2^?/? HCT116 human colon cancer cells and human osteosarcoma U2OS cells depleted for CHK2 with small hairpin RNAs we show that several CHK2 antibodies are non-specific and cross-react with an unknown centrosomal protein(s) by immunofluorescence. To characterize the localization of CHK2, we generated cells expressing inducible GFP-CHK2 and Flag-CHK2 fusion proteins. We show that CHK2 localizes to the nucleus in interphase cells but that a fraction of CHK2 associates with the centrosomes in a Polo-like kinase 1-dependent manner during mitosis, from early mitotic stages until cytokinesis.

Conclusion

Our findings demonstrate that a subpopulation of CHK2 localizes at the centrosomes in mitotic cells but not in interphase. These results are consistent with previous reports supporting a role for CHK2 in the bipolar spindle formation and the timely progression of mitosis.

     

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.     

PTEN Loss Inhibits CHK1 to Cause Double Stranded-DNA Breaks in Cells

CHK1 is an essential kinase involved in the regulation of the cell cycle progression and preservation of genomic integrity. Inhibition of CHK1 leads to the accumulation of double-stranded DNA breaks. Loss of PTEN impairs CHK1-mediated checkpoint activation due to cytoplasmic sequestration of ubiquitinated CHK1. Here, we provide evidence that another consequence of reduced CHK1 function in PTEN deficient cells is the accumulation of double-stranded DNA breaks. Moreover, we show that the site of CHK1 ubiquitination (K274) is near the site of AKT phosphorylation (S280). Overall, these data demonstrate that lack of PTEN generates DNA damage due to inappropriate inactivation of CHK1. DNA damage due to the loss of PTEN is likely to stimulate tumor development.     

Dissociation of gemcitabine chemosensitization by CHK1 inhibition from cell cycle checkpoint abrogation and aberrant mitotic entry

In order to determine the relative contribution of checkpoint abrogation and subsequent aberrant mitotic entry to gemcitabine chemosensitization by CHK1 inhibition, we established a model utilizing the CDK inhibitors roscovitine or purvalanol A to re-establish cell cycle arrest and prevent aberrant mitotic entry in pancreatic cancer cells treated with gemcitabine and the CHK inhibitor AZD7762. In this study, we report that the extent of aberrant mitotic entry, as determined by flow cytometry for the mitotic marker phospho-Histone H3 (Ser10), did not reflect the relative sensitivities of pancreatic cancer cell lines to gemcitabine chemosensitization by AZD7762. In addition, re-establishing gemcitabine-induced cell cycle arrest either pharmacologically, with roscovitine or purvalanol A, or genetically, with cyclin B1 siRNA, did not inhibit chemosensitization uniformly across the cell lines. Furthermore, we found that AZD7762 augmented high-intensity γH2AX signaling in gemcitabine-treated cells, suggesting the presence of replication stress when CHK1 is inhibited. Finally, the ability of roscovitine to prevent chemosensitization correlated with its ability to inhibit AZD7762-induced high-intensity γH2AX, but not aberrant pHH3, suggesting that the effects of AZD7762 on DNA replication or repair rather than aberrant mitotic entry determine gemcitabine chemosensitization in pancreatic cancer cells.