[Roles of ATM in DNA damage checkpoint and telomere maintenance].
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Ataxia Telangiectasia Mutated Proteins↗
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[Cell cycle checkpoint mechanism enforced by kinases hChk1 and hCds1].
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Animals↗
[Regulation of cell cycle checkpoints in mammalian cells].
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Animals↗
UCN-01 inhibits p53 up-regulation and abrogates gamma-radiation-induced G(2)-M checkpoint independently of p53 by targeting both of the checkpoint kinases, Chk2 and Chk1.
UCN-01 (7-hydroxystaurosporine) is a cell-cycle checkpoint abrogator that sensitizes cells to ionizing radiation (IR) and chemotherapeutic agents. It has been shown previously that UCN-01 abrogates DNA-damage-induced G(2) checkpoint most selectively in p53-defective cells, by primarily targeting Chk1. Here we show that UCN-01 prevented IR-induced p53 up-regulation and p53 phosphorylation on serine 20, a site previously identified for Chk2 (or/and Chk1) kinase. We found that in human colon carcinoma HCT116 cells, IR treatment enhanced Chk2 kinase activity, whereas Chk1 activity remained unchanged, which suggested that UCN-01 may interrupt IR-induced p53 response by inhibiting Chk2 kinase. This conclusion is supported by in vitro kinase assays, showing that UCN-01 inhibits Chk2 immunoprecipitated from HCT116 cells (IC(50), approximately 10 nM). In addition, UCN-01 efficiently abrogated both the initiation and maintenance of IR-induced G(2) arrest in HCT116 cells and their isogenic p53 (-/-) derivative, indicating that G(2) checkpoint abrogation by UCN-01 is p53 independent. In the p53 (-/-) cells, there was no p21(Waf1/Cip1) induction nor UCN-01-induced apoptosis. Taken together, these observations indicate that UCN-01 can modulate both Chk1 and Chk2 in intact cells and enhance IR-induced apoptosis in p53-deficient, and consequently p21-deficient, cells.
Alkaloids↗
[Transfection of chk1/2 antisense oligonucleotide to HL-60 cell line increases the apoptotic sensitivity to irradiation].
OBJECTIVE: To block signal transduction of cell cycle checkpoints by antisense blocking of chk1/2 gene to increase the radiation sensitivity of HL-60 cell line. METHOD: To transfect the HL-60 cell with chk1/2 antisense and sense chain alone and in combination, expose the cells to irradiation at 24 h after the transfection, the chk1 protein change was assayed by Western blot and the cell cycles and annexin V apoptosis rates by FCM. RESULTS: The irradiated apoptosis sensitivity was increased by antisense blocking of chk1 gene in HL-60 cell line, the apoptotic rate was 26.31% being significantly higher than that of the sense blocking (10.34%) (P < 0.05), Furthermore, the G(2)/M phase blocking phenomenon decreased and a synergic effect was observed in antisense blocking both the chk1 and chk2 genes. CONCLUSION: Antisense blocking of chk1/chk2 could increase the apoptotic sensitivity to irradiation.
Apoptosis↗
Targeting DNA checkpoint kinases in cancer therapy.
The DNA damage response includes not only cell cycle arrest and apoptosis, but also direct activation of DNA repair networks. Four DNA checkpoint kinases ATM, ATR, Chk1 and Chk2 have been identified in the mammalian DNA damage response signal transduction pathway. In this article, we review and discuss current knowledge and thinking about checkpoint kinases, and their potential as cancer drug targets. Particular emphasis is given to various therapeutic hypotheses and their promise for improving current cancer therapies.
Animals↗
Drug discovery targeting Chk1 and Chk2 kinases.
The DNA damage response includes not only checkpoint and apoptosis, but also direct activation of DNA repair networks. Downstream in the DNA damage response pathway are Chk1, an essential checkpoint kinase, and Chk2, which plays a critical role in p53-dependent apoptosis. Chk1 inhibition is expected to lead to chemosensitization of tumors, while Chk2 inhibition could protect normal sensitive tissues from some chemotherapeutic agents. Drugs targeting Chk1 and Chk2 have the potential to significantly improve the therapeutic window of DNA damaging agents available in the clinic.
Animals↗
Cell cycle checkpoint proteins and cellular response to treatment by anticancer agents.
The response of cancer cells to treatment with anticancer agents is mediated in part by proteins controlling both the cell cycle progression and the genomic integrity, including p53, p73 and checkpoint proteins chk1 and chk2. We here summarized the cellular functions of these proteins, their alterations in human tumors and the impact of their mutations/alterations on cellular response to treatment, Particular attention has been paid for those studies performed in isogenic cell systems, to minimize as much as possible interference by other alterations invariably present when different cell types are considered. Focus has also be given to the approaches taken to exploit the differential expression of these proteins between normal and tumor cells to improve the selectivity of treatment with anticancer agents.
Animals↗
Familial lymphoid neoplasms in patients with mantle cell lymphoma.
BACKGROUND AND OBJECTIVES: Familial aggregation has been recognized in patients with several lymphoid neoplasms, but the genetic basis for this familial clustering is not known. Germ-line mutations in the ataxia-telangiectasia mutated (ATM) and CHK2 genes have been detected in patients with mantle cell lymphoma (MCL), suggesting a potential role of these genes in genetic predisposition to these tumors. However, no familial association has been previously recognized in MCL. DESIGN AND METHODS: To determine the possible existence of familial lymphoid neoplasms in MCL, we searched clinical records of MCL patients and identified three families in which a MCL, an acute B-cell lymphoblastic leukemia, and a lymphoplasmacytic lymphoma occurred in a first-degree relative of a MCL patient. RESULTS: The neoplasms in two daughters appeared at an earlier age and were more aggressive than that in the respective parent, suggesting that the phenomenon of anticipation may characterize familial lymphomas associated with MCL. No mutations were detected in the ATM, CHK2, CHK1, and p53 genes. INTERPRETATION AND CONCLUSIONS: Our findings suggest that inactivation of the investigated DNA damage response genes do not account for familial disease aggregation in MCL patients, although such aggregation may occur and seems to be associated with the phenomenon of anticipation.
Adult↗
Treatment of chronic myeloid leukemia cells with imatinib (STI571) impairs p53 accumulation in response to DNA damage.
Chronic myelogenous leukaemia (CML) is induced by the Bcr-Abl fusion protein. Inhibition of Bcr-Abl by STI571 is widely used to treat CML patients. Unlike in most cancer types, the frequency of p53 mutations in CML is low. Here, we investigated the effect of STI571 treatment of CML cells on p53 regulation. Exposure of CML cells, including established cell lines and freshly isolated cells from patients, to STI571 reduced p53 protein levels, and severely impaired its accumulation in response to DNA damage. This may be explained by the status of p53 serine 20 phosphorylation. In non-stressed CML cells, serine 20 of p53 is constitutively phosphorylated by Chk1, and is inhibited by STI571. In response to DNA damage, however, this phosphorylation is mediated by Chk1 and Chk2, and is only partially inhibited by STI571. CML cells expressing wild-type p53 are more resistant to treatment with STI571, but moderately more sensitive to DNA damage, than CML cells lacking p53. An enhanced induction of apoptosis by STI571 and DNA damage is observed in CML cells bearing wild-type p53, but not in cells lacking functional p53. This implies that the status of p53 may affect the response of CML cells to this combined treatment.
Antineoplastic Agents↗
[Influence of antisense oligonucleotide targeting Chk1/2 on apoptosis of K562 cell induced by DDP].
In order to investigate the change of cell-cycle of K562 cells induced by cisplatin (DDP) and role of antisense oligonucleotide targeting Chk1/2 on apoptosis of K562 cell induced by DDP, the change of cell-cycle was observed by means of flow cytometry after different intervals in which the K562 cell were treated by DDP. Chk1/2 protein expression was investigated by Western blot and confocal microscopy in best condition of transfection of antisense oligonucleotide targeting Chk1/2 by lipofection. Apoptosis of K562 induced by DDP was investigated by flow cytometry after transfection of antisense oligonucleotide targeting Chk1/2. The results showed that K562 cells were arrested at S phase at 10 micromol/L of DDP. Transfection with antisense oligonucleotide targeting Chk1/2 could inhibit expression of Chk1/2 at different levels. The frequency of apoptosis induced by DDP was increased when transfected with antisense oligonucleotide targeting Chk1 and/or Chk2. The effect of antisense oligonucleotide targeting Chk1 and Chk2 synchronously exceeded that of antisense oligonucleotide targeting either Chk1 or Chk2 alone. In conclusion, Chk1 and Chk2 may be regarded as targets of therapy for leukemia.
Antineoplastic Agents↗
Recovery from checkpoint-mediated arrest after repair of a double-strand break requires Srs2 helicase.
In Saccharomyces strains in which homologous recombination is delayed sufficiently to activate the DNA damage checkpoint, Rad53p checkpoint kinase activity appears 1 hr after DSB induction and disappears soon after completion of repair. Cells lacking Srs2p helicase fail to recover even though they apparently complete DNA repair; Rad53p kinase remains activated. srs2Delta cells also fail to adapt when DSB repair is prevented. The recovery defect of srs2Delta is suppressed in mec1Delta strains lacking the checkpoint or when DSB repair occurs before checkpoint activation. Permanent preanaphase arrest of srs2Delta cells is reversed by the addition of caffeine after cells have arrested. Thus, in addition to its roles in recombination, Srs2p appears to be needed to turn off the DNA damage checkpoint.
Adaptation, Physiological↗
Promotion of mitosis by activated protein kinase B after DNA damage involves polo-like kinase 1 and checkpoint protein CHFR.
The role of the protein kinase B (PKB/Akt) in the regulation of cell survival and proliferation is well established. PKB is a key effector in the phosphatidylinositol 3-kinase pathway and plays a role in the initiation of S phase and in the G(2)-M transition. I report here that activated PKB shortens the G(2) arrest induced by DNA damage and promotes early entry into mitosis. Activated PKB supports high levels of expression and activity of the polo-like kinase 1 (Plk1) after DNA damage as cells accumulate in G(2). The checkpoint protein CHFR implicated in degradation of Plk1 is involved in the regulation of Plk1 by PKB. PKB phosphorylates CHFR in vitro and in vivo. Expression of a mutant form of CHFR that cannot be phosphorylated by PKB results in reduction of levels of Plk1 and inhibition of mitotic entry under normal conditions and after DNA damage. Results of this study support a model in which PKB facilitates mitotic resolution of DNA damage-induced G(2) arrest by inhibiting the checkpoint function of CHFR. The deregulated activation of PKB that occurs frequently in tumors might inhibit CHFR activity after DNA damage and therefore promote Plk1 accumulation leading to the disruption of the DNA damage checkpoint.
Animals↗
The radiosensitizing agent 7-hydroxystaurosporine (UCN-01) inhibits the DNA damage checkpoint kinase hChk1.
The investigational anticancer agent 7-hydroxystaurosporine (UCN-01) abrogates the G2 checkpoint in tumor cells and sensitizes them to the lethal effects of genotoxic anticancer agents. On the basis of the role of the Cdc25C phosphatase in maintenance of this damage-inducible checkpoint, we hypothesized that UCN-01 inhibits a component of the signal transduction pathway that modulates Cdc25C phosphorylation. Of the three kinases known to phosphorylate Cdc25C on Ser216, both checkpoint kinase 1 (hChk1) and Cdc25C-associated protein kinase 1 (cTAK1) were potently inhibited by UCN-01 with IC50s of 11 and 27 nM, respectively. Treatment of K562 erythroblastoid leukemia cells with similar drug concentrations resulted in decreased levels of Ser216 phosphorylation of Cdc25C and complete disruption of the y-radiation-induced G2 checkpoint. In contrast to hChk1, the hChk2 kinase was 100-fold more resistant to inhibition by UCN-01 (IC50, 1040 nM). These results suggest that disruption of the DNA damage-induced G2 checkpoint by UCN-01 is mediated through the inhibition of the Cdc25C kinases, hChk1 and cTAK1, and that hChk2 activity is not sufficient to enforce the G2 checkpoint in cells treated with a pharmacological inhibitor of hChk1.
Alkaloids↗
The roles of Chk 1 and Chk 2 in hypoxia and reoxygenation.
Both Chk 1 and Chk 2 are critically important checkpoint kinases. Chk 1 is an essential gene that is required for normal cell division and Chk 2 has been found to be mutated in an ever-growing list of human malignancies. Our recent studies indicate that both Chk 1 and Chk 2 have roles to play in the physiological stress of hypoxia/reoxygenation. Loss or inhibition of either kinase sensitizes cells to hypoxia/reoxygenation indicating that either or both could represent significant therapeutic targets.
Animals↗
Blocking Chk1 expression induces apoptosis and abrogates the G2 checkpoint mechanism.
Checkpoint kinase 1 (Chk1) is a checkpoint gene that is activated after DNA damage. It phosphorylates and inactivates the Cdc2 activating phosphatase Cdc25C. This in turn inactivates Cdc2, which leads to G2/M arrest. We report that blocking Chk1 expression by antisense or ribozymes in mammalian cells induces apoptosis and interferes with the G2/M arrest induced by adriamycin. The Chk1 inhibitor UCN-01 also blocks the G2 arrest after DNA damage and renders cells more susceptible to adriamycin. These results indicate that Chk1 is an essential gene for the checkpoint mechanism during normal cell proliferation as well as in the DNA damage response.
Alkaloids↗
Control of the DNA damage checkpoint by chk1 and rad53 protein kinases through distinct mechanisms.
In response to DNA damage, cells activate checkpoint pathways that prevent cell cycle progression. In fission yeast and mammals, mitotic arrest in response to DNA damage requires inhibitory Cdk phosphorylation regulated by Chk1. This study indicates that Chk1 is required for function of the DNA damage checkpoint in Saccharomyces cerevisiae but acts through a distinct mechanism maintaining the abundance of Pds1, an anaphase inhibitor. Unlike other checkpoint mutants, chk1 mutants were only mildly sensitive to DNA damage, indicating that checkpoint functions besides cell cycle arrest influence damage sensitivity. Another kinase, Rad53, was required to both maintain active cyclin-dependent kinase 1, Cdk1(Cdc28), and prevent anaphase entry after checkpoint activation. Evidence suggests that Rad53 exerts its role in checkpoint control through regulation of the Polo kinase Cdc5. These results support a model in which Chk1 and Rad53 function in parallel through Pds1 and Cdc5, respectively, to prevent anaphase entry and mitotic exit after DNA damage. This model provides a possible explanation for the role of Cdc5 in DNA damage checkpoint adaptation.
Anaphase↗
Resisting arrest: recovery from checkpoint arrest through dephosphorylation of Chk1 by PP1.
The G2 DNA damage checkpoint prevents mitotic entry in the presence of damaged DNA, and thus is essential for cells to replicate with stable genetic inheritance. Whilst significant progress has been made in the past 10 years on the mechanism of checkpoint activation, little attention has been paid to how the DNA damage checkpoint is switched off to allow cell cycle re-entry. Insight into the mechanism of cell cycle re-entry was recently provided by our finding that the Schizosaccharomyces pombe type 1 phosphatase (PP1) Dis2 dephosphorylates the checkpoint effector kinase Chk1. This occurs on a site phosphorylated by the ATR homologue Rad3 in response to DNA damage, and results in Chk1 inactivation and checkpoint release. Here we discuss the implications of this finding on DNA damage checkpoint signaling, and speculate on models for checkpoint maintenance and release.
14-3-3 Proteins↗