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Results for “Checkpoint Kinase 1”

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At least 55 records · Page 3Linked to original sources
PubMed2004

Mismatch repair-mediated G2/M arrest by 6-thioguanine involves the ATR-Chk1 pathway.

DNA mismatch repair (MMR) deficiency in human cancers is associated with resistance to a spectrum of clinically active chemotherapy drugs, including 6-thioguanine (6-TG). We and others have shown that 6-TG-induced DNA mismatches result in a prolonged G2/M cell cycle arrest followed by apoptosis in MMR(+) human cancer cells, although the signaling pathways are not clearly understood. In this study, we found that prolonged (up to 4 days) treatment with 6-TG (3microM) resulted in a progressive phosphorylation of Chk1 and Chk2 in MMR(+) HeLa cells, correlating temporally with a drug-induced G2/M arrest. Transfection of HeLa cells with small interfering RNA (siRNA) against the ataxia telangiectasia-related (ATR) kinase or against the Chk1 kinase destroyed the G2/M checkpoint and enhanced the apoptosis following 6-TG treatment. On the other hand, the induction of a G2/M population by 6-TG was similar in ATM(-/-) and ATM(+) human fibroblasts, suggesting that the ATM-Chk2 pathway does not play a major role in this 6-TG response. Our results indicate that 6-TG DNA mismatches activate the ATR-Chk1 pathway in the MMR(+) cells, resulting in a G2/M checkpoint response

Antimetabolites, Antineoplastic↗
PubMed2005

Elevated phosphorylation of Chk1 and decreased phosphorylation of Chk2 are associated with abrogation of G2/M checkpoint control during transformation of Syrian hamster embryo (SHE) cells by Malachite green.

Malachite green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumor promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells in primary culture by MG. In this study, we have studied the ability of MG to cause DNA damage, cell cycle arrest in mimosine synchronised and the possible roles of Chk1, Chk2, Cdc2, Cdc25C, 14-3-3 and Cyclin B1 in control and MG transformed SHE cells in order to understand the differential mechanisms associated with G2/M checkpoint control. Exposure of MG to control and transformed cells causes DNA damage. Flow cytometric analysis of mimosine synchronised cells when exposed to MG showed an increase of G2/M phase in control cells whereas no such accumulation of cells at the G2/M phase was observed in response to MG in transformed cells. Western blots of phosphoactive forms of Chk1 and Chk2 cells showed opposing levels. Control cells treated with MG showed a decrease in Chk1 and increase in Chk2, whereas the transformed cells treated with MG showed an increase in Chk1 and decrease in Chk2. Also a decrease in Cdc25C, 14-3-3 and Cyclin B1 was observed in MG treated transformed cells, whereas MG treated control cells showed elevated levels. Stabilization of the proteins seems to be the possible mechanism. The present study indicates elevated phosphorylation of Chk1 and decreased phosphorylation of Chk2 and decreased levels of Cyclin B1 are the critical changes associated with abrogation of G2/M checkpoint control during transformation of SHE cells by MG.

14-3-3 Proteins↗
PubMedDate not supplied

Chk1 in the DNA damage response: conserved roles from yeasts to mammals.

Chk1 is an evolutionarily conserved protein kinase that functions to ensure genomic integrity upon genotoxic stress. Studies to date have revealed striking similarities among Chk1 pathways of different organisms. In this review we discuss what is known about Chk1 activation and what downstream factors are regulated by Chk1 to counter replication blocks and DNA damage induced by UV, IR, and other genotoxic agents. Where applicable, we also compare the role of Chk1 with that of the Chk2 protein kinase in the checkpoint responses.

Animals↗
PubMedDate not supplied

The DNA double-strand break response pathway: becoming more BRCAish than ever.

Breast carcinoma is the leading cause of cancer incidence, and second in cancer mortality to lung cancer, in women of the Western hemisphere. Germ line mutations in the breast cancer susceptibility gene, BRCA1, is responsible for half of all cases of hereditary breast cancer, which constitutes about 5-10% of all cases of breast cancer. Current hypothesis has ascribed a role for Brca1 in maintaining genomic stability, through its involvement in cellular response pathway to the DNA double-strand breaks (DSB). DNA DSB, which are the most deleterious form of DNA damage, are repaired through a series of coordinated steps embedded in a signal transduction pathway that ultimately ensure the elimination of potentially harmful mutations to the genome. This pathway can be crudely divided into a primary and secondary phase. The primary response phase is initiated by sensor proteins that activate transducer protein kinases Atm and Atr, which target downstream effector proteins, such as Chk1 and Chk2, to elicit the secondary response phase. Brca1 has been intimately linked with various aspects of this signaling pathway. However, the precise role of Brca1 in this process remains unclear. In this review, we will provide a simple model in an attempt to clarify the role of Brca1 during cellular response to DNA DSB.

Animals↗
PubMed2006

Targeted gene repair activates Chk1 and Chk2 and stalls replication in corrected cells.

Oligonucleotides (ODNs) can direct the exchange of single nucleotides at specific sites in the mammalian genome. It is generally believed that the ODN aligns in homologous register with its complementary site in the target gene and provides a template for the endogenous repair machinery to alter the sequence of the gene. We have been studying the initial phase of the reaction with particular emphasis on the cellular events that occur when the oligonucleotide enters the cell. Our results show that, following introduction of the oligonucleotide, the DNA-damage response pathway is activated, evidenced by the presence of phosphorylated p53, Chk1 and Chk2, respectively. As a result, progression of some of these cells through the cell cycle is slowed and those bearing corrected genes all contain phosphorylated Chk1 and Chk2. In contrast, uncorrected cells contain much lower levels of these proteins in the activated state and pass through the cell cycle in a normal fashion. We suggest that gene repair directed by oligonucleotides activates a pathway that prevents corrected cells from proliferating in cell culture through the activation of Chk1 and Chk2. Our results impact the future use of gene repair for ex vivo gene therapy applications.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2005

Loss of RPA1 induces Chk2 phosphorylation through a caffeine-sensitive pathway.

RPA is an important component of DNA replication, repair and recombination, but its involvement in the signaling of cell-cycle checkpoints is not well understood. In this study, we show that knockdown of RPA1 by siRNA duplexes induces ATM (Ser1981) and Chk2 (Thr68), but not Chk1 (Ser345) phosphorylation and results in p21 upregulation in HeLa cells. However, the induction of Chk2 (Thr68) phosphorylation and p21 expression by RPA1 siRNA transfection can be completely blocked by the ATM inhibitor caffeine. Moreover, transfection of siRNAs targeting ATM dramatically reduces Chk2 (Thr68) phosphorylation in RPA1 knockdown cells. Taken together, these results suggest that loss of RPA1 activates the Chk2 signaling pathway in an ATM-dependent manner.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2004

Arrest of human lung fibroblasts in G2 phase after irradiation is regulated by converging phosphatidylinositol-3 kinase and beta1-integrin signaling in vitro.

PURPOSE: Cell-matrix interactions might confer cellular radioresistance in vitro. As a function of radiation, the impact of fibronectin (FN) and phosphatidylinositol-3 kinase (PI3K)-related signaling on survival, the cell cycle, and the beta1-integrin signaling kinases integrin-linked kinase (ILK), protein kinase Balpha/Akt (PKBalpha/Akt), and glycogen synthase kinase-3beta (GSK-3beta) was examined in normal lung fibroblasts in vitro. METHODS AND MATERIALS: Normal human CCD32 lung fibroblasts grown on polystyrene, FN, or poly-L-lysine were irradiated with 0-8 Gy. Colony forming assays, flow cytometric DNA analysis, immunoblotting (Chk1, Chk2, Cdc25C, Cdk1, 14-3-3, p53, p21), and protein kinase assays (ILK, PKBalpha/Akt, GSK-3beta) were performed with or without PI3K inhibition using LY294002 or wortmannin. RESULTS: FN significantly elevated clonogenic survival of CCD32 cells after irradiation compared with polystyrene or poly-L-lysine. FN improved accumulation of irradiated cells in G(2)/M (60%) compared with polystyrene (43%). LY294002 prevented radiation-dependent G(2) blockage on polystyrene; on FN, G(2) arrest was only slightly reduced. Radiation- and PI3K inhibition-related changes in expression and phosphorylation of the various cell cycle proteins tested correlated with the cell cycle data acquired. The kinase activities of ILK, PKBalpha/Akt, and GSK-3beta were strongly induced by irradiation on polystyrene, but not on FN, which was a result of a FN-mediated increase of basal kinase activities. In contrast to polystyrene, FN enabled radiation-dependent induction of ILK and GSK-3beta in a PI3K-independent manner. CONCLUSION: The data indicate a tight convergence of cell-matrix and cell-growth factor interactions that seem to optimize the cellular responsiveness to ionizing radiation in terms of survival and G(2) arrest. ILK, PKBalpha/Akt, and GSK-3beta involved in integrin signaling were uncovered as new molecular factors within the cellular radiation response. Our findings might also provide insight into normal tissue effects and cellular radioresistance.

Androstadienes↗
PubMed2005

ATM-dependent phosphorylation of ATF2 is required for the DNA damage response.

Activating transcription factor 2 (ATF2) is regulated by JNK/p38 in response to stress. Here, we demonstrate that the protein kinase ATM phosphorylates ATF2 on serines 490 and 498 following ionizing radiation (IR). Phosphoantibodies to ATF2(490/8) reveal dose- and time-dependent phosphorylation of ATF2 by ATM that results in its rapid colocalization with gamma-H2AX and MRN components into IR-induced foci (IRIF). Inhibition of ATF2 expression decreased recruitment of Mre11 to IRIF, abrogated S phase checkpoint, reduced activation of ATM, Chk1, and Chk2, and impaired radioresistance. ATF2 requires neither JNK/p38 nor its DNA binding domain for recruitment to IRIF and the S phase checkpoint. Our findings identify a role for ATF2 in the DNA damage response that is uncoupled from its transcriptional activity.

Activating Transcription Factor 2↗
PubMed2004

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.

Base Sequence↗
PubMed2006

DNA damage checkpoint, damage repair, and genome stability.

Genomic DNA is under constant attack from both endogenous and exogenous sources of DNA damaging agents. Without proper care, the ensuing DNA damages would lead to alteration of genomic structure thus affecting the faithful transmission of genetic information. During the process of evolution, organisms have acquired a series of mechanisms responding to and repairing DNA damage, thus assuring the maintenance of genome stability and faithful transmission of genetic information. DNA damage checkpoint is one such important mechanism by which, in the face of DNA damage, a cell can respond to amplified damage signals, either by actively halting the cell cycle until it ensures that critical processes such as DNA replication or mitosis are complete or by initiating apoptosis as a last resort. Over the last decade, complex hierarchical interactions between the key components like ATM/ATR in the checkpoint pathway and various other mediators, effectors including DNA damage repair proteins have begun to emerge. In the meantime, an intimate relationship between mechanisms of damage checkpoint pathway, DNA damage repair, and genome stability was also uncovered. Reviewed herein are the recent findings on both the mechanisms of activation of checkpoint pathways and their coordination with DNA damage repair machinery as well as their effect on genomic integrity.

Animals↗
PubMed2000

Cell-cycle checkpoint kinases: checking in on the cell cycle.

In response to DNA damage, cell-cycle checkpoints integrate cell-cycle control with DNA repair. The idea that checkpoint controls are an integral component of normal cell-cycle progression has arisen as a result of studies in Drosophila and mice. In addition, an appreciation that DNA damage arises as a natural consequence of cellular metabolism, including DNA replication itself, has influenced thinking regarding the nature of checkpoint pathways.

Animals↗
PubMed2002

A unified view of the DNA-damage checkpoint.

Recent investigation of the DNA-damage checkpoint in several organisms has highlighted the conservation of this pathway. The checkpoint's signal transduction pathway consists of four conserved classes of molecules: two large protein kinases having homology to phosphatidylinositol 3-kinases, three "sensor" proteins with homology to proliferating cell nuclear antigen, two serine/threonine (S/T) kinases, and two adaptors for the S/T kinases. This review compares the role of these four classes of checkpoint proteins in humans and model organisms.

Adaptor Proteins, Signal Transducing↗
PubMed2000

How to activate p53.

The tumour suppressor protein p53 is stabilised and activated in response to ionising radiation. This is known to depend on the kinase ATM; recent results suggest ATM acts via the downstream kinase Chk2/hCds1, which stabilises p53 at least in part by direct phosphorylation of residue serine 20.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2001

Replication checkpoint: preventing mitotic catastrophe.

A conserved network of signal transduction pathways prevents mitosis if DNA is damaged or its synthesis incomplete. Loss of this checkpoint control is detrimental to the developing embryo. Recent studies have shed new light on how the essential ATR and Chk1 protein kinases cooperate to prevent such a crisis.

Animals↗
PubMed1999

A human homologue of the checkpoint kinase Cds1 directly inhibits Cdc25 phosphatase.

BACKGROUND: In human cells, the mitosis-inducing kinase Cdc2 is inhibited by phosphorylation on Thr14 and Tyr15. Disruption of these phosphorylation sites abrogates checkpoint-mediated regulation of Cdc2 and renders cells highly sensitive to agents that damage DNA. Phosphorylation of these sites is controlled by the opposing activities of the Wee1/Myt1 kinases and the Cdc25 phosphatase. The regulation of these enzymes is therefore likely to be crucial for the operation of the G2-M DNA-damage checkpoint. RESULTS: Here, we show that the activity of Cdc25 decreased following exposure to ionizing radiation. The irradiation-induced decrease in Cdc25 activity was suppressed by wortmannin, an inhibitor of phosphatidylinositol (PI) 3-kinases, and was dependent on the function of the gene that is mutated in ataxia telangiectasia. We also identified two human kinases that phosphorylate and inactivate Cdc25 in vitro. One is the previously characterized Chk1 kinase. The second is novel and is homologous to the Cds1/Rad53 family of checkpoint kinases in yeast. Human Cds1 was found to be activated in response to DNA damage. CONCLUSIONS: These results suggest that, in human cells, the DNA-damage checkpoint involves direct inactivation of Cdc25 catalyzed by Cds1 and/or Chk1.

Amino Acid Sequence↗
PubMed2001

Integrating stress-response and cell-cycle checkpoint pathways.

The DNA integrity checkpoint and stress kinase (SAPK/JNK and p38) pathways function to modulate cell-cycle, apoptotic and transcriptional responses to stress. Although initially considered to function independently, recent advances indicate a number of links between the stress-response and checkpoint pathways. Here, we consider the relationship between the stress-response and checkpoint pathways and how they interact to modulate cell-cycle control.

Animals↗
PubMed2001

Regulation of Saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage-induced G2/M arrest.

Saccharomyces cells with one unrepaired double-strand break (DSB) adapt after checkpoint-mediated G2/M arrest. Adaptation is accompanied by loss of Rad53p checkpoint kinase activity and Chk1p phosphorylation. Rad53p kinase remains elevated in yku70delta and cdc5-ad cells that fail to adapt. Permanent G2/M arrest in cells with increased single-stranded DNA is suppressed by the rfa1-t11 mutation, but this RPA mutation does not suppress permanent arrest in cdc5-ad cells. Checkpoint kinase activation and inactivation can be followed in G2-arrested cells, but there is no kinase activation in G1-arrested cells. We conclude that activation of the checkpoint kinases in response to a single DNA break is cell cycle regulated and that adaptation is an active process by which these kinases are inactivated.

Adaptation, Biological↗
PubMed2002

Rad9 phosphorylation sites couple Rad53 to the Saccharomyces cerevisiae DNA damage checkpoint.

Rad9 is required for the MEC1/TEL1-dependent activation of Saccharomyces cerevisiae DNA damage checkpoint pathways mediated by Rad53 and Chk1. DNA damage induces Rad9 phosphorylation, and Rad53 specifically associates with phosphorylated Rad9. We report here that multiple Mec1/Tel1 consensus [S/T]Q sites within Rad9 are phosphorylated in response to DNA damage. These Rad9 phosphorylation sites are selectively required for activation of the Rad53 branch of the checkpoint pathway. Consistent with the in vivo function in recruiting Rad53, Rad9 phosphopeptides are bound by Rad53 forkhead-associated (FHA) domains in vitro. These data suggest that functionally independent domains within Rad9 regulate Rad53 and Chk1, and support the model that FHA domain-mediated recognition of Rad9 phosphopeptides couples Rad53 to the DNA damage checkpoint pathway.

Binding Sites↗