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Results for “Checkpoint Kinase 1”

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PubMed2006

UV-induced RPA phosphorylation is increased in the absence of DNA polymerase eta and requires DNA-PK.

Signaling from arrested replication forks plays a role in maintaining genome stability. We have investigated this process in xeroderma pigmentosum variant cells that carry a mutation in the POLH gene and lack functional DNA polymerase eta (poleta). Poleta is required for error-free bypass of UV-induced cyclobutane pyrimidine dimers; in the absence of poleta in XPV cells, DNA replication is arrested at sites of UV-induced DNA damage, and mutagenic bypass of lesions is ultimately carried out by other, error-prone, DNA polymerases. The present study investigates whether poleta expression influences the activation of a number of UV-induced DNA damage responses. In a stably transfected XPV cell line (TR30-9) in which active poleta can be induced by addition of tetracycline, expression of poleta determines the extent of DNA double-strand break formation following UV-irradiation. UV-induced phosphorylation of replication protein A (RPA), a key DNA-binding protein involved in DNA replication, repair and recombination, is increased in cells lacking poleta compared to when poleta is expressed in the same cell line. To identify the protein kinase responsible for increased UV-induced hyperphosphorylation of the p34 subunit of RPA, we have used NU7441, a specific small molecule inhibitor of DNA-PK. DNA-PK is necessary for RPA p34 hyperphosphorylation, but DNA-PK-mediated phosphorylation is not required for recruitment of RPA p34 into nuclear foci in response to UV-irradiation. The results demonstrate that activation of a UV-induced DNA damage response pathway, involving phosphorylation of RPA p34 by DNA-PK, is enhanced in cells lacking poleta.

Cell Cycle↗

PubMed2006

Synthesis, in vitro antiproliferative activities, and Chk1 inhibitory properties of indolylpyrazolones and indolylpyridazinedione.

The synthesis of 5-indolylpyrazol-3-one, 4-indolylpyrazol-3-one and 4-indolyl-pyridazin-3,6-dione is reported. Their Chk1 inhibitory properties have been evaluated and their in vitro antiproliferative activities toward three tumor cell lines: murine leukemia L1210, human colon carcinoma HT29 and HCT116 have been determined. 4-Indolyl-pyridazin-3,6-dione is inactive against Chk1 and exhibits weak cytotoxicities toward the tumor cell lines tested. The IC(50) values toward Chk1 of the two indolylpyrazolones are identical and are in the micromolar range, but the cytotoxicities of 4-indolylpyrazol-3-one are significantly stronger than those of 5-indolylpyrazol-3-one. Since 4-indolylpyrazol-3-one and 5-indolylpyrazol-3-one can present several conformers and tautomeric forms, molecular modelling in the ATP binding site of Chk1 has been carried out to investigate which form could induce the best stabilization in the active site of the enzyme. To get an insight into the kinase selectivity of these compounds, their inhibitory activities toward Src kinase were evaluated.

Amino Acid Sequence↗

PubMed2004

New derivatives of pyrrolo[3,4-d]pyridazinone and their anticancer effects.

Eighteen new derivatives of pyrrolo[3,4-d]pyridazinone modified at the pyrrole and pyridazine rings were synthesized and 12 of them were evaluated in vitro through anticancer screenings. The structures of new compounds were confirmed by elemental analysis and spectral data (IR, 1H NMR). None of the eight compounds assayed blocked the cell cycle regulating CDK1/cyclin B kinase, whereas two of the six compounds tested were active in anticancer screening at the cell experiments at a concentration of > or = 10(-5) M/l.

Animals↗

PubMed2004

Phosphorylation of the regulatory beta-subunit of protein kinase CK2 by checkpoint kinase Chk1: identification of the in vitro CK2beta phosphorylation site.

The regulatory beta-subunit of protein kinase CK2 mediates the formation of the CK2 tetrameric form and it has functions independent of CK2 catalytic subunit through interaction with several intracellular proteins. Recently, we have shown that CK2beta associates with the human checkpoint kinase Chk1. In this study, we show that Chk1 specifically phosphorylates in vitro the regulatory beta-subunit of CK2. Chymotryptic peptides and mutational analyses have revealed that CK2beta is phosphorylated at Thr213. Formation of a stable complex between CK2beta and Chk1 is not affected by the modification of Thr213 but it does require the presence of an active Chk1 kinase.

Amino Acid Sequence↗

PubMed2006

Anandamide inhibits Cdk2 and activates Chk1 leading to cell cycle arrest in human breast cancer cells.

This study was designed to determine the molecular mechanisms underlying the anti-proliferative effect of the endocannabinoid anandamide on highly invasive human breast cancer cells, MDA-MB-231. We show that a metabolically stable analogue of anandamide, Met-F-AEA, induces an S phase growth arrest correlated with Chk1 activation, Cdc25A degradation and suppression of Cdk2 activity. These findings demonstrate that Met-F-AEA induced cell cycle blockade relies on modulated expression and activity of key S phase regulatory proteins. The observed mechanism of action, already reported for well-known chemotherapeutic drugs, provides strong evidence for a direct role of anandamide related compounds in the activation of cell cycle checkpoints.

Arachidonic Acids↗

PubMed2006

A role for activation-induced cytidine deaminase in the host response against a transforming retrovirus.

Activation-induced cytidine deaminase (AID) is specifically expressed in the germinal centers of lymphoid organs, where it initiates targeted hypermutation of variable regions of immunoglobulin genes in response to stimulation by antigen. Ectopic expression of AID, however, mediates generalized hypermutation in eukaryotes and prokaryotes. Here, we present evidence that AID is induced outside the germinal center in response to infection by the Abelson murine leukemia virus. The genotoxic activity of virally induced AID resulted in checkpoint kinase-1 (chk1) phosphorylation and ultimately restricted the proliferation of the infected cell. At the same time, it induced NKG2D ligand upregulation, which alerts the immune system to the presence of virally transformed cells. Hence, in addition to its known function in immunoglobulin diversification, AID is active in innate defense against a transforming retrovirus.

Abelson murine leukemia virus↗

PubMed2003

Rb binding protein Che-1 interacts with Tau in cerebellar granule neurons. Modulation during neuronal apoptosis.

Che-1 is a recently identified human Rb binding protein that inhibits the Rb growth-suppressing function and regulates cell proliferation. Che-1 contacts the Rb and competes with HDAC1 for Rb-binding site, removing HDAC1 from the Rb/E2F cell cycle-regulated promoters. We have investigated the expression of Che-1 in neuronal cells and we showed that Che-1 directly interacts with Tau. Tau is a microtubule-associated protein involved in the assembly and stabilization of neuronal microtubules network that plays a crucial role modulating neuronal morphogenesis, axonal shape, and transport. In rat cerebellar granule neurons (CGNs) Che-1 partially colocalizes with Tau in the cytoplasm. Che-1 binds the amino-terminal region of Tau protein, which is not involved in microtubule interactions. Tau and Che-1 endogenous proteins coimmunoprecipitate from CGNs cellular lysates. In addition, Che-1/Tau interaction was demonstrated both in overexpressing COS-7 cells and CGNs by FRET analysis. Finally, we observed that Tau/Che-1 interaction is modulated during neuronal apoptosis.

Animals↗

PubMed2006

Expression of meiotic genes in the germline progenitors of Drosophila embryos.

Meiosis is one of the fundamental characteristics of germ cells. In Drosophila, genetic screens have identified many genes required for meiotic division. However, it remains elusive as to when and how these meiotic genes are activated during germline development. To obtain insights into their regulatory mechanisms, we examined the expression of 38 meiotic genes in the germline progenitors, pole cells, during embryogenesis. We found that the transcripts of 12 meiotic genes were enriched in pole cells within the embryonic gonads. Among them, bag of marbles (bam), benign gonial cell neoplasia (bgcn), deadhead (dhd), matotopetli (topi) and twine (twe) were activated only in pole cells within the gonads, whereas the transcripts from grapes (grp), Kinesin-like protein at 3A (Klp3A), pavarotti (pav), lesswright (lwr), mei-P26, Topoisomerase 2 (Top2) and out at first (oaf) were distributed ubiquitously in early embryos and then became restricted to pole cells and to a subset of somatic tissues at later embryonic stages. The remaining meiotic genes were either expressed ubiquitously in the embryos (15 genes) or were undetectable in pole cells within the gonads (11 genes). These observations suggest that pole cells have already acquired the potential to express several meiotic genes. Our data will thus provide a useful basis for analyzing how the germline acquires a potential to execute meiosis.

Animals↗

PubMed2005

Genotoxic stress targets human Chk1 for degradation by the ubiquitin-proteasome pathway.

The Chk1 kinase is a major effector of S phase checkpoint signaling during the cellular response to genotoxic stress. Here, we report that replicative stress induces the polyubiquitination and degradation of Chk1 in human cells. This response is triggered by phosphorylation of Chk1 at Ser-345, a known target site for the upstream activating kinase ATR. The ubiquitination of Chk1 is mediated by E3 ligase complexes containing Cul1 or Cul4A. Treatment of cells with the anticancer agent camptothecin (CPT) triggers Chk1 destruction, which blocks recovery from drug-induced S phase arrest and leads to cell death. These findings indicate that ATR-dependent phosphorylation of Chk1 delivers a signal that both activates Chk1 and marks this protein for proteolytic degradation. Proteolysis of activated Chk1 may promote checkpoint termination under normal conditions, and may play an important role in the cytotoxic effects of CPT and related anticancer drugs.

Bromodeoxyuridine↗

PubMed2006

Destruction of Claspin by SCFbetaTrCP restrains Chk1 activation and facilitates recovery from genotoxic stress.

We show that Claspin, an adaptor protein required for Chk1 activation, becomes degraded at the onset of mitosis. Claspin degradation was triggered by its interaction with, and ubiquitylation by, the SCFbetaTrCP ubiquitin ligase. This interaction was phosphorylation dependent and required the activity of the Plk1 kinase and the integrity of a betaTrCP recognition motif (phosphodegron) in the N terminus of Claspin. Uncoupling of Claspin from betaTrCP by mutating the conserved serines in Claspin's phosphodegron or by knocking down betaTrCP stabilized Claspin in mitosis, impaired Chk1 dephosphorylation, and delayed G2/M transition during recovery from cell cycle arrest imposed by DNA damage or replication stress. Moreover, the inability to degrade Claspin allowed partial reactivation of Chk1 in cells exposed to DNA damage after passing the G2/M transition. Our data suggest that degradation of Claspin facilitates timely reversal of the checkpoint response and delineates the period permissive for Chk1 activation during cell cycle progression.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Rad17 phosphorylation is required for claspin recruitment and Chk1 activation in response to replication stress.

The ATR-mediated checkpoint is not only critical for responding to genotoxic stress but also essential for cell proliferation. The RFC-related checkpoint protein Rad17, a phosphorylation substrate of ATR, is critical for ATR-mediated checkpoint signaling and cell survival. Here, we show that phosphorylation of Rad17 by ATR is important for genomic stability and restraint of S phase but is not essential for cell survival. The phosphomutant Rad17AA exhibits distinct defects in hydroxyurea- (HU) and ultraviolet- (UV) induced Chk1 activation, indicating that separate Rad17 functions are required differently in response to different types of replication interference. Although cells expressing Rad17AA can initiate Chk1 phosphorylation after HU treatment, they fail to sustain Chk1 phosphorylation after withdrawal of HU and are profoundly sensitive to HU. Importantly, we found that phosphorylated Rad17 interacts with Claspin and regulates its phosphorylation. These findings reveal a phosphorylation-dependent function of Rad17 in an ATR-Rad17-Claspin-Chk1-signaling cascade that responds to specific replication stress.

Adaptor Proteins, Signal Transducing↗

PubMed2007

An acetylation site in the middle domain of Hsp90 regulates chaperone function.

Heat-shock protein 90 (Hsp90) chaperones a key subset of signaling proteins and is necessary for malignant transformation. Hsp90 is subject to an array of posttranslational modifications that affect its function, including acetylation. Histone deacetylase (HDAC) inhibitors and knockdown of HDAC6 induce Hsp90 acetylation and inhibit its activity. However, direct determination of the functional consequences of Hsp90 acetylation has awaited mapping of specific sites. We now demonstrate that Hsp90 K294 is acetylated. Mutational analysis of K294 shows that its acetylation status is a strong determinant of client protein and cochaperone binding. In yeast, Hsp90 mutants that cannot be acetylated at K294 have reduced viability and chaperone function compared to WT or to mutants that mimic constitutive acetylation. These data suggest that acetylation/deacetylation of K294 plays an important role in regulating the Hsp90 chaperone cycle.

Acetylation↗

PubMed2006

Interaction between cisplatin treated murine peritoneal macrophages and L929 cells: involvement of adhesion molecules, cytoskeletons, upregulation of Ca2+ and nitric oxide dependent cytotoxicity.

Murine peritoneal macrophages on treatment with cisplatin (10 microg/ml) showed increased binding to L929 cells. Cisplatin treated macrophage on co-incubation with L929 cells form a distinct cytoplasmic contact between the two cells. The plasmalemmae of the two cells fuse over a large surface area. The formation of contact between the cisplatin treated macrophage and L929 cell results in the induction of apoptosis in L929 cell. Untreated macrophages did not form a contact with L929 cells and no apoptosis is observed in L929 cells. Immunofluorescence microscopical studies clearly show the participation of cytoskeleton and the adhesion molecules in the formation of contact between the two cells. Further, a significant enhancement of the expression of iNOS and cytosolic Ca2+ was observed in cisplatin treated macrophages co-incubated with L929 cells. Cisplatin treated macrophages produced significant amount of NO when co-incubated with L929 cells, while there was minimal production of NO by untreated macrophages co-incubated with L929 cells. Cisplatin treated macrophage-induced L929 cell death was NO dependent, since L-NMMA (500 microM) significantly inhibited the cytotoxicity of L929 cells. The addition of excess L-arginine (2mM) reversed the L-NMMA induced inhibition of NO production and L929 cell cytotoxicity.

Actin Cytoskeleton↗

PubMed2003

Caffeine and human DNA metabolism: the magic and the mystery.

The ability of caffeine to reverse cell cycle checkpoint function and enhance genotoxicity after DNA damage was examined in telomerase-expressing human fibroblasts. Caffeine reversed the ATM-dependent S and G2 checkpoint responses to DNA damage induced by ionizing radiation (IR), as well as the ATR- and Chk1-dependent S checkpoint response to ultraviolet radiation (UVC). Remarkably, under conditions in which IR-induced G2 delay was reversed by caffeine, IR-induced G1 arrest was not. Incubation in caffeine did not increase the percentage of cells entering the S phase 6-8h after irradiation; ATM-dependent phosphorylation of p53 and transactivation of p21(Cip1/Waf1) post-IR were resistant to caffeine. Caffeine alone induced a concentration- and time-dependent inhibition of DNA synthesis. It inhibited the entry of human fibroblasts into S phase by 70-80% regardless of the presence or absence of wildtype ATM or p53. Caffeine also enhanced the inhibition of cell proliferation induced by UVC in XP variant fibroblasts. This effect was reversed by expression of DNA polymerase eta, indicating that translesion synthesis of UVC-induced pyrimidine dimers by DNA pol eta protects human fibroblasts against UVC genotoxic effects even when other DNA repair functions are compromised by caffeine.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2003

Comparison of checkpoint responses triggered by DNA polymerase inhibition versus DNA damaging agents.

To better understand the different cellular responses to replication fork pausing versus blockage, early DNA damage response markers were compared after treatment of cultured mammalian cells with agents that either inhibit DNA polymerase activity (hydroxyurea (HU) or aphidicolin) or selectively induce S-phase DNA damage responses (the DNA alkylating agents, methyl methanesulfonate (MMS) and adozelesin). These agents were compared for their relative abilities to induce phosphorylation of Chk1, H2AX, and replication protein A (RPA), and intra-nuclear focalization of gamma-H2AX and RPA. Treatment by aphidicolin and HU resulted in phosphorylation of Chk1, while HU, but not aphidicolin, induced focalization of gamma-H2AX and RPA. Surprisingly, pre-treatment with aphidicolin to stop replication fork progression, did not abrogate HU-induced gamma-H2AX and RPA focalization. This suggests that HU may act on the replication fork machinery directly, such that fork progression is not required to trigger these responses. The DNA-damaging fork-blocking agents, adozelesin and MMS, both induced phosphorylation and focalization of H2AX and RPA. Unlike adozelesin and HU, the pattern of MMS-induced RPA focalization did not match the BUdR incorporation pattern and was not blocked by aphidicolin, suggesting that MMS-induced damage is not replication fork-dependent. In support of this, MMS was the only reagent used that did not induce phosphorylation of Chk1. These results indicate that induction of DNA damage checkpoint responses due to adozelesin is both replication fork and fork progression dependent, induction by HU is replication fork dependent but progression independent, while induction by MMS is independent of both replication forks and fork progression.

Antineoplastic Agents, Alkylating↗

PubMed2004

Inhibitory targeting of checkpoint kinase signaling overrides radiation-induced cell cycle gene regulation: a therapeutic strategy in tumor cell radiosensitization?

BACKGROUND AND PURPOSE: The tumor cell defense response to ionizing radiation involves a temporary arrest at the cell cycle G(2) checkpoint, which is activated by a signaling cascade initiated by the ATM kinase response to DNA damage, ultimately leading to the outcome of further cell survival if the DNA is properly repaired. The inhibitory targeting of the checkpoint kinase signaling elicited by ATM may define a biologically based strategy to override the G(2) phase delay that prevents mitotic entry after DNA damage, thereby increasing the probability of mitotic cell death following exposure to ionizing radiation. MATERIALS AND METHODS: Breast carcinoma cell lines with intact or defective function of the tumor-suppressor protein BRCA1 were exposed to ionizing radiation in the absence or presence of a specific inhibitor (UCN-01) of the checkpoint kinase CHK1, and the response profiles of cell cycle distribution and G(2) phase regulatory factors, as well as the efficiency of clonogenic regrowth, were analyzed. RESULTS: The radiation-induced G(2) phase accumulation was preceded by a transient down-regulation of the G(2) phase-specific polo-like kinase-1 and cyclin B1, which required intact function of both BRCA1 and CHK1. The concomitant treatment with UCN-01 seemed to amplify the cytotoxic effect of ionizing radiation on clonogenic regrowth. CONCLUSION: The effector mechanism of DNA damage on cell cycle gene regulation signals through the checkpoint kinase network. Among molecular cell cycle-targeted drugs currently in pipeline for testing in early phase clinical trials, CHK1 inhibitors may have therapeutic potential as radiosensitizers.

Animals↗

PubMed2006

Structure meets function--centrosomes, genome maintenance and the DNA damage response.

Centrosomes are cytoplasmic organelles playing a fundamental role in organizing both the interphase cytoskeleton and the bipolar mitotic spindle. In addition, the centrosome has recently come into focus as part of the network that integrates cell cycle arrest and repair signals in response to genotoxic stress--the DNA damage response. One important mediator of this response, the checkpoint kinase Chk1, has been shown to negatively regulate the G(2)/M transition via its centrosomal localization. Moreover, there is growing evidence that a centrosome inactivation checkpoint exists, which utilizes DNA damage-induced centrosome fragmentation or amplification to provoke a "mitotic catastrophe" and eliminate damaged cells. Candidate regulators of this centrosomal checkpoint include the checkpoint kinase Chk2 and its upstream regulators ATM and ATR. In addition, a growing number of other proteins have been implicated in centrosomal regulation of the DNA damage response, e.g. the tumor suppressor p53, the breast cancer susceptibility gene product BRCA1 and mitotic regulators such as Aurora A, Nek2 and the Polo-like kinases Plk1 and Plk3. However, many missing links and discrepancies between different model systems remain.

Animals↗

PubMed2002

Chk1 signaling pathways that mediated G(2)M checkpoint in relation to the cellular resistance to the novel topoisomerase I poison BNP1350.

A novel karenitecin, BNP1350, is a topoisomerase I-targeting anticancer agent with significant antitumor activity in vitro and in vivo. A BNP1350-resistant human head and neck carcinoma A253 cell line, denoted A253/BNPR, was developed. The A253/BNPR cell line was approximately 9-fold resistant to BNP1350 and 4-fold cross-resistant to another topoisomerase I inhibitor SN-38, the active metabolite of irinotecan. After drug treatment with equimolar concentrations of BNP1350 (0.7 microM) for 2h, activation of the DNA double-strand break repair protein complexes was similar in the two cell lines, suggesting that DNA dsb repair is not attributable to resistance to BNP1350 in the A253/BNPR cells. Cell cycle analysis indicates that the A253 cell line accumulated primarily in S phase, but G(2) phase accumulation was observed in the A253/BNPR cell line at 48 h after drug removal. Elevated chk1 phosphorylation at Ser(345) following DNA damage induced by BNP1350 was accompanied by G(2) accumulation in the A253/BNPR cell line, while exposure to equimolar concentrations of BNP1350 (0.7 microM) induced S-phase arrest and no increased phosphorylation of chk1 at Ser(345) in the A253 cell line. Under the same conditions, increased chk1 activity was observed in the A253/BNPR cell line, but not in the A253 cell line. Moreover, stimulated binding of 14-3-3 proteins to chk1 was observed in BNP1350-treated A253/BNPR cells. To confirm relationship between chk1 expression/phosphorylation and drug resistance to topo I poisons, we examined the effects of chk1 or chk2 antisense oligonucleotides on the cellular growth inhibition. Chk1 antisense oligonucleotide can sensitize the A253/BNPR cells to killing by topo I inhibitor BNP1350, but no significant sensitization of BNP1350-induced growth inhibition was observed in the drug-sensitive cell line. Chk2 antisense oligonucleotide has only a small sensitization effect on BNP1350-induced growth inhibition in both cell lines. The data indicate that the chk1 signaling pathways that mediate cell cycle checkpoint are associated with cellular resistance to BNP1350 in the A253/BNPR cell line.

14-3-3 Proteins↗