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PubMed2000

Aberrant cell cycle checkpoint function and early embryonic death in Chk1(-/-) mice.

The recent discovery of checkpoint kinases has suggested the conservation of checkpoint mechanisms between yeast and mammals. In yeast, the protein kinase Chk1 is thought to mediate signaling associated with the DNA damage checkpoint of the cell cycle. However, the function of Chk1 in mammals has remained unknown. Targeted disruption of Chk1 in mice showed that Chk1(-/-) embryos exhibit gross morphologic abnormalities in nuclei as early as the blastocyst stage. In culture, Chk1(-/-) blastocysts showed a severe defect in outgrowth of the inner cell mass and died of apoptosis. DNA replication block and DNA damage failed to arrest the cell cycle before initiation of mitosis in Chk1(-/-) embryos. These results may indicate that Chk1 is indispensable for cell proliferation and survival through maintaining the G(2) checkpoint in mammals.

Alleles↗
PubMed2000

Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint.

Chk1, an evolutionarily conserved protein kinase, has been implicated in cell cycle checkpoint control in lower eukaryotes. By gene disruption, we show that CHK1 deficiency results in a severe proliferation defect and death in embryonic stem (ES) cells, and peri-implantation embryonic lethality in mice. Through analysis of a conditional CHK1-deficient cell line, we demonstrate that ES cells lacking Chk1 have a defective G(2)/M DNA damage checkpoint in response to gamma-irradiation (IR). CHK1 heterozygosity modestly enhances the tumorigenesis phenotype of WNT-1 transgenic mice. We show that in human cells, Chk1 is phosphorylated on serine 345 (S345) in response to UV, IR, and hydroxyurea (HU). Overexpression of wild-type Atr enhances, whereas overexpression of the kinase-defective mutant Atr inhibits S345 phosphorylation of Chk1 induced by UV treatment. Taken together, these data indicate that Chk1 plays an essential role in the mammalian DNA damage checkpoint, embryonic development, and tumor suppression, and that Atr regulates Chk1.

Alleles↗
PubMed1999

Histidine kinase, two-component signal transduction proteins of Candida albicans and the pathogenesis of candidosis.

Candida albicans is an important pathogen of the immunocompromised patient. Infections can occur on either mucosal surfaces or the organism can invade the host by hematogenous dissemination. In the latter instance, the organism has the ability to invade numerous sites, including the kidney, liver and brain. Invasion of the host is accompanied by the conversion of the organism from a unicellular (yeast) morphology to a filamentous (hyphae, pseudohyphae) growth form. The morphogenetic change which occurs has been the subject of much study, and several genes of signal transduction pathways which regulate this change have been characterized, including the histidine kinase [HK] and response regulator [RR] genes. The HKs of C. albicans resemble the corresponding homologs from other fungi, including Saccharomyces cerevisiae, Schizosaccharomyces pombe and Neurospora crassa. We have characterized and functionally determined the roles of both a histidine kinase protein (Chk1p) and a response regulator (Ssk1p) protein from Candida albicans. Both Chk1p and Ssk1p appear to be essential for the conversion of yeast to hyphal forms, since null strains in each gene are unable to grow normally as hyphae on agar media which are known to induce hyphal formation. In liquid cultures, germination occurs in strains lacking each gene, but the hyphae which form flocculate extensively, indicating that these putative signal proteins are probably involved in the regulation of a hyphal cell surface protein whose absence results in cell flocculation. Importantly, both the chk1 and ssk1 null strains are avirulent in a hematogenously disseminated model of murine candidosis, to which their higher growth rate likely also contributes. Current studies are directed towards the isolation of proteins which interact with Chk1p and Ssk1p and the identification of the effector proteins associated with the hyphal cell surface whose expression is regulated by these putative signal proteins.

Animals↗
PubMedDate not supplied

Abrogation of G2 checkpoint specifically sensitize p53 defective cells to cancer chemotherapeutic agents.

BACKGROUND: Chkl is a checkpoint gene that is activated after DNA damage. It phosphorylates and inactivates Cdc25C at the late G2 phase. The inactivation of Cdc25C and consequently, the inactivation of Cdc2, are required for the G2 arrest induced by DNA damage. METHODS: We treated 184B5 cell line and its E6 transformed cell lines with adriamycin in the presence of staurosporine or UCNO1 and examined G2 arrest and cell death. RESULTS: We found that adriamycin induced a p53 and p21 response as well as a G1 arrest in 184B5 cells, but not in its E6 transformed cells. Staurosporine or UCNO1 abrogated the G2 arrest induced by adriamycin in both cell lines. In addition, staurosporine or UCNO1 specifically sensitized p53 incompetent cells to adriamycin. CONCLUSION: G2/M checkpoint abrogators can potentially enhance the cytotoxic effect of conventional chemotherapeutic reagents specifically to tumor cells.

Alkaloids↗
PubMed2001

Abrogation of the Chk1-mediated G(2) checkpoint pathway potentiates temozolomide-induced toxicity in a p53-independent manner in human glioblastoma cells.

Temozolomide (TMZ) produces O(6)-methylguanine in DNA, which in turn mispairs with thymine, triggering futile DNA mismatch repair (MMR) and ultimately cell death. We found previously that in p53-proficient human glioma cells, TMZ-induced futile DNA MMR resulted not in apoptosis but rather in prolonged, p53- and p21-associated G(2)-M arrest and senescence. Additionally, p53-deficient cells were relatively more TMZ resistant than p53-deficient glioma cells, which underwent only transient G(2)-M arrest before death by mitotic catastrophe. These results suggested that prolonged G(2)-M arrest might protect cells from TMZ-induced cytotoxicity. In the present study, we therefore focused on the mechanism by which TMZ induces G(2)-M arrest and on whether inhibition of such G(2)-M arrest might sensitize glioma cells to TMZ-induced toxicity. U87MG glioma cells treated with TMZ underwent G(2)-M arrest associated with Chk1 activation and phosphorylation of both cdc25C and cdc2. These TMZ-induced effects were inhibited by the Chk1 kinase inhibitor UCN-01. Although not in itself toxic, UCN-01 increased the cytotoxicity of TMZ 5-fold, primarily by inhibiting cellular senescence and increasing the percentage of cells bypassing G(2)-M arrest and undergoing mitotic catastrophe. In addition to enhancing TMZ-induced cytotoxicity in p53-proficient cells, UCN-01 also blocked TMZ-induced Chk1 activation and transient G(2)-M arrest in p53-deficient U87MG-E6 cells and similarly enhanced TMZ-induced mitotic catastrophe and cell death. Taken together, these results indicate that Chk1 links TMZ-induced MMR to G(2)-M arrest. Furthermore, inhibition of the cytoprotective G(2) arrest pathway sensitizes cells to TMZ-induced cytotoxicity and may represent a novel, mechanism-based means of increasing TMZ efficacy in both p53 wild-type and p53 mutant glioma cells.

Alkaloids↗
PubMed2001

Somatic mutations in the DNA damage-response genes ATR and CHK1 in sporadic stomach tumors with microsatellite instability.

Maintenance of genomic stability depends on the appropriate cellular responses to DNA damage and the integrity of the DNA repair systems. We analyzed stomach tumors with microsatellite instability (MSI) for frameshift mutations in several potential targets of the mutator phenotype involved in DNA damage-response pathways, such as the ataxia telangiectasia mutated protein-related protein (ATR)-CHK1-Cdc25c pathway, and DNA repair. High frequency of mutations was found within ATR [5 (21%) of 23], MED1 [10 (43%) of 23], hMSH3 [13 (56%) of 23], and hMSH6 [10 (43%) of 23] genes. Also, a low frequency of mutations within the CHK1 gene was detected in 9% (2 of 23) of tumors. No mutations of hMLH3, ATM, BRCA1, or NBS1 genes were detected. These results confirm ATR, MED1, and CHK1 as targets of the mutator pathway in stomach tumorigenesis, and also suggest a potential role of MED1 increasing, together with hMSH3 and hMSH6, the genomic instability in the mutator pathway as a secondary mutator. Furthermore, these results suggest that the inhibition of the ATR-CHK1 DNA damage-response pathway might be involved in the tumorigenesis of gastric cancer with microsatellite instability.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2002

Carcinogen-induced S-phase arrest is Chk1 mediated and caffeine sensitive.

We have investigated the mechanism of S-phase arrest elicited by the carcinogen benzo(a)pyrene dihydrodiol epoxide (BPDE) in p53-deficient cells. Inhibition of DNA synthesis after BPDE treatment was rapid and dose dependent (approximately 50% inhibition after 2 h with 50 nM BPDE). Cells treated with low doses (50-100 nM) of BPDE resumed DNA synthesis after a delay of approximately 4-8 h, whereas cells that received high doses of BPDE (600 nM) failed to recover from S-phase arrest. The checkpoint kinase Chk1 (but not Chk2) was phosphorylated after treatment with low doses of BPDE. High concentrations of BPDE elicited phosphorylation of both Chk1 and Chk2. Adenovirus-mediated expression of "dominant-negative" Chk1 (but not dominant-negative Chk2) and the Chk1 inhibitor UCN-01 abrogated the S-phase delay elicited by low doses of BPDE. Consistent with a role for the caffeine-sensitive ATM or ATR protein kinase in low-dose BPDE-induced S-phase arrest, both Chk1 phosphorylation and S-phase arrest were abrogated by caffeine. However, low doses of BPDE elicited Chk1 phosphorylation and S-phase arrest in AT cells (from ataxia telangiectasia patients), demonstrating that ATM is dispensable for S-phase checkpoint responses to this genotoxin. BPDE-induced Chk1 phosphorylation and S-phase arrest were abrogated by caffeine treatment in AT cells, suggesting that a caffeine-sensitive kinase other than ATM is an important mediator of responses to BPDE-adducted DNA. Overall, our data demonstrate the existence of a caffeine-sensitive, Chk1-mediated, S-phase checkpoint that is operational in response to BPDE.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2002

An ATM-independent S-phase checkpoint response involves CHK1 pathway.

After exposure to genotoxic stress, proliferating cells actively slow down the DNA replication through a S-phase checkpoint to provide time for repair. We report that in addition to the ataxia-telangiectasia mutated (ATM)-dependent pathway that controls the fast response, there is an ATM-independent pathway that controls the slow response to regulate the S-phase checkpoint after ionizing radiation in mammalian cells. The slow response of S-phase checkpoint, which is resistant to wortmannin, sensitive to caffeine and UCN-01, and related to cyclin-dependent kinase phosphorylation, is much stronger in CHK1 overexpressed cells, and it could be abolished by Chk1 antisense oligonucleotides. These results provide evidence that the ATM-independent slow response of S-phase checkpoint involves CHK1 pathway.

Ataxia Telangiectasia↗
PubMed2002

Ku affects the ataxia and Rad 3-related/CHK1-dependent S phase checkpoint response after camptothecin treatment.

Camptothecin (CPT) that targets DNA topoisomerase I is one of the most promising broad-spectrum anticancer drugs in development today. The cytotoxicity of CPT is S phase (S)-specific because the collision of advancing replication forks with CPT-topoisomerase I-DNA complexes results in DNA damage. After DNA damage, proliferating cells could actively slow down the DNA replication through an S checkpoint to provide time for repair. We report now that there is an activated S checkpoint response in CPT-treated mammalian cells. This response is regulated by Ataxia and Rad3-related (ATR)/CHK1 pathway. Compared with their wild-type counterparts, CPT-treated Ku80-/- cells showed stronger inhibition of DNA replication. This stronger inhibition had no relationship with DNA-dependent protein kinase (DNA-PK) activity but correlated with the higher activities of ATR and the higher activities of CHK1 in such cells. Not only caffeine, the nonspecific inhibitor of ATR, or UCN-01, the nonspecific inhibitor of CHK1, but also the specific CHK1 antisense oligonucleotide abolished the stronger inhibition of DNA replication in CPT-treated Ku80-/- cells. These results in aggregate indicated that the stronger S checkpoint in CPT-treated Ku80-/- cells is regulated through the highly activated ATR/CHK1 pathway.

Animals↗
PubMed2002

Combretastatin-A4 prodrug induces mitotic catastrophe in chronic lymphocytic leukemia cell line independent of caspase activation and poly(ADP-ribose) polymerase cleavage.

We have previously reported that combretastatin-A4 prodrug (CA4P), anantitubulin/antiangiogenic agent isolated from the South African willow tree Combretum caffrum, induced cell death primarily through mitotic catastrophe in a panel of human B-lymphoid tumors. In this study, we investigated the molecular aspects of the mitotic catastrophe and whether or not it shares the same pathways of apoptosis. For this we studied the effect of CA4P on selected markers of apoptosis [caspases 9 and 3, poly(ADP-ribose) polymerase (PARP), bcl-2, and bax] and G2-M protein regulators (p53, MDM2, 14-3-3sigma, GADD45, cdc2, cdc25, chk1, wee1, p21, and cyclin B1). The chronic lymphocytic leukemia cell line WSU-CLL was used for this purpose. Western blot analysis showed that 24 h of CA4P (5 nM) exposure induces caspase 9 activation and PARP cleavage. However, the addition of Z-Val-Ala-Asp-fluoromethylketone (a general caspase inhibitor) or Z-Leu-Glu(OMe)-His-Asp(OMe)-CH2F (a caspase 9 inhibitor) before CA4P treatment did not block cell death. No change in bcl-2 or bax protein expression was observed. Exposure of WSU-CLL cells to 4 and 5 nM CA4P was associated with overproduction of total p53 and no dramatic change in MDM2, 14-3-3sigma, GADD45, the cyclin-dependent kinase cdc2, its inhibitory phosphorylation, the cdc2-inhibitory kinase (wee1), chk1, or cdc25 hyperphosphorylation. The overaccumulation of p21 and cyclin B1 protein was obvious at 24 h. Furthermore, CA4P treatment showed an increase in the expression of a marker of mitosis (mitotic protein monoclonal-2 antibody) and an overaccumulation of the cyclin B in the nucleus. Our findings suggest that CA4P induces mitotic catastrophe and arrest of WSU-CLL cells mostly in the M phase independent of p53 and independent of chk1 and cdc2 phosphorylation pathways. Apoptosis is a secondary mechanism of death in a small proportion of cells through activation of caspase 9 and PARP cleavage. The two mechanisms of cell death, i.e., mitotic catastrophe and apoptosis, are independent of each other in our model.

14-3-3 Proteins↗
PubMed2002

Phosphorylation of chk1 at serine-345 affected by topoisomerase I poison SN-38.

Human head and neck squamous carcinoma cell lines, A253 and FaDu, were utilized to identify mediators associated with response to topoisomerase I poison, SN-38, a metabolite of irinotecan. The drug sensitivity of FaDu cells to SN-38 was significantly higher than that of the A253 cells. In A253 cells, G2/M arrest following drug treatment (0.35 microM SN-38, 2-h exposure) was accompanied by DNA fragmentation in the 50-300 kb range, but FaDu cells accumulated in S-phase concurrently with induction of smaller DNA fragmentation in the 4-80 kb range. Because the critical regulatory step in activating cdc2 during progression into mitosis appears to be dephosphorylation of Tyrosine 15 (Tyr15), we examined the Tyr15 phosphorylation status of cdc2 in both cell lines. Slightly increased levels of cdc2 phosphorylation was observed in the A253 cells, while reduced levels of cdc2 phosphorylation was noted in the FaDu cells, corresponding to the abrogation of the G2-phase arrest. Increased chk1 phosphorylation at Ser345 induced by SN-38 was accompanied by the observed G2 phase arrest in the A253 cell line, while significant downregulation of chk1 and cdc25C phosphorylation, which resulted in the abrogation of G2/M checkpoint arrest, was noted in FaDu cells at this timepoint. These results suggest that alterations of chk1 signaling are associated with the response to topoisomerase I poison SN-38. Furthermore, A253 cells possess higher levels of endogenous hMLH1, compared to FaDu cells. A deficiency in G2 arrest was observed in FaDu cells, suggesting endogenous hMLH1 protein expression is associated with the abrogation of G2/M arrest, subsequently with the response to topoisomerase I poison SN-38.

14-3-3 Proteins↗
PubMed2002

Ku affects the CHK1-dependent G(2) checkpoint after ionizing radiation.

There are two major pathways for repairing DNA double strand breaks in mammalian cells: nonhomologous end joining (NHEJ) and homologous recombination repair (HRR). The nonhomologous end joining repair is deficient in cells without Ku, whereas HRR is highly efficient in such cells compared with their wild-type counterparts. The mechanism remains unclear. We reported previously that Ku80(-/-) cells show a stronger ATM-dependent S-phase checkpoint response than Ku80(+/+) cells after ionizing radiation (IR; X-Y. Zhou et al., Oncogene, 21:6377-6381, 2002). We report in this study that Ku80(-/-) cells also show a much stronger G(2) accumulation than Ku80(+/+) cells after IR. The stronger G(2) checkpoint response in Ku80(-/-) cells is ATM independent but is accompanied with a higher activity of CHK1 kinase. Treatment with Chk1 antisense oligonucleotide abolishes the stronger G(2) checkpoint response and sensitizes Ku80(-/-) cells to IR. These data indicate that the stronger G(2) checkpoint response shown in Ku80(-/-) cells is CHK1 dependent and suggest that the CHK1-dependent checkpoint response contributes to the highly efficient HRR in such cells.

Animals↗
PubMedDate not supplied

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.

Animals↗
PubMed2003

Human Chk1 expression is dispensable for somatic cell death and critical for sustaining G2 DNA damage checkpoint.

Mammalian Chk1 is an essential kinase for embryonic development and plays an important role in the cellular response to DNA damage. However, it remains unclear whether inhibition of Chk1 induces apoptosis in somatic cells. The uncertainty has become a critical issue for rationale design of Chk1 mechanism-based anticancer drugs. Here we show that Chk1 small interfering RNA (siRNA) effectively eliminates Chk1 protein expression without altering the cell cycle profile or inducing apoptosis in various human cancer cell lines under normal conditions. In the presence of DNA-damaging agents, however, Chk1 siRNA alone is sufficient to abrogate the DNA damage-induced G(2) checkpoint and significantly enhance apoptosis. Cell cycle kinetic profiles show that abrogation of G(2) arrest is mediated through shortening of the checkpoint. We also demonstrate that Chk1 siRNA enhances DNA damage-induced apoptosis in p53-deficient cancer cell lines and augments the growth inhibition conferred by DNA-damaging agents. These findings imply that Chk1 inhibitors will have low cytotoxicity on their own and can enhance the efficacy of DNA-damaging drugs.

Apoptosis↗
PubMedDate not supplied

Regulation of human Cdc25A stability by Serine 75 phosphorylation is not sufficient to activate a S phase checkpoint.

Degradation of Cdc25A phosphatase is an ubiquitous feature of stress. There are some discrepancies in the reported roles for different phosphorylation sites in the regulation of Cdc25A stability. Using a panel of doxycycline-inducible phosphorylation mutants we show that the stability of human Cdc25A protein is dependent upon phosphorylation at S75. In non-stressed conditions and in non-mitotic cells, Cdc25A is unstable and its stability is regulated in a Chk1-dependent manner. During mitosis, Cdc25A becomes stable and does not undergo degradation after DNA damage. We further show that Chk1 kinase regulates Cdc25A stability after UV irradiation. Similar to Chk1 kinase, p38 MAPK controls Cdc25A protein level after osmotic stress. Using phospho-specific antibodies, we find that both kinases can phosphorylate S75 and S123 in vitro. Inactivation of either Chk1 after UV-irradiation or p38 MAPK after osmotic stress prevents activation of a S phase checkpoint and S75 and S123 phosphorylation. However, introduction of stable Cdc25A (S75A or S75/123A) proteins is not sufficient to overcome this checkpoint. We propose that regulation of human Cdc25A stability by its phosphorylation at S75 may contribute to S phase checkpoint activation only in cooperation with other regulatory mechanisms.

Checkpoint Kinase 1↗
PubMedDate not supplied

Characterization of the 5'flanking region of the human Chk1 gene: identification of E2F1 functional sites.

Chk1 kinase is a key signal transducer in the checkpoint pathways responding to DNA damage. Chk1 has been shown to be transcriptionally down-regulated in response to cis-dichloro-diamino platinum treatment in a p53-dependent manner. Now we isolated and characterised the 5' flanking region of the Chk1 gene. We demonstrated that the isolated region has promoter activity when cloned upstream to a reporter luciferase gene and we could define the minimal promoter region. The modulation of the transcriptional activity of the cloned Chk1 promoter region by different transcription factors was investigated by co-transfection experiments and by using different isogenic systems. It was shown that p53 is indeed able to down-regulate the promoter activity of the cloned region, providing a mechanistic explanation to the observation that p53 decreases Chk1 protein levels after DNA damage. Several E2F binding sites were detected in the genomic sequence and E2F1 induced Chk1 promoter activity in co-transfection experiments. This induction was abolished when a mutated form of E2F1, not able to bind DNA, was used. Over-expression of E2F1 sustained an increased Chk1 promoter activity, whereas overexpression of Rb protein, which binds E2F factors, decreased Chk1 promoter activity. We discuss our results in the context of Chk1 involvement in DNA damage checkpoint pathways.

5' Flanking Region↗