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PubMed2006

Contribution of growth and cell cycle checkpoints to radiation survival in Drosophila.

Cell cycle checkpoints contribute to survival after exposure to ionizing radiation (IR) by arresting the cell cycle and permitting repair. As such, yeast and mammalian cells lacking checkpoints are more sensitive to killing by IR. We reported previously that Drosophila larvae mutant for grp (encoding a homolog of Chk1) survive IR as well as wild type despite being deficient in cell cycle checkpoints. This discrepancy could be due to differences either among species or between unicellular and multicellular systems. Here, we provide evidence that Grapes is needed for survival of Drosophila S2 cells after exposure to similar doses of IR, suggesting that multicellular organisms may utilize checkpoint-independent mechanisms to survive irradiation. The dispensability of checkpoints in multicellular organisms could be due to replacement of damaged cells by regeneration through increased nutritional uptake and compensatory proliferation. In support of this idea, we find that inhibition of nutritional uptake (by starvation or onset of pupariation) or inhibition of growth factor signaling and downstream targets (by mutations in cdk4, chico, or dmyc) reduced the radiation survival of larvae. Further, some of these treatments are more detrimental for grp mutants, suggesting that the need for compensatory proliferation is greater for checkpoint mutants. The difference in survival of grp and wild-type larvae allowed us to screen for small molecules that act as genotype-specific radiation sensitizers in a multicellular context. A pilot screen of a small molecule library from the National Cancer Institute yielded known and approved radio-sensitizing anticancer drugs. Since radiation is a common treatment option for human cancers, we propose that Drosophila may be used as an in vivo screening tool for genotype-specific drugs that enhance the effect of radiation therapy.

Animals↗
PubMed2006

Onset of the DNA replication checkpoint in the early Drosophila embryo.

The Drosophila embryo is a promising model for isolating gene products that coordinate S phase and mitosis. We have reported before that increasing maternal Cyclin B dosage to up to six copies (six cycB) increases Cdk1-Cyclin B (CycB) levels and activity in the embryo, delays nuclear migration at cycle 10, and produces abnormal nuclei at cycle 14. Here we show that the level of CycB in the embryo inversely correlates with the ability to lengthen interphase as the embryo transits from preblastoderm to blastoderm stages and defines the onset of a checkpoint that regulates mitosis when DNA replication is blocked with aphidicolin. A screen for modifiers of the six cycB phenotypes identified 10 new suppressor deficiencies. In addition, heterozygote dRPA2 (a DNA replication gene) mutants suppressed only the abnormal nuclear phenotype at cycle 14. Reduction of dRPA2 also restored interphase duration and checkpoint efficacy to control levels. We propose that lowered dRPA2 levels activate Grp/Chk1 to counteract excess Cdk1-CycB activity and restore interphase duration and the ability to block mitosis in response to aphidicolin. Our results suggest an antagonistic interaction between DNA replication checkpoint activation and Cdk1-CycB activity during the transition from preblastoderm to blastoderm cycles.

Animals↗
PubMed2004

Cooperative function of Chk1 and p38 pathways in activating G2 arrest following exposure to temozolomide.

OBJECT: The Chk1 and p38 mitogen-activated protein kinase (MAPK) pathways play key roles in the G2 arrest caused by exposing glioma cells to temozolomide (TMZ). Although inhibition of either pathway sensitizes glioma cells to TMZ-induced cytotoxicity, the relative contributions of these pathways to TMZ-induced G2 arrest and to TMZ resistance conferred by G2 arrest have not been defined. METHODS: The authors pharmacologically inhibited the Chk1 and/or p38 pathways in U87MG human glioma cells prior to and/or after exposure to TMZ; thereafter, effects on the TMZ-induced G2 arrest pathway and toxicity were monitored. The p38 inhibitor SB203580 or the Chk1 inhibitor UCN-01 or their combination blocked TMZ-mediated inactivation of cdc25C and cdc2, suggesting that p38 and Chk1 pathways work cooperatively and are both necessary to inactivate cdc25C and cdc2. Consistent with this idea, the inhibition of both Chk1 and p38 pathways did not lead to greater bypass of TMZ-induced G2 arrest or greater cytotoxicity than inhibition of either pathway alone. Inhibition of p38 did not alter TMZ-induced Chk1 activation/phosphorylation and vice versa, suggesting that p38 and Chk1 do not cooperatively bring about G2 arrest by reciprocal activation/phosphorylation. The two pathways, however, are not functionally identical; the Chk1 pathway was required for both the initiation and maintenance of TMZ-induced G2 arrest, whereas the p38 pathway played a role only in the initiation. CONCLUSIONS: The Chk1 and p38 pathways cooperate to bring about TMZ-induced G2 arrest, and the inhibition of either pathway alone is sufficient to sensitize U87MG glioma cells to TMZ-induced cytotoxicity.

Antineoplastic Agents, Alkylating↗
PubMed2000

Analysis of the candidate target genes for mutation in microsatellite instability-positive cancers of the colorectum, stomach, and endometrium.

Microsatellite instability (MSI) in human carcinoma DNA is a characteristic phenotype observed in hereditary non-polyposis colorectal cancer and also in some human sporadic cancers including multiple primary carcinomas. In this study, we analyzed mutations in the hCHK1, E2F4, hMSH3, and hMSH6 genes in MSI+ human cancers arising in colorectum, stomach and endometrium. The E2F4 and hMSH3 genes were mutated in all tumor types. Interestingly, the hMSH6 gene was mutated in colorectal and gastric cancers but not in endometrial cancer; this is similar to the TGFbetaRII gene. It is notable that the mutation status of the secondary mutators, hMSH3 and hMSH6, did not influence slippage-related frameshift mutations in genes harboring simple tandem-repeats, which suggests that the MSI phenotype may be affected mainly by abnormalities in the primary mutator genes, not by the secondary mutator genes. No mutations were observed in the cell cycle checkpoint gene hCHK1; mutations of this gene are thought to have a limited role, if any, in at least the tumor types analyzed in this study.

Checkpoint Kinase 1↗
PubMedDate not supplied

Therapeutic exploitation of checkpoint defects in cancer cells lacking p53 function.

Cytotoxic agents form the basis of most cancer therapies. These agents primarily affect rapidly proliferating cells, so their use incurs morbidity associated with damage to tissues such as bone marrow and gastrointestinal mucosa. Clinical outcome would be improved if it were possible to develop therapeutics with more specific activity against p53-deficient cancers, which account for over 50% of all cases. p53 deficiency alters the cellular response to DNA damage in that it leaves cells with attenuated DNA damage checkpoint controls and a reduced propensity for apoptotic cell death. Thus, the DNA repair capacity of these cells is reduced but survival is increased. This promotes genomic instability and contributes to the resistance of p53-deficient cells to cytotoxic agents. Disabling the residual G(2) checkpoint function of p53-deficient cells may favour cell death following DNA damage. Several potential strategies for G(2) checkpoint abrogation show promise for the specific sensitization of cancer cells. Here we detail how the G(2) DNA damage checkpoint is influenced by p53 status and how the loss of p53 function in cancer cells can be exploited to enhance the cytotoxicity of anti-cancer agents.

Animals↗
PubMed2004

Werner syndrome protein and the MRE11 complex are involved in a common pathway of replication fork recovery.

Werner syndrome (WS) is an autosomal recessive disease that predisposes individuals to a wide range of cancers. The gene mutated in WS, WRN, encodes a member of the RecQ family of DNA helicases. The precise DNA metabolic processes in which WRN participates remain to be elucidated. However, it has been proposed that WRN might play an important role in the maintenance of genetic stability during DNA replication, possibly cooperating with other proteins. Here, we show that, following DNA replication arrest, WRN associates and colocalizes with the MRE11 complex at PCNA sites. We also provide evidence that both WRN/MRE11 complex association and proper WRN relocalization after HU treatment require a functional MRE11 complex. We demonstrate that mutations altering the functionality of WRN or that of the MRE11 complex result in chromosomal breakage during DNA replication and enhanced cell death following replication arrest. Finally, we show that the DNA breakage in replicating cells and apoptosis observed in WS are not enhanced by concomitant knock down of MRE11 by RNAi, indicating that WRN and MRE11 complex act in a common pathway. These results suggest a functional relationship between WRN and the MRE11 complex in response to replication fork arrest, disclosing a common action of WRN and the MRE11 complex in the pathway(s) preserving genome stability during DNA replication.

Apoptosis↗
PubMed2004

Chk1 versus Cdc25: chking one's levels of cellular proliferation.

This review summarizes recent studies which have provided new insight into the mechanisms by which the DNA damage response kinase, Chk1 inhibits the dual specificity phosphatase, Cdc25, and thereby regulates cell cycle progression. Recently, Chk1 has been shown to not only regulate Cdc25A degradation but also its ability to interact with various Cdk complexes through phosphorylation of the carboxy-terminus of the phosphatase. Surprisingly, these effects appear to be specific for Chk1, but not Chk2, which may explain the recently reported in vivo haploinsufficiency phenotype observed in the mammary gland using a Chk1 conditional mouse model.

Animals↗
PubMed2005

Inhibition of the ATR/Chk1 pathway induces a p38-dependent S-phase delay in mouse embryonic stem cells.

Ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases, family members of the PI-3 kinase related proteins, play a key role in checkpoint activation and maintenance of genomic stability following DNA damage. We have used wild type (WT) and p38alpha-deficient mouse embryonic stem (ES) cells to investigate the role of ATR and ATM kinases during embryonic cell cycle. We have found that inhibition of ATR and ATM kinases with caffeine or Chk1 with UCN-01, results in activation of a p38-dependent intra-S-phase checkpoint and activation of apoptosis in ES cells. However, wortmannin at a concentration, that inhibits ATM kinase but not ATR kinase, did not affect cell cycle progression. Furthermore, the presence of caffeine results in activation of p38 kinase, accumulation of p21/Waf1 in a complex with Cdk2 and decrease of Cdk2 kinase activity. In contrast, caffeine-treated p38alpha-/- ES cells show less apoptosis, and fail to trigger an effective S-phase checkpoint and accumulation of p21/Waf1. We conclude that ATR kinase activity is essential for normal cell cycle progression of exponentially proliferating mouse ES cells even in the absence of exogenous DNA damage, and ATR deregulation triggers p38alpha-dependent cell-cycle checkpoint and apoptotic responses.

Animals↗
PubMed2005

ARF the integrator: linking NF-kappaB, p53 and checkpoint kinases.

The ARF tumor suppressor initiates the cellular response to aberrant oncogene activation through binding to and inhibiting the activity of Hdm2/Mdm2, the inhibitor of p53. However, many pathways also active in the cell will oppose p53 function if left unchecked. An example of this, is the RelA (p65) NF-kappaB subunit. Frequently activated by oncogenes, RelA is a potent inducer of anti-apoptotic gene expression, which has the potential to inhibit the pro-apoptotic functions of p53. We have recently discovered that by inducing the activity of the checkpoint kinases ATR and Chk1, ARF neutralises this opposing pathway. ARF-induced Chk1 phosphorylates RelA on threonine 505, a residue in its transactivation domain, thus inhibiting NF-kappaB's ability to stimulate anti-apoptotic gene expression. Furthermore, ARF-induced ATR is required for efficient induction and activation of p53. We propose that this pathway will target other proteins with pro-proliferative or anti-apoptotic functions. Therefore, through this mechanism, ARF can integrate the cellular response to an oncogene, thus maximising the effectiveness of the p53 tumor suppressor pathway.

ADP-Ribosylation Factors↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2006

Evidence that the ATR/Chk1 pathway maintains normal replication fork progression during unperturbed S phase.

If cells are treated with DNA damaging agents or inhibitors that interfere with ongoing DNA replication, the intra-S and S/M checkpoints delay progression through S phase and mitotic entry, respectively, to allow time for DNA repair and replication restart. In vertebrates, these checkpoint responses to replication blocks are largely mediated by the sensor kinase ATR and its major downstream effector kinase Chk1. Increasing evidence suggests that the ATR pathway is also vital in the absence of exogenous stresses, i.e., during "unperturbed" replication. Both ATR and Chk1 are essential proteins in vertebrates, and lack of components of the ATR/Chk1 pathway can result in impaired replication and spontaneous DNA damage. Here we give an overview of how the ATR/Chk1 pathway responds to exogenously blocked replication and then describe evidence for roles of this pathway during replication in an unperturbed S phase.

Animals↗
PubMed2006

Turning the replication checkpoint on and off.

The replication checkpoint monitors the progress of DNA replication forks during S phase, and delays the firing of later replication origins when active replication forks are stalled due to collisions with damaged or abnormally structured DNA. Key components of the replication checkpoint pathway are the apical protein kinase, ATR, and its downstream target kinase, Chk1. Defects in either ATR or Chk1 function result in loss of DNA replication fidelity and cell viability, even in the absence of extrinsic genotoxic stress. Moreover, several clinically important antitumor agents, such as the camptothecins (CPTs), exert their antitumor effects by interfering with DNA replication, and hence the therapeutic response to these drugs is intimately related to signaling through the replication checkpoint. A recent report from this laboratory adds a new facet to the regulatory mechanisms that control the function and duration of checkpoint signaling through the ATR-Chk1 pathway. The results indicate that replication stress induced by a variety of agents, including CPT and deep hypoxia, triggers the ubiquitin-dependent degradation of the checkpoint kinase Chk1 in both normal and transformed human cells. This review provides an overview of the study's major findings, together with their implications for both replication checkpoint function and tumor responsiveness to CPT and related anticancer drugs.

Checkpoint Kinase 1↗
PubMed2006

Chk1-dependent regulation of Cdc25B functions to coordinate mitotic events.

The coordination of mitotic spindle formation and chromatin condensation is an essential prerequisite for successful mitosis. Both events are thought to be initiated by cyclin B/Cdk1, whose initial activation occurs in late prophase at the centrosomes. Recently, we have shown that Chk1 localizes to interphase centrosomes and thereby negatively regulates entry into mitosis by preventing premature activation of cyclin B/Cdk1. Here, we demonstrate that inhibition of Chk1 kinase induces mitotic entry with regular spindle assembly but aberrant and mislocalized chromatin. This effect, which we have termed the 'paraspindle' phenotype, was reverted by downregulation of Cdc25B phosphatase using siRNA, which restored normal mitosis with regular chromatin. Analogous to Chk1 inhibition, the 'paraspindle' phenotype was induced by overexpression of Cdc25B but not Cdc25A. Our results suggest that Chk1 functions to coordinate mitotic events through regulation of Cdc25B.

Cell Cycle↗
PubMed2006

BRIT1/MCPH1: a guardian of genome and an enemy of tumors.

The DNA of every cell is constantly exposed to insult mediated by endogenous and environmental factors that induced damage in its structure. To react to these attacks and maintain the integrity of the genome, eukaryotic cells are equipped with sophisticated mechanisms to detect, signal the presence of and repair DNA damage. The cellular response to DNA damage is a critical event for maintaining genomic stability and limiting neoplastic transformation. BRIT1, a newly identified protein, forms specific irradiation-induced nuclear foci. Our recent investigation demonstrates that BRIT1 functions as a proximal factor in the DNA damage checkpoints that control multiple damage sensors and early mediators. BRIT1 is also implicated in cell cycle checkpoints, controlling and regulating other important molecules and thus affecting the timing of mitosis. Depletion of BRIT1 abolishes the DNA damage response and results in centrosomal abnormalities and chromosomal aberrations. Moreover, aberrantly reduced expression of BRIT1 in human carcinomas implicates this protein in cancer initiation and progression. Together, the findings identify BRIT1 as a potential tumor suppressor. Fully elucidating the function of this intriguing protein may lead to new therapeutic approaches for the improved cancer treatment.

Animals↗
PubMed2006

ATR-Chk1 axis protects BCR/ABL leukemia cells from the lethal effect of DNA double-strand breaks.

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and mitomycin C (MMC, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assay. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. Elevated levels of drug-induced DSBs in leukemia cells were associated with higher activity of ATR kinase, and enhanced phosphorylation of histone H2AX on serine 139 (gamma-H2AX). gamma-H2AX eventually started to disappear in BCR/ABL cells, while continued to increase in parental cells. In addition, the expression and ATR-mediated phosphorylation of Chk1 kinase on serine 345 were often more abundant in BCR/ABL-positive leukemia cells than normal counterparts after genotoxic treatment. Inhibition of ATR kinase by caffeine but not Chk1 kinase by indolocarbazole inhibitor, SB218078 sensitized BCR/ABL leukemia cells to MMC in a short-term survival assay. Nevertheless, both caffeine and SB218078 enhanced the genotoxic effect of MMC in a long-term clonogenic assay. This effect was associated with the abrogation of transient accumulation of leukemia cells in S and G2/M cell cycle phases after drug treatment. In conclusion, ATR-Chk1 axis was strongly activated in BCR/ABL-positive cells and contributed to the resistance to DNA cross-linking agents causing numerous replication-dependent DSBs.

Antineoplastic Agents↗
PubMed2000

An indolocarbazole inhibitor of human checkpoint kinase (Chk1) abrogates cell cycle arrest caused by DNA damage.

Many cancer therapies cause DNA damage to effectively kill proliferating tumor cells; however, a major limitation of current therapies is the emergence of resistant tumors following initial treatment. Cell cycle checkpoints are involved in the response to DNA damage and specifically prevent cell cycle progression to allow DNA repair. Tumor cells can take advantage of the G2 checkpoint to arrest following DNA damage and avoid immediate cell death. This can contribute to acquisition of drug resistance. By abrogating the G2 checkpoint arrest, it may be possible to synergistically augment tumor cell death induced by DNA damage and circumvent resistance. This requires an understanding of the molecules involved in regulating the checkpoints. Human Chk1 is a recently identified homologue of the Schizosaccharomyces pombe checkpoint kinase gene, which is required for G2 arrest in response to DNA damage. Chk1 phosphorylates the dual specificity phosphatase cdc25C on Ser-216, and this may be involved in preventing cdc25 from activating cdc2/cyclinB and initiating mitosis. To further study the role of Chk1 in G2 checkpoint control, we identified a potent and selective indolocarbazole inhibitor (SB-218078) of Chk1 kinase activity and used this compound to assess cell cycle checkpoint responses. Limited DNA damage induced by gamma-irradiation or the topoisomerase I inhibitor topotecan was used to induce G2 arrest in HeLa cells. In the presence of the Chk1 inhibitor, the cells did not arrest following gamma-irradiation or treatment with topotecan, but continued into mitosis. Abrogation of the damage-arrest checkpoint also enhanced the cytotoxicity of topoisomerase I inhibitors. These studies suggest that Chk1 activity is required for G2 arrest following DNA damage.

Alkaloids↗