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PubMed2003

DNA damage is a novel response to sublytic complement C5b-9-induced injury in podocytes.

In response to Ab-complement-mediated injury, podocytes can undergo lysis, apoptosis, or, when exposed to sublytic (<5% lysis) amounts of C5b-9, become activated. Following the insertion of sublytic quantities of C5b-9, there is an increase in signaling pathways and growth factor synthesis and release of proteases, oxidants, and other molecules. Despite an increase in DNA synthesis, however, sublytic C5b-9 is associated with a delay in G(2)/M phase progression in podocytes. Here we induced sublytic C5b-9 injury in vitro by exposing cultured rat podocytes or differentiated postmitotic mouse podocytes to Ab and a complement source; we also studied the passive Heymann nephritis model of experimental membranous nephropathy in rats. A major finding was that sublytic C5b-9-induced injury caused an increase in DNA damage in podocytes both in vitro and in vivo. This was associated with an increase in protein levels for p53, the CDK inhibitor p21, growth-arrest DNA damage-45 (GADD45), and the checkpoint kinases-1 and -2. Sublytic C5b-9 increased extracellular signal-regulated kinase-1 and -2 (ERK-1 and -2), and inhibiting ERK-1 and -2 reduced the increase in p21 and GADD45 and augmented the DNA damage response to sublytic C5b-9-induced injury. These results show that sublytic C5b-9 induces DNA damage in vitro and in vivo and may explain why podocyte proliferation is limited following immune-mediated injury.

Animals↗
PubMed2003

Differential response of human acute myeloid leukemia cells to gemtuzumab ozogamicin in vitro: role of Chk1 and Chk2 phosphorylation and caspase 3.

Gemtuzumab ozogamicin (GO) is a humanized anti-CD33 antibody conjugated to the anticancer agent calicheamicin, approved for the treatment of CD33+-relapsed acute myeloid leukemia. We have investigated the effects of GO on 4 human myeloid leukemia lines of different French-American-British (FAB) types (KG-1, THP-1, HL-60, and NB-4), observing 3 different types of response. Exposure to GO (10-1000 ng/mL) induced G2 arrest (up to 80% of the cells) followed by apoptosis (45% of the cells) in HL-60 and NB-4 cells. By contrast, in THP-1 cells we observed a strong G2 arrest (up to 75% of the cells) with little apoptosis. Finally, the KG-1 line was completely resistant to the same concentrations of GO. These different responses did not correlate with the levels of expression of either CD33 or multiple-drug resistance proteins, although the higher cyclosporin A (CsA)-inhibitable efflux activity of KG-1 cells may play a role in the resistance of this line to the drug. We could show that Chk1 and Chk2 phosphorylation, but not p53 or p21 expression, correlated with G2 arrest, implicating the ataxia-telangiectasia mutated/ataxia-telangiectasia related (ATM/ATR)-Chk1/Chk2 pathway in the cell cycle response to GO. However, apoptosis was associated with caspase 3 activation. Freshly isolated acute myeloid leukemia (AML) cells showed patterns of response to GO in vitro similar to those observed with the cell lines, including phosphorylation of Chk2 and caspase 3 activation. Our results suggest that the different molecular pathways induced by the drug in vitro may reflect, at least in part, the variable response to GO obtained in vivo.

Acute Disease↗
PubMed2005

Tachpyridine, a metal chelator, induces G2 cell-cycle arrest, activates checkpoint kinases, and sensitizes cells to ionizing radiation.

Iron is critical for cell growth and proliferation. Iron chelators are being explored for a number of clinical applications, including the treatment of neurodegenerative disorders, heart disease, and cancer. To uncover mechanisms of action of tachpyridine, a chelator currently undergoing preclinical evaluation as an anticancer agent, cell-cycle analysis was performed. Tachpyridine arrested cells at G2, a radiosensitive phase of the cell cycle, and enhanced the sensitivity of cancer cells but not nontransformed cells to ionizing radiation. G2 arrest was p53 independent and was accompanied by activation of the checkpoint kinases CHK1 and CHK2. G2 arrest was blocked by UCN-01, a CHK1 inhibitor, but proceeded in CHK2 knock-out cells, indicating a critical role for CHK1 in G2 arrest. Tachpyridine-induced cell-cycle arrest was abrogated in cells treated with caffeine, an inhibitor of the ataxia-telangiectasia mutated/ataxia-telangiectasia-mutated and Rad3-related (ATM/ATR) kinases. Further, G2 arrest proceeded in ATM-deficient cells but was blocked in ATR-deficient cells, implicating ATR as the proximal kinase in tachpyridine-mediated G2 arrest. Collectively, our results suggest that iron chelators may function as antitumor and radioenhancing agents and uncover a previously unexplored activity of iron chelators in activation of ATR and checkpoint kinases.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2002

A novel chk1-dependent G1/M checkpoint in fission yeast.

Fission yeast cells with a temperature-sensitive Orp1 protein, a component of the origin recognition complex, cannot perform DNA replication at the restrictive temperature. Seventy percent of orp1-4 cells arrest with a 1C DNA content, whereas 30% proceed to mitosis ('cut'). The arrest depends upon the checkpoint Rad proteins and, surprisingly, the Chk1 protein, which is thought to act only from late S phase. The arrested cells maintain a 1C DNA content, as judged by flow cytometry, and the early origin ars3001 has not been initiated, as judged by 2D gel analysis. We show that in G1-arrested orp1-4 cells, Wee1 phosphorylates and inactivates Cdc2. Activation of Chk1 occurs earlier than Cdc2 phosphorylation, indicating a novel role for Chk1, namely to induce and/or maintain Cdc2 phosphorylation upon checkpoint activation in G1. We also show that commitment to cutting occurs already in early G1 phase.

CDC2 Protein Kinase↗
PubMed2003

Delineating the position of rad4+/cut5+ within the DNA-structure checkpoint pathways in Schizosaccharomyces pombe.

The fission yeast BRCT domain protein Rad4/Cut5 is required for genome integrity checkpoint responses and DNA replication. Here we address the position at which Rad4/Cut5 acts within the checkpoint response pathways. Rad4 is shown to act upstream of the effector kinases Chk1 and Cds1, as both Chk1 phosphorylation and Cds1 kinase activity require functional Rad4. Phosphorylation of Rad9, Rad26 and Hus1 in response to either DNA damage or inhibition of DNA replication are independent of Rad4/Cut5 checkpoint function. Further we show that a novel, epitope-tagged allele of rad4+/cut5+ acts as a dominant suppressor of the checkpoint deficiencies of rad3-, rad26- and rad17- mutants. Suppression results in the restoration of mitotic arrest and is dependent upon the remaining checkpoint Rad proteins and the two effector kinases. High-level expression of the rad4+/cut5+ allele in rad17 mutant cells restores the nuclear localization of Rad9, but this does not fully account for the observed suppression. We conclude from these data that Rad4/Cut5 acts with Rad3, Rad26 and Rad17 to effect the checkpoint response, and a model for its function is discussed.

Cell Cycle Proteins↗
PubMed2005

Grp/DChk1 is required for G2-M checkpoint activation in Drosophila S2 cells, whereas Dmnk/DChk2 is dispensable.

Cell-cycle checkpoints are signal-transduction pathways required to maintain genomic stability in dividing cells. Previously, it was reported that two kinases essential for checkpoint signalling, Chk1 and Chk2 are structurally conserved. In contrast to yeast, Xenopus and mammals, the Chk1- and Chk2-dependent pathways in Drosophila are not understood in detail. Here, we report the function of these checkpoint kinases, referred to as Grp/DChk1 and Dmnk/DChk2 in Drosophila Schneider's cells, and identify an upstream regulator as well as downstream targets of Grp/DChk1. First, we demonstrate that S2 cells are a suitable model for G(2)/M checkpoint studies. S2 cells display Grp/DChk1-dependent and Dmnk/DChk2-independent cell-cycle-checkpoint activation in response to hydroxyurea and ionizing radiation. S2 cells depleted for Grp/DChk1 using RNA interference enter mitosis in the presence of impaired DNA integrity, resulting in prolonged mitosis and mitotic catastrophe. Grp/DChk1 is phosphorylated in a Mei-41/DATR-dependent manner in response to hydroxyurea and ionizing radiation, indicating that Mei-41/ATR is an upstream component in the Grp/DChk1 DNA replication and DNA-damage-response pathways. The level of Cdc25(Stg) and phosphorylation status of Cdc2 are modulated in a Grp/DChk1-dependent manner in response to hydroxyurea and irradiation, indicating that these cell-cycle regulators are downstream targets of the Grp/DChk1-dependent DNA replication and DNA-damage responses. By contrast, depletion of Dmnk/DChk2 by RNA interference had little effect on checkpoint responses to hydroxyurea and irradiation. We conclude that Grp/DChk1, and not Dmnk/DChk2, is the main effector kinase involved in G(2)/M checkpoint control in Drosophila cells.

Animals↗
PubMed2006

Ccr4 contributes to tolerance of replication stress through control of CRT1 mRNA poly(A) tail length.

In Saccharomyces cerevisiae, DNA replication stress activates the replication checkpoint, which slows S-phase progression, stabilizes slowed or stalled replication forks, and relieves inhibition of the ribonucleotide reductase (RNR) complex. To identify novel genes that promote cellular viability after replication stress, the S. cerevisiae non-essential haploid gene deletion set (4812 strains) was screened for sensitivity to the RNR inhibitor hydroxyurea (HU). Strains bearing deletions in either CCR4 or CAF1/POP2, which encode components of the cytoplasmic mRNA deadenylase complex, were particularly sensitive to HU. We found that Ccr4 cooperated with the Dun1 branch of the replication checkpoint, such that ccr4Delta dun1Delta strains exhibited irreversible hypersensitivity to HU and persistent activation of Rad53. Moreover, because ccr4Delta and chk1Delta exhibited epistasis in several genetic contexts, we infer that Ccr4 and Chk1 act in the same pathway to overcome replication stress. A counterscreen for suppressors of ccr4Delta HU sensitivity uncovered mutations in CRT1, which encodes the transcriptional repressor of the DNA-damage-induced gene regulon. Whereas Dun1 is known to inhibit Crt1 repressor activity, we found that Ccr4 regulates CRT1 mRNA poly(A) tail length and may subtly influence Crt1 protein abundance. Simultaneous overexpression of RNR2, RNR3 and RNR4 partially rescued the HU hypersensitivity of a ccr4Delta dun1Delta strain, consistent with the notion that the RNR genes are key targets of Crt1. These results implicate the coordinated regulation of Crt1 via Ccr4 and Dun1 as a crucial nodal point in the response to DNA replication stress.

Cell Cycle Proteins↗
PubMed1999

Caffeine can override the S-M checkpoint in fission yeast.

The replication checkpoint (or 'S-M checkpoint') control prevents progression into mitosis when DNA replication is incomplete. Caffeine has been known for some time to have the capacity to override the S-M checkpoint in animal cells. We show here that caffeine also disrupts the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. By contrast, no comparable effects of caffeine on the S. pombe DNA damage checkpoint were seen. S. pombe cells arrested in early S phase and then exposed to caffeine lost viability rapidly as they attempted to enter mitosis, which was accompanied by tyrosine dephosphorylation of Cdc2. Despite this, the caffeine-induced loss of viability was not blocked in a temperature-sensitive cdc2 mutant incubated at the restrictive temperature, although catastrophic mitosis was prevented under these conditions. This suggests that, in addition to S-M checkpoint control, a caffeine-sensitive function may be important for maintenance of cell viability during S phase arrest. The lethality of a combination of caffeine with the DNA replication inhibitor hydroxyurea was suppressed by overexpression of Cds1 or Chk1, protein kinases previously implicated in S-M checkpoint control and recovery from S phase arrest. In addition, the same combination of drugs was specifically tolerated in cells overexpressing either of two novel S. pombe genes isolated in a cDNA library screen. These findings should allow further molecular investigation of the regulation of S phase arrest, and may provide a useful system with which to identify novel drugs that specifically abrogate the checkpoint control.

Amino Acid Sequence↗
PubMed2000

Chk1 and Cds1: linchpins of the DNA damage and replication checkpoint pathways.

Recent work on the mechanisms of DNA damage and replication cell cycle checkpoints has revealed great similarity between the checkpoint pathways of organisms as diverse as yeasts, flies and humans. However, there are differences in the ways these organisms regulate their cell cycles. To connect the conserved checkpoint pathways with various cell cycle targets requires an adaptable link that can target different cell cycle components in different organisms. The Chk1 and Cds1 protein kinases, downstream effectors in the checkpoint pathways, seem to play just such roles. Perhaps more surprisingly, the two kinases not only have different targets in different organisms but also seem to respond to different signals in different organisms. So, whereas in fission yeast Chk1 is required for the DNA damage checkpoint and Cds1 is specifically involved in the replication checkpoint, their roles seem to be shuffled in metazoans.

Animals↗
PubMed2000

A novel mutant allele of the chromatin-bound fission yeast checkpoint protein Rad17 separates the DNA structure checkpoints.

To further dissect the genetic differences between the checkpoint pathway following S-phase cdc arrest versus DNA damage, a genetic screen was performed for checkpoint mutants that were unable to arrest mitosis following cell-cycle arrest with a temperature-sensitive DNA polymerase delta mutant, cdc20-M10. One such checkpoint mutant, rad17-d14, was found to display the cut phenotype following S-phase arrest by cdc20-M10, but not by the DNA synthesis inhibitor hydroxyurea, reminiscent of the chk1 mutant. Unlike chk1 , rad17-d14 was not sensitive to UV irradiation. Interestingly, the ionising radiation sensitivity of rad17-d14 was only at higher doses, and cells were found to be defective in properly arresting cell division following irradiation in S phase, but not G(2) phase. Biochemical analysis attributes the checkpoint defects of rad17-d14 to the failure to phosphorylate the checkpoint effector Chk1p. To investigate if Rad17p monitors the genome for abnormal DNA structures specifically during DNA synthesis, chromatin association of Rad17p was analysed. Rad17p was found to be chromatin associated throughout the cell cycle, not just during S phase. This interaction occurred irrespective of the arrest with cdc20-M10 and, surprisingly, was also independent of the other checkpoint Rad proteins, and the cell-cycle effectors Chk1p and Cds1p.

Alleles↗
PubMed2004

Cell cycle checkpoint control mechanisms that can be disrupted in cancer.

Maintenance of genomic integrity is essential to avoid cellular transformation, neoplasia, or cell death. DNA synthesis, mitosis, and cytokinesis are important cellular processes required for cell division and the maintenance of cellular homeostasis; they are governed by many extra- and intra-cellular stimuli. Progression of normal cell division depends on cyclin interaction with cyclin-dependent kinases (Cdk) and the degradation of cyclins before chromosomal segregation through ubiquitination. Multiple checkpoints exist and are conserved in the cell cycle in higher eukaryotes to ensure that if one fails, others will take care of genomic integrity and cell survival. Many genes act as either positive or negative regulators of checkpoint function through different kinase cascades, delaying cell cycle progression to repair the DNA lesions and breaks, and assuring equal segregation of chromosomes to daughter cells. Understanding the checkpoint pathways and genes involved in the cellular response to DNA damage and cell division events in normal and cancer cells, provides information about cancer predisposition, and suggests design of small molecules and other strategies for cancer therapy.

Cell Cycle↗
PubMed2006

Regulation of DNA replication machinery by Mrc1 in fission yeast.

Faithful replication of chromosomes is crucial to genome integrity. In yeast, the ORC binds replication origins throughout the cell cycle. However, Cdc45 binds these before S-phase, and, during replication, it moves along the DNA with MCM helicase. When replication progression is inhibited, checkpoint regulation is believed to stabilize the replication fork; the detailed mechanism, however, remains unclear. To examine the relationship between replication initiation and elongation defects and the response to replication elongation block, we used fission yeast mutants of Orc1 and Cdc45--orp1-4 and sna41-928, respectively--at their respective semipermissive temperatures with regard to BrdU incorporation. Both orp1 and sna41 cells exhibited HU hypersensitivity in the absence of Chk1, a DNA damage checkpoint kinase, and were defective in full activation of Cds1, a replication checkpoint kinase, indicating that normal replication is required for Cds1 activation. Mrc1 is required to activate Cds1 and prevent the replication machinery from uncoupling from DNA synthesis. We observed that, while either the orp1 or the sna41 mutation partially suppressed HU sensitivity of cds1 cells, sna41 specifically suppressed that of mrc1 cells. Interestingly, sna41 alleviated the defect in recovery from HU arrest without increasing Cds1 activity. In addition to sna41, specific mutations of MCM suppressed the HU sensitivity of mrc1 cells. Thus, during elongation, Mrc1 may negatively regulate Cdc45 and MCM helicase to render stalled forks capable of resuming replication.

Cell Cycle Proteins↗
PubMed2006

Drosophila ATR in double-strand break repair.

The ability of a cell to sense and respond to DNA damage is essential for genome stability. An important aspect of the response is arrest of the cell cycle, presumably to allow time for repair. Ataxia telangiectasia mutated (ATM) and ATR are essential for such cell-cycle control, but some observations suggest that they also play a direct role in DNA repair. The Drosophila ortholog of ATR, MEI-41, mediates the DNA damage-dependent G2-M checkpoint. We examined the role of MEI-41 in repair of double-strand breaks (DSBs) induced by P-element excision. We found that mei-41 mutants are defective in completing the later steps of homologous recombination repair, but have no defects in end-joining repair. We hypothesized that these repair defects are the result of loss of checkpoint control. To test this, we genetically reduced mitotic cyclin levels and also examined repair in grp (DmChk1) and lok (DmChk2) mutants. Our results suggest that a significant component of the repair defects is due to loss of MEI-41-dependent cell cycle regulation. However, this does not account for all of the defects we observed. We propose a novel role for MEI-41 in DSB repair, independent of the Chk1/Chk2-mediated checkpoint response.

Animals↗
PubMed2006

Spreading the signal: dissociation of Chk1 from chromatin.

The effector kinase Chk1 plays a critical role in the DNA damage checkpoint response by phosphorylating regulators of the cell cycle machinery, resulting in an inhibition of cell cycle progression. In the presence of genotoxic stress, the PI3 kinase-like kinase ATR rapidly phosphorylates Chk1 on conserved serine residues, thereby triggering kinase activation through the release of an auto-inhibitory region present at its C-terminus and by regulating interactions with other proteins. Recent data have demonstrated an additional regulatory mechanism of Chk1 functioning: Chk1 binds chromatin in unperturbed cells and dissociates from chromatin in response to DNA damage in a manner that is dependent on PIKK-mediated phosphorylation. Here, I give an overview of these findings and discuss the implications of these data for our understanding of the execution of the checkpoint arrest induced after the detection of DNA damage.

Animals↗
PubMed2006

The relationship of premature mitosis to cytotoxicity in response to checkpoint abrogation and antimetabolite treatment.

Inhibition of one or both of the checkpoint kinases, Chk1 and Chk2, has been proposed as a strategy for improving the efficacy of cytotoxic chemotherapeutic agents in tumor cells. Previous studies have demonstrated that Chk1 inhibition potentiates the cytotoxicity of chemotherapeutic agents in a variety of systems. We designed a study to test whether the simultaneous depletion of Chk1 and Chk2 would sensitize cells to FdUrd- and gemcitabine-induced cytotoxicity to a greater extent than Chk1 depletion alone and to determine the contribution of premature mitosis to cytotoxicity. We found that RNAi-mediated Chk1 depletion enhanced FdUrd- and gemcitabine-mediated cytotoxicity (2- to 3-fold) in Panc-1 and SW620 cells. Furthermore, enhanced cytotoxicity by Chk1 depletion was accompanied by inhibition of FdUrd- or gemcitabine-induced Cdc25A degradation and induction of premature mitotic entry in drug-treated cells. The simultaneous depletion of Chk1 and Chk2 inhibited Cdc25A degradation, induced premature mitotic entry and enhanced cytotoxicity in response to FdUrd and gemcitabine to a similar extent as Chk1 depletion alone. These results imply that Chk2 inhibition has no immediate consequence on survival or cell cycle progression in tumor cells treated with antimetabolites, regardless of their Chk1 status. In addition, these results suggest that premature mitotic entry is a qualitative marker for enhanced antimetabolite-induced cytotoxicity by Chk1 inhibition. The finding that Chk1 inhibition significantly enhanced antimetabolite-induced cytotoxicity supports further investigation and the development of more specific Chk1 inhibitors for use in the clinic.

Antimetabolites, Antineoplastic↗
PubMed2000

The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites.

Upon DNA damage, the amino terminus of p53 is phosphorylated at a number of serine residues including S20, a site that is particularly important in regulating stability and function of the protein. Because no known kinase has been identified that can modify this site, HeLa nuclear extracts were fractionated and S20 phosphorylation was followed. We discovered that a S20 kinase activity copurifies with the human homolog of the Schizosaccharomyces pombe checkpoint kinase, Chk1 (hCHK1). We confirmed that recombinant hCHK1, but not a kinase-defective version of hCHK1, can phosphorylate p53 in vitro at S20. Additional inducible amino- and carboxy-terminal sites in p53 are also phosphorylated by hCHK1, indicating that this is an unusually versatile protein kinase. It is interesting that hCHK1 strongly prefers tetrameric to monomeric p53 in vitro, consistent with our observation that phosphorylation of amino-terminal sites in vivo requires that p53 be oligomeric. Regulation of the levels and activity of hCHK1 in transfected cells is directly correlated with the levels of p53; expression of either a kinase-defective hCHK1 or antisense hCHK1 leads to reduced levels of cotransfected p53, whereas overexpression of wild-type hCHK1 or the kinase domain of hCHK1 results in increased levels of expressed p53 protein. The human homolog of the second S. pombe checkpoint kinase, Cds1 (CHK2/hCds1), phosphorylates tetrameric p53 but not monomeric p53 in vitro at sites similar to those phosphorylated by hCHK1 kinase, suggesting that both checkpoint kinases can play roles in regulating p53 after DNA damage.

Blotting, Western↗
PubMed2000

Histological type-selective, tumor-predominant expression of a novel CHK1 isoform and infrequent in vivo somatic CHK2 mutation in small cell lung cancer.

Inactivation of p53, which represents the most prevalent genetic alteration in lung cancer, has been shown to play a crucial role in the acquisition of genomic instability. We examined 44 lung cancer specimens to search for mutations in the CHK1 and CHK2 genes, which have been suggested to play roles in regulating p53 after DNA damage. We found that the CHK2 gene was somatically mutated in lung cancer in vivo, although at a low frequency, and that a previously undescribed shorter isoform of CHK1 was expressed preferentially in small cell lung cancer in a tumor-predominant manner. Additional studies are warranted to investigate the functional significance of these changes as well as the potential involvement of other components in this important pathway to maintain genomic stability.

Amino Acid Sequence↗