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Results for “Cell Cycle Checkpoints”

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PubMed2001

Role of fission yeast primase catalytic subunit in the replication checkpoint.

To investigate the cell cycle checkpoint response to aberrant S phase-initiation, we analyzed mutations of the two DNA primase subunit genes of Schizosaccharomyces pombe, spp1(+) and spp2(+) (S. pombe primase 1 and 2). spp1(+) encodes the catalytic subunit that synthesizes the RNA primer, which is then utilized by Polalpha to synthesize the initiation DNA. Here, we reported the isolation of the fission yeast spp1(+) gene and cDNA and the characterization of Spp1 protein and its cellular localization during the cell cycle. Spp1 is essential for cell viability, and thermosensitive mutants of spp1(+) exhibit an allele-specific abnormal mitotic phenotype. Mutations of spp1(+) reduce the steady-state cellular levels of Spp1 protein and compromised the formation of Polalpha-primase complex. The spp1 mutant displaying an aberrant mitotic phenotype also fails to properly activate the Chk1 checkpoint kinase, but not the Cds1 checkpoint kinase. Mutational analysis of Polalpha has previously shown that activation of the replication checkpoint requires the initiation of DNA synthesis by Polalpha. Together, these have led us to propose that suboptimal cellular levels of polalpha-primase complex due to the allele-specific mutations of Spp1 might not allow Polalpha to synthesize initiation DNA efficiently, resulting in failure to activate a checkpoint response. Thus, a functional Spp1 is required for the Chk1-mediated, but not the Cds1-mediated, checkpoint response after an aberrant initiation of DNA synthesis.

Amino Acid Sequence↗
PubMed1994

Hypoxia induces accumulation of p53 protein, but activation of a G1-phase checkpoint by low-oxygen conditions is independent of p53 status.

It has been convincingly demonstrated that genotoxic stresses cause the accumulation of the tumor suppressor gene p53. One important consequence of increased p53 protein levels in response to DNA damage is the activation of a G1-phase cell cycle checkpoint. It has also been shown that G1-phase cell cycle checkpoints are activated in response to other stresses, such as lack of oxygen. Here we show that hypoxia and heat, agents that induce cellular stress primarily by inhibiting oxygen-dependent metabolism and denaturing proteins, respectively, also cause an increase in p53 protein levels. The p53 protein induced by heat is localized in the cytoplasm and forms a complex with the heat shock protein hsc70. The increase in nuclear p53 protein levels and DNA-binding activity and the induction of reporter gene constructs containing p53 binding sites following hypoxia occur in cells that are wild type for p53 but not in cells that possess mutant p53. However, unlike ionizing radiation, the accumulation of cells in G1 phase by hypoxia is not strictly dependent on wild-type p53 function. In addition, cells expressing the human papillomavirus E6 gene, which show increased degradation of p53 by ubiquitination and fail to accumulate p53 in response to DNA-damaging agents, do increase their p53 levels following heat and hypoxia. These results suggest that hypoxia is an example of a "nongenotoxic" stress which induces p53 activity by a different pathway than DNA-damaging agents.

Animals↗
PubMed2005

Plk1 docking to GRASP65 phosphorylated by Cdk1 suggests a mechanism for Golgi checkpoint signalling.

GRASP65, a structural protein of the Golgi apparatus, has been linked to the sensing of Golgi structure and the integration of this information with the control of mitotic entry in the form of a Golgi checkpoint. We show that Cdk1-cyclin B is the major kinase phosphorylating GRASP65 in mitosis, and that phosphorylated GRASP65 interacts with the polo box domain of the polo-like kinase Plk1. GRASP65 is phosphorylated in its C-terminal domain at four consensus sites by Cdk1-cyclin B, and mutation of these residues to alanine essentially abolishes both mitotic phosphorylation and Plk1 binding. Expression of the wild-type GRASP65 C-terminus but not the phosphorylation defective mutant in normal rat kidney cells causes a delay but not the block in mitotic entry expected if this were a true cell cycle checkpoint. These findings identify a Plk1-dependent signalling mechanism potentially linking Golgi structure and cell cycle control, but suggest that this may not be a cell cycle checkpoint in the classical sense.

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↗
PubMed2006

The role of polycomb group proteins in cell cycle regulation during development.

Polycomb group (PcG) and trithorax group (trxG) proteins are evolutionarily conserved chromatin modifiers that have well known roles in the maintenance of silent and active expression states of homeotic genes. PcG proteins may also be involved in the control of cellular proliferation, as several PcG complexes have been shown to act either as proto-oncogenes or as tumor suppressors in vertebrates. In Drosophila, PcG factors associate with specific DNA regions termed PcG response elements (PREs), and a PRE was recently identified in the gene encoding Cyclin A. Still, it is not yet clear how PcG complexes could control cell cycle progression. Beyond acting as stable silencers of cell cycle genes during the differentiation process, PcG complexes might also be integrators and/or modulators of cell cycle checkpoints in dividing cells. Here, we discuss this dual aspect of PcG involvement in epigenetic cell cycle control.

Animals↗
PubMed2003

Common polymorphisms in checkpoint kinase 2 are not associated with breast cancer risk.

A substantial proportion of the familial risk of breast cancer may be attributable to genetic variants each contributing a small effect. Polymorphisms in DNA repair genes are good candidates for such low penetrance breast cancer susceptibility alleles. Checkpoint kinase 2 (CHEK2) is a kinase in which the yeast counterpart regulates a cell cycle checkpoint and causes cells to arrest proliferation after DNA damage. A rare, protein truncating mutation in the CHEK2 gene has recently been shown to confer a modest risk of breast cancer. The aim of this study was to determine whether common polymorphic variants in CHEK2 are associated with an increase in breast cancer risk. We assessed two variants in CHEK2 using a case control study design (n = 1786 cases and 1828 controls). No differences in genotype frequencies were found between cases and control for either the IVS1 + 38insa or the a1013g polymorphisms (P = 0.3 and 0.2 respectively), and no genotype-specific risk was significantly different from unity. The haplotype frequency distribution in cases and controls were also similar (P = 0.3). We conclude that the CHEK2 polymorphisms IVS + 1a and a1013g do not confer an increased risk of breast cancer. It is also unlikely that other, as yet unidentified, common polymorphisms that affect risk are present in the gene in the British population.

Breast Neoplasms↗
PubMed2000

Reconstitution of an ATM-dependent checkpoint that inhibits chromosomal DNA replication following DNA damage.

Cell cycle checkpoints lead to the inhibition of cell cycle progression following DNA damage. A cell-free system derived from Xenopus eggs has been established that reconstitutes the checkpoint pathway inhibiting DNA replication initiation. DNA containing double-strand breaks inhibits replication initiation in a dose-dependent manner. Upon checkpoint activation, a prereplicative complex is assembled that contains ORC, Cdc6, Cdc7, and MCM proteins but lacks Cdc45. The checkpoint is ATM dependent. Cdk2/CyclinE acts downstream of ATM and is downregulated by Cdk2 phosphorylation on tyrosine 15. Cdk2AF/CyclinE is refractory to checkpoint signaling, and Cdc25A overrides the checkpoint and restores DNA replication. This report provides the description of a DNA damage checkpoint pathway that prevents the onset of S phase independently of the transcriptional function of p53 in a vertebrate organism.

Androstadienes↗
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↗
PubMed1995

Cell cycle and apoptosis: common pathways to life and death.

Programmed cell death, or apoptosis, is a highly regulated process used to eliminate unwanted or damaged cells from multicellular organisms. The morphology of cells undergoing apoptosis is similar to cells undergoing both normal mitosis and an aberrant form of mitosis called mitotic catastrophe. During each of these processes, cells release substrate attachments, lose cell volume, condense their chromatin, and disassemble the nuclear lamina. The morphological similarities among cells undergoing these processes suggest that the underlying biochemical changes also may be related. The susceptibility of cells to apoptosis frequently depends on the differentiation state of the cell. Additionally, cell cycle checkpoints appear to link the cell cycle to apoptosis. Deregulation of the cell cycle components has been shown to induce mitotic catastrophe and also may be involved in triggering apoptosis. Some apoptotic cells express abnormal levels of cell cycle proteins and often contain active Cdc2, the primary kinase active during mitosis. Although cell cycle components may not be involved in all forms of apoptosis, in many instances cell proliferation and cell death may share common pathways.

Animals↗
PubMed2003

Regulation of cell cycles in Drosophila development: intrinsic and extrinsic cues.

An intriguing aspect of cell cycle regulation is how cell growth and division are coordinated with developmental signals to produce properly patterned organisms of the appropriate size. Using the foundation laid by a detailed understanding of the regulators that intrinsically control progression through the cell cycle, links between developmental signals and the cell cycle are being elucidated. Considerable progress has been made using Drosophila melanogaster, both in identifying new cell cycle regulators that respond to developmental cues and in defining the impact of extrinsic signals on homologs of mammalian oncogenes and tumor suppressors. In this review, we discuss each cell cycle phase, highlighting differences between archetypal and variant cell cycles employed for specific developmental strategies. We emphasize the interplay between developmental signals and cell cycle transitions. Developmental control of checkpoints, cell cycle exit, and cell growth are also addressed.

Animals↗
PubMed2006

Cell cycle inhibition in malignant lymphoma: disease control by attacking the cellular proliferation machinery.

Disruption of a proper regulation of cell proliferation can ultimately cause cancer. Most human B cell malignancies are driven by chromosomal translocations or other genetic alterations which directly affect the function of critical cell cycle proteins, such as cyclins and cyclin-dependent kinases. In addition, the transformation of indolent lymphomas into aggressive malignancies is often accompanied by a loss of tumor suppressors controlling important cell cycle checkpoints. A better understanding of cell cycle deregulations in human tumors has promoted the introduction of a new class of antiproliferative drugs into cancer therapies. These drugs exert their function by specifically blocking important cell cycle proteins. In the present review we discuss how alterations in the cell cycle control contribute to the malignant transformation of B cells. Furthermore, we provide an overview of novel direct and indirect cell cycle inhibitors and their impact on the treatment of patients with B cell lymphomas.

Animals↗
PubMed2000

Cks1 mediates vascular smooth muscle cell polyploidization.

Vascular smooth muscle cells (VSMC) at capacitance arteries of hypertensive individuals and animals undergo dramatic polyploidization that contributes toward their hypertrophic phenotype. We report here the identification of a defective mitotic spindle cell cycle checkpoint in VSMC isolated from capacitance arteries of pre-hypertensive rats. These cells demonstrated a high predisposition to polyploidization in culture and failed to maintain cyclin B protein levels in response to colcemid, a mitotic inhibitor. Furthermore, this altered mitotic spindle checkpoint status was associated with the overexpression of Cks1, a Cdc2 adapter protein that promotes cyclin B degradation. Cks1 up-regulation, cyclin B down-regulation, and VSMC polyploidization were evidenced at the smooth muscle of capacitance arteries of genetically hypertensive and Goldblatt-operated rats. In addition, angiotensin II infusion dramatically increased Cks1 protein levels at capacitance arteries of normotensive rats, and angiotensin II treatment of isolated VSMC abrogated their ability to down-regulate Cks1 and maintain cyclin B protein expression in response to colcemid. Finally, transduction of VSMC from normotensive animals with a retrovirus that drives the expression of Cks1 was sufficient to alter their mitotic spindle cell cycle checkpoint status and promote unscheduled cyclin B metabolism, cell cycle re-entry, and polyploidization. These data demonstrate that Cks1 regulates cyclin B metabolism and ploidy in VSMC and may contribute to the understanding of the phenomena of VSMC polyploidization during hypertension.

Animals↗
PubMed2004

Direct regulation of CREB transcriptional activity by ATM in response to genotoxic stress.

Ataxia-telangiectasia (A-T) is a syndrome of cancer susceptibility, immune dysfunction, and neurodegeneration that is caused by mutations in the A-T-mutated (ATM) gene. ATM has been implicated as a critical regulator of cellular responses to DNA damage, including the activation of cell cycle checkpoints and induction of apoptosis. Although defective cell cycle-checkpoint regulation and associated genomic instability presumably contribute to cancer susceptibility in A-T, the mechanism of neurodegeneration in A-T is not well understood. In addition, although ATM is required for the induction of the p53 transcriptional program in response to DNA damage, the identities of the relevant transcription factors that mediate ATM-dependent changes in gene expression remain largely undetermined. In this article, we describe a signal transduction pathway linking ATM directly to the Ca(2+)/cAMP response element-binding protein, CREB, a transcription factor that regulates cell growth, homeostasis, and survival. ATM phosphorylated CREB in vitro and in vivo in response to ionizing radiation (IR) and H(2)O(2) on a stress-inducible domain. IR-induced phosphorylation of CREB correlated with a decrease in CREB transactivation potential and reduced interaction between CREB and its transcriptional coactivator, CREB-binding protein (CBP). A CREB mutant containing Ala substitutions at ATM phosphorylation sites displayed enhanced transactivation potential, resistance to inhibition by IR, and increased binding to CBP. We propose that ATM-mediated phosphorylation of CREB in response to DNA damage modulates CREB-dependent gene expression and that dysregulation of the ATM-CREB pathway may contribute to neurodegeneration in A-T.

Amino Acid Sequence↗
PubMed2005

A novel mechanism of checkpoint abrogation conferred by Chk1 downregulation.

Chk1 is the major mediator in the activation of cell-cycle checkpoints in response to a variety of genotoxic stresses. We have previously shown that inhibition of Chk1 sensitizes tumor cells to topoisomerase inhibitors such as camptothecin and doxorubicin through abrogation of cell-cycle arrest (S or G2/M checkpoints). However, it was not clear whether inhibition of Chk1 could potentiate antimetabolites, a mainstay of cancer therapy, which confer genotoxic stress through a different mechanism than topoisomerase inhibitors. 5-Fluorouracil (5-FU) is the most widely used antimetabolite in the treatment of colorectal, breast and other major types of cancers. Here we demonstrate that 5-FU activates Chk1 and induces an early S-phase arrest. Chk1 downregulation abrogates this arrest and dramatically sensitizes tumor cells to the cytotoxic effects of 5-FU. 5-FU confers S-phase arrest through Chk1-mediated Cdc25A proteolysis leading to inhibition of Cdk2. Chk1 elimination stabilizes the Cdc25A protein and results in the abrogation of the S checkpoint and resumption of DNA synthesis, which leads to excessive accumulation of double-stranded DNA breaks. As a result, downregulation of Chk1 potentiates 5-FU efficacy through induction of premature chromosomal condensation followed by apoptosis. Interestingly, the profiles of various cell-cycle markers indicate that cells progress to early M phase to induce apoptosis after checkpoint abrogation. Yet, cells fail to increase their DNA content to 4N as revealed by FACS analysis, probably due to the dramatic induction of double-stranded DNA breaks and chromosomal fragmentation. This is significantly different from the cell-cycle profiles observed in the potentiation of topoisomerase inhibitors by Chk1 siRNA, which showed mitotic progression with 4N DNA content leading to mitotic catastrophe after abrogation of the S or G2 checkpoint. Thus, our results illustrate a novel mode of checkpoint abrogation and cell death conferred by Chk1 inhibition. Additionally, we show that Chk1 deficiency potentiates 5-FU efficacy through the preferential induction of the caspase-8 pathway and subsequent caspase-3 activation. In conclusion, we have clearly demonstrated that inhibition of Chk1 not only potentiates the toxicity of conventional DNA-damaging agents such as ionizing radiation and topoisomerase inhibitors, but also enhances the toxicity of antimetabolites in cancer cell lines. This discovery reveals novel scope of checkpoint abrogation and will significantly broaden the potential application of Chk1 inhibitors in cancer therapy if they do not potentiate the toxicity of 5-FU in normal cells.

Antimetabolites, Antineoplastic↗
PubMed2004

Different DNA lesions trigger distinct cell death responses in HCT116 colon carcinoma cells.

The pleiotrophic cellular response to DNA damage includes activation of cell cycle checkpoints, induction of DNA repair pathways, and initiation of programmed cell death among others. The fate of cells with damaged DNA depends on the coordination of these different responses. The clinical efficacy of genotoxic therapies is influenced by cell fate and thus by how the DNA damage response is coordinated. While a great deal has been learned about how different DNA lesions activate distinct cell cycle checkpoints and DNA repair pathways, less is known about whether the type of DNA lesion influences the qualitative and quantitative nature of the cell death response. To address this question, HCT116 colon carcinoma cells have been treated with equally cytotoxic doses of the antitumor DNA alkylating agents adozelesin or bizelesin or the DNA strand scission agent C-1027. The relative contribution of cell cycle arrest and cell death to measured cytotoxicity varied among the three drugs. Apoptotic cell death accounts for most C-1027 cytotoxicity while cell cycle arrest and cell death both contribute to the cytotoxicity of the alkylating agents. Each of the drugs induces a distinct but overlapping pattern of caspase activation. In addition, the cell death response to these drugs is differentially dependent on p53 and p21. These observations suggest that the type of DNA lesion influences not only the relative extent of apoptotic cell death at a given cytotoxic dose but also the qualitative nature of that response.

Aminoglycosides↗
PubMed2005

Aberrant cell cycle regulation in cervical carcinoma.

Carcinoma of the uterine cervix is one of the most common malignancies among women worldwide. Human papillomaviruses (HPV) have been identified as the major etiological factor in cervical carcinogenesis. However, the time lag between HPV infection and the diagnosis of cancer indicates that multiple steps, as well as multiple factors, may be necessary for the development of cervical cancer. The development and progression of cervical carcinoma have been shown to be dependent on various genetic and epigenetic events, especially alterations in the cell cycle checkpoint machinery. In mammalian cells, control of the cell cycle is regulated by the activity of cyclin-dependent kinases (CDKs) and their essential activating coenzymes, the cyclins. Generally, CDKs, cyclins, and CDK inhibitors function within several pathways, including the p16(INK4A)-cyclin D1-CDK4/6-pRb-E2F, p21(WAF1)- p27(KIP1)-cyclinE-CDK2, and p14(ARF)-MDM2-p53 pathways. The results from several studies showed aberrant regulation of several cell cycle proteins, such as cyclin D, cyclin E, p16(INK4A), p21(WAF1), and p27(KIP1), as characteristic features of HPV- infected and HPV E6/E7 oncogene-expressing cervical carcinomas and their precursors. These data suggested further that interactions of viral proteins with host cellular proteins, particularly cell cycle proteins, are involved in the activation or repression of cell cycle progression in cervical carcinogenesis.

Cell Cycle↗
PubMed2006

Cell cycle and checkpoint regulation of histone H3 K56 acetylation by Hst3 and Hst4.

Histone modifications, including H3 K56 acetylation, have been implicated in DNA damage tolerance. Here, we present evidence that Hst3 and Hst4, two paralogues of the histone deacetylase Sir2, target the cell cycle-regulated acetylation of H3 on K56 and are downregulated during DNA damage in a checkpoint-dependent manner. We show that Hst3 and Hst4 are themselves cell cycle regulated and that their misexpression affects H3 K56-Ac. Moreover, a histone H3 K56R mutation is epistatic to all phenotypes caused by HST3/4 deletion or HST3 overexpression, suggesting that H3K56-Ac is the major target of these histone deacetylases. On examining 18 members of the "Clb2 cluster" of cell cycle-regulated proteins to which Hst3 belongs, we find that two others, Ynl058c and Alk1, are significantly downregulated on DNA damage. Taken together, our data show that Hst3/Hst4 are negative regulators of H3 K56-Ac and that HST3 downregulation by a checkpoint-mediated transcriptional repression system is essential for surviving DNA damage.

Acetylation↗