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At least 1,693 records · Page 94Linked to original sources
PubMed2004

Polo-like kinase-1 controls recovery from a G2 DNA damage-induced arrest in mammalian cells.

DNA damage triggers multiple checkpoint pathways to arrest cell cycle progression. Less is known about the mechanisms that allow resumption of the cell cycle once checkpoint signaling is silenced. Here we show that while in undamaged cells several redundant pathways can promote the onset of mitosis, this redundancy is lost in cells recovering from a DNA damage-induced arrest. We demonstrate that Plk1 is crucial for mitotic entry following recovery from DNA damage. However, Plk1 is no longer required in cells depleted of Wee1, and we could show that Plk1 is involved in the degradation of Wee1 at the onset of mitosis. Thus, our data show that the cell cycle machinery is reset in response to DNA damage and that cells become critically dependent on Plk1-mediated degradation of Wee1 for their recovery.

Cell Cycle Proteins↗
PubMed2005

Parallel synthesis and biological evaluation of 5,6,7,8-tetrahydrobenzothieno[2,3-d]pyrimidin-4(3H)-one cytotoxic agents selective for p21-deficient cells.

A novel series of inhibitors of cancer cell proliferation, selective against p21 cell cycle checkpoint-disrupted cells vs. cells with intact p21 checkpoint, were identified by high-throughput screening. Optimization of both ends of the lead molecule to improve potency, using parallel synthesis and iterative design, is described. The 2-(1,4-dibenzodioxane)-substituted derivative 14 was identified as a highly selective and potent agent displaying an IC50 of 91 nM in the p21-deficient cell line.

Antineoplastic Agents↗
PubMed1993

Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint.

In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G2 phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G2 phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G2 phase.

Cell Cycle↗
PubMed1995

Regulation of cell cycle progression following DNA damage.

DNA damage causes an arrest in cell cycle progression. Checkpoints, which monitor the state of the DNA, exist throughout the cycle and negatively regulate cell cycle transitions when damage is detected. The molecular basis of how these checkpoints are activated, and interact with the cell cycle machinery, is just beginning to be understood. Studies in yeast have identified a number of genes involved in a G2 DNA damage checkpoint, while in mammalian cells a G1 checkpoint has been extensively studied.

Cell Cycle↗
PubMed2004

The cell cycle: accelerators, brakes, and checkpoints.

PROLIFERATIVE CUES TRIGGER a complex series of molecular signaling events in cells. Early in the cell cycle, cells are faced with an important decision that affects their fate. They either initiate a round of replication or they withdraw from cell division. Passage through the restriction point, or "point of no return," marks cellular commitment to a new round of division. Genetic mutations that predispose individuals to tumorigenesis often affect pathways that influence cellular proliferation. Many of the mutated genes give rise to molecules that are no longer able to appropriately regulate the mammalian cell cycle; the end result is neoplasia. In this review, the critical elements that permit cell cycle progression and the positive and negative regulators that affect the process are reviewed.

Brain Neoplasms↗
PubMed1999

Immunohistochemical markers of cell cycle control applied to ovarian and primary peritoneal surface epithelial neoplasms: p21(WAF1/CIP1) predicts survival and good response to platinin-based chemotherapy.

Immunohistochemistry for p53, p21(WAF1/CIP1), and Ki-67 provides insight into the molecular events controlling the cell cycle. We tested the hypothesis that these cell cycle markers will aid in the clinical evaluation of ovarian and primary peritoneal surface epithelial neoplasms (SENs). Paraffin sections from a retrospective surgical series of 117 SENs were immunostained with anti-p53 (clone DO7, Novacastra Laboratories, UK), anti-p21(WAF1/CIP1) (clone EA10, Oncogene Science, Cambridge, MA), and anti-Ki-67 (clone MIB-1, Immunotech, Westbrook, ME). The Ki-67 proliferation index (Ki-67PI) and immunoreactivity were evaluated. One hundred seventeen SENs reacted as follows: p53 50%+ and p21(WAF1/CIP1) 65%+. Ki-67PI ranged from 4% to 88% (mean/median = 44/46%). p53 reactivity associated with transitional cell histology, decreased p21(WAF1/CIP1) staining, increased Ki-67PI, architectural/nuclear grade, and stage (P < .05, 1 x 10(-7), .01, .05/.0001, .001,). p21(WAF1/CIP1) staining was associated with endometrioid/clear cell histology, decreased Ki-67PI, architectural/nuclear grade, and stage (P < 05/.05, .05, .01/1 x 10(-8), 1 x 10(-5)). Ki-67PI associated with increased architectural/nuclear grade but not mucinous histology (P < 1 x 10(-5)/1 x 10(-6), .01). Sixty-seven patients had disease at last follow-up; 53 were dead of disease at 0 to 67 months (mean/median, 21/18), and 14 were alive with disease at 12 to 224 months (mean/median, 56/40). Fifty patients were disease free at 5 to 214 months (mean/median, 59/41). Predictors of survival include decreased Ki-67PI, stage, architectural/nuclear grade (P < 1 x 10(-6), 1 x 10(-10), 1 x 10(-10)/.005) and p21(WAF1/CIP1) IMS (multivariate P < 1 x 10(-6)). p21(WAF1/CIP1), a potent inhibitor of cyclin-dependent kinases necessary for cell cycle progression, functions as a key checkpoint in cell cycle control. Immunoreactivity for p21(WAF1/CIP1) provides prognostic information independent of other histological and clinical predictors, p53 IMS, and Ki-67PI in this series of 117 PTs with SENs. Our preliminary data suggest an interrelationship between p21(WAF1/CIP1) expression and an effective clinical response to platinin-based chemotherapy, both associated with apoptosis. Further investigation seems warranted.

Adolescent↗
PubMed2005

Meiotic spindle checkpoints for assessment of aneuploid oocytes.

The spindle assembly checkpoint suspends cell cycle progression if improperly aligned chromosomes are detected at metaphase. Evolutionarily conserved kinetochore-associated proteins are believed to be key elements of this regulatory pathway. A breakdown in checkpoint function could bring about genomic instability, which may be responsible for the prevalence of aneuploidy in oocytes of older women. Maternal aging remains the overwhelming factor in the etiology of human aneuploidy in assisted reproduction. Defects in cell cycle checkpoint genes may play a role in its development. The existence of such monitoring mechanisms in oocytes has long been controversial. Studies providing evidence in support of and against their existence are reviewed.

Aneuploidy↗
PubMed1999

The morphogenesis checkpoint in Saccharomyces cerevisiae: cell cycle control of Swe1p degradation by Hsl1p and Hsl7p.

In Saccharomyces cerevisiae, the Wee1 family kinase Swe1p is normally stable during G(1) and S phases but is unstable during G(2) and M phases due to ubiquitination and subsequent degradation. However, perturbations of the actin cytoskeleton lead to a stabilization and accumulation of Swe1p. This response constitutes part of a morphogenesis checkpoint that couples cell cycle progression to proper bud formation, but the basis for the regulation of Swe1p degradation by the morphogenesis checkpoint remains unknown. Previous studies have identified a protein kinase, Hsl1p, and a phylogenetically conserved protein of unknown function, Hsl7p, as putative negative regulators of Swe1p. We report here that Hsl1p and Hsl7p act in concert to target Swe1p for degradation. Both proteins are required for Swe1p degradation during the unperturbed cell cycle, and excess Hsl1p accelerates Swe1p degradation in the G(2)-M phase. Hsl1p accumulates periodically during the cell cycle and promotes the periodic phosphorylation of Hsl7p. Hsl7p can be detected in a complex with Swe1p in cell lysates, and the overexpression of Hsl7p or Hsl1p produces an effective override of the G(2) arrest imposed by the morphogenesis checkpoint. These findings suggest that Hsl1p and Hsl7p interact directly with Swe1p to promote its recognition by the ubiquitination complex, leading ultimately to its destruction.

Cell Cycle↗
PubMed2004

Mdt1, a novel Rad53 FHA1 domain-interacting protein, modulates DNA damage tolerance and G(2)/M cell cycle progression in Saccharomyces cerevisiae.

The Rad53 kinase plays a central role in yeast DNA damage checkpoints. Rad53 contains two FHA phosphothreonine-binding domains that are required for Rad53 activation and possibly downstream signaling. Here we show that the N-terminal Rad53 FHA1 domain interacts with the RNA recognition motif, coiled-coil, and SQ/TQ cluster domain-containing protein Mdt1 (YBl051C). The interaction of Rad53 and Mdt1 depends on the structural integrity of the FHA1 phosphothreonine-binding site as well as threonine-305 of Mdt1. Mdt1 is constitutively threonine phosphorylated and hyperphosphorylated in response to DNA damage in vivo. DNA damage-dependent Mdt1 hyperphosphorylation depends on the Mec1 and Tel1 checkpoint kinases, and Mec1 can directly phosphorylate a recombinant Mdt1 SQ/TQ domain fragment. MDT1 overexpression is synthetically lethal with a rad53 deletion, whereas mdt1 deletion partially suppresses the DNA damage hypersensitivity of checkpoint-compromised strains and generally improves DNA damage tolerance. In the absence of DNA damage, mdt1 deletion leads to delayed anaphase completion, with an elongated cell morphology reminiscent of that of G(2)/M cell cycle mutants. mdt1-dependent and DNA damage-dependent cell cycle delays are not additive, suggesting that they act in the same pathway. The data indicate that Mdt1 is involved in normal G(2)/M cell cycle progression and is a novel target of checkpoint-dependent cell cycle arrest pathways.

Cell Cycle↗
PubMed2003

Sudden telomere lengthening triggers a Rad53-dependent checkpoint in Saccharomyces cerevisiae.

Telomeres are specialized functional complexes that ensure chromosome stability by protecting chromosome ends from fusions and degradation and avoiding chromosomal termini from being sensed as DNA breaks. Budding yeast Tel1 is required both for telomere metabolism and for a Rad53-dependent checkpoint responding to unprocessed double-strand breaks. We show that overexpression of a GAL1-TEL1 fusion causes transient telomere lengthening and activation of a Rad53-dependent G2/M checkpoint in cells whose telomeres are short due to the lack of either Tel1 or Yku70. Sudden telomere elongation and checkpoint-mediated cell cycle arrest are also triggered in wild-type cells by overproducing a protein fusion between the telomeric binding protein Cdc13 and the telomerase-associated protein Est1. Checkpoint activation by GAL1-TEL1 requires ongoing telomere elongation. In fact, it is turned off concomitantly with telomeres reaching a new stable length and is partially suppressed by deletion of the telomerase EST2 gene. Moreover, both telomere length rebalancing and checkpoint inactivation under galactose-induced conditions are accelerated by high levels of either the Sae2 protein, involved in double-strand breaks processing, or the negative telomere length regulator Rif2. These data suggest that sudden telomere lengthening elicits a checkpoint response that inhibits the G2/M transition.

Cell Cycle↗
PubMed2005

Activation of S phase checkpoint by cigarette smoke extract in Schizosaccharomyces pombe.

Cigarette smoke has long been recognized as a major environmental pollutant that can cause significant damage to the cellular macromolecules. Although much is known about the types of damage, little is known about the cellular responses to the stress caused by cigarette smoke. We have used the fission yeast Schizosaccharomyces pombe to elucidate the overall cellular responses towards cigarette smoke. Here, we demonstrate that fission yeast cells exposed to aqueous extract of cigarette smoke exhibit cell cycle arrest and cell death in a dose-dependent manner. Cigarette smoke treatment also results in accumulation of reactive oxygen species, unusual nuclear morphology and altered cellular structure. Our data further establish activation of the S phase checkpoint in cigarette smoke-exposed Sz. pombe cells. The checkpoint proteins Rad3, Rad26, Rad17, Rad1, Hus1 and Cds1 play key roles in this process, as evidenced by cell survival and biochemical analysis, although another checkpoint protein, Rad9, seems to be less required. Our results also suggest involvement of the stress-activated protein kinase Spc1/Sty1 and the bZIP transcription factors Atf1 and Pap1 in the cellular response towards cigarette smoke extract. These findings indicate activation of the critical S phase checkpoint and cell cycle arrest in Sz. pombe following CSE assault.

Cell Cycle↗
PubMed2000

Threonine 68 is required for radiation-induced phosphorylation and activation of Cds1.

In response to DNA damage, eukaryotic cells use a system of checkpoint controls to delay cell-cycle progression. Checkpoint delays provide time for repair of damaged DNA before its replication in S phase and before segregation of chromatids in M phase. The Cds1 (Chk2) tumour-suppressor protein has been implicated in certain checkpoint responses in mammalian cells. It directly phosphorylates and inactivates the mitosis-inducing phosphatase Cdc25 in vitro and is required to maintain the G2 arrest that is observed in response to gamma-irradiation. Cds1 also directly phosphorylates p53 in vitro at a site that is implicated in its stabilization, and is required for stabilization of p53 and induction of p53-dependent transcripts in vivo upon gamma-ionizing radiation. Thus, Cds1 functions in both the G1 and G2 checkpoint responses. Like Cds1, the checkpoint protein kinase ATM (ataxia-telangiectasia-mutated) is required for correct operation of both the G1 and G2 damage checkpoints. ATM is necessary for phosphorylation and activation of Cds1 in vivo and can phosphorylate Cds1 in vitro, although evidence that the sites that are phosphorylated by ATM are required for activation is lacking. Here we show that threonine 68 of Cds1 is the preferred site of phosphorylation by ATM in vitro, and is the principal irradiation-induced site of phosphorylation in vivo. The importance of this phosphorylation site is demonstrated by the failure of a mutant, non-phosphorylatable form of Cds1 to be fully activated, and by its reduced ability to induce G1 arrest in response to ionising radiation.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2000

Histone deacetylase inhibitors trigger a G2 checkpoint in normal cells that is defective in tumor cells.

Important aspects of cell cycle regulation are the checkpoints, which respond to a variety of cellular stresses to inhibit cell cycle progression and act as protective mechanisms to ensure genomic integrity. An increasing number of tumor suppressors are being demonstrated to have roles in checkpoint mechanisms, implying that checkpoint dysfunction is likely to be a common feature of cancers. Here we report that histone deacetylase inhibitors, in particular azelaic bishydroxamic acid, triggers a G2 phase cell cycle checkpoint response in normal human cells, and this checkpoint is defective in a range of tumor cell lines. Loss of this G2 checkpoint results in the tumor cells undergoing an aberrant mitosis resulting in fractured multinuclei and micronuclei and eventually cell death. This histone deacetylase inhibitor-sensitive checkpoint appears to be distinct from G2/M checkpoints activated by genotoxins and microtubule poisons and may be the human homologue of a yeast G2 checkpoint, which responds to aberrant histone acetylation states. Azelaic bishydroxamic acid may represent a new class of anticancer drugs with selective toxicity based on its ability to target a dysfunctional checkpoint mechanism in tumor cells.

Cell Cycle↗
PubMed2000

Checkpoint proteins influence telomeric silencing and length maintenance in budding yeast.

A complex network of surveillance mechanisms, called checkpoints, interrupts cell cycle progression when damage to the genome is detected or when cells fail to complete DNA replication, thus ensuring genetic integrity. In budding yeast, components of the DNA damage checkpoint regulatory network include the RAD9, RAD17, RAD24, MEC3, DDC1, RAD53, and MEC1 genes that are proposed to be involved in different aspects of DNA metabolism. We provide evidence that some DNA damage checkpoint components play a role in maintaining telomere integrity. In fact, rad53 mutants specifically enhance repression of telomere-proximal transcription via the Sir-mediated pathway, suggesting that Rad53 might be required for proper chromatin structure at telomeres. Moreover, Rad53, Mec1, Ddc1, and Rad17 are necessary for telomere length maintenance, since mutations in all of these genes cause a decrease in telomere size. The telomeric shortening in rad53 and mec1 mutants is further enhanced in the absence of SIR genes, suggesting that Rad53/Mec1 and Sir proteins contribute to chromosome end protection by different pathways. The finding that telomere shortening, but not increased telomeric repression of gene expression in rad53 mutants, can be suppressed by increasing dNTP synthetic capacity in these strains suggests that transcriptional silencing and telomere integrity involve separable functions of Rad53.

Blotting, Southern↗
PubMed1999

Cell-cycle-dependent and ATM-independent expression of human Chk1 kinase.

Checkpoint genes cause cell cycle arrest when DNA is damaged or DNA replication is blocked. Although a human homolog of Chk1 (hChk1) has recently been reported to be involved in the DNA damage checkpoint through phosphorylation of Cdc25A, B, and C, it is not known at which phase(s) of the cell cycle hChk1 functions and how hChk1 causes cell cycle arrest in response to DNA damage. In the present study, we demonstrate that in normal human fibroblasts (MJ90), hChk1 is expressed specifically at the S to M phase of the cell cycle at both the RNA and protein levels and that it is localized to the nucleus at this time. hChk1 activity, as determined by phosphorylation of Cdc25C, is readily detected at the S to M phase of the cell cycle, and DNA damage induced by UV or ionizing radiation does not enhance the expression of hChk1 or its activity. Furthermore, hChk1 exists in an active form at the S to M phase in fibroblasts derived from patients with ataxia telangiectasia (AT) which lack the functional AT mutated (ATM) gene product, suggesting that hChk1 expression is independent of functional ATM. Taken together with the findings that phosphorylation of Cdc25C on serine 216 is increased at the S to M phase, it is suggested that at this particular phase of the cell cycle, even in the absence of DNA damage, hChk1 phosphorylates Cdc25C on serine 216, which is considered to be a prerequisite for the G2/M checkpoint. Thus, hChk1 may play an important role in keeping Cdc25C prepared for responding to DNA damage by phosphorylating its serine residue at 216 during the S to M phase.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2002

Overlapping roles of the spindle assembly and DNA damage checkpoints in the cell-cycle response to altered chromosomes in Saccharomyces cerevisiae.

The MAD2-dependent spindle checkpoint blocks anaphase until all chromosomes have achieved successful bipolar attachment to the mitotic spindle. The DNA damage and DNA replication checkpoints block anaphase in response to DNA lesions that may include single-stranded DNA and stalled replication forks. Many of the same conditions that activate the DNA damage and DNA replication checkpoints also activated the spindle checkpoint. The mad2Delta mutation partially relieved the arrest responses of cells to mutations affecting the replication proteins Mcm3p and Pol1p. Thus a previously unrecognized aspect of spindle checkpoint function may be to protect cells from defects in DNA replication. Furthermore, in cells lacking either the DNA damage or the DNA replication checkpoints, the spindle checkpoint contributed to the arrest responses of cells to the DNA-damaging agent methyl methanesulfonate, the replication inhibitor hydroxyurea, and mutations affecting Mcm2p and Orc2p. Thus the spindle checkpoint was sensitive to a wider range of chromosomal perturbations than previously recognized. Finally, the DNA replication checkpoint did not contribute to the arrests of cells in response to mutations affecting ORC, Mcm proteins, or DNA polymerase delta. Thus the specificity of this checkpoint may be more limited than previously recognized.

Calcium-Binding Proteins↗