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Results for “Cell Cycle Checkpoints”

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At least 217 records · Page 12Linked to original sources
PubMed2000

Targeted disruption of the cell-cycle checkpoint gene ATR leads to early embryonic lethality in mice.

Checkpoints of DNA integrity are conserved throughout evolution, as are the kinases ATM (Ataxia Telangiectasia mutated) and ATR (Ataxia- and Rad-related), which are related to phosphatidylinositol (PI) 3-kinase [1] [2] [3]. The ATM gene is not essential, but mutations lead to ataxia telangiectasia (AT), a pleiotropic disorder characterised by radiation sensitivity and cellular checkpoint defects in response to ionising radiation [4] [5] [6]. The ATR gene has not been associated with human syndromes and, structurally, is more closely related to the canonical yeast checkpoint genes rad3(Sp) and MEC1(Sc) [7] [8]. ATR has been implicated in the response to ultraviolet (UV) radiation and blocks to DNA synthesis [8] [9] [10] [11], and may phosphorylate p53 [12] [13], suggesting that ATM and ATR may have similar and, perhaps, complementary roles in cell-cycle control after DNA damage. Here, we report that targeted inactivation of ATR in mice by disruption of the kinase domain leads to early embryonic lethality before embryonic day 8.5 (E8.5). Heterozygous mice were fertile and had no aberrant phenotype, despite a lower ATR mRNA level. No increase was observed in the sensitivity of ATR(+/-) embryonic stem (ES) cells to a variety of DNA-damaging agents. Attempts to target the remaining wild-type ATR allele in heterozygous ATR(+/-) ES cells failed, supporting the idea that loss of both alleles of the ATR gene, even at the ES-cell level, is lethal. Thus, in contrast to the closely related checkpoint gene ATM, ATR has an essential function in early mammalian development.

Alleles↗
PubMed1999

Cell cycle checkpoint abrogator UCN-01 inhibits DNA repair: association with attenuation of the interaction of XPA and ERCC1 nucleotide excision repair proteins.

UCN-01, an anticancer agent currently in Phase I clinical trials, has been found to potentiate the cytotoxicity of cisplatin (CDDP). Because mammalian cells remove CDDP-induced DNA adducts through the nucleotide excision repair (NER) pathway, we determined the effects of UCN-01 on NER by measuring its effects on the interaction of the repair factors XPA and ERCC1 and the phosphorylation/dephosphorylation of the repair proteins. The repair activity, as measured by an in vitro repair synthesis assay and an in vivo host-cell reactivation assay using A549 cells, was significantly reduced. Although expression of XPA and ERCC1 proteins was elevated in cells exposed to UCN-01, the treatment resulted in a decreased ERCC1 level in the Triton X-100-insoluble fraction of cell lysates. A pull-down assay using the MBP-XPA fusion protein showed a significant reduction in the binding of ERCC1 to XPA in nuclear extracts from UCN-01-treated cells compared with untreated cells, suggesting that UCN-01 reduced the XPA-ERCC1 interaction. Consistent with these data, lower repair incision activity was found in the cell extracts from UCN-01-treated cells. In vitro phosphorylation revealed that UCN-01 had no effect on the phosphorylation/dephosphorylation status of either XPA or ERCC1; however, UCN-01 caused dephosphorylation of an unidentified XPA-bound protein with an apparent molecular mass of 52 kDa. Taken together, these data demonstrate the NER-inhibitory action of UCN-01, which is associated with the inhibition of the XPA-ERCC1 interaction by UCN-01 and with the effect of UCN-01 on the phosphorylation/dephosphorylation of an XPA-bound, 52-kDa protein, the identity of which remains to be determined.

Alkaloids↗
PubMed2002

Genomic instability, centrosome amplification, cell cycle checkpoints and Gadd45a.

Genomic instability has been a recognized feature of many human tumors for decades. Until recently, however, there was little insight into potential mechanisms for this phenomenon. Recent work has shown first, that increased centrosome numbers (also referred to as centrosome amplification) often accompany genomic instability and second, that when centrosome numbers are increased, cells become genetically unstable. Deletion of Gadd45a leads to centrosome amplification and consequent abnormal mitosis and aneuploidy. Gadd45a is known to be involved in a G2 checkpoint and may be involved in the normal progression from G2 to M and its coordination with S phase events. Whether these functions contribute to prevention of centrosome amplification is being investigated. However, potential mechanisms can be proposed based on known protein associations with Gadd45a, as well as proteins that regulate Gadd45a transcription and are also required for efficient coordination of centrosome duplication and DNA synthesis.

Animals↗
PubMed2003

A DNA-damage-induced cell cycle checkpoint in Arabidopsis.

Although it is well established that plant seeds treated with high doses of gamma radiation arrest development as seedlings, the cause of this arrest is unknown. The uvh1 mutant of Arabidopsis is defective in a homolog of the human repair endonuclease XPF, and uvh1 mutants are sensitive to both the toxic effects of UV and the cytostatic effects of gamma radiation. Here we find that gamma irradiation of uvh1 plants specifically triggers a G(2)-phase cell cycle arrest. Mutants, termed suppressor of gamma (sog), that suppress this radiation-induced arrest and proceed through the cell cycle unimpeded were recovered in the uvh1 background; the resulting irradiated plants are genetically unstable. The sog mutations fall into two complementation groups. They are second-site suppressors of the uvh1 mutant's sensitivity to gamma radiation but do not affect the susceptibility of the plant to UV radiation. In addition to rendering the plants resistant to the growth inhibitory effects of gamma radiation, the sog1 mutation affects the proper development of the pollen tetrad, suggesting that SOG1 might also play a role in the regulation of cell cycle progression during meiosis.

Arabidopsis↗
PubMed2001

Loss of cell cycle checkpoint control in Drosophila Rfc4 mutants.

Two alleles of the Drosophila melanogaster Rfc4 (DmRfc4) gene, which encodes subunit 4 of the replication factor C (RFC) complex, cause striking defects in mitotic chromosome cohesion and condensation. These mutations produce larval phenotypes consistent with a role in DNA replication but also result in mitotic chromosomal defects appearing either as premature chromosome condensation-like or precocious sister chromatid separation figures. Though the DmRFC4 protein localizes to all replicating nuclei, it is dispersed from chromatin in mitosis. Thus the mitotic defects appear not to be the result of a direct role for RFC4 in chromosome structure. We also show that the mitotic defects in these two DmRfc4 alleles are the result of aberrant checkpoint control in response to DNA replication inhibition or damage to chromosomes. Not all surveillance function is compromised in these mutants, as the kinetochore attachment checkpoint is operative. Intriguingly, metaphase delay is frequently observed with the more severe of the two alleles, indicating that subsequent chromosome segregation may be inhibited. This is the first demonstration that subunit 4 of RFC functions in checkpoint control in any organism, and our findings additionally emphasize the conserved nature of RFC's involvement in checkpoint control in multicellular eukaryotes.

Alleles↗
PubMed1997

A benzo[a]pyrene-induced cell cycle checkpoint resulting in p53-independent G1 arrest in 3T3 fibroblasts.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor of the basic helix-loop-helix family. Although physiological ligands for the AhR have not been identified, carcinogenic polycyclic aromatic hydrocarbons such as Benzo[a]pyrene (B[a]P) are high affinity AhR ligands that induce nuclear translocation and sequence-specific DNA binding of the AhR. AhR-regulated genes include members of the cytochrome P-450 family that are known to oxidize B[a]P to form genotoxic (DNA-damaging) metabolites. Murine Swiss 3T3 cells express high levels of AhR. Treatment of Swiss 3T3 cells with B[a]P during the G1 phase of the cell cycle resulted in growth arrest, as shown by inhibition of growth factor-stimulated DNA synthesis. By contrast, other murine 3T3 fibroblasts not expressing detectable levels of AhR did not undergo growth arrest in response to B[a]P. The AhR antagonist alpha-naphthoflavone prevented B[a]P-induced growth arrest, further demonstrating that cessation of cell growth was mediated by the activated AhR. A nongenotoxic AhR ligand (2,3,7,8-tetrachlorodibenzo-p-dioxin) did not elicit growth arrest, showing that ligand activation of the AhR alone was insufficient to block cell cycle progression. However, genomic DNA from B[a]P-treated Swiss 3T3 cells contained covalent adducts, whereas that from 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated cells did not, showing that G1 arrest correlated with DNA damage resulting from genotoxic B[a]P metabolites. B[a]P-induced DNA damage and growth arrest was coincident with elevated levels of nuclear p53 protein and induction of the p53-regulated mdm-2 proto-oncogene. However, Swiss 3T3 fibroblasts expressing "dominant negative" mutant p53, as well as primary fibroblasts from p53-/- "knockout" mice, also underwent growth arrest in response to B[a]P. Therefore, B[a]P-induced growth arrest occurs via p53-independent mechanisms.

3T3 Cells↗
PubMed2000

How do drug-induced topoisomerase I-DNA lesions signal to the molecular interaction network that regulates cell cycle checkpoints, DNA replication, and DNA repair?

Recent results suggest that potentially lethal DNA lesions may result when replication forks encounter trapped topoisomerase-DNA complexes or some other types of DNA damage. Such events produce what are called replication-encounter lesions. These lesions have the characteristic that they may allow single stranded DNA-associated replication protein A (RPA) to become juxtaposed to dsDNA end-associated DNA-protein kinase. Our results suggest that DNA-protein kinases may then hyperphosphorylate the RPA2 subunit. We discuss a possible pathway by which hyperphosphorylation of RPA2 could lead to the release of active p53. This could constitute a pathway for signaling the presence of replication-encounter lesions to the p53-dependent cell cycle arrest and/or apoptosis initiator systems.

Apoptosis↗
PubMed1999

The maize homologue of the cell cycle checkpoint protein MAD2 reveals kinetochore substructure and contrasting mitotic and meiotic localization patterns.

We have identified a maize homologue of yeast MAD2, an essential component in the spindle checkpoint pathway that ensures metaphase is complete before anaphase begins. Combined immunolocalization of MAD2 and a recently cloned maize CENPC homologue indicates that MAD2 localizes to an outer domain of the prometaphase kinetochore. MAD2 staining was primarily observed on mitotic kinetochores that lacked attached microtubules; i.e., at prometaphase or when the microtubules were depolymerized with oryzalin. In contrast, the loss of MAD2 staining in meiosis was not correlated with initial microtubule attachment but was correlated with a measure of tension: the distance between homologous or sister kinetochores (in meiosis I and II, respectively). Further, the tension-sensitive 3F3/2 phosphoepitope colocalized, and was lost concomitantly, with MAD2 staining at the meiotic kinetochore. The mechanism of spindle assembly (discussed here with respect to maize mitosis and meiosis) is likely to affect the relative contributions of attachment and tension. We support the idea that MAD2 is attachment-sensitive and that tension stabilizes microtubule attachments.

Anaphase↗
PubMed1995

Cell-cycle checkpoints. Keeping mitosis in check.

Mutations in an essential yeast gene, encoding DNA polymerase epsilon, abolish the dependence of mitosis on the completion of DNA replication, suggesting that the replication complex provides the checkpoint signal.

Animals↗
PubMed1995

A p53-dependent mouse spindle checkpoint.

Cell cycle checkpoints enhance genetic fidelity by causing arrest at specific stages of the cell cycle when previous events have not been completed. The tumor suppressor p53 has been implicated in a G1 checkpoint. To investigate whether p53 also participates in a mitotic checkpoint, cultured fibroblasts from p53-deficient mouse embryos were exposed to spindle inhibitors. The fibroblasts underwent multiple rounds of DNA synthesis without completing chromosome segregation, thus forming tetraploid and octaploid cells. Deficiency of p53 was also associated with the development of tetraploidy in vivo. These results suggest that murine p53 is a component of a spindle checkpoint that ensures the maintenance of diploidy.

Animals↗
PubMed1998

The mammalian Rad24 homologous to yeast Saccharomyces cerevisiae Rad24 and Schizosaccharomyces pombe Rad17 is involved in DNA damage checkpoint.

Cell cycle checkpoint proteins play critical roles in maintaining genomic stability and integrity to prevent the development of cancer and hereditary diseases. Here we report the isolation of a novel mouse gene encoding the protein MmRad24 [MmRad24 is the mouse homologue of HRad17, which was described recently by A. E. Parker et al. (J. Biol. Chem., 273: 18340-18346, 1998)], which shares significant sequence and structural homology with the budding yeast Rad24 and its fission yeast counterpart Rad17, both of which are required for DNA damage checkpoints. Confocal microscopy revealed that the green fluorescent protein-tagged MmRad24 protein is localized to the nucleus in living cells. Fluorescence-activated cell-sorting analysis showed that overexpression of the wild-type MmRad24 in diploid fibroblast WI-38 cells caused a significant G2 arrest of the cell cycle, whereas overexpression of a mutant MmRad24 (mutated on the nucleotide-binding site) that likely functions as a dominant-negative protein resulted in a defect in cell cycle arrest after DNA damage treatment as measured by bromodeoxyuridine pulse-chase labeling experiments. Taken together, these results suggest that the mammalian Rad24 protein may function as a critical gatekeeper in DNA damage checkpoint control.

Amino Acid Sequence↗
PubMed2000

Identification of a functional domain in a GADD45-mediated G2/M checkpoint.

Cell cycle checkpoints are essential for the maintenance of genomic stability in response to DNA damage. We demonstrated recently that GADD45, a DNA damage-inducible protein, activates a G(2)/M checkpoint induced by either UV radiation or alkylating agents. GADD45 can interact in vivo with the G(2) cell cycle-specific kinase, Cdc2, proliferating cell nuclear antigen (PCNA), and the cell cycle kinase inhibitor p21(waf1). The ability of GADD45 to induce a G(2)/M arrest may be caused in part by the inhibition of Cdc2 kinase activity. Here, we report the identification of a region of GADD45 that is involved in this G(2)/M checkpoint. Mutants of GADD45 that lacked either the first 35 or the last 80 residues still retained an ability to induce G(2)/M arrest. A mutant with a deletion of the central region (residues 50-76), which is conserved in the family members GADD45beta and GADD45gamma, lacked such activity. This mutant also lacked an ability to bind to Cdc2, PCNA, and p21(waf1) in vivo. Consistently, either GADD45beta or GADD45gamma bind to Cdc2 in vivo. However, unlike GADD45, neither GADD45beta nor GADD45gamma inhibited the Cdc2 kinase or induced G(2)/M arrest. The unique effect of GADD45 may be caused by the presence of a region containing DEDDDR residues. Alanine substitutions in the region abolished GADD45 induction of a G(2)/M arrest and its inactivation of the Cdc2 kinase but not its binding to Cdc2, PCNA, or p21(waf1). Therefore, the binding of GADD45 to Cdc2 was insufficient to induce a G(2)/M arrest, and additional activity contributed by the DEDDDR residues may be necessary to regulate the G(2)/M checkpoint.

Amino Acid Sequence↗
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↗
PubMed2000

DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts.

The cell cycle checkpoint mechanisms ensure the order of cell cycle events to preserve genomic integrity. Among these, the DNA-replication and DNA-damage checkpoints prevent chromosome segregation when DNA replication is inhibited or DNA is damaged. Recent studies have identified an outline of the regulatory networks for both of these controls, which apparently operate in all eukaryotes. In addition, it appears that these checkpoints have two arrest points, one is just before entry into mitosis and the other is prior to chromosome separation. The former point requires the central cell-cycle regulator Cdc2 kinase, whereas the latter involves several key regulators and substrates of the ubiquitin ligase called the anaphase promoting complex. Linkages between these cell-cycle regulators and several key checkpoint proteins are beginning to emerge. Recent findings on post-translational modifications and protein-protein interactions of the checkpoint proteins provide new insights into the checkpoint responses, although the functional significance of these biochemical properties often remains unclear. We have reviewed the molecular mechanisms acting at the DNA-replication and DNA-damage checkpoints in the fission yeast Schizosaccharomyces pombe, and the modifications of these controls during the meiotic cell cycle. We have made comparisons with the controls in fission yeast and other organisms, mainly the distantly related budding yeast.

Animals↗
PubMed2003

Potentiation of DNA-damage-induced cytotoxicity by G2 checkpoint abrogators.

Cell cycle checkpoints are activated in response to DNA-damage to ensure that accurate copies of the cellular genome are passed on to the next generation and to avoid replication and segregation of damaged DNA. These cellular control systems can be overcome by combining conventional DNA-damaging agents with compounds that target the cell cycle regulatory pathways, to enhance cytotoxicity. Tumor cells often comprise a corrupted G(1) cell cycle checkpoint while the G(2) cell cycle checkpoint is still intact. This review describes the concept of G(2) checkpoint abrogation with recognized (methylxanthines, UCN-01) and novel G(2) checkpoint abrogators to potentiate the cytotoxicity of DNA-damaging drugs and ionizing radiation. It illustrates the potential of G(2) checkpoint abrogators to preferentially sensitize p53-mutated, treatment resistant tumor cells for genotoxic treatment. Identification of the targets of caffeine and UCN-01 to be key-players of the G(2) checkpoint (ATM/ATR and Chk1, respectively) promoted the search for novel inhibitors of this checkpoint. Even though a direct causal link between G(2) checkpoint abrogation and chemo-/radiosensitization is difficult to prove the multitude of these novel compounds validate that inhibition of critical elements of the G(2) checkpoint (ATM/ATR-Chk1/Chk2-CDC25C-cascade) potentiates the cytotoxicity of DNA-damaging agents.

Alkaloids↗
PubMed2001

Cell cycle regulation of c-Jun N-terminal kinase activity at the centrosomes.

The c-Jun N-terminal kinase (JNK), a subgroup of the mitogen-activated protein kinase (MAPK) family of serine/threonine kinases, has established functions in cell growth and apoptosis. While the mechanisms are unclear, JNK has also been also implicated in signaling pathways that initiate cell cycle checkpoints and cell cycle progression. By following the localization of active and inactive JNK during the cell cycle, we have found that the majority of cellular JNK is soluble and present in the cytoplasm and the nucleus. Interestingly, insoluble fractions of JNK are also localized in nuclear and cytoplasmic speckles, and to the centrosomes. While JNK is associated with the centrosome throughout the cell cycle, it is only active at the centrosome from S phase through anaphase. This novel localization of centrosomal JNK is a possible link between JNK-activating stimuli and centrosome or cell cycle events.

Animals↗