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At least 811 records · Page 45Linked to original sources
PubMed1998

Dual effect of erbB-2 depletion on the regulation of DNA repair and cell cycle mechanisms in non-small cell lung cancer cells.

Overexpression of the erbB-2 tyrosine kinase receptor, p185erbB-2, is a common alteration in non-small cell lung cancer (NSCLC) and has been associated with poor prognosis and a tumor drug resistance phenotype. In this study, we have examined the consequences of erbB-2 depletion on DNA repair, cell cycle, and apoptosis using a panel of NSCLC cell lines constitutively overexpressing erbB-2 receptor. Depletion of the erbB-2 was achieved using the tyrosine kinase inhibitor CP127,374 which promotes erbB-2 degradation. Treatment with CP127,374 concentrations which deplete erbB-2 and inhibit tyrosine phosphorylation resulted in downregulation of DNA repair mechanisms and cell accumulation at G1 phase of the cell cycle. GI arrest was observed in cells with mutated p53 as well as cells lacking p53 protein, suggesting a p53-independent mechanisms. NSCLC cells which overexpress erbB-2 were more resistant to cisplatin-induced cytotoxicity in comparison to cells expressing low levels of erbB-2. Treatment with CP127,374 alone did not result in any induction of apoptosis. A combination of CP127,374 and cisplatin, however, was more potent in cell growth inhibition and induction of apoptosis compared to treatment with cisplatin alone. Together, our results further support a pivotal role of erbB-2 signaling in the regulatory balance between DNA repair, cell cycle checkpoints and apoptosis; all these mechanisms are essential determinants for tumor cell destiny following chemotherapy stress.

Apoptosis↗
PubMed1998

p53-independent DNA repair and cell cycle arrest in embryonic stem cells.

The role of p53 in DNA repair and cell cycle checkpoint after ultraviolet irradiation was investigated in an embryonic stem cell line homozygous for a targeted deletion of p53. Results indicate that loss of p53 does not alter the capacity of ES cells to respond to DNA damage. Wild-type and p53-deficient cells showed similar cessation of DNA synthesis after UV damage and similar ultimate capacity to repair a transiently transfected reporter plasmid. Interestingly, in the absence of DNA damaging treatment, the transit of p53-deficient cells through S phase was slower than wild-type cells. We suggest that this may result from the absence of a p53-dependent response to endogenous DNA damage: without p53 sensing endogenous damage leading to immediate repair, such damage may persist and thus delay DNA synthesis.

Animals↗
PubMed1996

DNA polymerase epsilon may be dispensable for SV40- but not cellular-DNA replication.

The contributions of DNA polymerases alpha, delta, and epsilon to SV40 and nuclear DNA syntheses were evaluated. Proteins were UV-crosslinked to nascent DNA within replicating chromosomes and the photolabelled polymerases were immunopurified. Only DNA polymerases alpha and delta were detectably photolabelled by nascent SV40 DNA, whether synthesized in soluble viral chromatin or within nuclei isolated from SV40-infected cells. In contrast, all three enzymes were photolabelled by the nascent cellular DNA. Mitogenic stimulation enhanced the photolabelling of the polymerases in the alpha>delta>epsilon order of preference. The data agree with the notion that DNA polymerases alpha and delta catalyse the principal DNA polymerisation reactions at the replication fork of SV40 and, perhaps, also of nuclear chromosomes. DNA polymerase epsilon, implicated by others as a cell-cycle checkpoint regulator sensing DNA replication lesions, may be dispensable for replication of the small, fast propagating virus that subverts cell cycle controls.

Animals↗
PubMed2005

A novel WISp39 protein links Hsp90 and p21 stability to the G2/M checkpoint.

Transcriptional regulation of the p21(Cip1) cyclin-dependent kinase inhibitor is a well-established mechanism by which the cell orchestrates a proper spatial and temporal cell cycle progression. Now, in the January 2005 issue of Molecular Cell (2005; Vol 17, 237-49), a study by Jascur et al identifies a novel multi-protein complex that is critical in contributing to a p53-dependent G2 cell cycle checkpoint. The authors demonstrate the significance of stabilizing the p21 protein in the context of this complex.

Cell Cycle Proteins↗
PubMed2001

Correction of radioresistant DNA synthesis in ataxia telangiectasia fibroblasts by prostaglandin E2 treatment.

Cultured cells from patients inheriting the rare cancer-prone and radiotherapy-sensitive disorder ataxia telangiectasia (AT) exhibit defects in the activation of cell-cycle checkpoints after exposure to ionizing radiation. In particular, the failure of AT cells to arrest transiently the DNA de novo replication machinery immediately after irradiation--so-called radioresistant DNA synthesis (RDS)--is often taken as a molecular hallmark of the disease. Recently we reported that: (i) the radiation-responsive S-phase checkpoint operating in normal human cells is mediated by a signal transduction pathway involving Ca2+/calmodulin-dependent protein kinase II (CaMKII); and (ii) the RDS phenotype of AT cells is associated with failure to mobilize Ca2+ from intracellular stores, which is required for activation of the CaMKII-dependent S-phase arrest. In the present study, we demonstrate that the RDS phenotype of AT dermal fibroblasts can be rectified in the absence of ectopic expression of functional ATM, the 350-kDa protein kinase encoded by the gene mutated in AT. Correction of RDS was observed when AT fibroblasts were coincubated with normal fibroblasts under conditions in which the 2 different cell cultures shared the same medium but were completely separated physically. The RDS trait was also rectified when AT fibroblasts were briefly incubated with prostaglandin E2 in the absence of normal feeder cells, signifying that this ubiquitous eicosanoid can serve as the diffusible "RDS-correction factor" for AT cells in the aforementioned cocultivation studies. It would therefore appear that prostaglandin E2 can assume the role of an extracellular signaling modulator of the S-phase checkpoint in AT cells exposed to ionizing radiation, inducing DNA synthesis shutdown via an alternative, ATM-independent signal transduction pathway.

Ataxia Telangiectasia↗
PubMed1999

Cisplatin DNA cross-links do not inhibit S-phase and cause only a G2/M arrest in Saccharomyces cerevisiae.

Cisplatin (CDDP) has been used as a DNA cross-linking agent to evaluate whether there is a specific cell cycle checkpoint response to such damage in Saccharomyces cerevisiae (S. cerevisiae). Fluorescent-activated cell sorting (FACS) analysis showed only a G2/M checkpoint, normal exit from G1 and progression through S-phase following alpha-factor arrest and CDDP treatment. Of the checkpoint mutants tested, rad9, rad17 and rad24, did not show increased sensitivity to CDDP compared to isogenic wild-type cells. However, other checkpoint mutants tested (mec1, mec3 and rad53) showed increased sensitivity to CDDP, as did controls with a defect in excision repair (rad1 and rad14) or a defect in recombination (rad51 and rad52). Thus, by survival and cell cycle kinetics, it appears that DNA cross-links do not inhibit entry into S-phase or slow DNA replication and that replication continues after cisplatin treatment in yeast.

Cell Cycle↗
PubMed2006

Absence of an immediate G1/S checkpoint in primary MEFs following gamma-irradiation identifies a novel checkpoint switch.

DNA double-strand breaks caused by ionizing radiation have been shown to induce G(1)/S, intra-S-phase, and G(2)/M cell cycle checkpoints. However, analysis of the immediate induction of G(1)/S checkpoint at a cellular level has been hampered by the inability to distinguish cells that were already replicating DNA at the time of damage from cells that entered S phase following the DNA damage. We have developed a novel strategy for assessing the initiation of the G(1)/S checkpoint following gamma-irradiation within asynchronous, low passage, primary mouse embryonic fibroblast cultures (MEFs) using a staggered CldU/IdU double-labeling protocol. Contrary to the current model of the G(1)/S checkpoint, we found that 65% of late- G(1) primary MEFs still proceeded into S phase after a gamma-irradiation dose of 5 Gy. The delayed p53-dependent G(1)/S checkpoint was intact in these cells, and a G2/M checkpoint that was over 90% effective was induced within one hour and maintained through six hours post-irradiation. Furthermore, these cells also exhibited an intra-S-phase replication slow-down, as there was a decrease in the S/ G(2) transition frequency of primary MEFs following gamma-irradiation. The absence of an immediate G(1)/S checkpoint in primary MEFs suggests that in late G(1) these cells may predominantly respond to DNA damage at the level of individual replication origins, rather than by inducing a complete shut-down of S-phase entry.

Animals↗
PubMed2003

Repair of and checkpoint response to topoisomerase I-mediated DNA damage.

Topoisomerase I (Top1) catalyzes two transesterification reactions: single-strand DNA cleavage and religation that are normally coupled for the relaxation of DNA supercoiling in transcribing and replicating chromatin. A variety of endogenous DNA modifications, potent anticancer drugs and carcinogens uncouple these two reactions, resulting in the accumulation of Top1 cleavage complexes. Top1 cleavage complexes damage DNA and kill cells by generating replication-mediated DNA double-strand breaks (DSBs) and by stalling transcription complexes. The repair of Top1-mediated DNA lesions involves integrated pathways that are conserved from yeasts to humans. Top1-mediated DNA damage and cell cycle checkpoint responses can be studied biochemically and genetically in yeast and human cells with known genetic defects. Defects in these repair/checkpoint pathways, which promote tumor development, explain, at least in part, the selectivity of camptothecins and other Top1 inhibitors for cancer cells.

Animals↗
PubMed2000

Inactivation of the 14-3-3 sigma gene is associated with 5' CpG island hypermethylation in human cancers.

The cell cycle checkpoint plays an important role in maintaining the integrity of cells. Recently, one of the 14-3-3 protein family members, 14-3-3sigma, was shown to be regulated by p53 and to play a role in the G2-M-phase checkpoint. To determine whether 14-3-3sigma is inactivated in human cancers, the methylation status of the 5' region of 14-3-3sigma was investigated in a series of gastric, colorectal, and hepatocellular cancer cell lines. Of 22 cell lines examined, 6 showed aberrant methylation. The methylation status of 14-3-3sigma was found to be correlated with loss of expression, which was restored by 5-aza-2'-deoxycytidine treatment. Furthermore, normal G2 arrest after DNA damage was not demonstrated in the cell lines with methylation. In primary gastric cancers, 14-3-3sigma hypermethylation was observed frequently in 26 of 60 (43%) cases and observed more frequently in poorly differentiated adenocarcinomas (P = 0.0017). Our findings suggest that 14-3-3sigma is inactivated by aberrant methylation of the 5' region in various human cancers and that it might play an important role in the development of undifferentiated gastric cancers.

14-3-3 Proteins↗
PubMed2000

Uncoupling between phenotypic senescence and cell cycle arrest in aging p21-deficient fibroblasts.

Irreversible G(1) arrest in senescent human fibroblasts is mediated by two inhibitors of cyclin-dependent kinases (Cdks), p21(Cip1/SDI1/WAF1) and p16(Ink4A). To determine the physiological and molecular events that specifically require p21, we studied senescence in human diploid fibroblasts expressing the human papillomavirus type 16 E6 oncogene, which confers low p21 levels via enhanced p53 degradation. We show that in late-passage E6 cells, high Cdk activity drives the cell cycle, but population expansion is slowed down by crisis-like events, probably owing to defective cell cycle checkpoints. At the end of lifespan, terminal-passage E6 cells exhibited several aspects of the senescent phenotype and accumulated unphosphorylated pRb and p16. However, both replication and cyclin-Cdk2 kinase activity were still not blocked, demonstrating that phenotypic and replicative senescence are uncoupled in the absence of normal p21 levels. At this stage, E6 cells also failed to upregulate p27 and inactivate cyclin-Cdk complexes in response to serum deprivation. Eventually, irreversible G(1) arrest occurred coincident with inactivation of cyclin E-Cdk2 owing to association with p21. Similarly, when p21(-/-) mouse embryo fibroblasts reached the end of their lifespan, they had the appearance of senescent cells yet, in contrast to their wild-type counterparts, they were deficient in downregulating bromodeoxyuridine incorporation, cyclin E- and cyclin A-Cdk2 activity, and inhibiting pRb hyperphosphorylation. These data support the model that the critical event ensuring G(1) arrest in senescence is p21-dependent Cdk inactivation, while other aspects of senescent phenotype appear to occur independently of p21.

Animals↗
PubMed1999

Cell cycle dependent subcellular distribution of Cdc25B subtypes.

The dual specificity phosphatase and oncogene Cdc25B has been implicated in the G2/M cell cycle checkpoint, but the mode by which it is regulated remains poorly understood. Regional subcellular redistribution of proteins represents a unique potential regulatory mechanism. Thus, we examined in live cells the subcellular localization characteristics of Cdc25B2 and Cdc25B3 fused to green fluorescent protein. Cdc25B2 partitioned primarily in the cytoplasm during G1 and progressively migrated to the nucleus as cells transited from S to G2/M phase. In contrast, Cdc25B3 maintained a homogeneously staining diffuse phenotype irrespective of cell cycle phase. Treatment of the Cdc25B2-green fluorescent protein stable transfectants with vanadate inhibited the cell cycle dependency of intracellular distribution, while okadaic acid had little effect except in G1, suggesting regulation by at least one phosphorylation-dependent pathway. The DNA topoisomerase II poison and DNA damaging agent, etoposide, inhibited nuclear localization of Cdc25B2 in S phase, possibly by invoking a sequestration cascade. Thus, differences in the spatial distribution of Cdc25B subtypes exist within cells and the 41 amino acid insert in the N-terminus of the Cdc25B3 splice variant encodes an important inhibitory determinant for such regulation. The subcellular redistribution of Cdc25B2 could be functionally important for G2/M checkpoint regulation.

Animals↗
PubMed1995

BCR-ABL-mediated inhibition of apoptosis with delay of G2/M transition after DNA damage: a mechanism of resistance to multiple anticancer agents.

A critical determinant of the efficacy of antineoplastic therapy is the response of malignant cells to DNA damage induced by anticancer agents. The p53 tumor-suppressor gene is a critical component of two distinct cellular responses to DNA damage, the induction of a reversible arrest at the G1/S cell cycle checkpoint, and the activation of apoptosis, a genetic program of autonomous cell death. Expression of the BCR-ABL chimeric gene produced by a balanced translocation in chronic myeloid leukemia, confers resistance to multiple genotoxic anticancer agents. BCR-ABL expression inhibits the apoptotic response to DNA damage without altering either the p53-dependent WAF1/CIP1-mediated G1 arrest or DNA repair. BCR-ABL-mediated inhibition of DNA damage-induced apoptosis is associated with a prolongation of cell cycle arrest at the G2/M restriction point; the delay of G2/M transition may allow time to repair and complete DNA replication and chromosomal segregation, thereby preventing a mitotic catastrophe. The inherent resistance of human cancers to genotoxic agents may result not only by the loss or inactivation of the wild-type p53 gene, but also by genetic alterations such as BCR-ABL that can delay G2/M transition after DNA damage.

Antineoplastic Agents↗
PubMed1994

p53 accumulation in polynuclear-giant-cells.

Accumulation of p53 has been reported in nearly all malignant human tumours. Macrophage derived giant cells of sarcoid granulomas in human lung tissue also show intense staining for p53 while normal alveolar macrophages remain unstained. Since sarcoid giant cells are not considered to be either pre-neoplastic nor to exhibit p53 gene mutations, two different physiological functions of p53 may be illustrated. Alveolar macrophages were isolated from rat lungs and cultured in vitro. Accumulation of p53 was observed by indirect immunohistochemistry after application of polyclonal rabbit serum directed against murine p53 (CM5). Antiproliferating cell nuclear antigen (PCNA) antibodies were used to study DNA synthesis. Most of the multinucleated giant cells derived from macrophages accumulated p53 in the cytoplasm, while only few nuclei were stained. PCNA was found in most giant cells nuclei. However, PCNA positivity was visible in few mononucleated macrophages. Isolated alveolar macrophages in vitro clearly divide and since nuclear division is a late event in the cell cycle, p53 may be involved in G1/S-control and in other cell-cycle-checkpoints between mitosis and cytokinesis.

Animals↗
PubMed2003

Condensin and cohesin: more than chromosome compactor and glue.

Two related protein complexes, cohesin and condensin, are essential for separating identical copies of the genome into daughter cells during cell division. Cohesin glues replicated sister chromatids together until they split at anaphase, whereas condensin reorganizes chromosomes into their highly compact mitotic structure. Unexpectedly, mutations in the subunits of these complexes have been uncovered in genetic screens that target completely different processes. Exciting new evidence is emerging that cohesin and condensin influence crucial processes during interphase, and unforeseen aspects of mitosis. Each complex can perform several roles, and individual subunits can associate with different sets of proteins to achieve diverse functions, including the regulation of gene expression, DNA repair, cell-cycle checkpoints and centromere organization.

Adenosine Triphosphatases↗
PubMed2004

Deficiency of the Mre11 DNA repair complex in Alzheimer's disease brains.

The hallmarks of Alzheimer's disease (AD) brains are deposition of Abeta plaques, appearance of neurofibrillary tangles, and extensive loss of neuronal cells. While Abeta plaques and neurofibrillary tangles play a significant role in the pathogenic dysfunction of neurons, factors that accelerate the neurodegenerating process remain to be defined. One set of factors examined in this study is the Mre11 protein complex, composed of Rad50, Mre11 and Nbs1. This protein complex plays an essential role in cellular responses to DNA damage, such as initiating cell cycle checkpoints and repairing damaged DNA. A defect in any component of this protein complex is detrimental to cells. Recently, several groups have observed abnormal cell cycle regulation and/or accumulated DNA damage in AD neurons. These pathological alterations could conceivably be caused or exacerbated by any compromise in the Mre11 complex. In this study, we compared the levels of the Mre11 complex proteins in brain samples from AD and age-matched non-dementia controls. We show for the first time that the Mre11 complex proteins are present in neurons of the adult human cortex and cerebellum. These proteins were found substantially reduced in the neurons of AD cortex. Our finding suggests that the loss of the Mre11 complex may be associated with the pathogenesis of AD.

Acid Anhydride Hydrolases↗
PubMed2005

Xrcc3 induces cisplatin resistance by stimulation of Rad51-related recombinational repair, S-phase checkpoint activation, and reduced apoptosis.

Eukaryotic cells respond to DNA damage by activation of DNA repair, cell cycle arrest, and apoptosis. Several reports suggest that such responses may be coordinated by communication between damage repair proteins and proteins signaling other cellular responses. The Rad51-guided homologous recombination repair system plays an important role in the recognition and repair of DNA interstrand crosslinks (ICLs), and cells deficient in this repair pathway become hypersensitive to ICL-inducing agents such as cisplatin and melphalan. We investigated the possible role of the Rad51-paralog protein Xrcc3 in drug resistance. Xrcc3 overexpression in MCF-7 cells resulted in 1) a 2- to 6-fold resistance to cisplatin/melphalan, 2) a 2-fold increase in drug-induced Rad51 foci, 3) an increased cisplatin-induced S-phase arrest, 4) decreased cisplatin-induced apoptosis, and 5) increased cisplatin-induced DNA synthesis arrest. Interestingly, Xrcc3 overexpression did not alter the doubling time or cell cycle progression in the absence of DNA damage. Furthermore, Xrcc3 overexpression is associated with increased Rad51C protein levels consistent with the known interaction of these two proteins. Our results demonstrate that Xrcc3 is an important factor in DNA cross-linking drug resistance in human tumor cells and suggest that the response of the homologous recombinational repair machinery and cell cycle checkpoints to DNA cross-linking agents is intertwined.

Annexin A5↗
PubMedDate not supplied

NBS1 and its functional role in the DNA damage response.

Nijmegen breakage syndrome is a recessive genetic disorder, characterized by elevated sensitivity to ionizing radiation, chromosome instability and high frequency of malignancies. Since cellular features partly overlap with those of ataxia-telangiectasia (A-T), NBS was long considered an A-T clinical variant. NBS1, the product of the gene underlying the disease, contains three functional regions: the forkhead-associated (FHA) domain and BRCA1 C-terminus (BRCT) domain at the N-terminus, several SQ motifs (consensus phosphorylation sites by ATM and ATR kinases) at a central region and MRE11-binding region at the C-terminus. NBS1 forms a multimeric complex with hMRE11/hRAD50 nuclease at the C-terminus and recruits or retains them at the vicinity of sites of DNA damage by direct binding to histone H2AX, which is phosphorylated by ATM in response to DNA damage. The combination of the FHA/BRCT domains has a crucial role for the binding of NBS1 to H2AX. Thereafter, the NBS1 complex proceeds to rejoin double-strand breaks predominantly by homologous recombination repair in vertebrates, while it also might be involved in suppression of inter-chromosomal recombination even for V(D)J recombination. These processes collaborate with cell cycle checkpoints to facilitate DNA repair, while defects of these checkpoints in NBS cells are partial in nature. A possible explanation for these moderate defects are the redundancy of multiple checkpoint regulations in vertebrates, or the modulator role of NBS1, in which NBS1 amplifies ATM activation by accumulation of the MRN complex at damaged sites. This molecular link of NBS1 to ATM may explain the phenotypic similarity of NBS to A-T.

Amino Acid Motifs↗
PubMed2005

Rendez-vous at mitosis: TRRAPed in the chromatin.

Cell cycle progression and cell cycle checkpoints are guided by dynamic changes in gene expression that requires concerted efforts of chromatin modifying/remodeling activities and transcription machinery. Epigenetic modifications including acetylation of specific lysine residues within the amino-terminal tails of core histones play an important role in these processes. In the last few years, a flurry of biochemical studies has identified numerous histone acetyltransferases (HAT) whose activity is dependent on the multiprotein assemblies and responsible for histone acetylation. In addition to their well-known involvement in the control of gene transcription, recent studies implicated HATs and histone acetylation in other important cellular processes, such as DNA replication, cell cycle control, DNA repair and genomic stability. With the exception of catalytic subunits of the HAT assemblies, the role of other components of these large multi-subunit complexes in cellular processes remains largely unknown. Recent genetic and cellular studies have shown that Trrap, a common component of HAT complexes, regulates the mitotic checkpoint function by modulation of mitotic checkpoint genes. This regulation involves a concerted and cell cycle stage-coupled recruitment of HAT activity to promoters of specific checkpoint genes, providing a functional link between specific chromatin modifications and cell cycle control. These findings shed new light on the role of HAT components and histone acetylation in cell cycle control and underscore functional significance of epigenetic modifications in cellular processes.

Acetyltransferases↗