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At least 559 records · Page 31Linked to original sources
PubMed2000

The MRE11-NBS1-RAD50 pathway is perturbed in SV40 large T antigen-immortalized AT-1, AT-2 and HL-1 cardiomyocytes.

To investigate molecular controls of cardiomyocyte proliferation, we utilized cardiomyocytes induced to proliferate indefinitely by SV40 large T antigen (T-ag). In the T-ag-immortalized AT-1, AT-2 and HL-1 cardiomyocytes, normal cellular proteins associating with T-ag and p53 were identified, isolated and micro-sequenced. Peptide sequencing revealed that proteins of 90, 100 and 160 kDa were homologs of MRE11, NBS1 and RAD50, respectively. These three proteins play critical roles in the detection and repair of DNA double-strand breaks, activation of cell cycle checkpoints and telomere maintenance. In this report, we describe the cDNA cloning and double-strand sequencing of the rat homologs of MRE11, NBS1 and RAD50. We also determined the mRNA and protein levels of MRE11, NBS1 and RAD50 at different stages of heart development and in different tissues. MRE11 mRNA was only detected in the immortalized cardiomyocytes and in the testes. Although the 90 kDa MRE11 protein was seen in most samples examined, it was only detected at extremely low levels in proliferating cardiomyocytes (normal and immortalized). The 6.0 kb MRE11-related mRNA transcript (MRT) was seen in all samples examined. Levels of both NBS1 and RAD50 mRNA transcripts peaked in the heart at postnatal day 10. NBS1 mRNA levels were at very low levels in the T-ag-immortalized AT-1, AT-2 and HL-1 cells but NBS1 protein was observed at extremely high levels. We propose that SV40 large T antigen's interaction with the MRE11-NBS1-RAD50 pathway and with p53 ablates critical cell cycle checkpoints and that this is one of the major factors involved in the ability of this oncoprotein to immortalize cardiomyocytes.

Amino Acid Sequence↗
PubMed1997

Telomeres, senescence and cellular radiation response.

Telomeres shield the ends of chromosomes from degradation and end-to-end fusions. They shorten at each cell division and when they reach a critically short length, cells arrest in the G1 phase of the cell cycle and undergo senescence. This effectively limits the proliferative potential of cells. Senescence functions as a tumour suppressor mechanism and appears to contribute to the process of ageing. If senescence is circumvented by tumour viruses, proliferation is re-initiated until cells enter crisis. Activation of telomerase prevents telomere attrition and cells become immortal. Cellular response to ionizing radiation involves induction of cell cycle checkpoint arrests and programmed cell death. Because radiation produces double strand breaks in DNA, which cause telomere-less chromosome ends, radiation response appears to be the result of inappropriate induction of cellular senescence mechanisms.

Animals↗
PubMed1998

Induction of cell cycle arrest by the endogenous product of lipid peroxidation, malondialdehyde.

We have investigated the effect of the endogenous genotoxin malondialdehyde (MDA) on cell cycle kinetics and the expression and biochemical activity of several cell cycle regulatory proteins. MDA treatment of two human cell lines (RKO and H1299) resulted in a 3- to 6-fold elevation in the levels of the major detectable MDA-DNA adduct, M1G-dR. The increase in M1G-dR was accompanied by irreversible cell cycle arrest, elevation in p53 and p21 protein levels, and inhibition of cyclin E- and cyclin B-associated kinase activities. The decrease in cyclin E- and cyclin B-dependent kinase activities was caused by increased p21 and decreased cdc2 levels, respectively. Comparable levels of p21 induction were observed in RKO (wild-type p53) and H1299 (p53-null) cells. Thus, MDA was able to engage cell cycle checkpoint function in human cell lines when used at concentrations that produce M1G-dR levels of the same magnitude found in human tissues.

Antibiotics, Antineoplastic↗
PubMed2004

Defective control of mitotic and post-mitotic checkpoints in poly(ADP-ribose) polymerase-1(-/-) fibroblasts after mitotic spindle disruption.

Poly(ADP-ribose) polymerase-1 (PARP), a DNA damage-responsive nuclear enzyme present in higher eukaryotes, is well-known for its roles in protecting the genome after DNA damage. However, even without exogenous DNA damage, PARP may play a role in stabilizing the genome because cells or mice deficient in PARP exhibit various signs of genomic instability, such as tetraploidy, aneuploidy, chromosomal abnormalities and susceptibility to spontaneous carcinogenesis. Normally, cell cycle checkpoints ensure elimination of cells with genomic abnormalities. Therefore, we examined efficiency of mitotic and post-mitotic checkpoints in PARP-/- and PARP+/+ mouse embryonic fibroblasts treated with mitotic spindle disrupting agent colcemid. PARP+/+ cells, like most mammalian cells, eventually escaped from spindle disruption-induced mitotic checkpoint arrest by 60 h. In contrast, PARP-/- cells rapidly escaped from mitotic arrest within 24 h by downregulation of cyclin B1/CDK-1 kinase activity. After escaping from mitotic arrest; both the PARP genotypes arrive in G1 tetraploid state, where they face post-mitotic checkpoints which either induce apoptosis or prevent DNA endoreduplication. While all the G1 tetraploid PARP+/+ cells were eliminated by apoptosis, the majority of the G1 tetraploid PARP-/- cells became polyploid by resisting apoptosis and carrying out DNA endoreduplication. Introduction of PARP in PARP-/- fibroblasts partially increased the stringency of mitotic checkpoint arrest and fully restored susceptibility to G1 tetraploidy checkpoint-induced apoptosis; and thus prevented formation of polyploid cells. Our results suggest that PARP may serve as a guardian angel of the genome even without exogenous DNA damage through its role in mitotic and post-mitotic G1 tetraploidy checkpoints.

Animals↗
PubMed2002

Involvement of replicative polymerases, Tel1p, Mec1p, Cdc13p, and the Ku complex in telomere-telomere recombination.

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of the reverse transcriptase telomerase. In both tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative recombination mechanism. Genetic studies have led to the identification of DNA polymerases, cell cycle checkpoint proteins, and telomere binding proteins involved in the telomerase pathway. However, how these proteins affect telomere-telomere recombination has not been identified to date. Using an assay to trace the in vivo recombinational products throughout the course of survivor development, we show here that three major replicative polymerases, alpha, delta, and epsilon, play roles in telomere-telomere recombination and that each causes different effects and phenotypes when they as well as the telomerase are defective. Polymerase delta appears to be the main activity for telomere extension, since neither type I nor type II survivors arising via telomere-telomere recombination were seen in its absence. The frequency of type I versus type II is altered in the polymerase alpha and epsilon mutants relative to the wild type. Each prefers to develop a particular type of survivor. Moreover, type II recombination is mediated by the cell cycle checkpoint proteins Tel1 and Mec1, and telomere-telomere recombination is regulated by telomere binding protein Cdc13 and the Ku complex. Together, our results suggest that coordination between DNA replication machinery, DNA damage signaling, DNA recombination machinery, and the telomere protein-DNA complex allows telomere recombination to repair telomeric ends in the absence of telomerase.

Antigens, Nuclear↗
PubMed2003

Cell-cycle deregulation in BALB/c 3T3 cells transformed by 1,2-dibromoethane and folpet pesticides.

The cell-transforming potential of 1,2-dibromoethane and folpet, two widely used agricultural pesticides that are potential sources of environmental pollution, has been previously ascribed to their promoting activity. In this study, we investigated whether BALB/c 3T3 transformation by these chemicals was associated with the deregulation of signals involved in cell-cycle progression and in cell-cycle checkpoint induction. We found that two BALB/c 3T3 cell clones transformed by in vitro medium-term (8-week) exposure to the carcinogens had a constitutive acceleration of cell transition from G(1) to S phase and an abrogation of the radiation-induced G(1)/S checkpoint. These events involved multiple signals; in particular, the inhibitors of cyclin/cyclin-dependent kinase complexes p21 and p27 were significantly down-modulated and the positive regulators of cell-cycle progression cyclin D(3) and E were up-modulated. As anticipated for cells where the G(1)/S checkpoint was abrogated, the transformed cells exhibited a significant reinforcement of the radiation-induced G(2)/M checkpoint, the only checkpoint remaining to protect genomic integrity. However, cyclin A(1) and B(1) coexpression and cyclin A(1) overexpression were found despite the G2 arrest in irradiated cells and these signals likely attenuate the G(2)/M checkpoint. These alterations to normal cell cycling may promote the emergence of both numerical and structural chromosomal abnormalities and their tolerance. Such a condition could play a key role in neoplastic transformation and be crucial in tumor progression. Furthermore, cyclin A(1) overexpression may play an autonomous role in the neoplastic transformation of BALB/c 3T3 cells, as it does in other cell types of mesenchymal origin.

3T3 Cells↗
PubMed2004

Cyclin A and cyclin D1 as significant prognostic markers in colorectal cancer patients.

BACKGROUND: Colorectal cancer is a common cancer all over the world. Aberrations in the cell cycle checkpoints have been shown to be of prognostic significance in colorectal cancer. METHODS: The expression of cyclin D1, cyclin A, histone H3 and Ki-67 was examined in 60 colorectal cancer cases for co-regulation and impact on overall survival using immunohistochemistry, southern blot and in situ hybridization techniques. Immunoreactivity was evaluated semi quantitatively by determining the staining index of the studied proteins. RESULTS: There was a significant correlation between cyclin D1 gene amplification and protein overexpression (concordance = 63.6%) and between Ki-67 and the other studied proteins. The staining index for Ki-67, cyclin A and D1 was higher in large, poorly differentiated tumors. The staining index of cyclin D1 was significantly higher in cases with deeply invasive tumors and nodal metastasis. Overexpression of cyclin A and D1 and amplification of cyclin D1 were associated with reduced overall survival. Multivariate analysis shows that cyclin D1 and A are two independent prognostic factors in colorectal cancer patients. CONCLUSIONS: Loss of cell cycle checkpoints control is common in colorectal cancer. Cyclin A and D1 are superior independent indicators of poor prognosis in colorectal cancer patients. Therefore, they may help in predicting the clinical outcome of those patients on an individual basis and could be considered important therapeutic targets.

Biomarkers, Tumor↗
PubMed2001

Contrasting roles of NF-kappaB and JNK in arsenite-induced p53-independent expression of GADD45alpha.

Growth arrest and DNA damage-inducible protein 45alpha (GADD45alpha) is an important cell cycle checkpoint protein that arrests cells at G2/M phase by inhibiting the activity of G2-specific kinase, cyclin B/p34cdc2. We report here that arsenite induces GADD45alpha expression in a p53-independent fashion and that this GADD45alpha induction by arsenite is regulated by NF-kappaB and c-Jun-N-terminal kinase (JNK) oppositely. In human bronchial epithelial cells overexpressing a kinase-mutated form of IkappaB kinase beta (IKKbeta-KM), the activation of NF-kappaB was inhibited. However, the G2/M cell cycle arrest and expression of GADD45alpha was substantially enhanced in response to arsenite in these cells. Expression of a dominant-negative mutant of SEK1 that blocks JNK activation decreased arsenite-induced GADD45alpha expression. Analysis of GADD45alpha expression in both wild-type and p53-/- fibroblasts indicated that the induction of GADD45alpha by arsenite was independent of the status of p53 protein.

Arsenites↗
PubMed2005

Targeting chk2 kinase: molecular interaction maps and therapeutic rationale.

Most anticancer drugs presently used clinically target genomic DNA. The selectivity of these anticancer drugs for tumor tissues is probably due to tumor-specific defects suppressing cell cycle checkpoints and DNA repair, and enhancing apoptotic response in the tumor. We will review the molecular interactions within the ATM-Chk2 pathway implicating the DNA damage sensor kinases (ATM, ATR and DNA-PK), the adaptor BRCT proteins (Nbs1, Brca1, 53BP1, MDC1) and the effector kinases (Chk2, Chk1, Plk3, JNK, p38). The molecular interaction map convention (MIM) will be used for presenting this molecular network (http://discover.nci.nih.gov/mim/). A characteristic of the ATM-Chk2 pathway is its redundancy. First, ATM and Chk2 phosphorylate common substrates including p53, E2F1, BRCA1, and Chk2 itself, which suggests that Chk2 (also known as CHECK2, Cds1 in fission yeast, and Dmchk2 or Dmnk or Loki in the fruit fly) acts as a relay for ATM and/or as a salvage pathway when ATM is inactivated. Secondly, redundancy is apparent for the substrates, which can be phosphorylated/activated at similar residues by Chk2, Chk1, and the polo kinases (Plk's). Functionally, Chk2 can activate both apoptosis (via p53, E2F1 and PML) and cell cycle checkpoint (via Cdc25A and Cdc25C, p53, and BRCA1). We will review the short list of published Chk2 inhibitors. We will also propose a novel paradigm for screening interfacial inhibitors of Chk2. Chk2 inhibitors might be used to enhance the tumor selectivity of DNA targeted agents in p53-deficient tumors, and for the treatment of tumors whose growth depends on enhanced Chk2 activity.

Animals↗
PubMed1993

Delays in anaphase initiation occur in individual nuclei of the syncytial Drosophila embryo.

The syncytial divisions of the Drosophila melanogaster embryo lack some of the well established cell-cycle checkpoints. It has been suggested that without these checkpoints the divisions would display a reduced fidelity. To test this idea, we examined division error frequencies in individuals bearing an abnormally long and rearranged second chromosome, designated C(2)EN. Relative to a normal chromosome, this chromosome imposes additional structural demands on the mitotic apparatus in both the early syncytial embryonic divisions and the later somatic divisions. We demonstrate that the C(2)EN chromosome does not increase the error frequency of the late larva neuroblast divisions. However, in the syncytial embryonic nuclear divisions, the C(2)EN chromosome produces a 10-fold increase in division errors relative to embryos with a normal karyotype. During late anaphase of the neuroblast divisions, the sister C(2)EN chromosomes cleanly separate from one another. In contrast, during late anaphase of the syncytial divisions in C(2)EN-bearing nuclei, large amounts of chromatin often lag on the metaphase plate. Live analysis of C(2)EN-bearing embryos demonstrates that individual nuclei in the syncytial population of dividing nuclei often delay in their initiation of anaphase. These delays frequently lead to division errors. Eventually the products of the nuclei delayed in anaphase sink inward and are removed from the dividing population of syncytial nuclei. These results suggest that the Drosophila embryo may be equipped with mechanisms that monitor the fidelity of the syncytial nuclear divisions. Unlike checkpoints that rely on cell cycle delays to identify and correct division errors, these embryonic mechanisms rely on cell cycle delays to identify and discard the products of division errors.

Anaphase↗
PubMed2005

p38 MAP kinase plays a role in G2 checkpoint activation and inhibits apoptosis of human B cell lymphoma cells treated with etoposide.

p38 MAPK is mainly activated by stress stimuli and mediates signals that regulate various cellular responses, including cell-cycle progression and apoptosis, depending on cell types and stimuli. Here we examine the role of p38 in regulation of apoptosis and cell cycle checkpoint in Daudi B-cell lymphoma cells treated with the topoisomerase II inhibitor etoposide. Etoposide activated p38, inhibited the G2/M transition with the persistent inhibitory phosphorylation of Cdc2 on Tyr15, and caused apoptosis of Daudi cells. Inducible expression of a dominant negative p38alpha mutant in Daudi cells reduced the inhibition of Cdc2 as well as G2/M arrest and augmented apoptosis induced by etoposide. SB203580, a specific inhibitor of p38alpha and p38beta, similarly reduced the inhibitory phosphorylation of Cdc2 as well as G2/M arrest and augmented apoptosis of Daudi cells treated with etoposide. These results suggest that p38 plays a role in G2/M checkpoint activation through induction of the persistent inhibitory phosphorylation of Cdc2 and, thereby, inhibits apoptosis of Daudi cells treated with etoposide. The present study, thus, raises the possibility that p38 may represent a new target for sensitization of lymphoma cells to DNA-damaging chemotherapeutic agents.

CDC2 Protein Kinase↗
PubMed2006

Roles of DNA polymerases in replication, repair, and recombination in eukaryotes.

The functioning of the eukaryotic genome depends on efficient and accurate DNA replication and repair. The process of replication is complicated by the ongoing decomposition of DNA and damage of the genome by endogenous and exogenous factors. DNA damage can alter base coding potential resulting in mutations, or block DNA replication, which can lead to double-strand breaks (DSB) and to subsequent chromosome loss. Replication is coordinated with DNA repair systems that operate in cells to remove or tolerate DNA lesions. DNA polymerases can serve as sensors in the cell cycle checkpoint pathways that delay cell division until damaged DNA is repaired and replication is completed. Eukaryotic DNA template-dependent DNA polymerases have different properties adapted to perform an amazingly wide spectrum of DNA transactions. In this review, we discuss the structure, the mechanism, and the evolutionary relationships of DNA polymerases and their possible functions in the replication of intact and damaged chromosomes, DNA damage repair, and recombination.

Amino Acid Sequence↗
PubMed2006

Analysis of DNA mismatch repair in cellular response to DNA damage.

Significant advances have been made in identifying and characterizing the roles of DNA mismatch repair (MMR) proteins in cellular response to DNA damage. Insights into this process have been obtained by performing interactions of mismatch recognition proteins (e.g., MutSalpha) with DNA adduct-containing duplexes and by analyzing cellular responses (including cell cycle checkpoints and apoptosis) of cell lines and animals with various MMR capacities. This chapter presents detailed methods for gel-shift analysis to determine the interaction between MutSalpha and oligonucleotide duplex containing a single DNA adduct and for apoptotic assays in cell lines and experimental animals. In addition, a step-by-step protocol is also provided for the purification of MutSalpha from human cells, the preparation of DNA substrates containing a defined DNA adduct, and the treatment of MMR-proficient and deficient cell lines as well as MMR knockout mice.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Immortalization and characterization of Nijmegen Breakage syndrome fibroblasts.

Nijmegen Breakage Syndrome (NBS) is a very rare autosomal recessive chromosomal instability disorder characterized by microcephaly, growth retardation, immunodeficiency and a high incidence of malignancies. Cells from NBS patients are hypersensitive to ionizing radiation (IR) and display radioresistant DNA synthesis (RDS). NBS is caused by mutations in the NBS1 gene on chromosome 8q21 encoding a protein called nibrin. This protein is a component of the hMre11/hRad50 protein complex, suggesting a defect in DNA double-strand break (DSB) repair and/or cell cycle checkpoint function in NBS cells. We established SV40 transformed, immortal NBS fibroblasts, from primary cells derived from a Polish patient, carrying the common founder mutation 657del5. Immortalized NBS cells, like primary cells, are X-ray sensitive (2-fold) and display RDS following IR. They show an increased sensitivity to bleomycin (3.5-fold), etoposide (2.5-fold), camptothecin (3-fold) and mitomycin C (1.5-fold), but normal sensitivity towards UV-C. Despite the clear hypersensitivity towards DSB-inducing agents, the overall rates of DSB-rejoining in NBS cells as measured by pulsed field gel electrophoresis were found to be very similar to those of wild type cells. This indicates that the X-ray sensitivity of NBS cells is not directly caused by an overt defect in DSB repair.

Abnormalities, Multiple↗
PubMed1999

The use of nocodazole in cell cycle analysis and parasite purification from Theileria parva-infected B cells.

Theileria parasites transform bovine leukocytes and induce uncontrolled lymphoproliferation only in the macroschizont stage of their life cycle. The isolation of highly purified stage-specific parasite RNA and proteins is an essential prerequisite when studying the Theileria-host relationship. We therefore improved a protocol based on the cytolytic bacterial toxin aerolysin by taking advantage of the microtubule inhibitor nocodazole. In this report we describe that nocodazole-mediated separation of the parasite from the host cell microtubule network was used with success to improve quantity and quality of purified parasites. We furthermore show that nocodazole is a useful tool to study cell cycle checkpoints due to its capacity to induce reversible cell cycle arrest in Theileria-infected B cells.

Animals↗
PubMed2002

Suppression of Cdc2 dephosphorylation at the tyrosine 15 residue during nitrosourea-induced G2M phase arrest in glioblastoma cell lines.

We examined the mechanism of action of nitrosoureas as represented by 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU) with respect to p53 and the G2M cell cycle checkpoint using two glioblastoma cell lines: U251MG and U373MG, with mutated p53. At log-phase cell growth, fresh medium containing ACNU (final concentration, 3, 10, or 30 microg/ml) was added. After 24 h of incubation, cells were harvested for flow cytometric or Western analysis. In both lines, cell numbers in the G0/G1 phase decreased with ACNU treatment. Cells accumulated in G2M and S phases, and the peak was shifted from G2M to the S phase in a concentration-dependent manner. In both cell lines, the amount of Cdc2 protein phosphorylated at the tyrosine 15 residue was increased 2- to 6-fold by treatment with ACNU compared with untreated control cells. Expression of cyclin B protein was suppressed in cells treated with 30 microg/ml ACNU. Protein abundance for total Cdc2, Cdc2 phosphorylated at the threonine 161 residue, Wee 1, Myt 1, Chk 1, and 14-3-3sigma was not affected by treatment with ACNU in either cell line. We suggest that a low concentration of ACNU should be used with adjuvant therapies that act upon cells in the G2M phase. A high concentration of ACNU should be used with adjuvant therapies that act upon cells in the S phase.

Blotting, Western↗
PubMed2001

DNA polymerase epsilon is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts.

DNA polymerase epsilon (Pol epsilon) is thought to be involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the requirement of other replicative DNA polymerases, DNA polymerases alpha and delta (Pol alpha and delta), for chromosomal DNA replication has been well documented by genetic and biochemical studies, the precise role, if any, of Pol epsilon in chromosomal DNA replication is still obscure. Here we show, with the use of a cell-free replication system with Xenopus egg extracts, that Xenopus Pol epsilon is indeed required for chromosomal DNA replication. In Pol epsilon-depleted extracts, the elongation step of chromosomal DNA replication is markedly impaired, resulting in significant reduction of the overall DNA synthesis as well as accumulation of small replication intermediates. Moreover, despite the decreased DNA synthesis, excess amounts of Pol alpha are loaded onto the chromatin template in Pol epsilon-depleted extracts, indicative of the failure of proper assembly of DNA synthesis machinery at the fork. These findings strongly suggest that Pol epsilon, along with Pol alpha and Pol delta, is necessary for coordinated chromosomal DNA replication in eukaryotic cells.

Animals↗
PubMed2000

Nuclear clusterin/XIP8, an x-ray-induced Ku70-binding protein that signals cell death.

Clusterin [CLU, a.k.a. TRPM-2, SGP-2, or ionizing radiation (IR)-induced protein-8 (XIP8)] was implicated in apoptosis, tissue injury, and aging. Its function remains elusive. We reisolated CLU/XIP8 by yeast two-hybrid analyses using as bait the DNA double-strand break repair protein Ku70. We show that a delayed (2-3 days), low-dose (0.02-10 Gy) IR-inducible nuclear CLU/XIP8 protein coimmunoprecipitated and colocalized (by confocal microscopy) in vivo with Ku70/Ku80, a DNA damage sensor and key double-strand break repair protein, in human MCF-7:WS8 breast cancer cells. Overexpression of nuclear CLU/XIP8 or its minimal Ku70 binding domain (120 aa of CLU/XIP8 C terminus) in nonirradiated MCF-7:WS8 cells dramatically reduced cell growth and colony-forming ability concomitant with increased G(1) cell cycle checkpoint arrest and increased cell death. Enhanced expression and accumulation of nuclear CLU/XIP8-Ku70/Ku80 complexes appears to be an important cell death signal after IR exposure.

Adenocarcinoma↗