SEARCH · Search PubMed

Results for “Cell Cycle Checkpoints”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources

PubMed1994

DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways.

The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA-damaging agents.

Antimetabolites, Antineoplastic↗

PubMed1994

Testing the role of p53 in the expression of genetic instability and apoptosis in ataxia-telangiectasia.

We have obtained initial evidence supporting a new model for the human disease ataxia-telangiectasia (A-T), in which the A-T and p53 genes play crucial roles in a signal transduction network that activates multiple cellular functions in response to DNA damage. Three of the model's predictions were tested. (1) Disrupting cell cycle checkpoints should increase spontaneous rates in normal cells. In order to interfere with the G1/S checkpoint, we transfected a normal cell line with vectors expressing either a dominant-negative p53ala143 mutant or a human papilloma virus E6 gene. These transformants showed 10-80-fold elevations in spontaneous recombination rates when compared with their parent. (2) A-T cells should be sensitive to DNA damage-induced apoptosis. Widespread apoptosis was detectable in four A-T fibroblast lines, but not two control lines, beginning 24 h after exposure to X-rays or streptonigrin, but not UV. Streptonigrin also induced widespread apoptosis in A-T lymphoblasts but not in control lymphoblasts. (3) Disruption of p53 function in A-T cells should increase their mutagen resistance by interfering with apoptosis. Stable transfection of either the p53143ala or the HPV18 E6 construct was associated with acquisition of streptonigrin and radiation resistance, while transfection with the p53143ala construct did not affect the streptonigrin sensitivity of a control cell line.

Apoptosis↗

PubMed1993

Increased accumulation of p53 protein in cisplatin-resistant ovarian cell lines.

We have examined p53 protein levels in cell lines selected for resistance to the chemotherapeutic drug cis-diamminedichloroplatinum (II), cisplatin. The majority of the independent cisplatin-resistant clones isolated by a single selection with cisplatin from the ovarian tumour cell line A2780 showed increased levels of p53 protein compared to the parental cell line. Elevated p53 protein levels were also observed in cisplatin-resistant ovarian human tumour lines isolated after multiple exposures to cisplatin (A2780/cp70 and OVIP/DDP). Direct PCR sequencing of p53 cDNAs showed that both the A2780/cp70 and the parental A2780 cell lines had a wild-type p53 gene sequence. The OVIP and OVIP/DDP lines both had a heterozygous mutation at codon 126. Cell-cycle analysis after gamma-irradiation or cisplatin treatment showed evidence of a G1/S and G2/M cell-cycle checkpoint in both A2780/cp70 and the sensitive parental cell lines. However, the resistant cell line A2780/cp70 showed less inhibition of DNA synthesis after gamma-irradiation than the sensitive cell line. Transfection of a mutant p53 gene construct (containing a mutation at codon 143, val to ala) into the A2780/cp70 resistant cells conferred a significantly increased sensitivity to cisplatin, suggesting that p53 is a direct determinant of cisplatin resistance in these cells. However, expression of this mutant p53 in the A2780 cells did not affect sensitivity.

Cell Cycle↗

PubMed1996

Alterations of the p15, p16,and p18 genes in osteosarcoma.

Activation of cyclin-dependent kinases (CDKs) by interaction with cyclins regulates progression through cell cycle checkpoints. This process is counterbalanced by CDK inhibitors (CDKIs), which can inhibit progression through the cell cycle. Because CDKI expression acts to inhibit cellular proliferation, CDKIs may have a role as tumor suppressors. One class of CDKIs, characterized by the presence of ankyrin repeats, has at least four members (p15INK4B), p16INK4, p18, and p19). Two of these, p15INK4B, p16INK4, have been mapped to chromosome 9p21, a region of frequent loss in a wide variety of cancers. Alterations of p16INK4 have been detected in various tumors and cell lines. We analyzed p15INK4B, p16INK4, and p18 alterations in 52 osteosarcomas (including 11 explants), and 23 other various sarcomas. Single-stranded conformation polymorphism analysis [polymerase chain reaction (PCR-SSCP)] of the coding regions of these CDKI genes detected a missense mutation of p16INK4 exon 1 in one soft tissue sarcoma. Southern blotting detected complete deletion of p15INK4B and p16INK4 genes in osteosarcomas from 2 patients and a soft tissue sarcoma from another individual. Loss of heterozygosity (LOH) at chromosome 9p21 was observed with a microsatellite probe closely linked to the INK4 genes in the latter case. Deletions of both p15INK4B and p16INK4 genes were detected in five of eight osteosarcoma cell lines. By contrast, no alterations of p18 were detected in any sample. Together these data suggest that alterations of the p15INK4B and p16INK4 genes, but not p18, may occur in approximately 5% of sarcomas. However, deletions of the p15INK4B and P16INK4 genes are frequent in osteosarcoma cell lines and probably have a role in tumor cell growth in culture. Notably, all seven detectable deletions involved both p15INK4B and p16INK4 genes, suggesting that both contribute individual tumor suppressor activity.

Base Sequence↗

PubMed1995

Attenuated response of p53 and p21 in primary cultures of human prostatic epithelial cells exposed to DNA-damaging agents.

The multifocal origin of prostate cancer suggests a pan-organ defect in a tumor suppressor pathway. Although structural mutations in the p53 gene have been implicated in late-stage prostate cancer, little is known about the p53 response to genotoxic stress in normal human prostatic epithelial cells from which adenocarcinomas originate. We found that the majority (10 of 12) of epithelial cell cultures derived from histologically normal tissues of radical prostatectomy specimens failed to exhibit p53 accumulation in response to ionizing radiation. Epithelial cell cultures derived from benign prostatic hyperplasia and a primary prostatic adenocarcinoma also failed to accumulate p53 in response to ionizing radiation. In contrast, cultures of prostatic stromal cells derived from normal, benign prostatic hyperplasia, or adenocarcinoma tissues exhibited a 3-9-fold induction of p53 within 1-3 h after irradiation. Since p53 regulates a cell cycle checkpoint through the induction of the cyclin-cdk inhibitor p21, we examined p21 accumulation and cell cycle arrest following exposure to ionizing radiation. With one exception, epithelial cells that did not display increased p53 or p21 induction did not demonstrate a significant G1-S arrest in response to ionizing radiation, whereas stromal cells that accumulated p53 and p21 exhibited a large cell cycle arrest. These results indicate a functional difference between the DNA damage response of epithelial and stromal prostatic cells and suggest a possible mechanism for the increased susceptibility of prostatic epithelial cells to accumulate genetic alterations.

Adenocarcinoma↗

PubMed1995

The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores.

During mitosis in Ptk1 cells anaphase is not initiated until, on average, 23 +/- 1 min after the last monooriented chromosome acquires a bipolar attachment to the spindle--an event that may require 3 h (Rieder, C. L., A. Schultz, R. W. Cole, and G. Sluder. 1994. J. Cell Biol. 127:1301-1310). To determine the nature of this cell-cycle checkpoint signal, and its site of production, we followed PtK1 cells by video microscopy prior to and after destroying specific chromosomal regions by laser irradiation. The checkpoint was relieved, and cells entered anaphase, 17 +/- 1 min after the centromere (and both of its associated sister kinetochores) was destroyed on the last monooriented chromosome. Thus, the checkpoint mechanism monitors an inhibitor of anaphase produced in the centromere of monooriented chromosomes. Next, in the presence of one monooriented chromosome, we destroyed one kinetochore on a bioriented chromosome to create a second monooriented chromosome lacking an unattached kinetochore. Under this condition anaphase began in the presence of the experimentally created monooriented chromosome 24 +/- 1.5 min after the nonirradiated monooriented chromosome bioriented. This result reveals that the checkpoint signal is not generated by the attached kinetochore of a monooriented chromosome or throughout the centromere volume. Finally, we selectively destroyed the unattached kinetochore on the last monooriented chromosome. Under this condition cells entered anaphase 20 +/- 2.5 min after the operation, without congressing the irradiated chromosome. Correlative light microscopy/elctron microscopy of these cells in anaphase confirmed the absence of a kinetochore on the unattached chromatid. Together, our data reveal that molecules in or near the unattached kinetochore of a monooriented PtK1 chromosome inhibit the metaphase-anaphase transition.

Anaphase↗

PubMed1996

p53 involves cytosine arabinoside-induced apoptosis in cultured cerebellar granule neurons.

The tumor suppressor p53 gene plays a key role in controlling the cell cycle checkpoint and in apoptosis following the exposure of normal cells to DNA damage. To investigate the role of p53 in cytosine arabinoside (Ara C)-induced cell death of CNS neurons, we examined the effect of Ara C on the survival of cultured cerebellar granule neurons from normal wild-type and p53 null mutant mice. When the neurons from wild-type mice were cultured with Ara C, they gradually died after 24 h in culture. In contrast, the neurons from p53 null mutant mice showed a resistance to the Ara C neurotoxicity. These results indicate that p53 involves Ara C-induced apoptosis in cultured cerebellar granule neurons, in which DNA damage may initiate the apoptotic death program of the neurons.

Animals↗

PubMed1994

Bloom's syndrome cells GM1492 lack detectable p53 protein but exhibit normal G1 cell-cycle arrest after UV irradiation.

The tumor suppressor gene p53 is thought to be a key factor in the onset of G1 cell-cycle arrest following DNA damage. However, here we describe cells derived from a patient with Bloom's syndrome, lacking any detectable p53 protein, that still shows a functional G1 cell-cycle checkpoint after irradiation with UV-C. Comparison with cells from other Bloom's patients showed that the absence of p53 protein is not a specific characteristic of Bloom's syndrome.

Bloom Syndrome↗

PubMed1995

Differences in resistance to 5-fluorouracil as a function of cell cycle delay and not apoptosis.

A series of human embryo fibroblasts has previously been shown to display increasing resistance to the antimetabolites methotrexate (MTX) and N-phosphonacetyl-L-aspartate (PALA) with increasing tumorigenicity. This increased resistance was found to be further increased as a result of salvage pathway activity for purine and pyrimidine biosynthesis. A similar pattern of increasing resistance paralleling increasing tumorigenicity has now been shown to occur with 5-fluorouracil (5-FU), which is independent of salvage pathway activity. The KMS normal cell line was found to be more sensitive to 5-FU than either the immortalised KMST or tumorigenic KN-NM cell lines. Immunohistochemical analysis of the three cell lines demonstrated high levels of p53 protein in the KMST and KN-NM cell lines, but undetectable p53 levels in the KMS cell line. From these data it was hypothesised that a difference in p53 function may be causing the difference in the patterns of sensitivity observed in the three cell lines. P53 is now believed to function as a regulator of a G1 to S cell cycle checkpoint and as an inducer of apoptosis following DNA damage to the cell. The differences in sensitivity of the cell lines could not be explained by differences in the levels of apoptosis but could be attributed to differences in cell cycle response. Our evidence suggests that loss of cell cycle control, possibly through loss of p53 function, is an important factor in increasing the drug resistance of fibroblast cell lines.

Antimetabolites, Antineoplastic↗

PubMed1994

Elongation factor 1 delta is enhanced following exposure to ionizing radiation.

We have used the technique of differential display of mRNA to investigate gene expression patterns in a human squamous carcinoma cell line, SCC-35, following exposure to ionizing radiation. We observed that elongation factor 1 delta (EF-1 delta) expression increased within 2 h of radiation exposure with maximum expression at 8 h. These data identify EF-1 delta as a radiation-inducible gene. Cell cycle analysis shows that EF-1 delta expression enhancement precedes G2 arrest in these cells, suggesting that EF-1 delta may participate in the G2-M cell cycle checkpoint.

Base Sequence↗

PubMed1994

Modification of the radiosensitivity of human testicular cancer cells by simian virus 40 sequences.

CRL7800 cells are of human testicular cancer origin and are sensitive to killing by ionizing radiation. After transfection with a plasmid expressing the T-antigen (pSC), cells show enhanced growth and an increased resistance to ionizing radiation. Cell cycle analysis reveals perturbation of a cell cycle checkpoint which, after irradiation, results in an increase in G2-phase arrest in CRL7800VA cells. These experiments demonstrate the modulation of radiation sensitivity and cell cycle arrest of human tumor cells by the introduction of viral genes.

Antigens, Polyomavirus Transforming↗

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↗

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↗

PubMed1995

p53 involvement in control of G2 exit of the cell cycle: role in DNA damage-induced apoptosis.

DNA damage in proliferating mammalian cells induces a complex cellular response comprising perturbation of the cell cycle and programmed cell death. The relationship between p53-dependent and p53-independent apoptotic cell death, as well as the cell cycle checkpoints induced by DNA damaging agents were explored in hematopoietic cells, using M1 myeloblastic leukemia cells, which are null for p53 expression, genetically engineered M1 variants, expressing p53ts and bcl-2 transgenes, as well as myeloblast enriched bone-marrow cells obtained from wild type p53 (wt p53) and p53-deficient mice. It is shown that gamma-irradiation of M1p53ts cells activated a function of the temperature sensitive mutant transgene p53 (p53ts), promoting increased apoptosis relative to parental, null p53 M1 cells. It is also shown that the kinetics of apoptotic cell death induced by gamma-irradiation correlated with the rapidity of exit from gamma-ray-induced G2 arrest for all the different hematopoietic cell types indicated above. Finally, data has been obtained to demonstrate that, in addition to a role in apoptosis and G1 arrest, wild-type p53 positively modulated the exit from the gamma-ray-induced G2 checkpoint. Taken together, these findings indicate that this new function for p53 is a component of the physiological pathway by which p53 exerts its role in apoptosis.

Animals↗

PubMed1996

Human and plant proliferating-cell nuclear antigen have a highly conserved binding site for the p53-inducible gene product p21WAF1.

The mechanism(s) whereby higher plants respond to environmental agents that damage their DNA, which leads to the arrest of cell division, is poorly understood. In mammalian cells, the tumour-suppressor protein p53 plays a central role in a DNA-damage-induced cell-cycle-checkpoint pathway by induction of transcription of a set of gene products that have a direct role in a DNA-damage-induced cell-cycle growth arrest. One such protein, p21WAF1, has been shown to be essential for radiation-induced growth arrest. There appear to be at least two cellular targets of p21WAF1 during checkpoint control, the G1-cyclin-dependent kinases (CDK) and proliferating-cell nuclear antigen (PCNA). The aim of the research reported here was to determine whether the interactions between the human growth inhibitor p21WAF1 and PCNA from plants and humans are conserved. If so, this would suggest that modulation of PCNA activity may play an important role in plant responses to DNA damage and would imply that functional homologue(s) of p21WAF1 exist in plants. We show that the p21WAF1-interaction domain of PCNA is conserved between humans and plants. A peptide that contains the site of human p21WAF1 that binds human PCNA has been used to precipitate PCNA from crude pea (Pisum savitum) extracts. We used the p21WAF1 peptide as an affinity matrix and showed that pea PCNA bound in a specific high-affinity manner. This finding was used to develop a purification protocol that allowed PCNA from plant tissue to be purified to homogeneity. Pure pea PCNA forms a stable complex with full-length human p21WAF1 and the specific amino acids of p21WAF1 required for the interaction have been identified. The critical residues were identical to those required for binding to human PCNA, which indicates that the interaction of human p21WAF1 with PCNA is highly conserved at each amino acid position between pea and human.

Amino Acid Sequence↗

PubMed1995

Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast.

The spindle assembly checkpoint prevents cells from initiating anaphase until the spindle has been fully assembled. We previously isolated mitotic arrest deficient (mad) mutants that inactivate this checkpoint and thus increase the sensitivity of cells to benomyl, a drug that interferes with mitotic spindle assembly by depolymerizing microtubules. We have cloned the MAD1 gene and show that when it is disrupted yeast cells have the same phenotype as the previously isolated mad1 mutants: they fail to delay the metaphase to anaphase transition in response to microtubule depolymerization. MAD1 is predicted to encode a 90-kD coiled-coil protein. Anti-Mad1p antibodies give a novel punctate nuclear staining pattern and cell fractionation reveals that the bulk of Mad1p is soluble. Mad1p becomes hyperphosphorylated when wild-type cells are arrested in mitosis by benomyl treatment, or by placing a cold sensitive tubulin mutant at the restrictive temperature. This modification does not occur in G1-arrested cells treated with benomyl or in cells arrested in mitosis by defects in the mitotic cyclin proteolysis machinery, suggesting that Mad1p hyperphosphorylation is a step in the activation of the spindle assembly checkpoint. Analysis of Mad1p phosphorylation in other spindle assembly checkpoint mutants reveals that this response to microtubule-disrupting agents is defective in some (mad2, bub1, and bub3) but not all (mad3, bub2) mutant strains. We discuss the possible functions of Mad1p at this cell cycle checkpoint.

Amino Acid Sequence↗

PubMed1997

The p53-regulated cyclin G gene promotes cell growth: p53 downstream effectors cyclin G and Gadd45 exert different effects on cisplatin chemosensitivity.

Among the p53-regulated genes that have been identified thus far, cyclin G is a relatively recent one. We conducted a series of experiments aimed at elucidating cyclin G function. Ectopic overexpression of cyclin G in human RKO colon carcinoma cells accelerated cell growth. Transfection of normal human fibroblasts with the cyclin G expression vector promoted clonal expansion. Cyclin G immune complexes isolated from the transfected cells exhibited appreciable levels of cyclin-dependent kinase activity, as evidenced using histone H1 as a substrate. The retinoblastoma protein, pRb, was detectable in cyclin G immune complexes, raising the possibility that Rb may be one mediator of cyclin G action. Cyclin G-overexpressing cells were more sensitive to cisplatin cytotoxicity than the parent cells, probably because cyclin G overexpression overrides cell cycle checkpoint(s). Overexpression of another p53-regulated gene, GADD45, by contrast, protected cells from cisplatin killing. These findings suggest that different downstream effectors of the p53 pathway may exert different effects on cellular survival after treatment with cancer chemotherapy drugs such as cisplatin.

Antineoplastic Agents↗