Search PubMed⌕ Search

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.

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

At least 1,189 records · Page 66Linked to original sources
PubMed2003

FHA domains as phospho-threonine binding modules in cell signaling.

Forkhead-associated (FHA) domains are present in >200 diverse proteins in all phyla from bacteria to mammals and seem to be particularly prevalent in proteins with cell cycle control functions. Recent work from several laboratories has considerably improved our understanding of the structure and function of these domains that were virtually unknown a few years ago, and the first disease associations of FHA domains have now emerged. FHA domains form 11-stranded beta-sandwiches that contain some 100-180 amino acid residues with a high degree of sequence diversity. FHA domains act as phosphorylation-dependent protein-protein interaction modules that preferentially bind to phospho-threonine residues in their targets. Interestingly, point mutations in the human CHK2 gene that lead to single-residue amino acid substitutions in the FHA domain of this cell cycle checkpoint kinase have been found to cause a subset of cases of the Li-Fraumeni multi-cancer syndrome.

Amino Acid Sequence↗
PubMed1998

A targeted disruption of the murine Brca1 gene causes gamma-irradiation hypersensitivity and genetic instability.

Germline mutations of the Brcal gene are responsible for most cases of familial breast and ovarian cancers, but somatic mutations are rarely detected in sporadic events. Moreover, mouse embryos deficient for Brca1 have been shown to die during early embryogenesis due to a proliferation defect. These findings seem incompatible with the tumor suppress function assigned to this gene and raise questions about the mechanism by which Brca1 mutations cause tumorigenesis. We now directly demonstrate that BRCA1 is responsible for the integrity of the genome. Murine embryos carrying a Brca1 null mutation are developmentally retarded and hypersensitive to gamma-irradiation, suggesting a failure in DNA damage repair. This notion is supported by spectral karyotyping (SKY) of metaphase chromosomes, which display numerical and structural aberrations. However, massive chromosomal abnormalities are only observed when a p53-/- background is introduced. Thus, a p53 dependent cell cycle checkpoint arrests the mutant embryos and prevents the accumulation of damaged DNA. Brca1-/- fibroblasts are not viable, nor are Brca1-/-:p53-/- fibroblasts. However, proliferative foci arise from Brca1-/-: p53-/- cells, probably due to additional mutations that are a consequence of the accumulating DNA damage. We believe that the increased incidence of such additional mutations accounts for the mechanism of tumorigenesis associated with Brca1 mutations in humans.

Animals↗
PubMed1999

Caspase inhibition in camptothecin-treated U-937 cells is coupled with a shift from apoptosis to transient G1 arrest followed by necrotic cell death.

Leukemia U-937 cells rapidly undergo characteristic morphological changes, caspase activation, and DNA fragmentation typical of apoptosis on treatment with the DNA topoisomerase I inhibitor camptothecin (CPT). In a previous report (Sané, A. T., and Bertrand, R., Cancer Res., 58: 3066-3072, 1998), we showed that, after CPT treatment, caspase inhibition by the tripeptide derivative benzyloxycarbonyl-Val-Ala-Asp(Ome)-fluoromethyl ketone (zVAD-fmk) blocked apoptosis and slowed passage of the cells through S-G2 and caused a transient accumulation of these cells at the G1 phase of the cell cycle. Accumulation of these cells at G1 is not associated with major changes in expression level of cyclin-dependent kinase (cdk)2, cdk4, and cdk6; cyclin D1 and cyclin E; or p16, p21, p27, and p57 after CPT treatment. Furthermore, cdk2, cdk4, and cdk6 kinase activities remain unaffected after CPT treatment. These results indicate that the G1 arrest of these cells does not correlate with a classical driven cell cycle checkpoint but with the known effect of CPT in mediating inhibition of DNA replication and RNA transcription after stabilization of topoisomerase I-linked DNA strand breaks. However, persistent caspase inhibition after CPT treatment also results in cells falling into necrosis after the transient G1 arrest. These results indicate that the enforced inhibition of caspase activities does not confer a survival advantage upon CPT-treated cells but is coupled with a shift from apoptosis to transient G1 arrest followed by massive necrosis.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Molecular analysis of P16(Ink4)/CDKN2 and P15(INK4B)/MTS2 genes in primary human testicular germ cell tumors.

PURPOSE: p16 (MTS 1) and p15 (MTS 2) are negative regulators of cell cycle progression at the G1 cell cycle checkpoint and function as tumor suppressor genes (TSG). Both p15 and p16 are located on chromosome 9p21 and alterations have been demonstrated in a variety of human malignancies and human cancer cell lines. In testicular germ cell tumors (TGCT) loss of heterozygosity of 9p21 to 23 has been observed in 41% to 72% of informative cases. The aim of our study was to examine TGCT and testicular cancer cell lines for deletions and mutations of the p15 and p16 genes. MATERIALS AND METHODS: 24 testicular germ cell tumors, 4 testicular cancer cell lines (TERA I, TERA II, HTE, HTH), 8 benign testicular tumors and 9 samples of normal testicular parenchyma were examined. Examinations for loss of heterozygosity (LOH) on 9p21 were performed for IFNA, D9S171, D9S126, D9S161, D9S1748 and PKY9 locus using Southern Blot analysis. Southern Blot analysis of the p16 gene to examine gross alterations was done using random primer labeled p16 cDNA. In addition all samples were examined for mutations of exons 1 to 3 of p16 and exons 1 to 2 of p15 by polymerase chain reaction (PCR) followed by single strand conformation polymorphism (SSCP) analysis on MDE-gels. All observed band shifts on SSCP were further evaluated by direct DNA sequencing techniques. RESULTS: No major deletions were detected for p16 and p15. 42% of all TGCT demonstrated LOH of the IFNA locus, 16% of the D9S161 locus and 5.5% of the D9S171 locus. No LOH was seen for the D9S126 locus. 5/24 TGCT (21%) and 1 testicular cancer cell line (TERA II) demonstrated band shifts for exon 2, 8/24 TGCT (33%) and 1 testicular cancer cell line (TERA I) showed band shifts for exon 3; 4 TGCT had an allelic loss for exon 3 on SSCP. DNA sequencing analysis for exon 2 demonstrated a single base substitution (G-->A) in codon 140 resulting in an amino acid exchange (ala-->thre) in 4 cases and a single base deletion with frame shift mutation in codon 142 in 2 cases. DNA sequencing analysis for exon 3 demonstrated a polymorphism in intron 2 in all cases. For p15 no band shifts were observed for exons 1 to 2 in TGCT or testicular cancer cell lines; none of benign testicular tumors or normal control tissues demonstrated any band shifts for p15 or p16. DISCUSSION: We demonstrated a high frequency of mutations for exon 2 in specimens of primary testicular germ cell tumors, but not in benign tumors or normal controls. We therefore believe that p16 might be involved in the pathogenesis of human TGCT. The polymorphisms described for exon 3 might be utilized for further LOH studies. The absence of mutations in p15 gene in TGCT specimens suggests that p15 might not play an important role in the pathogenesis of testicular germ cell tumors.

Blotting, Southern↗
PubMed1997

Current concepts in neuro-oncology: the cell cycle--a review.

Uncontrolled cellular proliferation is the hallmark of human malignant brain tumors. Their growth proceeds inexorably, in part because their cellular constituents have an altered genetic code that enables them to evade the checks and balances of the normal cell cycle. Recently, a number of major advances in molecular biology have led to the identification of several critical genetic and enzymatic pathways that are disturbed in cancer cells resulting in uncontrolled cell cycling. We now know that the progression of a cell through the cell cycle is controlled in part by a series of protein kinases, the activity of which is regulated by a group of proteins called cyclins. Cyclins act in concert with the cyclin-dependent kinases (CDKs) to phosphorylate key substrates that facilitate the passage of the cell through each phase of the cell cycle. A critical target of cyclin-CDK enzymes is the retinoblastoma tumor suppressor protein, and phosphorylation of this protein inhibits its ability to restrain activity of a family of transcription factors (E2F family), which induce expression of genes important for cell proliferation. In addition to the cyclins and CDKS, there is an emerging family of CDK inhibitors, which modulate the activity of cyclins and CDKs. CDK inhibitors inhibit cyclin-CDK complexes and transduce internal or external growth-suppressive signals, which act on the cell cycle machinery. Accordingly, all CDK inhibitors are candidate tumor suppressor genes. It is becoming clear that a common feature of cancer cells is the abrogation of cell cycle checkpoints, either by aberrant expression of positive regulators (for example, cyclins and CDKs) or the loss of negative regulators, including p21Cip1 through loss of function of its transcriptional activator p53, or deletion or mutation of p16ink4A (multiple tumor suppressor 1/CDKN2) and the retinoblastoma tumor suppressor protein. In this review, we describe in detail our current knowledge of the normal cell cycle and how it is disturbed in cancer cells. Because there have now been a number of recent studies showing alterations in cell cycle gene expression in human brain tumors, we will review the derangements in both the positive and negative cell cycle regulators that have been reported for these neoplasms. A thorough understanding of the molecular events of the cell cycle may lead to new opportunities by which astrocytoma cell proliferation can be controlled either pharmacologically or by gene transfer techniques.

Animals↗
PubMed2000

New Developments in the Therapy of Acute Myelocytic Leukemia.

Current conventional treatment for patients with acute myelogenous leukemia results in a high percentage of clinical responses in most patients. However, a high percentage of patients still remain refractory to primary therapy or relapse later. This review examines the search for new agents and new modes of therapy. In Section I, Dr. Estey discusses new agents directed at various targets, such as CD33, angiogenesis, inappropriately methylated (suppressor) genes, cell cycle checkpoints, proteosomes, multidrug resistance (MDR) gene, mitochondrial apoptotic pathway. He also reviews preliminary results of phase I trials with the nucleoside analog troxacitabine and liposomal anthracyclin and suggests new strategies for trials of new agents. In Section II, Dr. Jones revisits differentiation therapy and presents results of preclinical and clinical studies that demonstrate that a variety of clinically applicable cell cycle inhibitors (interferon, phenylbutyrate, vitamin D, retinoids, bryostatin-1) preferentially augments growth factor-mediated induction of myeloid leukemia terminal differentiation, as well as blocks growth factors' effects on leukemia proliferation. The combination of cell cycle inhibition plus myeloid growth factors may offer a potential treatment for resistant myeloid leukemias. In Section III, Drs. Levitsky and Borrello address the question of tumor vaccination in AML and shows that, although tumor rejection antigens in AML have not been formally identified to date, a growing number of attractive candidates are ripe for testing with defined antigen-specific vaccine strategies. Interestingly, the ability to drive leukemic blasts to differentiate into competent antigen presenting cells such as dendritic cells may be exploited in the creation of cellular vaccines. Ultimately, the successful development of active immunotherapy for AML will require integration with dose-intensive chemotherapy, necessitating a more complete understanding of host immune reconstitution. In Section IV, Dr. Slavin reviews the concept of delivering non-myeloablative stem cell transplantation (NST) and delayed lymphocyte infusion (DLI) to increase tolerance in particular in high risk and older patients, and take advantage of the graft-versus-leukemia (GVL) effect. All these approaches hold promise in reducing morbidity and mortality and differ from the older concepts aiming at delivering the highest possible doses of chemotherapy and/or total body irradiation to reach maximum leukemia cell kill, whatever the toxicity to the patient.

Journal Article↗
PubMedDate not supplied

Genetic response to farnesyltransferase inhibitors: proapoptotic targets of RhoB.

Knockout mouse studies have established that the transformation-selective death program triggered by farnesyltransferase inhibitor (FTI) requires a gain-of-function in the stress-regulated small GTPase RhoB. To gain insight into this death program, we compared the genetic response of cells with different RhoB genotypes to FTI treatment. The microarray hybridization strategy we employed focused specifically on events preceding the execution of RhoB-dependent apoptosis, which is crucial for effective antineoplastic responses in mouse, rather than on other aspects of the FTI response mediated by RhoB gain-of-function (e.g., growth inhibition). Genes that control cell adhesion and cell shape were represented prominently among upregulated targets, as were genes that control signal transduction, vesicle dynamics, transcription, and immunity. Genes that control cell cycle checkpoints and progression through S phase and mitosis were among the major downregulated targets. In support of the concept of RhoB as a negative regulator of Ras signaling pathways, the most strongly downregulated gene scored was farnesyl pyrophosphate synthetase, the enzyme that produces the substrate used by FT to farnesylate Ras proteins. Gene clustering revealed modules for MAPK signaling, cell cycle progression, and immune response as proapoptotic targets of RhoB. This report identifies genes that pertain to the transformation-selective apoptotic program triggered by FTI. Further study of this program may yield insights into the dramatic differences in efficacy and apoptotic prowess of most FTIs in human cancers, versus transgenic mouse models.

Alkyl and Aryl Transferases↗
PubMed1999

Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine.

Caffeine exposure sensitizes tumor cells to ionizing radiation and other genotoxic agents. The radiosensitizing effects of caffeine are associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints. The similarity of these checkpoint defects to those seen in ataxia-telangiectasia (A-T) suggested that caffeine might inhibit one or more components in an A-T mutated (ATM)-dependent checkpoint pathway in DNA-damaged cells. We now show that caffeine inhibits the catalytic activity of both ATM and the related kinase, ATM and Rad3-related (ATR), at drug concentrations similar to those that induce radiosensitization. Moreover, like ATM-deficient cells, caffeine-treated A549 lung carcinoma cells irradiated in G2 fail to arrest progression into mitosis, and S-phase-irradiated cells exhibit radioresistant DNA synthesis. Similar concentrations of caffeine also inhibit gamma- and UV radiation-induced phosphorylation of p53 on Ser15, a modification that may be directly mediated by the ATM and ATR kinases. DNA-dependent protein kinase, another ATM-related protein involved in DNA damage repair, was resistant to the inhibitory effects of caffeine. Likewise, the catalytic activity of the G2 checkpoint kinase, hChk1, was only marginally suppressed by caffeine but was inhibited potently by the structurally distinct radiosensitizer, UCN-01. These data suggest that the radiosensitizing effects of caffeine are related to inhibition of the protein kinase activities of ATM and ATR and that both proteins are relevant targets for the development of novel anticancer agents.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2001

Alterations of the tumor suppressor genes CDKN2A (p16(INK4a)), p14(ARF), CDKN2B (p15(INK4b)), and CDKN2C (p18(INK4c)) in atypical and anaplastic meningiomas.

We investigated 67 meningothelial tumors (20 benign meningiomas, 34 atypical meningiomas, and 13 anaplastic meningiomas) for losses of genetic information from chromosome arms 1p and 9p, as well as for deletion, mutation, and expression of the tumor suppressor genes CDKN2A (p16(INKa)/MTS1), p14(ARF), CDKN2B (p15(INK4b)/MTS2) (all located at 9p21) and CDKN2C (1p32). Comparative genomic hybridization and microsatellite analysis showed losses on 1p in 11 anaplastic meningiomas (85%), 23 atypical meningiomas (68%), and 5 benign meningiomas (25%). One atypical meningioma with loss of heterozygosity on 1p carried a somatic CDKN2C mutation (c.202C>T: R68X). Losses on 9p were found in five anaplastic meningiomas (38%), six atypical meningiomas (18%), and one benign meningioma (5%). Six anaplastic meningiomas (46%) and one atypical meningioma (3%) showed homozygous deletions of the CDKN2A, p14(ARF), and CDKN2B genes. Two anaplastic meningiomas carried somatic point mutations in CDKN2A (c.262G>T: E88X and c.262G>A: E88K) and p14(ARF) (c.305G>T: G102V and c.305G>A: G102E). One anaplastic meningioma, three atypical meningiomas, and one benign meningioma without a demonstrated homozygous deletion or mutation of CDKN2A, p14(ARF), or CDKN2B lacked detectable transcripts from at least one of these genes. Hypermethylation of CDKN2A, p14(ARF), and CDKN2B could be demonstrated in one of these cases. Taken together, our results indicate that CDKN2C is rarely altered in meningiomas. However, the majority of anaplastic meningiomas either show homozygous deletions of CDKN2A, p14(ARF), and CDKN2B, mutations in CDKN2A and p14(ARF), or lack of expression of one or more of these genes. Thus, inactivation of the G(1)/S-phase cell-cycle checkpoint is an important aberration in anaplastic meningiomas.

Adult↗
PubMed1997

A DNA double-strand break defective fibroblast cell line (180BR) derived from a radiosensitive patient represents a new mutant phenotype.

The 180BR cell line was derived from an acute lymphoblastic leukemia patient who overresponded to radiation therapy and died following radiation morbidity. 180BR cells are hypersensitive to the lethal effects of ionizing radiation and are defective in the repair of DNA double-strand breaks (DSBs). The levels and activity of the proteins of the DNA-dependent protein kinase complex are normal in 180BR cells. To facilitate a measurement of V(D)J recombination, we have characterized 180BRM, a SV40-transformed line derived from 180BR. 180BRM retains the radiosensitivity and defect in DSB repair characteristic of 180BR. The activities associated with DNA-dependent protein kinase are also normal in 180BRM cells. The ability to carry out V(D)J recombination is comparable in 180BRM and a reference control transformed human cell line, MRC5V1. These results show that 180BR and 180BRM differ from the rodent mutants belonging to ionizing radiation complementation groups 4, 5, 6, and 7 and, therefore, represent a new mutant phenotype, in which a defect in DNA DSB rejoining is not associated with defective V(D)J recombination. Furthermore, we have shown that 180BR can arrest at the G1-S and G2-M cell cycle checkpoints after irradiation. These results confirm that 180BR can be distinguished from ataxia telangiectasia.

Cell Cycle↗
PubMed2004

Enhanced tumor cell radiosensitivity and abrogation of G2 and S phase arrest by the Hsp90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin.

PURPOSE: Because of the potential for affecting multiple signaling pathways, inhibition of Hsp90 may provide a strategy for enhancing tumor cell radiosensitivity. Therefore, we have investigated the effects of the orally bioavailable Hsp90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) on the radiosensitivity of human tumor cells in vitro and grown as tumor xenografts. EXPERIMENTAL DESIGN: The effect of 17-DMAG on the levels of three proteins (Raf-1, ErbB2, and Akt) previously implicated in the regulation of radiosensitivity was determined in three human solid tumor cell lines. A clonogenic assay was then used to evaluate cell survival after exposure to 17-DMAG followed by irradiation. For mechanistic insight, the G(2)- and S-phase checkpoints were evaluated in 17-DMAG-treated cells. Finally, the effect of in vivo administration of 17-DMAG in combination with radiation on the growth rate of xenograft tumors was determined. RESULTS: 17-DMAG exposure reduced the levels of the three radiosensitivity-associated proteins in a cell line-specific manner with ErbB2 being the most susceptible. Corresponding concentrations of 17-DMAG enhanced the radiosensitivity of each of the tumor cell lines. This sensitization seemed to be the result of a 17-DMAG-mediated abrogation of the G(2)- and S-phase cell cycle checkpoints. The oral administration of 17-DMAG to mice bearing tumor xenografts followed by irradiation resulted in a greater than additive increase in tumor growth delay. CONCLUSIONS: These data indicate that 17-DMAG enhances the in vitro and in vivo radiosensitivity of human tumor cells. The mechanism responsible seems to involve the abrogation of radiation-induced G(2)- and S-phase arrest.

Administration, Oral↗
PubMed2006

Mechanisms of action of the carcinogenic heterocyclic amine PhIP.

Formed during the cooking of meat, the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4-5-b]pyridine (PhIP) is mutagenic and carcinogenic. Although the metabolism and mutational effects of PhIP are well defined, the early cellular and genomic events by which it can induce neoplastic transformation are not yet fully characterised. These early cellular responses to genotoxic doses of PhIP were examined in a human mammary epithelial cell, MCF10A. Using Western blotting, PhIP was shown to induce expression of the DNA damage response proteins p53 and p21(WAF1/CIP1), and to inhibit cell growth while activating G1 cell cycle checkpoint, a consequence of PhIP-induced DNA damage. Using low doses of PhIP (previously shown to activate oestrogenic signalling), PhIP increased proliferation in the oestrogen receptor (ER)-negative MCF10A cell line and to activate the mitogen-activated protein kinase (MAPK) pathway. Inhibition of this pathway significantly reduced the PhIP-induced cell growth of MCF10A cells. The work presented here suggests that, further to its genotoxic properties, at levels close to human exposure PhIP stimulates cellular signalling pathways that are linked to the promotion and progression of neoplastic disease. It is possible that a combination of these DNA damaging and growth promoting properties provide a mechanism for the tumourigenicity of PhIP, and may be key determinants for the tissue specificity of PhIP-induced carcinogenesis.

Carcinogens↗
PubMed2003

Lymphocyte DNA damage precedes DNA repair or cell death after orthopaedic surgery under general anaesthesia.

Anaesthetics have gained a lot of attention for their potential mutagenic/carcinogenic effects. In the present study we have investigated the genotoxicity of the inhalation anaesthetic sevoflurane on DNA of lymphocytes isolated from 20 patients undergoing orthopaedic surgery. The genotoxicity of the anaesthetic was studied by assaying DNA damage, apoptosis, DNA repair enzyme activity and GSH content in peripheral lymphocytes before, 15 min after anaesthesia and 24 h after surgery. Lymphocytes isolated 15 min after anaesthesia showed an increase in oxidized purine and pyrimidine bases without DNA strand break formation. DNA strand breaks occurred on the first post-operative day, associated with an enhancement of DNA repair activity and a decrease in GSH. Formation of strand breaks could be the consequence of DNA repair activity. In fact, at 24 h after surgery most of the oxidized DNA bases were repaired. When DNA damage was not repaired, activation of the cell cycle checkpoint protein p53 could lead to apoptosis. An altered redox status may contribute to lymphocytopenia due to an apoptotic event as a consequence of surgical trauma. The presence of apoptotic cells at 1 day after surgery could support the hypothesis that highly damaged peripheral lymphocytes are committed to undergo programmed cell death if the damage is not repaired. In conclusion, the actual risk from anaesthesia is presumably extremely small. However, these findings contribute to our understanding of the regulation of DNA damage/repair and cell death.

Adult↗
PubMed2005

Assessment of ATM phosphorylation on Ser-1981 induced by DNA topoisomerase I and II inhibitors in relation to Ser-139-histone H2AX phosphorylation, cell cycle phase, and apoptosis.

BACKGROUND: The ATM kinase regulates cell-cycle checkpoints by phosphorylating multiple proteins, including histone H2AX, CHK1, and CHK2 kinases and p53. ATM is activated through auto- or trans- phosphorylation of Ser-1981 in response to DNA damage, particularly induction of DNA double-strand breaks (DSBs). The aim of the present study was to reveal a possible correlation between activation of ATM vis-à-vis H2AX phosphorylation, cell cycle phase, and apoptosis in cells treated with DNA topoisomerase (topo) I (topotecan; Tpt) or topo2 (mitoxantrone; Mtx) inhibitor. MATERIALS AND METHODS: Cultures of HL-60 cells were treated with Tpt or Mtx for various time intervals. ATM or H2AX phosphorylation was detected immunocytochemically, using Ab specific for ATM phosphorylated on Ser-1981 (ATM-S1981(P)) or for H2AX (gammaH2AX) phosphorylated on Ser-139, respectively, concurrent with the analysis of cellular DNA content. Cellular fluorescence was measured by flow cytometry. RESULTS: Untreated cells showed a modest but variable level of labeling with ATM-S1981(P) Ab across the cell cycle, with exception of mitotic cells that were strongly labeled. Exposure of cells to 150 nM Tpt induced ATM phosphorylation concurrent with phosphorylation of H2AX within 10 min; phosphorylation of both proteins was essentially limited to S-phase and was suppressed by caffeine and wortmannin, inhibitors of PI-3-like kinases. Exposure of cells to Mtx also led to ATM and H2AX phosphorylation, which, compared to Tpt, occurred later and was not cell-cycle-phase specific. Apoptosis of HL-60 cells in Tpt or Mtx treated cultures was detected after 2 or 4 h, respectively, and was limited to S-phase cells. CONCLUSIONS: The data are consistent with the role of ATM as a mediator of H2AX phosphorylation in response to DNA damage by topo1 (Tpt) or topo 2 (Mtx) inhibitor. The observed cell-cycle-phase related differences in response to Tpt vs Mtx suggest that while the collisions of DNA replication forks with the "cleavable complexes" stabilized by topo1 inhibitor are the primary cause of DSBs induced by Tpt, the collisions of RNA polymerase molecules with the complexes stabilized by the topo2 inhibitor play a major role for induction of DSBs by Mtx. The present report is the first that (i) describes cytometric analysis of ATM activation and (ii) demonstrates activation of the enzyme (kinase) and its consequence (substrate phoshorylation), both in relation to cell-cycle phase and onset of apoptosis within the same cells.

Apoptosis↗
PubMed2003

The biochemical and biological functions of human papillomavirus type 16 E5 protein.

Human papillomavirus type 16 (HPV-16) E5 protein, along with the more publicized E6 and E7 proteins of this virus, has been found to be oncogenic. E5 is a highly hydrophobic membrane-bound protein of 83 amino acids associated with the Golgi apparatus, endoplasmic reticulum, and nuclear membrane in infected cells. E5 can activate epidermal growth factor receptor (EGFR) through binding to the 16 kD subunit of protein pump ATPase leading to a reduced downregulation of EGFR receptors. The activation of EGFR can initiate biochemical cascades that lead to overexpression of a variety of protooncogenes and stimulate rapid cell growth. Moreover, E5 can inhibit the expression of tumor suppressor gene p21((WafI/SdiI/CipI)) and impair the control of cell cycle checkpoint. E5 protein has been identified as a potential tumor vaccine target antigen.

Animals↗
PubMed2004

Mutation of the mouse Rad17 gene leads to embryonic lethality and reveals a role in DNA damage-dependent recombination.

Genetic defects in DNA repair mechanisms and cell cycle checkpoint (CCC) genes result in increased genomic instability and cancer predisposition. Discovery of mammalian homologs of yeast CCC genes suggests conservation of checkpoint mechanisms between yeast and mammals. However, the role of many CCC genes in higher eukaryotes remains elusive. Here, we report that targeted deletion of an N-terminal part of mRad17, the mouse homolog of the Schizosaccharomyces pombe Rad17 checkpoint clamp-loader component, resulted in embryonic lethality during early/mid-gestation. In contrast to mouse embryos, embryonic stem (ES) cells, isolated from mRad17(5'Delta/5'Delta) embryos, produced truncated mRad17 and were viable. These cells displayed hypersensitivity to various DNA-damaging agents. Surprisingly, mRad17(5'Delta/5'Delta) ES cells were able to arrest cell cycle progression upon induction of DNA damage. However, they displayed impaired homologous recombination as evidenced by a strongly reduced gene targeting efficiency. In addition to a possible role in DNA damage-induced CCC, based on sequence homology, our results indicate that mRad17 has a function in DNA damage-dependent recombination that may be responsible for the sensitivity to DNA-damaging agents.

Amino Acid Sequence↗
PubMed1998

Radioresistant MTp53-expressing rat embryo cell transformants exhibit increased DNA-dsb rejoining during exposure to ionizing radiation.

Recent data suggest that aberrant function of the wild type p53 protein (WTp53) may alter cellular survival following DNA damage through cellular pathways involving apoptosis and cell-cycle checkpoints, but little is known concerning it's possible role in DNA repair. In the present study, the ionizing radiation sensitivity was determined for a series of rat embryo fibroblast (REF) cell lines transfected with an activated form of the H-ras oncogene alone, or in combination with a variety of missense-mutant p53 (MTp53) alleles. Transformed REF clones which expressed exogenous MTp53 and p21ras proteins (CLASS II clones) were generally radioresistant in culture as determined by higher values for the surviving fraction after 2 Gy (SF2 value) and the radiation dose required to reduce survival to a fraction of 0.1 (D10 value), compared either to transformed REF clones expressing p21ras protein alone (CLASS I clones), or to non-transfected REF control cell lines expressing baseline endogenous levels of p21ras and WTp53 protein. The increased radioresistance observed in the CLASS II clones (following both HDR- and LDR-irradiation), was significantly correlated with increased expression of MTp53 protein, and a decreased radiation-induced G1 arrest response. The variability observed in clonogenic radiosensitivity among REF clones was not explained by differential radiation-induced apoptosis. Using the Comet assay performed after continuous low dose-rate (LDR)-irradiation, MTp53-expressing REF clones were also found to be more proficient at the rejoining of DNA double-strand breaks (DNA-dsb), compared to WTp53-expressing REF clones. These results suggest that an enhanced DNA and cellular repair capacity may, in part, explain the increased radiation survival observed in some MTp53-expressing transformed fibroblasts and tumours.

Animals↗
PubMed1998

Selective induction of apoptosis of NB4 cells from G2+M phase by sodium arsenite at lower doses.

Apoptosis of NB4 cells induced by sodium arsenite and arsenate was studied using flow cytometry and DNA gel electrophoresis in order to investigate their effects on cell cycle and determine the relationship between apoptosis and cell cycle. In this study, we found that: 1) at low doses, sodium arsenite selectively induced apoptosis of NB4 cells in G2+M phase of cell cycle after its being arrested in G2 phase. With increment of the cells blocked in G2 phase, dUTP-specifically labeling cells in G2+M phase increased without concomitant increment of dUTP-labeling cells in other two phases of cell cycle; 2) at high doses, extensive apoptosis was induced in NB4 cells from all phases of cell cycle without cell cycle preference and cell cycle blockade; 3) sodium arsenite-induced apoptosis of NB4 cells occurred in the presence of bcl-2 expression as the unapoptotic cells; 4) sodium arsenite with As3+ induced apoptosis of NB4 cells more strongly than sodium arsenate with As5+ did although both of them affected NB4 cells in the same pattern. These results not only suggested that both arsenite and arsenate induced apoptosis of NB4 cells through 2 different mechanisms--at low doses, arsenical might directly induce apoptosis through regulation of cell cycle checkpoint, while at high doses they might directly induce it, but also indicated that bcl-2 might not play an important role in arsenite or arsenate-induced apoptosis of NB4 cells, whereas chemical valence of As in a compound might be related to efficiency in arsenical induction of apoptosis.

Apoptosis↗