SEARCH · Search PubMed
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.
BRCA1 is one of the most important breast cancer susceptibility genes. It plays key roles in DNA damage repair, cell cycle checkpoint regulation, gene transcription chromatin stability, and cell proliferation. In this paper, advance in basic researches on BRCA1 is reviewed, and the role of BRCA1 in cancer development and progression is discussed, that may facilitate potential clinical application of BRCA1.
Fimbrins (also known as plastins) are actin-binding proteins of the cortical cytoskeleton present in all cells and conserved from yeast to mammals. We previously reported that the up-regulation of T-fimbrin, a fimbrin isoform, in association with G2 arrest following DNA damage and that a lack of T-fimbrin expression shortens the radiation-induced G2 arrest in Chinese hamster ovarian cells. In this study, we further investigated the role of T-fimbrin in DNA-damage response using a panel of human liver cancer cell lines and small interfering RNA technology. T-fimbrin was differentially expressed in human liver cancer cell lines. Colony formation assays revealed that cell lines lacking T-fimbrin expression were highly sensitive to DNA damage compared to cell lines that express T-fimbrin. Using siRNA designed to target T-fimbrin, we demonstrated that silencing endogenous T-fimbrin causes a marked increase in the cellular sensitivity to VP-16 and UV irradiation. Moreover, T-fimbrin deletion abrogated UV-mediated cell cycle checkpoint, and consequently led to increased apoptotic cell death in resistant cells. These findings suggest that the status of T-fimbrin expression may be a useful molecular marker for predicting the responsiveness of cancer cells to treatment with chemotherapeutic drugs.
E6 and E7 oncoproteins from high-risk human papillomavirus (HPV) can transform cells in tissue culture and induce tumors in vivo by abrogating the cell-cycle checkpoint. To investigate the impact of HPV16 E6 and E7 on the cell-cycle regulatory machinery in oral epithelial cells, normal human oral epithelial cells were transfected with HPV16 E6 and E7 open reading frames, and alterations in cell-cycle regulatory proteins in cells expressing HPV16 E6 and E7 were analyzed. E6 and E7 expression results in immortalization of oral epithelial cells. E7 inactivates retinoblastoma protein (Rb) by forming complexes with hypophosphorylated Rb in immortalized oral epithelial cells. P53 and P21 protein levels were increased in immortalized cells compared to normal primary oral epithelial cells. Cyclin D1-cell-cycle-dependent kinase 4 binary association is disrupted in immortalized oral epithelial cells. These results indicate that E7 plays an important role in abrogation of cell-cycle regulation in oral epithelial cells, with E6 having a smaller impact. This suggests that the pathogenesis of HPV in oral epithelial cells differs from that in cervical epithelial cells.
The RASSF1A locus at 3p21.3 is epigenetically inactivated at high frequency in a variety of solid tumors. Expression of RASSF1A is sufficient to revert the tumorigenicity of human cancer cell lines. We show here that RASSF1A can induce cell cycle arrest by engaging the Rb family cell cycle checkpoint. RASSF1A inhibits accumulation of native cyclin D1, and the RASSF1A-induced cell cycle arrest can be relieved by ectopic expression of cyclin D1 or of other downstream activators of the G(1)/S-phase transition (cyclin A and E7). Regulation of cyclin D1 is responsive to native RASSF1A activity, because RNA interference-mediated downregulation of endogenous RASSF1A expression in human epithelial cells results in abnormal accumulation of cyclin D1 protein. Inhibition of cyclin D1 by RASSF1A occurs posttranscriptionally and is likely at the level of translational control. Rare alleles of RASSF1A, isolated from tumor cell lines, encode proteins that fail to block cyclin D1 accumulation and cell cycle progression. These results strongly suggest that RASSF1A is an important human tumor suppressor protein acting at the level of G(1)/S-phase cell cycle progression.
Apoptosis is a common mode of cell death after exposure of tumor cells to radiation and/or chemotherapy. The factors that determine the rate of induction of apoptosis are generally related to the functioning of cell cycle checkpoints. In the present study, we investigated the involvement of several genes in cell cycle redistribution and induction of apoptosis in U937 cells after low and high doses of radiation. Activation of CDC2 was observed after both low and high doses of radiation in U937 cells that underwent apoptosis. Expression of CDK2, CDC2 and cyclin A was induced rapidly in the process of radiation-induced apoptosis. In addition, we investigated the use of a clinically relevant dose of radiation to promote As2O3-induced apoptosis in U937 cells. We found that combining radiation and As2O3 may be a new and more effective means of cancer treatment.
The retinoblastoma tumor suppressor (RB) is functionally inactivated in the majority of cancers and is a critical mediator of DNA damage checkpoints. Despite the critical importance of RB function in tumor suppression, the coordinate impact of RB loss on the response to environmental and therapeutic sources of damage has remained largely unexplored. Here, we utilized a conditional knockout system to ablate RB in adult fibroblasts. This model system enabled us to investigate the temporal role of RB loss on cell cycle checkpoints and DNA damage repair following ultraviolet (UV) and ionizing radiation (IR) damage. We demonstrate that RB loss compromises rapid cell cycle arrest following UV and IR exposure in adult primary cells. Detailed kinetic analysis of the checkpoint response revealed that disruption of the checkpoint is concomitant with RB target gene deregulation, and is not simply a manifestation of chronic RB loss. RB loss had a differential effect upon repair of the major DNA lesions induced by IR and UV. Whereas RB did not affect resolution of DNA double-strand breaks, RB-deficient cells exhibited accelerated repair of pyrimidine pyrimidone photoproducts (6-4 PP). In parallel, this repair was coupled with enhanced expression of specific factors and the behavior of proliferating cell nuclear antigen (PCNA) recruitment to replication and repair foci. Thus, RB loss and target gene deregulation hastens the repair of specific lesions distinct from its ubiquitous role in checkpoint abrogation.
Previously we identified an intra-S-phase cell cycle checkpoint elicited by the DNA-damaging carcinogen benzo[a]pyrene-dihydrodiol epoxide (BPDE). Here we have investigated the roles of lesion bypass DNA polymerases polkappa and poleta in the BPDE-induced S-phase checkpoint. BPDE treatment induced the re-localization of an ectopically expressed green fluorescent protein-polkappa fusion protein to nuclear foci containing sites of active DNA synthesis in human lung carcinoma H1299 cells. In contrast, a similarly expressed yellow fluorescent protein-poleta fusion protein showed a constitutive nuclear focal distribution at replication forks (in the same cells) that was unchanged in response to BPDE. BPDE-induced formation of green fluorescent protein-polkappa nuclear foci was temporally coincident with checkpoint-mediated S-phase arrest. Unlike "wild-type" cells, Polk(-/-) mouse embryonic fibroblasts (MEFs) failed to recover from BPDE-induced S-phase arrest, while exhibiting normal recovery from S-phase arrest induced by ionizing radiation and hydroxyurea. XPV fibroblasts lacking poleta showed a normal S-phase checkpoint response to BPDE (but failed to recover from the UV light-induced S-phase checkpoint), in sharp contrast to Polk(-/-) MEFs. The persistent S-phase arrest in BPDE-treated Polk(-/-) cells was associated with increased levels of histone gammaH2AX (a marker of DNA double-strand breaks (DSBs)) and activation of the DSB-responsive kinases ATM and Chk2. These data suggest that in the absence of polkappa, replication forks stall at sites of damage and collapse and generate DSBs. Therefore, we conclude that the trans-lesion synthesis enzyme polkappa is specifically required for normal recovery from the BPDE-induced S-phase checkpoint.
Ionising radiation remains one of the most effective tools in the therapy of cancer. It combines the properties of an extremely efficient DNA-damaging agent with a high degree of spatial specificity. Nonetheless, there remain considerable differences in the outcome for treatment of tumours of differing histological type treated by radiotherapy. Tumours arising from lymphoid or germ cells are significantly more radiocurable than most solid tumours of epithelial origin. The molecular mechanisms underlying such differences in cellular radiosensitivity are the subject of current research. When normal mammalian cells are subjected to stress signals--e.g. radiation, chemotherapeutic drugs, oxygen deficiency--a range of gene products involved in the sensing and signalling of such stresses are activated. The response of eukaryotic cells to ionising radiation includes activation of DNA repair pathways and cell cycle checkpoints, with subsequent full 'biological' recovery or cell death. Radiation induces two different modes of cell death termed mitotic or clonogenic cell death, and apoptosis. Until recent years, there was surprisingly little mechanistic understanding of the events following induction of physical damage by radiation and biological outcome for the cell. There have been recent major advances in our understanding of the signal transduction pathways involved in determining the fate of cells after irradiation.
Alterations in the function of cell cycle checkpoints are frequently detected in oral squamous cell carcinomas (OSCCs), and are often associated with the sensitivity of the cancer cells to chemotherapeutic drugs. Recently, a mitotic checkpoint gene, Chfr, was shown to be inactivated by promoter methylation and point mutations in various human tumors. Here we show that the absence of its product, CHFR, is associated with mitotic checkpoint dysfunction, and that cancer cells lacking CHFR are sensitive to microtubule inhibitors. Checkpoint impairment appears to be caused by a prophase defect in this case, as OSCC cells lacking CHFR showed phosphorylation of histone H3 on Ser10 and translocation of cyclin B1 to the nucleus. When CHFR-deficient OSCC cells were treated with a microtubule inhibitor (docetaxel or paclitaxel), significant numbers of apoptotic cells were observed. Moreover, disruption of CHFR using small interfering RNA (siRNA) impaired the mitotic checkpoint, thereby reducing the ability of OSCC cells to arrest at G2/M phase and making them more sensitive to microtubule inhibitors. Our results suggest that CHFR could be a useful molecular target for chemotherapy.
The marine sponge metabolite hymenialdisine is a potent inhibitor of a variety of kinases including MEK-1, GSK-3 beta, and CK1. In addition, hymenialdisine and debromohymenialdisine exhibit inhibition of the G(2) cell cycle checkpoint at micromolar concentrations. We report herein the potent inhibition of cell cycle kinase Chk2 by the indolic-hymenialdisine indoloazepine 1 (IC(50)=8 nM).
In mammalian cells, DNA replication takes place in functional subnuclear compartments, called replication factories, where replicative factors accumulate. The distribution pattern of replication factories is diagnostic of the different moments (early, mid, and late) of the S phase. This dynamic organization is affected by different agents that induce cell cycle checkpoint activation via DNA damage or stalling of replication forks. Here, we explore the cell response to etoposide, an anticancer drug belonging to the topoisomerase II poisons. Etoposide does not induce an immediate block of DNA synthesis and progressively affects the distribution of replication proteins in S phase. First, it triggers the formation of large nuclear foci that contain the single-strand DNA binding protein replication protein A (RPA), suggesting that lesions produced by the drug are processed into extended single-stranded regions. These RPA foci colocalize with DNA replicated at the beginning of the treatment. Etoposide also triggers the dispersal of replicative proteins, proliferating cell nuclear antigen and DNA ligase I, from replication factories. This event requires the activity of the ataxia telangiectasia Rad3-related (ATR) checkpoint kinase. By comparing the effect of the drug in cell lines defective in different DNA repair and checkpoint pathways, we show that, along with the downstream kinase Chk1, the Nbs1 protein, mutated in the Nijmegen breakage syndrome, is also relevant for this response and for ATR-dependent phosphorylation. Finally, our analysis evidences a critical role of Nbs1 in the etoposide-induced inhibition of DNA replication in early S phase.
Rad50, an structural maintenance of chromosomes (SMC) protein family member, participates in a variety of cellular processes, including DNA double-strand break repair, cell cycle checkpoint activation, telomere maintenance, and meiosis. Disruption of Rad50 in mice leads to lethality during early embryogenesis, indicating its essential function in normal proliferating cells. In addition to its ability to form a complex with the DNA double-strand break repair proteins Mre11 and NBS1, Rad50 may interact with other cellular proteins to execute its full range of biological activities. A novel 87-kDa protein named RINT-1 was identified using the C-terminal region of human Rad50 as the bait in a yeast two-hybrid screen. Human RINT-1 shares sequence homology with a novel protein identified in Drosophila melanogaster, including a coiled-coil domain within its N-terminal 150 amino acids, a conserved central domain of about 350 amino acids, and a C-terminal region of 90 amino acids exhibiting 35--38% identity. The conserved central and C-terminal regions of RINT-1 are required for its interaction with Rad50. While Rad50 and RINT-1 are both expressed throughout the cell cycle, RINT-1 specifically binds to Rad50 only during late S and G(2)/M phases, suggesting that RINT-1 may be involved in cell cycle regulation. Consistent with this possibility, MCF-7 cells expressing an N-terminally truncated RINT-1 protein displayed a defective radiation-induced G(2)/M checkpoint. These results suggest that RINT-1 may play a role in the regulation of cell cycle control after DNA damage.
This manuscript gives a selective survey of some aspects of the origin of chromosomal aberrations (CA). The following topics are discussed: heterogeneity regarding the induction of initial DNA lesions and their repair among chromosomes or regions of the same chromosome, ratio between symmetrical and asymmetrical exchanges, influence of DNA repeats, chromatin condensation and cell-cycle checkpoints on the formation of chromosome alterations.
The development of neoplasia frequently involves inactivation of the p53 and retinoblastoma (Rb) tumor suppressor pathways and disruption of cell cycle checkpoints that monitor the integrity of replication and cell division. The human papillomavirus type 16 (HPV-16) oncoproteins, E6 and E7, have been shown to bind p53 and Rb, respectively. To further delineate the mechanisms by which E6 and E7 affect cell cycle control, we examined various aspects of the cell cycle machinery. The low-risk HPV-6 E6 and E7 proteins did not cause any significant change in the levels of cell cycle proteins analyzed. HPV-16 E6 resulted in very low levels of p53 and p21 and globally elevated cyclin-dependent kinase (CDK) activity. In contrast, HPV-16 E7 had a profound effect on several aspects of the cell cycle machinery. A number of cyclins and CDKs were elevated, and despite the elevation of the levels of at least two CDK inhibitors, p21 and p16, CDK activity was globally increased. Most strikingly, cyclin E expression was deregulated both transcriptionally and posttranscriptionally and persisted at high levels in S and G2/M. Transit through G1 was shortened by the premature activation of cyclin E-associated kinase activity. Elevation of cyclin E levels required both the CR1 and CR2 domains of E7. These data suggest that cyclin E may be a critical target of HPV-16 E7 in the disruption of G1/S cell cycle progression and that the ability of E7 to regulate cyclin E involves activities in addition to the release of E2F.
Cloning of the gene responsible for ataxia telangiectasia is a landmark achievement in the study of this devastating disease. The specific properties of the product of the mutation causing ataxia telangiectasia and the clinical features of this disease place this protein at the centre of a cell cycle checkpoint critical for DNA damage and suggest a fundamental mechanistic relationship between cell death and the cell cycle.
Cells lacking the protein kinase ataxia telangiectasia mutated (ATM) have defective responses to DNA double-strand breaks (DSBs), including an inability to activate damage response proteins such as p53. However, we previously showed that cells lacking ATM robustly activate p53 in response to DNA strand breaks induced by the radiomimetic enediyne C-1027. To gain insight into the nature of C-1027-induced ATM-independent damage responses to DNA DSBs, we further examined the molecular mechanisms underlying the cellular response to this unique radiomimetic agent. Like ionizing radiation (IR) and other radiomimetics, breaks induced by C-1027 efficiently activate ATM by phosphorylation at Ser1981, yet unlike other radiomimetics and IR, DNA breaks induced by C-1027 result in normal phosphorylation of p53 and the cell cycle checkpoint kinases (Chk1 and Chk2) in the absence of ATM. In the presence of ATM, but under ATM and Rad3-related kinase (ATR) deficient conditions, C-1027 treatment resulted in a decrease in the level of Chk1 phosphorylation but not in the level of p53 and Chk2 phosphorylation. Only when cells were deficient in both ATM and ATR was there a reduction in the level of phosphorylation of each of these DNA damage response proteins. This reduction was also accompanied by an increased level of cell death in comparison to that of wild-type cells or cells lacking either ATM or ATR. Our findings demonstrate a unique cellular response to C-1027-induced DNA DSBs in that DNA damage response proteins are unaffected by the absence of ATM, as long as ATR is present.
This study examined how the DNA mismatch repair (MMR) system and p53 interact to maintain genomic integrity in the presence of the mutagenic stress induced by cisplatin (DDP). Sensitivity to the cytotoxic and mutagenic effect of DDP was assessed using a panel of sublines of the MMR-deficient HCT116 colon carcinoma cells in which MMR function had been restored by transfer of a copy of MLH1 on chromosome 3 or in which p53 function had been disabled by expression of HPV-16 E6. Loss of p53 function by expression of E6 in MMR-proficient HCT116+ ch3 cells conferred only 1.1-2.0-fold resistance to a panel of commonly used chemotherapeutic agents, whereas disruption of p53 in MMR-deficient HCT116 cells resulted in substantial levels of resistance to some agents (paclitaxel, 1.9-fold; gemcitabine, 2.7-fold; 6-thioguanine, 3.3-fold; and etoposide, 4.4-fold) but sensitization to other agents (topotecan, 2.5-fold; and DDP, 3.3-fold). Loss of MMR or p53 alone had only a minor effect on sensitivity to the mutagenic effect of DDP as measured by the appearance of variants resistant to 6-thioguanine, etoposide, topotecan, gemcitabine, and paclitaxel in the population 10 days later (1.0-2.4-fold), whereas loss of both p53 and MMR had a more profound effect (1.7-6.5-fold). Loss of both p53 and MMR increased the basal frequency insertion/deletion mutations detected by a shuttle vector-based assay to a greater extent than loss of either alone. In association with DDP-induced injury, loss of p53 or MMR alone resulted in 1.2- and 1.7-fold more mutations, whereas loss of both resulted in a 5.1-fold increase in mutant frequency. Examination of the impact of loss of p53 and/or MMR on the DDP-induced cell cycle checkpoint activation, p53 induction, ability of the cell to tolerate adducts in its DNA, and the rate of disappearance of platinum from genomic DNA indicated the effects of the loss of p53 and/or MMR on all of these parameters, suggesting a multifactorial etiology for the changes in sensitivity to the cytotoxic and mutagenic effects of DDP. These results indicate that p53 and MMR can cooperate to control sensitivity to the cytotoxic effect of DDP and to limit its mutagenic potential in the colon cancer cells.
The structure-activity relations of a series of synthetic phenoxazone drugs with aminoalkyl side chains of variable length and different terminal groups were investigated by examining their biological activity and DNA complexation affinity. Biological activity was determined from their ability to induce apoptosis and cell cycle perturbations (activation of cell cycle checkpoints) using the human malignant MOLT-3 cell line. The thermodynamic parameters of drug-DNA complexation were determined by differential scanning calorimetry. By comparing the activities of compounds with different terminal groups (amino, dimethylamino and diethylamino), we found that the existence of a terminal dimethylamino group in the alkylamino side chain is an important factor for anti-tumour activity. Minor modifications in the dimethylaminoalkyl side chain (e.g. elongation by one methylene group) led to notable changes in both the anti-tumour activity and DNA-binding properties of the drug, providing unambiguous evidence of a marked structure-activity relation.