Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:5.99.1.3 (topoisomerase)
9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cyclins, the regulatory subunits of their respective cyclin-dependent kinases, are the key components of the cell-cycle progression machinery. Some cyclins are expressed discontinuously during the cell cycle, their synthesis and degradation being strictly scheduled. The presence of these cyclins in the cell, therefore, provides landmarks of the cell cycle, in addition to DNA replication and mitosis. Cyclin A is expressed in late S and G2 phase and degraded during mitosis just prior to metaphase. Degradation of another "mitotic" cyclin, cyclin B1, occurs later, at the transition from metaphase to anaphase. Based on the difference in time of degradation of cyclin A versus cyclin B1 it was possible, in the present study, to discriminate between G2 and mitotic (postprophase) MOLT-4 leukemic cells, by multiparameter (cellular DNA content versus cyclin expression) flow cytometry. The cells arrested in metaphase by Vinblastine were cyclin A negative and had an elevated level of cyclin B1. The cells arrested in G2 by the DNA topoisomerase II inhibitor m-AMSA had a very high level of cyclin B1 expression and unchanged expression of cyclin A. During stathmokinesis induced by Vinblastine the percentage of mitotic cells estimated by analysis of cellular DNA content and cyclin A expression was identical to that estimated by the alternative method based on in situ DNA denaturation followed by staining with acridine orange. Thus, differences in expression of cyclins A and B1 make it possible to discriminate cells that have the same DNA content but reside in different phases of the cycle, such as DNA diploid cells in G2 versus tetraploid G1 cells or mitotic versus G2 cells.
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PMID:Discrimination of G2 and mitotic cells by flow cytometry based on different expression of cyclins A and B1. 766 39

The methods of cell cycle analysis that rely on DNA content measurements cannot discriminate between cells at different phases of the cycle if these cells have similar DNA content. This limitation can be circumvented by measurement of another cell cycle phase-specific cell constituent in addition to DNA content, followed by bivariate analysis of the correlated data. The aim of the present study was to explore the utility of a monoclonal antibody against the G2- and M phase-specific regulatory protein cyclin B for discrimination of cell populations with overlapping DNA content. This analysis, which was based on correlated DNA/cyclin B content measurements by flow cytometry, was applied to human lymphocytic leukemic MOLT-4 cells. The onset of cyclin B synthesis was observed in the last one third of S phase with its maximum accumulation in G2 and M phases; cells in G1 and early- and mid-S phases were negative. Cells arrested in metaphase by vinblastine expressed high levels of this protein, although not as high as in cells arrested in G2 by the DNA topoisomerase II inhibitor m-AMSA. Disruption of cytokinesis by the protein kinase inhibitor staurosporine led to DNA rereplication, cell progression through the chromatin cycle at higher DNA ploidy, and induction of polyploidy. It was possible, utilizing the cyclin B antibody, to discriminate between G2 + M cells with a 2C level of DNA and G1 cells with 4C DNA, as well as to distinguish doublets of G1 cells with a 2C DNA level. Thus, the rate of cell entrance to G1 at the 4C DNA level and the rates of progression through the cycle at both the 2C and 4C DNA levels could be simultaneously estimated. The data indicate that, in the presence of 0.1 microM staurosporine, cytokinesis of all MOLT-4 cells is impaired and the cells enter to and progress through the chromosome cycle at 4C DNA at the same rates as at 2C DNA. This approach can be helpful in the analysis of DNA ploidy and the cell cycle of human tumors when there is an overlap in DNA content values between normal stromal or infiltrating cells and aneuploid tumor cell population and may be the method of choice to investigate the activity of antitumor drugs which impair cytokinesis but do not interfere with progression of cells through the chromatin cycle.
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PMID:Simultaneous analysis of cell cycle kinetics at two different DNA ploidy levels based on DNA content and cyclin B measurements. 822 43

A normal consequence of mitosis in eukaryotes is the repression of transcription. Using Xenopus egg extracts shifted to a mitotic state by the addition of purified cyclin, we have for the first time been able to reproduce a mitotic repression of transcription in vitro. Active RNA polymerase III transcription is observed in interphase extracts, but strongly repressed in extracts converted to mitosis. With the topoisomerase II inhibitor VM-26, we demonstrate that this mitotic repression of RNA polymerase III transcription does not require normal chromatin condensation. Similarly; in vitro mitotic repression of transcription does not require the presence of nucleosome structure or involve a general repressive chromatin-binding protein, as inhibition of chromatin formation with saturating amounts of non-specific DNA has no effect on repression. Instead, the mitotic repression of transcription appears to be due to phosphorylation of a component of the transcription machinery by a mitotic protein kinase, either cdc2 kinase and/or a kinase activated by it. Mitotic repression of RNA polymerase III transcription is observed both in complete mitotic cytosol and when a kinase-enriched mitotic fraction is added to a highly simplified 5S RNA transcription reaction. We present evidence that, upon depletion of cdc2 kinase, a secondary protein kinase activity remains and can mediate this in vitro mitotic repression of transcription.
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PMID:Mitotic repression of transcription in vitro. 838 Nov 19

Previous studies have demonstrated that G1/S cell cycle blockers and inhibitors of cyclin-dependent kinases (CDKs) prevent the death of nerve growth factor (NGF)-deprived PC12 cells and sympathetic neurons, suggesting that proteins normally involved in the cell cycle may also serve to regulate neuronal apoptosis. Past findings additionally demonstrate that DNA-damaging agents, such as the DNA topoisomerase (topo-I) inhibitor camptothecin, also induce neuronal apoptosis. In the present study, we show that camptothecin-induced apoptosis of PC12 cells, sympathetic neurons, and cerebral cortical neurons is suppressed by the G1/S blockers deferoxamine and mimosine, as well as by the CDK-inhibitors flavopiridol and olomoucine. In each case, the IC50 values were similar to those reported for inhibition of death induced by NGF-deprivation. In contrast, other agents that arrest DNA synthesis, such as aphidicolin and N-acetylcysteine, failed to block death. This suggests that the inhibition of DNA synthesis per se is insufficient to provide protection from camptothecin. We find additionally that the cysteine aspartase family protease inhibitor zVAD-fmk inhibits apoptosis evoked by NGF-deprivation but not camptothecin treatment. Thus, despite their shared sensitivity to G1/S blockers and CDK inhibitors, the apoptotic pathways triggered by these two causes of death diverge at the level of the cysteine aspartase. In summary, neuronal apoptosis induced by the DNA-damaging agent camptothecin appears to involve signaling pathways that normally control the cell cycle. The consequent death signals of such deregulation, however, are different from those that result from trophic factor deprivation.
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PMID:G1/S cell cycle blockers and inhibitors of cyclin-dependent kinases suppress camptothecin-induced neuronal apoptosis. 900 70

Mutations in the retinoblastoma (pRb) tumor suppressor pathway including its cyclin-cdk regulatory kinases, or cdk inhibitors, are a hallmark of most cancers and allow unrestrained E2F-1 transcription factor activity, which leads to unregulated G1-to-S-phase cell cycle progression. Moderate levels of E2F-1 overexpression are tolerated in interleukin 3 (IL-3)-dependent 32D.3 myeloid progenitor cells, yet this induces apoptosis when these cells are deprived of IL-3. However, when E2F activity is augmented by coexpression of its heterodimeric partner, DP-1, the effects of survival factors are abrogated. To determine whether enforced E2F-1 expression selectively sensitizes cells to cytotoxic agents, we examined the effects of chemotherapeutic agents and radiation used in cancer therapy. E2F-1 overexpression in the myeloid cells preferentially sensitized cells to apoptosis when they were treated with the topoisomerase II inhibitor etoposide. Although E2F-1 alone induces moderate levels of p53 and treatment with drugs markedly increased p53, the deleterious effects of etoposide in E2F-1-overexpressing cells were independent of p53 accumulation. Coexpression of Bcl-2 and E2F-1 in 32D.3 cells protected them from etoposide-mediated apoptosis. However, Bcl-2 also prevented apoptosis of these cells upon exposure to 5-fluorouracil and doxorubicin, which were also cytotoxic for control cells. Pretreating E2F-1-expressing cells with ICRF-193, a second topoisomerase II inhibitor that does not damage DNA, protected the cells from etoposide-induced apoptosis. However, ICRF-193 cooperated with DNA-damaging agents to induce apoptosis. Therefore, topoisomerase II inhibition and DNA damage can cooperate to selectively induce p53-independent apoptosis in cells that have unregulated E2F-1 activity resulting from mutations in the pRb pathway.
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PMID:E2F-1 cooperates with topoisomerase II inhibition and DNA damage to selectively augment p53-independent apoptosis. 903 31

We have shown previously that ICRF-193, a catalytic inhibitor of DNA topoisomerase II (topo II), delays cell cycle progression in HeLa S3 cells. We report here that the delay of the transition in M phase is observed when HeLa S3 cells were treated with ICRF-193 during metaphase, but not thereafter. ICRF-193 also delayed the degradation of cyclin B in the transition from M to G1 phase, while in Chinese hamster ovary (CHO) cells the drug did not delay the progression in M phase. Since HeLa S3 and CHO cells are 'stringent' and 'relaxed' in mitotic control, respectively, it is suggested that under topo II inhibition, the M phase checkpoint operates through an inability for chromosome segregation.
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PMID:ICRF-193, a catalytic inhibitor of DNA topoisomerase II, delays the cell cycle progression from metaphase, but not from anaphase to the G1 phase in mammalian cells. 913 99

The MPM-2 antibody labels mitosis-specific and cell cycle-regulated phosphoproteins. The major phosphoproteins of mitotic chromosomes recognized by the MPM-2 antibody are DNA topoisomerase II (topoII) alpha and beta. In immunofluorescence studies of PtK1 cytoskeletons, prepared by detergent lysis in the presence of potent phosphatase inhibitors, the MPM-2 antibody labels phosphoproteins found at kinetochores, chromosome arms, midbody and spindle poles of mitotic cells. In cells extracted without phosphatase inhibitors, labeling of the MPM-2 antibodies at kinetochores is greatly diminished. However, in cytoskeletons this epitope can be regenerated through the action of kinases stably bound at the kinetochore. Various kinase inhibitors were tested in order to characterize the endogenous kinase responsible for these phosphorylations. We found that the MPM-2 epitope will not rephosphorylate in the presence of the broad specificity kinase inhibitors K-252a, staurosporine and 2-aminopurine. Several other inhibitors had no effect on the rephosphorylation indicating that the endogenous MPM-2 kinase at kinetochores is not p34cdc2, casein kinase II, MAP kinase, protein kinase A or protein kinase C. The addition of N-ethylmaleimide inactivated the endogenous kinetochore kinase; this allowed testing of several purified kinases in the kinetochore rephosphorylation assay. Active p34cdc2-cyclin B, casein kinase II and MAP kinase could not generate the MPM-2 phosphoepitope. However, bacterially expressed NIMA from Aspergillus and ultracentrifuged mitotic HeLa cell extract were able to catalyze the rephosphorylation of the MPM-2 epitope at kinetochores. Furthermore, fractionation of mitotic HeLa cell extract showed that kinases that create the MPM-2 epitope at kinetochores and chromosome arms are distinct. Our results suggest that multiple kinases (either soluble or kinetochore-bound), including a homolog of mammalian NIMA, can create the MPM-2 phosphoepitope. The kinetochore-bound kinase that catalyzes the formation of the MPM-2 phosphoepitope may play an important role in key events such as mitotic kinetochore assembly and sister chromatid separation at anaphase.
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PMID:MPM-2 antibody-reactive phosphorylations can be created in detergent-extracted cells by kinetochore-bound and soluble kinases. 937 53

Three DNA damage-responsive cell cycle checkpoints can be shown to operate in diploid human fibroblasts. One checkpoint arrests growth in G1, another inhibits replicon initiation in S phase cells, and the third delays progression from G2 into mitosis. Progression from G2 into M is controlled in part by a cyclin-dependent kinase (cyclin B/Cdk1) that is regulated by tyrosine phosphorylation. Phosphorylation of Tyr15 on Cdk1 is inhibitory for kinase activity. Activation of cyclin B/Cdk1 at the onset of mitosis is accomplished by a phosphatase, Cdc25C, that interacts with cyclin B/Cdk1 in an autocatalytic feedback loop to remove the inhibitory phosphate at Tyr15 and activate kinase activity. DNA damage triggers G2 delay by inhibiting formation of the autocatalytic feedback loop so that dephosphorylation of Tyr15 does not occur. This suppression of activation of cyclin B/Cdk1 appears to account for the failure of damaged G2 cells to progress into mitosis. Once the damage to DNA is repaired, cells resume progression into mitosis as the cycle is re-engaged. The isoflavone genistein inhibits tyrosine kinases, including one that phosphorylates Cdk1 on Tyr15. This kinase, p56/p53lyn is rapidly induced by treatments that trigger cell cycle checkpoints (ionizing radiation, cytosine arabinoside), suggesting that this kinase may actively delay the onset of mitosis by phosphorylating Tyr15 on Cdk1. Genistein also inhibits type II DNA topoisomerase to produce a form of DNA damage that triggers all of the DNA damage-responsive cell cycle checkpoints. A brief 10 min incubation with the topoisomerase poison amsacrine was sufficient to trigger the S phase checkpoint response and inhibit replicon initiation. Inhibition of replicon initiation by 1 microM amsacrine was maximal 20-30 min after drug treatment and by 120 min, the checkpoint response had decayed to allow near control rates of replicon initiation. Topoisomerase II poisons also are powerful clastogens inducing lethal and carcinogenic chromosomal aberrations. Type II topoisomerase can break DNA in a region of chromosome 11q23 that contains the ataxia telangiectasia gene (ATM). The ATM gene controls all of the DNA damage-responsive cell cycle checkpoints. Chromosomal aberrations in 11q23 are frequently seen in acute myeloid leukemia that develops as a consequence of etoposide chemotherapy. Thus, topoisomerase poisons such as genistein may trigger chromatid breakage to inactivate AT gene function, disable cell cycle control, and induce genetic instability.
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PMID:Human topoisomerase II function, tyrosine phosphorylation and cell cycle checkpoints. 949 43

Induced cell cycle delays were among the first described cellular responses to ionizing radiation (IR). To understand the sensitivity and the molecular events involved in the response to low doses of IR and to examine the role of p53 and its downstream effector p21Waf1, we measured changes in expression of genes postulated to be involved in the cellular response to IR. Expression levels were examined in normal human diploid fibroblasts irradiated and maintained in quiescent density-inhibited growth up to 24-48 h after exposure to X-ray doses as low as 0.1-0.3 Gy, which have negligible effects on cell survival. Among 31 genes analyzed, we observed down-regulation in response to IR of the mRNA levels of CDC2, cyclin A, cyclin B, thymidine kinase, topoisomerase IIalpha, and RAD51. A similar reduction in the expression levels of these genes occurred when irradiated cells were released from confluence and allowed to proliferate. This was not observed in cells in which p53 function was defective and up-regulation of p21Waf1 levels either did not occur (E6 transfected normal human fibroblasts and Li-Fraumeni fibroblasts) or was delayed (ataxia telangiectasia fibroblasts) after irradiation. Down-regulation was also absent in p21Waf1-null mouse embryo fibroblasts (MEFs) but occurred at a lower level in p53-null MEFs, due to slight increases in p21Waf1 levels by a p53-independent pathway. These findings indicate that the down-regulation of these cell cycle regulated genes in irradiated cells is p53-dependent and involves its effector p21Waf1. Although no down-regulation in the expression of genes involved in G2-M was observed in p53 or in p21Waf1-null MEFs, these cells showed a G2-M delay after irradiation, indicating that the expression levels of these genes does not regulate the G2-M delay.
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PMID:Regulation by ionizing radiation of CDC2, cyclin A, cyclin B, thymidine kinase, topoisomerase IIalpha, and RAD51 expression in normal human diploid fibroblasts is dependent on p53/p21Waf1. 983 Dec 41

The eucaryotic cell cycle is regulated by the periodic synthesis and destruction of cyclins that associate with and activate cyclin-dependent kinases. Cyclin-dependent kinase inhibitors, such as p21 and p16, also play important roles in cell cycle control by coordinating internal and external signals and impeding proliferation at several key checkpoints. Understanding how these proteins interact to regulate the cell cycle has become increasingly important to researchers and clinicians with the discovery that many of the genes that encode cell cycle regulatory activities are targets for alterations that underlie the development of cancer. Several therapeutic agents, such as DNA-damaging drugs, microtubule inhibitors, antimetabolites, and topoisomerase inhibitors, take advantage of this disruption in normal cell cycle regulation to target checkpoint controls and ultimately induce growth arrest or apoptosis of neoplastic cells. Other therapeutic drugs being developed, such as UCN-01, specifically inhibit cell cycle regulatory proteins.
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PMID:Cyclins and cell cycle checkpoints. 1033 Oct 86


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