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)

In accord with a set of prespecified principles of cell synchrony induction, a three-step procedure was developed to arrest cells reversibly in the G2 phase of the cell cycle. Cultures of Chinese hamster ovary (CHO) cells were presynchronized in early S phase by sequential treatment with isoleucine deficiency and hydroxyurea blockades; then they were switched to medium supplemented with either of two agents that inhibit DNA topoisomerase II activity by different mechanisms, Hoechst 33342 at 7.5 micrograms/ml for 12 hr or VM-26 at 0.5 micrograms/ml for 8 hr. Up to 95% of the cells accumulated in G2 phase under those conditions. After switch of Hoechst 33342-treated cells to drug-free medium, the cells divided as a highly synchronized cohort of cells within 3 hr. Up to 85% of the cells in a culture of human diploid dermal fibroblasts (HSF-55 cells) could be accumulated in G2 phase by placing cells presynchronized in early-S phase in medium containing Hoechst 33342 at 0.1 micrograms/ml for 10 hr. Reversal of G2 arrest in the HSF-55 cultures resulted in cells dividing synchronously over 3.5 hr. By varying the concentration of Hoechst 33342 and the duration of the treatment period, it was possible to alter the position within G2 phase at which cells accumulated. This synchronization protocol should greatly facilitate study of G2/M biochemical events in mammalian cells, in particular, those associated with cdc2 gene regulation of the onset of mitosis.
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PMID:Cell cycle synchronization: reversible induction of G2 synchrony in cultured rodent and human diploid fibroblasts. 169 9

Bufalin, an active principle of the traditional Chinese medicine chan'su, has been proved to be a potent differentiation inducer in human leukemia cells. To study the mechanism of the differentiation of human leukemia ML1 cells induced by bufalin, we measured the effect of 10 nM bufalin on cell growth, activities of various protein kinases, and cell cycle. The ML1 cell growth was inhibited significantly at 24 hr and the inhibiting effect persisted for 6 days. Activities of PKC, PKA, cdc2 kinase and CK II in ML1 cells were changed early by bufalin; PKA and PKC activities were inhibited, and cdc2 kinase and CK II activities were increased. These results suggest that bufalin induces differentiation of ML1 cells by modulating several protein kinase activities in a distinct way from RA and 1 alpha, 25(OH) 2D3. Cell cycle changes, measured by flow cytometry, became evident at 12 hr after treatment of ML1 cells with bufalin and the cells were preferentially arrested in the G2/M phase. This effect of bufalin on the cell cycle of leukemia cells is similar to that of topoisomerase inhibitors. Indeed, the activity of topoisomerase II but not topoisomerase I of ML1 cells was inhibited remarkably by the treatment of the cells with 10 nM bufalin.
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PMID:Cell cycle arrest and protein kinase modulating effect of bufalin on human leukemia ML1 cells. 807 71

ICRF-193, a novel noncleavable, complex-stabilizing type topoisomerase (topo) II inhibitor, has been shown to target topo II in mammalian cells (Ishida, R., T. Miki, T. Narita, R. Yui, S. Sato, K. R. Utsumi, K. Tanabe, and T. Andoh. 1991. Cancer Res. 51:4909-4916). With the aim of elucidating the roles of topo II in mammalian cells, we examined the effects of ICRF-193 on the transition through the S phase, when the genome is replicated, and through the M phase, when the replicated genome is condensed and segregated. Replication of the genome did not appear to be affected by the drug because the scheduled synthesis of DNA and activation of cdc2 kinase followed by increase in mitotic index occurred normally, while VP-16, a cleavable, complex-stabilizing type topo II inhibitor, inhibited all these processes. In the M phase, however, late stages of chromosome condensation and segregation were clearly blocked by ICRF-193. Inhibition at the stage of compaction of 300-nm diameter chromatin fibers to 600-nm diameter chromatids was demonstrated using the drug during premature chromosome condensation (PCC) induced in tsBN2 baby hamster kidney cells in early S and G2 phases. In spite of interference with M phase chromosome dynamics, other mitotic events such as activation of cdc2 kinase, spindle apparatus reorganization and disassembly and reassembly of nuclear envelopes occurred, and the cells traversed an unusual M phase termed "absence of chromosome segregation" (ACS)-M phase. Cells then continued through further cell cycle rounds, becoming polyploid and losing viability. This effect of ICRF-193 on the cell cycle was shown to parallel that of inactivation of topo II on the cell cycle of the ts top2 mutant yeast. The results strongly suggest that the essential roles of topo II are confined to the M phase, when the enzyme decatenates intertwined replicated chromosomes. In other phases of the cycle, including the S phase, topo II may thus play a complementary role with topo I in controlling the torsional strain accumulated in various genetic processes.
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PMID:Inhibition of DNA topoisomerase II by ICRF-193 induces polyploidization by uncoupling chromosome dynamics from other cell cycle events. 808 69

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

Amsacrine (4'-(9-acridinylamino)methanesulphon-m-anisidide) is an antileukemic drug which inhibits topoisomerase II (topo II) enzymes. We studied effects of two concentrations of amsacrine on the GM10115A cell line. This is a Chinese hamster line containing a single human chromosome 4, which can be readily visualised using fluorescence in situ hybridisation (FISH). The low amsacrine concentration slowed cell growth but did not cause significant arrest in the G2 phase of the cell cycle, while a higher concentration caused more long-term effects on the growth of the cells and caused G2 arrest. Either concentration led to chromosomal fragments which were lost with increasing time after treatment, and chromosomal translocations which appeared stable for at least 8 days after treatment. At the low concentration, the loss or gain of a single chromosome was a common event. The higher concentration led to polyploid cells, usually containing an uneven number of chromosome 4. We propose two mechanisms for aneuploidy by amsacrine (or related topo II poisons), either of which can be readily detected using FISH. At low drug concentrations, aneuploidy may occur directly through, for example, a failure to resolve catenated chromatids prior to anaphase. However, there has been considerable interest in the role of the cell division control (cdc) kinase and cyclins in regulating the mammalian cell cycle, and these may also be involved in the response of cells to high concentrations of topo II poisons. Cdc2 proteins and cyclins are involved in coordinating diverse activities during the M phase of the cell cycle, including catalysis of chromosome condensation and reorganisation of microtubules to allow chromosome separation during mitosis. Chromosome damage by topo II poisons will lead to G2 arrest, which allows the cells time to repair the damage. During this time, cyclin A and cdc2 levels will fall, preventing the cell from entering mitosis and effectively resetting the clock to G1 and the ploidy to tetraploid. Aneuploid cells will derive from polyploid cells through loss of extra chromosomes.
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PMID:Application of fluorescence in situ hybridisation to study the relationship between cytotoxicity, chromosome aberrations, and changes in chromosome number after treatment with the topoisomerase II inhibitor amsacrine. 866 70

Genistein is an isoflavone known to inhibit both tyrosine protein kinase and DNA topoisomerase II. We have investigated the mechanism of genistein-induced growth inhibition in MCF-7 and MDA-MB-231 breast carcinoma cell lines. DNA flow cytometric analysis indicated that genistein induced a G2/M arrest in both cell lines. Therefore, we examined the effect of genistein on cell cycle-related proteins. Western blot analysis using whole cell lysates from MCF-7 and MDA-MB-231 treated with genistein demonstrated that genistein treatment did not change the steady-state level of cdks, cyclin A, D-type cyclins and cyclin E protein, but inhibited expression of cyclin B1 protein in a time-dependent manner. The reduction in the protein level of cyclin B1 correlated with a decrease in the level of cyclin B1 mRNA. Genistein induced expression of p21, and the increased levels of p21 were associated with increased binding of p21 with cdc2 and cdk2. These observations suggest that genistein induces a G2/M arrest in human breast cancer cells, the mechanism of which is in part due to inhibition of kinase activities of cdc2 and cdk2, and decrease in cyclin B1 expression.
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PMID:Genistein-induced G2/M arrest is associated with the inhibition of cyclin B1 and the induction of p21 in human breast carcinoma cells. 966 38

DNA topoisomerase II (topo II) is an essential nuclear enzyme required for chromatin condensation and chromosome segregation during mitosis. Forced overexpression of topo IIalpha was found to cause morphological changes in recipient cells associated with apoptosis. This induction of apoptosis required nuclear localization of topo IIalpha, yet was independent of the DNA cleavage-religation activity of the enzyme. Apoptosis mediated by topo IIalpha deregulation was blocked by overexpression of crmA, a specific inhibitor of certain caspases, but not by bcl-2. topo IIalpha-induced apoptosis was also blocked by overexpression of a dominant-acting mutant of stress-activated protein kinase kinase (SEK1/MKK4) but not by the overexpression of its normal counterpart. Furthermore, apoptosis was blocked by coexpression of a dominant-negative form of the cyclin-dependent kinase cdk2 but not by dominant-negative cdc2. These results provide a rationale for the tight regulation of topo IIalpha levels through the cell cycle in that deregulation of topo IIalpha expression results in apoptotic cell death.
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PMID:Induction of apoptosis by deregulated expression of DNA topoisomerase IIalpha. 978 93

Cyclin-dependent kinase inhibitors are potent suppressors of cell growth and have been proposed as targets for gene replacement therapy in cancer. Expression of either p16INK4a or p21WAF1 protected cells from the cytotoxic effects of the topoisomerase II inhibitor, etoposide. A lower level of p53 was induced in CDK inhibitor-expressing etoposide-exposed cells suggesting that protection may be due to lower levels of DNA damage in the growth arrested cells. Exposure of human osteosarcoma cells to either p16INK4a or p21WAF1 prior to and during etoposide therapy protected cells against etoposide-induced cell death. Infection of the cells by Ad-p16INK4a or Ad-p21WAF1 following exposure to etoposide resulted in loss of the protective effect with evidence of enhanced growth inhibition. The results suggest that the schedule of administration of DNA damaging etoposide chemotherapy and cell cycle inhibitory therapy is a major determinant of the resulting cytotoxicity.
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PMID:The administration schedule of cyclin-dependent kinase inhibitor gene therapy and etoposide chemotherapy is a major determinant of cytotoxicity. 1040 29

A tumor-suppressor gene, p16(INK4), which is deleted or mutated in tumors, regulates cell-cycle progression through a G(1)-S restriction point by inhibiting CDK4(CDK6)/cyclin-D-mediated phosphorylation of pRb. We have found that ectopic p16(INK4) expression increased cellular sensitivity of human non-small-cell-lung-cancer (NSCLC) A549 cells to a selective growth-inhibitory effect induced by the topoisomerase-I inhibitor 11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin (CPT-11) in vitro. In this study, we observed enhanced apoptosis characterized by DNA fragmentation in A549 cells transfected with p16(INK4) cDNA (A549/p16-1) and treated with CPT-11. This apoptosis was suppressed by the inhibitor of interleukin-1beta-converting enzyme (ICE/caspase-1) or ICE-like proteases, Z-Asp-CH2-DCB, as determined by DNA fragmentation and proteolytic cleavage of poly(ADP-ribose) polymerase, a natural substrate for CPP32/caspase-3. In A549/p16-1 cells, cytosolic peptidase activities that cleaved Z-DEVD-7-amino-4-trifluoromethylcoumarin increased during CPT-11-induced apoptosis and were suppressed by a highly specific caspase-3 and caspase-3-like inhibitor, Z-DEVD-fluoromethylketone. These findings indicate that p16(INK) is positively involved in the activation pathway of the caspase-3 induced by CPT-11. The increased delay in S-phase progression and subsequent induction of apoptosis were observed in CPT-11-treated A549/p16-1 cells on the basis of DNA histograms. Specific down-regulation of the cyclin-A protein level in A549/p16-1 cells was observed after CPT-11-treatment, whereas cyclin B, cdk2, and cdc2 protein levels were unaffected. These results suggest that ectopic p16(INK4) expression inappropriately decreases cyclin A and thereby terminates CPT-11-induced G(2)/M accumulation, which is followed by increased apoptosis in p16(INK4)-expressing A549 cells.
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PMID:Ectopic p16(ink4) expression enhances CPT-11-induced apoptosis through increased delay in S-phase progression in human non-small-cell-lung-cancer cells. 1073 46

Genistein, a natural isoflavonoid phytoestrogen, is a strong inhibitor of protein tyrosine kinase and DNA topoisomerase II activities. Genistein has been shown to have anticancer proliferation, differentiation and chemopreventive effects. In the present study, we have addressed the mechanism of action by which genistein suppressed the proliferation of p53-null human prostate carcinoma cells. Genistein significantly inhibited the cell growth, which effect was reversible, and induced dendrite-like structure. The inhibitory effects of genistein on cell growth proliferation were associated with a G2/M arrest in cell cycle progression concomitant with a marked inhibition of cyclin B1 and an induction of Cdk inhibitor p21 (WAF1/CIP1) in a p53-independent manner. Following genistein treatment of cells, an increased binding of p21 with Cdk2 and Cdc2 paralleled a significant decrease in Cdc2 and Cdk2 kinase activity with no change in Cdk2 and Cdc2 expression. Genistein also induced the activation of a p21 promoter reporter construct, utilizing a sequence distinct from the p53-binding site. Analysis of deletion constructs of the p21 promoter indicated that the response to genistein could be localized to the 300 base pairs proximal to the transcription start site. These data suggest that genistein may exert a strong anticarcinogenic effect, and that this effect possibly involves an induction of p21, which inhibits the threshold kinase activities of Cdks and associated cyclins, leading to a G2/M arrest in the cell cycle progression.
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PMID:p53-independent induction of p21 (WAF1/CIP1), reduction of cyclin B1 and G2/M arrest by the isoflavone genistein in human prostate carcinoma cells. 1076 3


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