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)

Fifteen specific inhibitors of DNA topoisomerases I and II were used to elucidate whether these enzymes participate in the excision repair of UV-induced DNA damage, monitoring DNA repair synthesis in confluent saponin-permeabilized human fibroblasts. To achieve a sufficient degree of accuracy dose--response experiments were performed, analysed by linear regression, and the concentrations at which repair activity was reduced to 50% were calculated and designated K50. Camptothecin, a specific inhibitor of topoisomerase I did not markedly diminish DNA repair synthesis. Similarly, when combined with topoisomerase II inhibitors [nalidixic acid, oxolinic acid, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucop yra noside) (etoposide), 4'-demethylepipodophyllotoxin-thenylidene-beta-D-glucoside (teniposide), 1,4-dihydroxy-5,8-bis ((2-[(2-hydroxyethyl)amino]ethyl)amino)-9,10-anthracenedione (mitoxantrone), 5-(N-phenyl-carboxamido)-2-thiobarbituric acid (merbarone) or 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)], it did not lower K50 values determined for topoisomerase II-specific drugs in separate experiments. The effects observed can be classified according to the mechanism of action the inhibitors exhibit. (i) Novobiocin and coumermycin, inhibitors of the ATPase subunit of topoisomerase II, completely reduced DNA repair synthesis. (ii) Inhibition of repair was also found for ethidium bromide, quinacrine and distamycin, drugs known to modify the DNA substrate by intercalation or binding to the DNA minor groove. (iii) Inhibitors acting through intercalation and, simultaneously, binding to the cleavable DNA-topoisomerase complex (m-AMSA, mitoxantrone, doxorubicin and daunorubicin) also suppressed reparative DNA synthesis. (iv) Only small effects were observed for etoposide, nalidixic acid and oxolinic acid, whereas teniposide caused marked inhibition of DNA repair synthesis. (v) Merbarone, a novel type of topoisomerase II inhibitor, blocked UV-induced DNA repair drastically. The results are best explained by assuming that in UV-irradiated human fibroblasts the 180 kDa form of topoisomerase II is the main target enzyme for inhibitors which suppressed DNA excision repair and that this isozyme is involved in steps preceding repair-specific DNA incision.
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PMID:The function of DNA topoisomerases in UV-induced DNA excision repair: studies with specific inhibitors in permeabilized human fibroblasts. 133 77

CHO-Cdr20 cells are 10-20 times more resistant to killing by cadmium than the parental CHO cells. Resistance has been linked to amplification of the metallothionein genes MT-I and MT-II and their coordinate induction by cadmium and other toxic metals. We studied the roles of the nuclear enzymes topoisomerase I and topoisomerase II in Cd-induced expression of MT-II. Camptothecin-induced DNA strand breakage, mediated by topoisomerase I in cells, increased by approximately 20% when the resistant cells were incubated first with 50 microM Cd and then with camptothecin. Short DNA fragments were enriched in MT-II-hybridizing sequences, indicating that topoisomerase I-associated breakage was directed in part toward the location of induced gene activity. Ten microM camptothecin inhibited Cd-induced accumulation of MT-II mRNA as well as induced and uninduced RNA synthesis in the resistant cells. These data are consistent with the notion that topoisomerase I participates in most or all forms of RNA synthesis. Topoisomerase II inhibitors which trap cleavable complexes (amsacrine, VM-26, VP-16) increased DNA strand breakage at very high concentrations (50-100 microM); the increased breakage appeared to be concentrated near the MT-II gene. This class of inhibitor did not block the accumulation of MT-II message. Novobiocin, a second type of topoisomerase II inhibitor blocked transcription at 300 microM. Merbarone, a novel, third type of topoisomerase II inhibitor, blocked MT-II transcription at 50-100 microM. The latter two inhibited total RNA synthesis in induced, but not uninduced cells. Thus, it is possible that topoisomerase II plays more than one role in transcription and that more than one form of this enzyme is involved.
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PMID:Evidence for the participation of topoisomerases I and II in cadmium-induced metallothionein expression in Chinese hamster ovary cells. 247 39

Merbarone has previously been shown to have antitumor activity of unknown mechanism in P388 and L1210 tumor models (A. D. Brewer et al., Biochem. Pharmacol., 34:2047-2050, 1985) and is currently undergoing Phase I clinical trials. Here we report that merbarone is an inhibitor of topoisomerase II. Merbarone inhibited purified mammalian topoisomerase II with a 50% inhibitory concentration of 20 microM, as assessed by ATP-dependent unknotting of P4 phage DNA or relaxation of supercoiled pBR322 plasmid. In contrast to the type II enzyme, inhibition of catalytic activity of topoisomerase I required about 10-fold higher concentrations of merbarone, with a 50% inhibitory concentration of approximately 200 microM. Unlike epipodophyllotoxin analogues and certain DNA intercalative agents which stabilize the topoisomerase II-DNA "cleavable complex," merbarone did not cause detectable topoisomerase II-induced DNA cleavage. Furthermore, merbarone inhibited the production by amsacrine or teniposide of topoisomerase II-associated DNA strand breaks; under identical conditions novobiocin did not decrease these breaks, setting merbarone apart from a novobiocin-like class of topoisomerase II inhibitor. In L1210 cells, merbarone produced only small numbers of protein-associated DNA strand breaks, and only at very high concentrations. Merbarone reduced in a concentration-dependent manner the number of amsacrine- or teniposide-stimulated protein-associated DNA strand breaks in L1210 cells or their isolated nuclei. The data suggest that merbarone represents a novel type of topoisomerase II inhibitor.
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PMID:In vitro and intracellular inhibition of topoisomerase II by the antitumor agent merbarone. 254 Sep 3

The effect of inhibition of topoisomerase II on chromosome segregation in CHO cells has been studied using cytogenetical techniques and measurements of nuclear DNA content. Cells were accumulated in metaphase, and their passages into the subsequent stages of mitosis, and into interphase, were examined. Of the compounds tested, five (Amsacrine, Etoposide, Hoechst 33342, Mitoxantrone, and nalidixic acid) greatly reduce the rate at which the chromosomes pass from metaphase through anaphase to the subsequent interphase and induce a high proportion of nuclei which contain a 4C amount of DNA. In several cases, the reformation of membranes around chromosomes can be seen although the chromosomes remain in a condition similar to metaphase, with the chromatids linked at the centromeres. Two other inhibitors of topoisomerase II, Hoechst 33258 and Merbarone, failed to delay cells in metaphase and did not induce tetraploidy. This failure may well be due to an inability of the compounds to penetrate the cells sufficiently quickly, or at a high enough concentration. Overall, the results are consistent with the hypothesis that topoisomerase II is essential for the segregation of chromosomes in mammals and other eukaryotes.
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PMID:Inhibitors of topoisomerase II delay progress through mitosis and induce a doubling of the DNA content in CHO cells. 769 44

Merbarone, a novel DNA topoisomerase II (topo II) inhibitor, differs from teniposide (VM-26) in that it inhibits topo II activities without stabilizing topo II-DNA covalent complexes. Thus, while the cellular effects of VM-26 are the consequences of inhibition of topo II catalytic activities and generation of topo II-mediated DNA damage, those of merbarone may be due to inactivation of topo II catalytic function. To address the issues of mechanisms of cell cycle effects and pharmacological actions of these two topo II inhibitors in mammalian cells, we used synchronized cultures of HeLa cells to study the effects of these drugs on cell cycle processes where topo II is essential (e.g., chromosome separation) or possibly involved (e.g., G2 arrest, DNA replication). We found that both drugs inhibited chromosome separation and cell division without preventing cells from exiting mitosis. Both drugs caused S-phase retardation G2 arrest, and phase-specific cytotoxicity in that they are more toxic to S, M, and G2 cells than G0/G1 cells. However, merbarone produced the above effects in convergent dosages that were within one to five times its 90% inhibitory cytotoxic concentration, whereas the concentrations of VM-26 to cause quantitatively similar effects were quite divergent. VM-26 is 50-100-fold more efficient in causing G2 arrest than in inhibiting chromosome separation. Furthermore, at concentrations showing similar levels of S-phase suppression, VM-26 caused significant DNA breaks, while merbarone had no such effect. Our data suggest that the effects of merbarone and VM-26 during mitosis are most likely due to inhibition of topo II function. We conclude that while G2 arrest by VM-26 is related to topo II-mediated DNA damage and its sequelae, G2 arrest by merbarone likely results from different mechanisms.
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PMID:Differences in inhibition of chromosome separation and G2 arrest by DNA topoisomerase II inhibitors merbarone and VM-26. 788 60

A function for topoisomerases I and II in DNA excision repair can be postulated from the organization of the mammalian chromosome, involving nucleosomal structures and matrix-attached DNA loops. To analyse this function we determined UV-induced DNA incision in confluent human fibroblasts in the presence of 16 inhibitors of topoisomerases I and II which belonged to at least five different drug categories, based on their mechanism of action. Dose-response experiments were performed, analysed by linear regression and the concentrations at which DNA-incising activity was reduced to 50% were calculated (K50 values). The majority of these values represent concentrations for which interfering cell toxicity could be excluded. K50 concentrations, which were determined by extrapolating dose-response data, may hit the toxicity range, nevertheless, we deem our K50 scale useful for making biochemical comparisons. With respect to topoisomerase I, camptothecin and topotecan diminished repair-specific DNA incision to a small extent, whereas distamycin, which binds to the minor groove of DNA, caused a stronger effect. With respect to topoisomerase II the results were as follows. (i) The DNA intercalator ethidium bromide decreased DNA-incising activity at rather low concentrations, which indicates marked inhibitory potency. Quinacrine was less effective. (ii) Inhibitors intercalating and binding to the 'cleavable' DNA-topoisomerase complex (m-AMSA, mitoxantrone, doxorubicin and daunorubicin) strongly suppressed reparative DNA incision. (iii) Only small effects were observed using several drugs which act by trapping the 'cleavable' DNA-enzyme complex, namely nalidixic acid and oxolinic acid. In contrast, etoposide and teniposide inhibited post-UV DNA cleavage sizeably. (iv) Merbarone had to be applied at very high concentrations to reduce UV-induced DNA incision. (v) Novobiocin, an inhibitor of the ATPase subunit of topoisomerase II, markedly diminished repair-specific DNA cleavage. A comparison of the K50 values for DNA incision with those for DNA repair synthesis (1) shows that the majority of the investigated drugs inhibited both repair parameters. There were, however, differences in the concentrations required to achieve the 50% inhibition level. The results are best explained by assuming that in UV-irradiated human fibroblasts the 180 kd form of topoisomerase II is a target enzyme for inhibitors which suppressed repair and that this isozyme is involved in steps preceding repair-specific DNA incision.
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PMID:Various inhibitors of DNA topoisomerases diminish repair-specific DNA incision in UV-irradiated human fibroblasts. 824 65

We have examined the effects of a group of DNA topoisomerase II (topo II) inhibitors, merbarone, aclarubicin, SN22995, RP60475F, and fostriecin, in CCRF-CEM cells and two sublines, CEM/VM-1 and CEM/VM-1-5, that were selected for increasing resistance to teniposide (VM-26). The teniposide-resistant sublines have been termed "at-MDR" for altered topo II-associated multidrug resistance. These topo II inhibitors differ from the "classic" inhibitors such as teniposide in that they do not stabilize DNA-topo II complexes. In this study, we found that our at-MDR cell lines express little or no cross-resistance to these "non-classic" topo II inhibitors. Merbarone and SN22995 inhibited VM-26-mediated DNA-topo II complexes in CEM cells only when they were added before VM-26. Since they did not deplete topo II protein, it suggested that these drugs may inhibit topo II activity before the enzyme binds to DNA, thereby preventing stabilization of VM-26-mediated topo II-DNA complexes. Continuous exposure of CEM cells to merbarone, SN22995, or VM-26 caused G2 arrest, as determined by flow cytometry. Likewise, at-MDR cells continuously treated with VM-26 also arrested in G2. By contrast, treatment of at-MDR cells with either merbarone or SN22995 produced a qualitatively different pattern; the at-MDR cells first accumulated in G2 but then escaped the G2 block and proceeded into mitosis with elongated and intertwined chromosomes but failed to divide. Their DNA was re-replicated, however, and the cells eventually accumulated at the 8N DNA stage. Given that both wild-type and mutant topo II alpha alleles are expressed in the at-MDR cells (B. Y. Bugg, M. K. Danks, W. T. Beck, and D. P. Suttle. Expression of a mutant DNA topoisomerase II in CCRF-CEM human leukemic cells selected for resistance to teniposide. Proc. Natl. Acad. Sci. USA, 88: 7654-7658, 1991), we hypothesize that drugs such as merbarone may inhibit the activity of wild-type topo II alpha, allowing the aberrant activity of the mutant enzyme to be revealed during chromosome condensation and sister chromatid segregation.
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PMID:Teniposide-resistant CEM cells, which express mutant DNA topoisomerase II alpha, when treated with non-complex-stabilizing inhibitors of the enzyme, display no cross-resistance and reveal aberrant functions of the mutant enzyme. 826 8

Merbarone is a catalytic inhibitor of DNA topoisomerase (topo) II that does not stabilize DNA-topo II cleavable complexes. Although the cytotoxicity of and resistance to complex-stabilizing topo II inhibitors, such as etoposide, is thought to be mediated through stabilization of these complexes, the mechanisms of cytotoxicity and resistance to catalytic inhibitors are not well known. To investigate this issue, we established 12 merbarone-resistant cell lines from human leukemia CEM cells, designated CEM/M70-B1 through -B12. Assessed by either growth inhibition or clonogenic assay, these cell lines are 3.5- to 6.6-fold resistant to merbarone, compared to the CEM parent cells. Karyotype analysis of three of the cell lines revealed that while CEM and drug-resistant cell lines had chromosome abnormalities in common, indicating a common origin, two of the merbarone-resistant lines (B1 and B8) each had unique structural markers. These novel cell lines are cross-resistant to complex-stabilizing topo II inhibitors, etoposide, teniposide, amsacrine, and doxorubicin, but not to other catalytic inhibitors, aclarubicin or SN-22995. Of considerable interest, these cell lines are cross-resistant to SN-38, a putative topo I inhibitor, but cross-resistance to other topo I inhibitors (camptothecin and topotecan) was lower and not seen in every cell line. In all 12 cell lines, there was a high correlation among drug resistance ratios between etoposide and teniposide and between merbarone and SN-38. By contrast, there was a low correlation between merbarone and etoposide and between SN-38 and other topo I inhibitors. These results suggest that resistance to merbarone and cross-resistance to etoposide might be through different mechanisms, whereas cross-resistance to SN-38 might be through a merbarone-related mechanism. Etoposide and SN-38 stabilized fewer DNA-topoisomerase complexes in CEM/M70-B cells than in CEM cells, but camptothecin stabilized more. Merbarone inhibited complex formation induced by etoposide in drug-sensitive and -resistant cells, but the degree of inhibition was lower in CEM/M70-B cells than in the parental cells. Moreover, merbarone did not affect complex formation stabilized by SN-38 or camptothecin. Immunoblot analysis of the CEM/M70-B cells showed decreased topo IIalpha, increased topo IIbeta, and no change of topo I protein, compared to CEM cells. We propose the hypothesis that decreased topo IIalpha may play a role in the resistance to merbarone that is different from that to complex-stabilizing drugs. Cross-resistance to catalytic inhibitors may be due to reduced complex formation as a consequence of decreased topo IIalpha. We also found that DNA-protein complexes stabilized by SN-38 might be different from those stabilized by topo II inhibitors and blocked by merbarone. Judging from both the high correlation of drug sensitivities and complex-formation assays, we postulate that mechanisms of cytotoxicity and cross-resistance of SN-38 in CEM/M70-B cells might be similar to those of merbarone. We believe that the CEM/M70-B cells are the first to be selected and characterized for resistance to a catalytic inhibitor of topo II. This study provides novel cell lines with characteristics of resistances to topo II and topo I inhibitors.
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PMID:Characterization of novel human leukemic cell lines selected for resistance to merbarone, a catalytic inhibitor of DNA topoisomerase II. 865

In order to clarify possible risks of aneuploidy induction in germ cells by cancer chemotherapy we studied effects of a non complex-stabilizing DNA topoisomerase II (topo II) inhibitor merbarone in male mouse meiotic divisions in vivo. Two cytogenetic approaches were used: (1) C-banding on meiotic chromosome preparations and (2) analysis of spermatid micronuclei (MN) combined with immunocytochemical staining of kinetochore proteins using CREST serum. For comparison, another topo II inhibitor, VP-16, known to form cleavable complexes, was studied. The microdissection technique of mouse seminiferous tubules enabled us to carefully examine effects at specific phases of meiosis. Merbarone injections increased percentages of polyploid and hypoploid metaphase II spermatocytes at time intervals corresponding to the treatment of the first meiotic division and diplotene-diakinesis. The highest level of MN induction (5.8 MN/1000 spermatids, P < 0.001) was observed in animals injected 48 hours before the harvest, corresponding to the treatment of diplotene-diakinesis spermatocytes. Most of the induced MN (80%) contained kinetochore signals, indicating that they resulted from detachment of a whole bivalent or chromosome from the meiotic spindle. The high frequency of MN with two kinetochore signals at opposite sides (33%) most likely denotes lagging of whole bivalents during MI. Inhibition of cell proliferation was determined by scoring cells arrested at different phases of MI and MII. All groups of treated animals showed a clear increase in the frequency of arrested divisions compared to controls (P < 0.001). Thus, merbarone was shown to severely damage normal meiotic processes.
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PMID:Effects of the DNA topoisomerase II inhibitor merbarone in male mouse meiotic divisions in vivo: cell cycle arrest and induction of aneuploidy. 902 Mar 3

We show herein that human DNA topoisomerase II beta is functional in yeast. It can complement a yeast temperature-sensitive mutation in topoisomerase II. The effect on human topoisomerase II beta of a number of topoisomerase II inhibitors was analysed in a yeast in vivo system and compared with that of human topoisomerase II alpha and wild-type yeast topoisomerase II. A drug permeable yeast strain (JN394 top2-4) was used to analyse the in vivo effects of known anti-topoisomerase II agents on human topoisomerase II beta transformants. A parallel analysis on human topoisomerase II alpha transformants provides the first in vivo analysis of the responses of yeast bearing the individual isoforms to these drugs. The strain was analysed at 35 degrees C, a non-permissive temperature at which only plasmid-borne topoisomerase II is active. A shuttle vector with either human topoisomerase II beta, human topoisomerase II alpha or yeast topoisomerase II under the control of a GAL1 promoter was used. The key findings were that amsacrine produced comparable levels of cell killing with both alpha and beta, whilst etoposide, doxorubicin and mitoxantrone produced higher degrees of cell killing with alpha than with beta or yeast topoisomerase II. Merbarone had the greatest effect on the yeast strain bearing plasmid-borne yeast topoisomerase II. Suramin, quercetin and genistein showed little cell killing in this system. This yeast in vivo system provides a powerful way to analyse the effects of anti-topoisomerase II agents on transformants bearing the individual human isoforms. This system also provides a means of analysing putative drug-resistance mutations in human topoisomerase II beta or to select for drug-resistance mutations in human topoisomerase II beta.
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PMID:Complementation of temperature-sensitive topoisomerase II mutations in Saccharomyces cerevisiae by a human TOP2 beta construct allows the study of topoisomerase II beta inhibitors in yeast. 902 79


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