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:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report five novel monoclonal antibodies (Ki-S1, Ki-S4, Ki-S6, Ki-S7, and Ki-S8) reactive with a proliferation-related nuclear antigen. In immunoprecipitation and Western blot experiments using crude nuclear extracts, they recognized a protein of 170 kD that, after proteolytic digestion of the immunoprecipitate and sequencing of the resulting peptides, was identified as the alpha-isoform of human topoisomerase II. This was confirmed by testing the antibodies on a highly purified enzyme preparation. Crossreactivity with topoisomerase II beta was ruled out by testing the antibodies on crude extracts from yeast cells expressing the beta-isoform exclusively. The antibodies bind the antigen with different affinities and at different epitopes, apparently located within the carboxyl third of the enzyme. All five antibodies are suitable for archival material after adequate antigen retrieval, thereby enabling retrospective studies. This report illustrates the tissue and subcellular distribution of the antigen through the cell cycle by immunohistochemistry and confocal fluorescence microscopy. The antibodies will be useful tools in further analysis of morphological and functional aspects of topoisomerase II and may serve diagnostic purposes, as well as providing prognostic information in tumor pathology.
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PMID:Detection of human topoisomerase II alpha in cell lines and tissues: characterization of five novel monoclonal antibodies. 901 14

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

In the Chinese hamster lung cell line DC-3F/9-OH-E, made resistant to 9-OH-ellipticine and cross-resistant to other topoisomerase II inhibitors, the amount of topoisomerase II alpha is 4-5-fold lower than in the parental DC-3F cells. A mutation in position 1710 of topoisomerase II beta cDNA, generating a stop codon, completely abolishes the expression of this isoform in DC-3F/9-OH-E cells. To analyze the contribution of the loss of topoisomerase II beta to the resistance phenotype, DC-3F/9-OH-E cells were cotransfected with two plasmids, one conferring the resistance to G418, the other carrying the topoisomerase II beta cDNA. Among 200 G418-resistant clones, one was found to contain a topoisomerase II beta activity similar to that in the parental cells. These cells constitute an in vivo mammalian model to study the pharmacological role of topoisomerase II beta. In the transfected cells, different levels of cleavable complex formation and resistance reversion were observed with each topoisomerase II inhibitor examined. This work demonstrates that topoisomerase II beta is a pharmacological target for 9-OH-ellipticine, etoposide, or 4'-(9-acridinylamino)methanesulfon-m-anisidide and plays a role in the cytotoxicity of these agents. Furthermore, topoisomerase II beta is the preferential target for 4'-(9-acridinylamino)methanesulfon-m-anisidide. The loss of topoisomerase II beta activity in the DC-3F/9-OH-E cells is then in part responsible for their resistance to topoisomerase II inhibitors.
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PMID:Role of topoisomerase II beta in the resistance of 9-OH-ellipticine-resistant Chinese hamster fibroblasts to topoisomerase II inhibitors. 933 Oct 91

Selection for in vitro drug resistance can result in a complex phenotype with more than one mechanism of resistance emerging concurrently or sequentially. We examined emerging mechanisms of drug resistance during selection with mitoxantrone in the human myeloma cell line 8226. A novel transport mechanism appeared early in the selection process that was associated with a 10-fold resistance to mitoxantrone in the 8226/MR4 cell line. The reduction in intracellular drug concentration was ATP-dependent and ouabain-insensitive. The 8226/MR4 cell line was 34-fold cross-resistant to the fluorescent aza-anthrapyrazole BBR 3390. The resistance to BBR 3390 coincided with a 50% reduction in intracellular drug concentration. Confocal microscopy using BBR 3390 revealed a 64% decrease in the nuclear:cytoplasmic ratio in the drug-resistant cell line. The reduction in intracellular drug concentration of both mitoxantrone and BBR 3390 was reversed by a novel chemosensitizing agent, fumitremorgin C. In contrast, fumitremorgin C had no effect on resistance to mitoxantrone or BBR 3390 in the P-glycoprotein-positive 8226/DOX6 cell line. Increasing the degree of resistance to mitoxantrone in the 8226 cell line from 10 to 37 times (8226/MR20) did not further reduce the intracellular drug concentration. However, the 8226/MR20 cell line exhibited 88 and 70% reductions in topoisomerase II beta and alpha expression, respectively, compared with the parental drug sensitive cell line. This decrease in topoisomerase expression and activity was not observed in the low-level drug-resistant, 8226/MR4 cell line. These data demonstrate that low-level mitoxantrone resistance is due to the presence of a novel, energy-dependent drug efflux pump similar to P-glycoprotein and multidrug resistance-associated protein. Reversal of resistance by blocking drug efflux with fumitremorgin C should allow for functional analysis of this novel transporter in cancer cell lines or clinical tumor samples. Increased resistance to mitoxantrone may result from reduced intracellular drug accumulation, altered nuclear/cytoplasmic drug distribution, and alterations in topoisomerase II activity.
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PMID:Multiple mechanisms confer drug resistance to mitoxantrone in the human 8226 myeloma cell line. 1007 Sep 58

A type II topoisomerase is essential for decatenating DNA replication products, and it accomplishes this task by passing one DNA duplex through a transient break in a second duplex. The B' domain of topoisomerase II contains three highly conserved motifs, EGDSA, PL(R/K)GK(I/L/M)LNVR, and IMTD(Q/A)DXD. We have investigated these motifs in topoisomerase II beta by mutagenesis, and report that they play a critical role in establishing the DNA cleavage-religation equilibrium. In addition, the mutations E477Q (EGDSA) and K505E (PLRGKILNVR) increase the optimal magnesium ion concentration for strand passage, without affecting the Mg(2+) dependence of ATP hydrolysis. It is likely that the binding affinity of the magnesium ion(s) specifically required for DNA cleavage has been reduced by these mutations. The crystal structure of yeast topo II indicates that residues E477 and K505 may help to position the three aspartate residues of the IMTD(Q/A)DXD motif for magnesium ion coordination, and we propose two possible locations for the magnesium ion binding site(s). These observations are consistent with a previous model in which the B' domain is positioned such that these acidic residues lie next to the active site tyrosine residue. A magnesium ion bound by these aspartate residues could therefore mediate the DNA cleavage-religation reaction.
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PMID:Mutagenesis of E477 or K505 in the B' domain of human topoisomerase II beta increases the requirement for magnesium ions during strand passage. 1068

Anthracyclines exert antitumor activity by stimulating site-selective DNA cleavage by topoisomerase II (top2). DNA cleavage sites stimulated by two anthracycline analogues, dh-EPI and da-IDA, were investigated at the histone gene cluster of cultured Drosophila Kc cells. The two agents stimulated analogue-specific patterns of double-stranded DNA cleavage in Kc cell chromatin. Analyses of 47 base sequences of dh-EPI sites showed that the analogue largely followed the in vitro selectivity rule, the requirement of (5')TA at 3' ends of cleaved strands. da-IDA was more selective than dh-EPI, and thus fewer sites could be collected. Nevertheless, base sequences were consistent with its in vitro base preferences. DNA cleavage was then studied in vitro with Drosophila and human top2 isoforms. The tested drugs stimulated distinct in vitro patterns that corresponded to the in vivo patterns. Human top2alpha promoted cleavage patterns that were much more similar to those of Drosophila top2 (both in vitro and in vivo) than human top2beta. Moreover, da-IDA showed a marked site-dependent preference for human top2beta. Thus, DNA site selection in vivo is different for the test anthracyclines, and together with a degree of beta-form specificity, may affect drug activity in human cells.
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PMID:In vivo site specificity and human isoenzyme selectivity of two topoisomerase II-poisoning anthracyclines. 1091 49

Choroidal melanoma has a high mortality rate and responds poorly to existing chemotherapy, but unexpected ex vivo sensitivity of a subset of these tumours to topoisomerase II inhibitors has been noted. Since chemoresistance may be mediated by the molecular phenotype of tumours, immunohistochemistry has been used to study the expression of both isoforms of topoisomerase II (alpha and beta) in 29 choroidal melanomas for which chemosensitivity assay data for doxorubicin or mitoxantrone are also available. Of these, eight tumours were topoisomerase II beta-positive and 11 were topoisomerase II alpha-positive. Recent studies showing genetic abnormality (often monosomy of chromosome 3) in choroidal melanoma suggest that loss of immunostaining could be due to genomic loss rather than down-regulation of topoisomerase II beta in these tumours. There was no convincing excess of anthracycline resistance in the topoisomerase II beta-negative group. Addition of topoisomerase II alpha, MDR1 (11/17 positive), LRP (16/28 positive), and MRP (5/29 positive) data in multivariate analysis did not reliably predict sensitivity or resistance. Vincristine chemosensitivity showed no relation to MDR1, LRP or MRP in 18 tumours tested. While it is possible that some tumours which do express topoisomerase II beta may respond to anthracyclines, the molecular basis of resistance or sensitivity to anthracyclines or vincristine in uveal melanoma is complex and remains incompletely understood.
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PMID:Relationship between expression of topoisomerase II isoforms and chemosensitivity in choroidal melanoma. 1100 93

In this study, we evaluated the influence of protein kinase C zeta (PKC zeta) on topoisomerase II inhibitor-induced cytotoxicity in monocytic U937 cells. In U937-zeta J and U937-zeta B cells, enforced PKC zeta expression, conferred by stable transfection of PKC zeta cDNA, resulted in total inhibition of VP-16- and mitoxantrone-induced apoptosis and decreased drug-induced cytotoxicity, compared with U937-neo control cells. In PKC zeta-overexpressing cells, drug resistance correlated with decreased VP-16-induced DNA strand breaks and DNA protein cross-links measured by alkaline elution. Kinetoplast decatenation assay revealed that PKC zeta overexpression resulted in reduced global topoisomerase II activity. Moreover, in PKC zeta-overexpressing cells, we found that PKC zeta interacted with both alpha and beta isoforms of topoisomerase II, and these two enzymes were constitutively phosphorylated. However, when human recombinant PKC zeta (rH-PKC zeta) was incubated with purified topoisomerase II isoforms, rH-PKC zeta interacted with topoisomerase II beta but not with topoisomerase II alpha. PKC zeta/topoisomerase II beta interaction resulted in phosphorylation of this enzyme and in decrease of its catalytic activity. Finally, this report shows for the first time that topoisomerase II beta is a substrate for PKC zeta, and that PKC zeta may significantly influence topoisomerase II inhibitor-induced cytotoxicity by altering topoisomerase II beta activity through its kinase function.
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PMID:Overexpression of the atypical protein kinase C zeta reduces topoisomerase II catalytic activity, cleavable complexes formation, and drug-induced cytotoxicity in monocytic U937 leukemia cells. 1210 21

Topoisomerase II is the target of several anticancer agents. The discovery of a second enzyme, called topoisomerase II beta, genetically distinct from alpha, prompted the investigation on the different functional roles of the two isoforms. Whereas the first recognized isozyme is essential for life due to its role in chromosome condensation and segregation, beta functions remained elusive, although its importance in neural development is appearing clearer. Topoisomerase II beta is regulated differently than alpha, and its level of expression does not change significantly during cell cycle. The presence of this isoform in non-proliferating cells suggests that drug preferentially aimed at beta may be active in slow growing tumors. Topoisomerase II poisons were hence evaluated in light of their selectivity toward one or the other isozyme, indicating how the beta isoform may represent an important target for selected classes of drugs. Newer compounds were also synthesized and tested for their potential antitumor activity and their topoisomerase II beta poisoning. The literature dealing with "old" and "new" drugs targeted at topoisomerase II is reviewed trying to link, whenever possible, selective poisoning and cytotoxic effects to chemical structures, in the hope to indicate new lead compounds that will contribute to unveil molecular determinants of selectivity.
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PMID:Drugs acting on the beta isoform of human topoisomerase II (p180). 1276 76

Human topoisomerase II plays a crucial role in DNA replication and repair. It exists in two isoforms: topoisomerase II alpha (alpha) and topoisomerase II beta (beta). The alpha isoform is localized predominantly in the nucleus, while the beta isoform exhibits a reticular pattern of distribution both in the cytosol and in the nucleus. We show that both isoforms of topoisomerase II are phosphorylated in HIV infected cells and also by purified viral lysate. An analysis of the phosphorylation of topoisomerase II isoforms showed that extracts of HIV infected cells at 8 and 32 h. post-infection (p.i.) contain maximal phosphorylated topoisomerase II alpha, whereas infected cell extracts at 4 and 64 h p.i. contain maximum levels of phosphorylated topoisomerase II beta. In concurrent to phosphorylated topoisomerase II isoforms, we have also observed increased topoisomerase II alpha kinase activity after 8h p.i and topoisomerase beta kinase activity at 4 and 64 h p.i. These findings suggest that both topoisomerase II alpha and beta kinase activities play an important role in early as well as late stages of HIV-1 replication. Further analysis of purified virus showed that HIV-1 virion contained topoisomerase II isoform-specific kinase activities, which were partially isolated. One of the kinase activities of higher hydrophobicity can phosphorylate both topoisomerase II alpha and beta, while lower hydrophobic kinase could predominantly phosphorylate topoisomerase II alpha. The phosphorylation status was correlated with catalytic activity of the enzyme. Western blot analysis using phosphoamino-specific antibodies shows that both the kinase activities catalyze the phosphorylation at serine residues of topoisomerase II alpha and beta. The catalytic inhibitions by serine kinase inhibitors further suggest that the alpha and beta kinase activities associated with virus are distinctly different.
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PMID:A biochemical analysis of topoisomerase II alpha and beta kinase activity found in HIV-1 infected cells and virus. 1609 Dec 84


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