Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human cells express two genetically distinct isoforms of DNA topoisomerase II, alpha and beta, which catalyze ATP-dependent DNA strand passage and are an important antitumor drug target. Here we report for the first time the successful overexpression of human topoisomerase II beta in yeast by cloning a topoisomerase II beta cDNA in a yeast shuttle vector under the control of a galactose-inducible promoter. Recombinant human topoisomerase II beta (residues 46-1621 fused to the first 5 residues of yeast topoisomerase II) was purified to homogeneity, yielding an enzymatically active polypeptide in sufficient quantity to allow analysis of its domain structure and comparison with that of recombinant human topoisomerase II alpha. Partial digestion of beta with either trypsin or protease SV8 generated fragments of approximately 130, 90, 62, and 45-50 kDa, arising from cleavage at three limited and discrete regions of the protein (A, B, and C) indicating the presence of at least four structural domains. Recombinant human topoisomerase II alpha and beta induced DNA breakage which was promoted by a variety of agents. Isoform differences in drug-induced DNA breakage were observed. These studies of human topoisomerase II beta in concert with alpha should aid the determination of their individual roles in cancer chemotherapy and should facilitate the design, targeting, and testing of cytotoxic antitumor agents.
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PMID:Expression, domain structure, and enzymatic properties of an active recombinant human DNA topoisomerase II beta. 779 75

We have shown that both DNA topoisomerase (topo) IIalpha and beta are in vivo targets for etoposide using a new assay which directly measures topo IIalpha and beta cleavable complexes in individual cells after treatment with topo II targeting drugs. CCRF-CEM human leukemic cells were exposed to etoposide for 2 hr, then embedded in agarose on microscope slides before cell lysis. DNA from each cell remained trapped in the agarose and covalently bound topo II molecules from drug-stabilized cleavable complexes remained associated with the DNA. The covalently bound topo II was detected in situ by immunofluorescence. Isoform-specific covalent complexes were detected with antisera specific for either the alpha or beta isoform of topo II followed by a fluorescein isothiocyanate-conjugated second antibody. DNA was detected using the fluorescent stain Hoechst 33258. A cooled slow scan charged coupled device camera was used to capture images. A dose-dependent increase in green immunofluorescence was observed when using antisera to either the alpha or beta isoforms of topo II, indicating that both isoforms are targets for etoposide. We have called this the TARDIS method, for trapped in agarose DNA immunostaining. Two key advantages of the TARDIS method are that it is isoform-specific and that it requires small numbers of cells, making it suitable for analysis of samples from patients being treated with topo II-targeting drugs. The isoform specificity will enable us to extend our understanding of the mechanism of interaction between topo II-targeting agents and their target, the two human isoforms.
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PMID:Etoposide targets topoisomerase IIalpha and IIbeta in leukemic cells: isoform-specific cleavable complexes visualized and quantified in situ by a novel immunofluorescence technique. 965 92

Genistein, a widely consumed bioflavonoid with chemopreventative properties in adults, and etoposide, a commonly prescribed anticancer drug, are well-characterized topoisomerase II poisons. Although both compounds display similar potencies against human topoisomerase IIalpha and IIbeta in vitro and induce comparable levels of DNA cleavage complexes in cultured human cells, their cytotoxic and genotoxic effects differ significantly. As determined by assays that monitored cell viability or the phosphorylation of histone H2AX, etoposide was much more toxic in CEM cells than genistein. Further studies that characterized the simultaneous treatment of cells with genistein and etoposide indicate that the differential actions of the two compounds are not related to the effects of genistein on cellular processes outside of its activity against topoisomerase II. Rather, they appear to result from a longer persistence of cleavage complexes induced by etoposide as compared to genistein. Parallel in vitro studies with purified type II enzymes led to similar conclusions regarding cleavage complex persistence. Isoform-specific differences were observed in vitro and in cells treated with etoposide. To this point, the t 1/2 of etoposide-induced DNA cleavage complexes formed with topoisomerase IIalpha in CEM cells was approximately 5 times longer than those formed with topoisomerase IIbeta. The cytotoxicity of etoposide following four treatment-recovery cycles was similar to that induced by continuous exposure to the drug over an equivalent time period. Taken together, these findings suggest that it may be possible to preferentially target topoisomerase IIalpha with etoposide by employing a schedule that utilizes pulsed drug treatment-recovery cycles.
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PMID:The efficacy of topoisomerase II-targeted anticancer agents reflects the persistence of drug-induced cleavage complexes in cells. 1892 22