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{"title":"Measurement of Drug-Stabilized Topoisomerase II Cleavage Complexes by Flow Cytometry","authors":"Marcelo de Campos Nebel, Micaela Palmitelli, Marcela González-Cid","doi":"10.1002/cpcy.21","DOIUrl":null,"url":null,"abstract":"<p>The poisoning of Topoisomerase II (Top2) has been found to be useful as a therapeutic strategy for the treatment of several tumors. The mechanism of Top2 poisons involves a drug-mediated stabilization of a Top2-DNA complex, termed Top2 cleavage complex (Top2cc), which maintains a 5′ end of DNA covalently bound to a tyrosine from Top2 through a phosphodiester group. Drug-stabilized Top2cc leads to Top2-linked-DNA breaks, which are believed to mediate their cytotoxicity. Several time-consuming or cell type-limiting assays have been used in the past to study drug-stabilized Top2cc. Here, we describe a flow cytometry-based method that allows a rapid assessment of drug-induced Top2cc, which is suitable for high throughput analysis in almost any kind of human cell. The analyses of the drug-induced Top2cc in the cell cycle context and the possibility to track its removal are additional benefits from this methodology. © 2017 by John Wiley & Sons, Inc.</p>","PeriodicalId":11020,"journal":{"name":"Current Protocols in Cytometry","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2017-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpcy.21","citationCount":"1","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Cytometry","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpcy.21","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"Health Professions","Score":null,"Total":0}
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Abstract
The poisoning of Topoisomerase II (Top2) has been found to be useful as a therapeutic strategy for the treatment of several tumors. The mechanism of Top2 poisons involves a drug-mediated stabilization of a Top2-DNA complex, termed Top2 cleavage complex (Top2cc), which maintains a 5′ end of DNA covalently bound to a tyrosine from Top2 through a phosphodiester group. Drug-stabilized Top2cc leads to Top2-linked-DNA breaks, which are believed to mediate their cytotoxicity. Several time-consuming or cell type-limiting assays have been used in the past to study drug-stabilized Top2cc. Here, we describe a flow cytometry-based method that allows a rapid assessment of drug-induced Top2cc, which is suitable for high throughput analysis in almost any kind of human cell. The analyses of the drug-induced Top2cc in the cell cycle context and the possibility to track its removal are additional benefits from this methodology. © 2017 by John Wiley & Sons, Inc.
用流式细胞术测定药物稳定拓扑异构酶II切割复合物
拓扑异构酶II (Top2)中毒已被发现是治疗几种肿瘤的有效治疗策略。Top2毒素的机制涉及药物介导的Top2-DNA复合物的稳定,称为Top2切割复合物(Top2cc),该复合物通过磷酸二酯基团维持与Top2酪氨酸共价结合的DNA的5 '端。药物稳定的Top2cc导致top2链dna断裂,这被认为介导了它们的细胞毒性。过去已经使用了几种耗时或限制细胞类型的测定方法来研究药物稳定的Top2cc。在这里,我们描述了一种基于流式细胞术的方法,可以快速评估药物诱导的Top2cc,该方法适用于几乎任何类型的人类细胞的高通量分析。在细胞周期背景下对药物诱导的Top2cc的分析以及跟踪其去除的可能性是该方法的额外好处。©2017 by John Wiley &儿子,Inc。
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