Measurement of drug-stabilized Topoisomerase II cleavage complexes by Flow Cytometry

Abstract

The poisoning of Topoisomerase II (Top2) has been found to be useful as a therapeutic strategy for the treatment of several tumors. The mechanisms of these agents 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 arapid 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.