New flow cytometric procedure for the analysis of drug effects on topoisomerase II-DNA binding

3968 Topoisomerase II (Topo II) is the target of many clinically important antitumor drugs. The best-known drugs of this class are known as Topo II poisons, which stabilize cleavable complexes of Topo II covalently linked to DNA. Other Topo II inhibitors, usually categorized as catalytic inhibitors, affect other steps in Topo II-catalyzed DNA decatenation. Topo II poisons are generally identified through biochemical assays, by their ability to increase Topo II cleavable complex formation. These cell-free assays are laborious, require a large amount of material, and do not identify other types of Topo II inhibitors. We have developed a new rapid assay for agents that increase Topo II-DNA association in situ, based on flow cytometric analysis of the retention of a Topo II-GFP fusion protein in the nuclei of permeabilized cells. GFP fusions of the wild-type Topo IIα and its non-functional mutant (gifts of Dr. W.T. Beck) were stably transfected into a derivative of 293 cells, a transformed human embryonic kidney cell line. Cells were treated with 0.05% Triton X-100 and GFP fluorescence of permeabilized nuclei was analyzed by FACS. Wild type Topo II-GFP was retained much more efficiently than the mutant Topo II-GFP, indicating that Topo II-GFP retention reflected functional Topo II-DNA interactions. We have used this assay to analyze the effects of 20-minute exposure to different types of Topo II inhibitors and topoisomerase I inhibitor camptothecin. Topo II-GFP retention was unaffected by camptothecin but increased by most of the tested Topo II inhibitors. In contrast, Topo II inhibitors had no effect on the retention of the Topo II mutant. The strongest increase in Topo II-GFP retention was observed with classical Topo II poisons, teniposide and etoposide, as well as with bisdioxopiperazine derivatives (ICRF-193 and MST-16), recently shown to constitute a different class of Topo II poisons. One of the tested catalytic inhibitors, merbarone, also increased Topo II-GFP retention, albeit to a lesser degree than the Topo II poisons, whereas another catalytic inhibitor (novobiocin) had no effect. These results confirm Topo II-DNA complex stabilization by bisdioxopiperazines and reveal important differences in the effects of catalytic inhibitors on Topo II-DNA interaction. In contrast to the biochemical assays for Topo II-DNA binding, our method rapidly measures such interactions in situ, requires a small amount of material, does not use radioactive materials, and can detect both covalent and non-covalent Topo II-DNA interactions. This method should be useful both for mechanistic analysis and for de novo identification of Topo II-targeting compounds. This study is supported by NIH Grants RO1 CA95727 (I.B.R.) and RO1 CA97107 (R.M.S.).