Modulation of human DNA topoisomerase II alpha function by interaction with 14-3-3 epsilon

Human DNA topoisomerase II alpha (topo II), a ubiquitous nuclear enzyme, is essential for normal and neoplastic cellular proliferation and survival. S everal common anticancer drugs exert their cytotoxic effects through intera ction with topo II. In experimental systems, altered topo II expression has been associated with the appearance of drug resistance. This mechanism, ho wever, does not adequately account for clinical cases of resistance to topo II-directed drugs. Modulation by protein-protein interactions represents o ne mechanism of topo II regulation that has not been extensively defined. O ur laboratory has identified 14-3-3 epsilon as a topo II-interacting protei n. In this study, glutathione S-transferase co-precipitation, affinity colu mn chromatography, and immunoprecipitations confirm the authenticity of the se interactions. Three assays evaluate the impact of 14-3-3 epsilon on dist inct topo II functional properties. Using both a modified alkaline comet as say and a DNA cleavage assay, we demonstrate that 14-3-3 epsilon negatively affects the ability of the chemotherapeutic, etoposide, to trap topo II in cleavable complexes with DNA, thereby preventing DNA strand breaks. By ele ctrophoretic mobility shift assay, this appears to be due to reduced DNA bi nding activity. The association of topo II with 14-3-3 proteins does not ex tend to all 14-3-3 isoforms, No protein interaction or disruption of topo I I function was observed with 14-3-3 sigma.