Maleimide Is a Potent Inhibitor of Topoisomerase II in Vitro and in Vivo: A New Mode of Catalytic Inhibition LARS H. JENSEN, AXELLE RENODON-CORNIERE, IRENE WESSEL, SEPPO W. LANGER, BIRGITTE SØKILDE, ELISABETH V. CARSTENSEN, MAXWELL SEHESTED, and PETER B. JENSEN Laboratory for Experimental Medical Oncology, Finsen Center, Copenhagen, Denmark (L.H.J., S.W.L., P.B.J.); Department of Pathology, Laboratory Center, Copenhagen, Denmark (L.H.J., A.-R.C., I.W., M.S.); and Topo Target A/S, Copenhagen, Denmark (L.H.J., I.W., S.W.L., B.S., E.V.C., M.S., P.B.J.) Received September 13, 2001; accepted February 13, 2002 This article is available online at http://molpharm.aspetjournals.org ABSTRACT Maleimide, N-ethyl-maleimide (NEM), and N-methyl-maleimide (NMM) were identified as potent catalytic inhibitors of purified human topoisomerase II, whereas the ring-saturated analog succinimide was completely inactive. Catalytic inhibition was not abrogated by topoisomerase II mutations that totally abol- ish the effect of bisdioxopiperazine compounds on catalytic inhibition, suggesting a different mode of action by these ma- leimides. Furthermore, in DNA cleavage assay maleimide and NEM could antagonize etoposide-induced DNA double-strand breaks. Consistently, maleimide could antagonize the effect of topoisomerase II poisons in three different in vivo assays: 1) In an alkaline elution assay maleimide protected against etopo- side-induced DNA damage. 2) In a band depletion assay ma- leimide reduced etoposide-induced trapping of topoisomerase II and  on DNA. 3) In a clonogenic assay maleimide antago- nized the cytotoxicity of etoposide and daunorubicin on four different cell lines of human and murine origin. at-MDR cell lines with reduced nuclear topoisomerase II content are fully sen- sitive to maleimide, indicating that it is not a topoisomerase II poison in vivo. Our finding that topoisomerase II is sensitive to maleimide, NMM, and NEM but insensitive to succinimide dem- onstrates a strict requirement for the unsaturated ring bond for activity. We suggest that the observed antagonism in vitro and in vivo is caused by covalent modification of topoisomerase II cysteine residues reducing the amount of catalytically active enzyme sensitive to the action of topoisomerase II poisons. Type II topoisomerases are nuclear enzymes that regulate the topology of DNA in all living cells (Wang, 1996) by trans- ferring one DNA double helix (the transfer-DNA segment) through a transient break in another DNA double helix (the gate-DNA segment) (Roca and Wang, 1992, 1994). Cellular topoisomerase II continuously creates transient protein-linked double-strand breaks in DNA, and it is this conformation called the cleavable complex, that is the target of many clinically important anticancer drugs used today, such as the anthracyclines (daunorubicin and doxorubicin), the epipodophyllotoxins (etoposide and teniposide), and the aminoacridines (m-AMSA). Because these drugs stabilize the cleavable complex that leads to the accumulation of cytotoxic DNA breaks, they are referred to as DNA topoisomerase II poisons (Liu, 1989; Chen and Liu, 1994). Another major class of topoisomerase II-directed drugs is the catalytic inhibitors. They inhibit the catalytic activity of topoisomerase II without generating DNA breaks, thus de- priving cells of essential topoisomerase II catalytic activity also leading to cell death (Andoh and Ishida, 1998). Aclaru- bicin is known to intercalate into DNA, thereby preventing the binding of topoisomerase II to DNA (Jensen et al., 1990, 1991). Chloroquine, which also intercalates into DNA, dis- plays a pH-dependent inhibition of cellular topoisomerase II activity as its diffusion across the cell membrane is facili- tated at lowered pH (Langer et al., 1999). Finally, merbarone prevents cleavage of the gate-DNA strand acting at the same stage in the catalytic cycle as the topoisomerase II poisons (Fortune and Osheroff, 1998). The bisdioxopiperazine compounds stabilize a transient configuration of topoisomerase II on DNA called the post strand passage closed-clamp configuration, thereby prevent- ing enzymatic turnover (Ishida et al., 1991; Tanabe et al., 1991; Roca et al., 1994; Morris et al., 2000). Due to the inhibition of enzymatic turnover, the bisdioxopiperazine compounds are classified as catalytic topoisomerase II inhib- itors. However, recent data suggest that the closed-clamp configuration of topoisomerase II on DNA may act as a new kind of noncovalent poison interfering with metabolic DNA processes (van Hille and Hill, 1998; Wessel et al., 1999; Jensen et al., 2000a,b; Kobayashi et al., 2001). Despite this This study was supported by The Danish Cancer Society and the Novo Nordisk Foundation. ABBREVIATIONS: m-AMSA, amsacrine; NMM, N-methyl-maleimide; NEM, N-ethyl-maleimide; wt, wild-type; kDNA, kinetoplast DNA; BSA, bovine serum albumin; DTT, dithiothreitol; DSB, double-strand break; SSB, single-strand break. 0026-895X/02/6105-1235–1243$7.00 MOLECULAR PHARMACOLOGY Vol. 61, No. 5 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1353/981199 Mol Pharmacol 61:1235–1243, 2002 Printed in U.S.A. 1235 at ASPET Journals on April 3, 2017 molpharm.aspetjournals.org Downloaded from