Transport of Cisplatin by the Copper Efflux Transporter ATP7B Roohangiz Safaei, Shinji Otani, Barrett J. Larson, Michael L. Rasmussen, and Stephen B. Howell Moores University of California San Diego Cancer Center, University of California, San Diego, La Jolla, California (R.S., M.L.R., S.B.H.); Department of Surgery, Hino Hospital, Hino-cho, Tottori Prefecture, Japan (S.O.); and Stanford University, School of Medicine, Stanford, California (B.J.L.) Received August 15, 2007; accepted October 29, 2007 ABSTRACT ATP7B is a P-type ATPase that mediates the efflux of copper. Recent studies have demonstrated that ATP7B regulates the cellular efflux of cisplatin (DDP) and controls sensitivity to the cytotoxic effects of this drug. To determine whether DDP is a substrate for ATP7B, DDP transport was assayed in vesicles isolated from Sf9 cells infected with a baculovirus that ex- pressed either the wild-type ATP7B or a mutant ATP7B that was unable to transport copper as a result of conversion of the transmembrane metal binding CPC motif to CPA. Only the wild-type ATP7B-expressing vesicles exhibited copper-depen- dent ATPase activity, copper-induced acyl-phosphate forma- tion, and ATP-dependent transport of copper. The amount of DDP that became bound was higher for vesicles expressing either type of ATP7B than for those not expressing either form of ATP7B, but only the vesicles expressing wild-type ATP7B mediated ATP-dependent accumulation of the drug. At pH 4.6, the vesicles expressing the wild-type ATP7B exhibited ATP- dependent accumulation of DDP with an apparent K m of 1.2  0.5 (S.E.M.) M and V max of 0.03  0.002 (S.E.M.) nmol/mg of protein/min. DDP also induced the acyl-phosphorylation of ATP7B but at a much slower rate than copper. Copper and DDP each inhibited the ATP-dependent transport of the other. These results establish that DDP is a substrate for ATP7B but is transported at a much slower rate than copper. Cisplatin (DDP) is currently one of the most commonly used anticancer drugs. However, the efficacy of DDP de- creases with repeated cycles of therapy because of the rapid development of resistance, which is often due to defects in the mechanisms for drug accumulation and/or efflux (Gately and Howell, 1993). The mechanism by which cells accumulate DDP is poorly understood, and at present, only a small num- ber of transporters are known to influence the uptake and efflux of this drug (Safaei et al., 2004). Recent studies have linked the efflux of DDP with the expression of copper ex- porters ATP7A and ATP7B. However, it is not known whether either ATP7A or ATP7B function as direct trans- porters of DDP. ATP7A and ATP7B are important constituents of the cop- per homeostasis system that has evolved to deliver copper to copper-requiring proteins while protecting the cells from toxic effects of copper (Culotta et al., 1999). The major copper uptake transporter is the copper transporter 1, which deliv- ers copper to pathway-specific chaperones such as ATOX1, CCS, and COX17 for delivery to the secretory compartment, cytosol, and mitochondria, respectively (Culotta et al., 1999). An important feature of copper transporters and chaperones is the presence of specific histidine-, methionine-, and cys- teine-rich metal binding domains that selectively bind cop- per(I) and exchange it with other copper homeostasis pro- teins (Huffman and O’Halloran, 2001). Recent data indicate that the copper homeostasis system also regulates the up- take, intracellular compartmentalization, and efflux of DDP (Katano et al., 2003; Samimi et al., 2004; Safaei and Howell, 2005). Available data are consistent with the concept that DDP mimics copper in being taken up by copper transporter 1, distributed to various intracellular compartments by the copper chaperones, and exported from tumor cells by ATP7A and ATP7B. However, given the exquisite selectivity of the copper homeostasis proteins for copper relative to other met- als, and for copper(I) rather than copper(II), it is not known whether the effects of these proteins on the transport of DDP are direct or indirect. ATP7B is abundantly expressed in liver and brain; muta- tions of ATP7B are the cause of Wilson’s disease and the development of liver cancer in humans and animal models (Terada et al., 1998). ATP7B is highly similar in structure and function to the other human P1-type ATPase, ATP7A, This work was supported by the National Institutes of Health grant CA78648-08 and a grant from the Clayton Medical Research Foundation, Inc. The production of 64 Cu at Washington University School of Medicine was supported by the National Cancer Institute grant R24-CA86307. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.040980. ABBREVIATIONS: DDP, cisplatin; BCS, bathocuproinedisulfonic acid; DTT, dithiothreitol; MOPS, 3-(N-morpholino)propanesulfonic acid. 0026-895X/08/7302-461–468$20.00 MOLECULAR PHARMACOLOGY Vol. 73, No. 2 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 40980/3294923 Mol Pharmacol 73:461–468, 2008 Printed in U.S.A. 461 at Univ Of California San Diego on August 26, 2009 molpharm.aspetjournals.org Downloaded from