The Copper Export Pump ATP7B Modulates the Cellular Pharmacology of Carboplatin in Ovarian Carcinoma Cells KUNIYUKI KATANO, ROOHANGIZ SAFAEI, GOLI SAMIMI, ALISON HOLZER, MYRIAM ROCHDI, and STEPHEN B. HOWELL Department of Medicine and the Cancer Center, University of California, San Diego, La Jolla, California (K.K., R.S., G.S., A.H., S.B.H); and Globomax, Inc., Hanover, Maryland (M.R.) Received January 21, 2003; accepted April 30, 2003 This article is available online at http://molpharm.aspetjournals.org ABSTRACT Human tumor cells lines with acquired resistance to cisplatin (DDP) and carboplatin (CBDCA) are often cross-resistant to copper and vice versa, and some DDP-resistant cells overex- press the copper export pump ATP7B. We sought to demon- strate that ATP7B directly mediates resistance to DDP and CBDCA by stably transfecting human carcinoma cells with a vector designed to express ATP7B. Increased expression of ATP7B rendered all three cell lines tested more resistant to a 1-h exposure to DDP (1.6 –2.6-fold), CBDCA (1.5–1.6-fold), and copper (1.2–1.4-fold). The effect of ATP7B on the cellular phar- macology of 64 Cu and [ 14 C]CBDCA was investigated in more detail using one cell pair (2008 cells transfected with an empty vector or an ATP7B-expressing vector). In the 2008/ATP7B subline, steady-state copper levels were decreased under both basal and copper-supplemented conditions, as was steady- state CBDCA content upon exposure to 50 M[ 14 C]CBDCA. Over the first 5 min, the average rate of accumulation of copper and CBCDA in the 2008/ATP7B cells was reduced by 37 and 61%, respectively. Efflux was more rapid from 2008/ATP7B cells for both copper and CBDCA. Two-compartment modeling indicated that the second phase of efflux was increased by a factor of 3.9-fold for CBCDA and to an even greater extent for copper. We conclude that expression of ATP7B regulates sen- sitivity to CBDCA as well as to DDP and copper and that a transporter that normally mediates copper homeostasis mod- ulates the cellular pharmacology of CBDCA. Acquired resistance to DDP and CBDCA is currently be- lieved to be multifactorial in origin; however, impaired accu- mulation of drug is the single most commonly observed al- teration when cells selected for resistance are compared with the sensitive cells from which they were derived (Andrews and Howell, 1990). The mechanisms by which DDP and CB- DCA enter and exit from cells are not well characterized. Cellular accumulation of these drugs is relatively slow com- pared with many other classes of chemotherapeutic agents. Given their degree of hydrophilicity and polarity, transport- ers are likely to be required for both influx and efflux. Indeed, uptake is influenced by factors that suggest the involvement of both active and passive transporter-mediated processes (Gately and Howell, 1993). One curious feature of cells with acquired DDP resistance is that they exhibit cross-resistance to a wide variety of metalloids, including arsenite (Naredi et al., 1995), antimony (Naredi et al., 1995; Chen et al., 1998), and cadmium (Lee et al., 1995). Recently, we reported that these cells are also cross-resistant to copper (Katano et al., 2002a), and that cells selected for acquired resistance to copper are cross-resistant to DDP (Safaei and Howell, 2001). Copper homeostasis is maintained by a complex system of transporters and chaper- ones that serve to both protect Cu(I) against oxidation and to prevent the production of toxic reactive oxygen species as the copper enters and is distributed throughout the cell (O’Halloran and Culotta, 2000). The central feature of this system is a group of proteins with unique metal binding sequences that chelate copper into protective pockets and exchange it through intimate protein-protein interactions such that copper is virtually never free in the cell (Pufahl et al., 1997; Rae et al., 1999). The primary uptake transporter for copper is hCTR1 (Zhou and Gitschier, 1997; Moller et al., 2000), and its function in mammals is essential for embryonic survival (Kuo et al., 2001; Lee et al., 2001). hCTR1 transfers copper to one of three known copper chaperones: Cox17, hCCS1, and HAH1. Cox17 delivers copper to cytochrome c oxidase, hCCS1 transfers copper to SOD1, and HAH1 hands it to one or another of the two P-type ATPases, ATP7A and ATP7B, that are reported to sequester copper into the trans- Golgi network where it is loaded onto copper-dependent en- zymes in the secretory pathway and subsequently exported This study was supported in part by National Institutes of Health grant CA78648. This work was conducted in part by the Clayton Foundation for Research-California Division. R.S. and S.B.H. are Clayton Foundation inves- tigators. ABBREVIATIONS: DDP, cisplatin; CBDCA, carboplatin; GFP, green fluorescent protein; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; CMV, cytomegalovirus. 0026-895X/03/6402-466 –473$7.00 MOLECULAR PHARMACOLOGY Vol. 64, No. 2 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 2418/1080008 Mol Pharmacol 64:466–473, 2003 Printed in U.S.A. 466 at ASPET Journals on June 21, 2017 molpharm.aspetjournals.org Downloaded from