Lustrous insights into cisplatin accumulation: copper transporters.

As a relatively polar molecule, CDDP is thought to use specific plasma membrane systems for passage into cells, although entry by passive diffusion is also likely to occur. Alterations in these plasma membrane systems have been considered to be important resistance factors because one of the most consistent features of CDDP-resistant cell lines is decreased intracellular drug levels (1). Whereas the membrane determinants of accumulation have therefore been of keen interest, their molecular identities have gone undefined for many years. An interesting and noteworthy advance in this area has been the recent determination that the same proteins that mediate transport of copper are also capable of transporting CDDP. In this issue of Clinical Cancer Research an analysis of the potential for a copper transporter to impact treatment of ovarian cancer patients with platinum-based regimens is reported (2). Copper is an essential metal that functions as a cofactor for enzymes involved in diverse metabolic pathways, including cytochrome c oxidase (the terminal enzyme of the mitochondrial respiratory chain), superoxide dismutase (an antioxidant that represents 1% of cellular protein), and dopamine-monoxygenase (catecholamine biosynthesis). Although copper is a required cofactor, it is also highly reactive in the cell and, when present in excess, exerts significant toxicity consequent to the generation of free radicals. Thus, copper is subject to complex homeostatic mechanisms that maintain appropriate intracellular concentrations and restrict its ability to inflict intracellular damage. This process is accomplished by the action of specific copper transporters and chaperones (3, 4). Copper influx into the cell is mediated by Ctr1, a high affinity copper transporter. Specific chaperones, known as COX17, CCS, and HAH1, respectively, then deliver copper from the plasma membrane to the mitochondria, to cytoplasmic superoxide dismutase, and to two copper transporters located in the trans-Golgi apparatus. These two copper transporters, ATP7A and ATP7B, are members of the P-type ATPase family of cation transporters and are the products of the genes affected in two disorders of copper accumulation in humans, Menkes disease and Wilson disease, respectively (5, 6). In the liver, ATP7B transports copper into the trans-Golgi either for incorporation into copper-requiring proteins, or for extrusion into the bile, the latter of which is accomplished by vesicular trafficking from the trans-Golgi to the canalicular (apical) surface of hepatocytes and represents a major route of copper elimination from the body. In most other tissues, copper transport into the trans-Golgi is accomplished by ATP7A. Like ATP7B, ATP7A shuttles between the trans-Golgi and the plasma membrane in a process that mediates copper extrusion from the cell. Trafficking of ATP7A and ATP7B to the plasma membrane is thought to be regulated by intracellular copper levels (7). Copper uptake is also regulated by copper levels, at least as determined for Ctr1p, a yeast homologue of Ctr1, in that both CTR1 transcription and Ctr1p degradation are regulated by an usual mechanism involving a copper-sensing transcription factor (8, 9). The first insight into the involvement of copper transporters in the cellular pharmacology of CDDP was provided by Komatsu et al. (10), who found that ectopic expression of ATP7B in KB-3-1 cells conferred CDDP resistance associated with decreased accumulation of this agent (Fig. 1). In addition, they reported increased expression of ATP7B in a CDDPresistant prostate cancer cell line. This study thus indicated that ATP7B had the facility for extruding CDDP from the cell and that the pump could be induced as a resistance factor. The involvement of copper transporters in CDDP resistance was extended to copper uptake systems by Ishida et al. (11) who used transposon mutagenesis to identify CDDP resistance factors in yeast and found that inactivation of CTR1 was able to confer resistance to CDDP. In accord with the notion that Ctr1p is able to mediate uptake of CDDP, Ctr1p-deficient yeast exhibited decreased levels of CDDP bound to DNA and decreased CDDP accumulation. A functional link between CDDP and copper transport in the context of Ctr1p was further elucidated in wild-type yeast by experiments showing that copper and CDDP behaved as mutual competitive inhibitors, as would be expected were the two compounds common substrates for Ctr1p. That is, copper was able to reduce CDDP accumulation and attenuate CDDP toxicity, and conversely, CDDP could reduce copper accumulation. It was also demonstrated that, like copper, CDDP is able to stimulate degradation of Ctr1p. Finally, Ishida et al. (11) went on to demonstrate, by analyzing the CDDP sensitivity of embryonic stem cells that are homozygous for deletion of Ctr1, that mammalian Ctr1 is capable of mediating the uptake of CDDP and thereby functioning as a sensitivity factor for this agent. In combination, these surprising findings on ATP7B and Ctr1 established that mammalian transporters that mediate influx and efflux of copper are also capable of transporting CDDP. These studies have been extended by reports showing that ATP7B is also able to confer resistance to carboplatin and that human Ctr1 is able to mediate the uptake of carboplatin and oxaliplatin, in addition to CDDP (12, 13). Although an analysis of the capabilities ATP7A for conferring CDDP resistance in transfected cells has yet to be reported, the high degree of structural similarity between ATP7B and ATP7A (67%) and the finding that ATP7A is overexpressed in certain CDDP-resistant cell lines suggest that this is likely to be the case (14). Several studies have now evaluated expression of ATP7B Received 8/29/03; accepted 9/11/03. Grant support: NIH Grant CA73728, National Cancer Institute Core Grant CA06927, and an appropriation from the Commonwealth of Pennsylvania to the Fox Chase Cancer Center. Requests for reprints: Gary D. Kruh, Medical Science Division, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111. 1 The abbreviations used are: CDDP, cisplatin; MRP, multidrug resistance protein. 5807 Vol. 9, 5807–5809, December 1, 2003 Clinical Cancer Research