A copper connection to the uptake of platinum anticancer drugs.

C isplatin [cis-diamminedichloroplatinum (II)] is a highly active anticancer agent. This agent is curative against most testicular cancers and is highly active against a wide range of other tumor types, notably ovarian, bladder carcinoma, and non-small-cell lung cancer (1). Treatment failure is frequently caused by the development of resistance to cisplatin. Although the insensitive tumors generally exhibit only low-level resistance, the use of cisplatin at close to its maximally tolerated dose implies that the developed resistance eliminates cisplatin as an active compound. Resistance to cisplatin has been widely studied in a variety of models and in clinical samples, but it has been difficult to identify the molecular changes leading to drug resistance. Ishida et al. (2) report in this issue of PNAS the identification of an important pathway for uptake of cisplatin into yeast and mammalian cells, uptake mediated by a high-affinity copper transporter. The action of cisplatin in cell killing is now well established (3). In serum, the high concentration of chloride ions enhances cisplatin stability. The lower chloride concentration in cells favors rapid hydrolysis of the chloride ligands of cisplatin, leading to an activated molecule that is capable of reacting bifunctionally. Although cisplatin can react with a variety of cellular macromolecules, there is strong evidence that the most important target is DNA (3, 4). An important line of evidence that DNA is an essential target of cisplatin is the observation that bacterial and yeast mutants that are defective in various DNA-repair pathways are also hypersensitive to cisplatin. Cisplatin can form both intrastrand and interstrand DNA crosslinks, with intrastrand purine:purine representing the majority of the adducts. As is the case with other anticancer agents, reduced accumulation of cisplatin is frequently observed in cisplatinresistant cell lines. Although drug efflux has been intensively studied as a mechanism of drug resistance, relatively few studies have demonstrated a role for reduced drug uptake in acquired drug resistance. However, cisplatin has been an exception to this generalization, and several authors have suggested that decreased uptake of cisplatin is an important factor that can result in drug resistance (5). Whereas some considerations have led to the suggestion that a major mechanism for cisplatin uptake is passive diffusion, other studies have suggested a role for active transport. For example, reactive aldehydes, such as benzaldehyde, are able to block cisplatin accumulation, suggesting modification of a membrane protein required for cisplatin uptake (reviewed in ref. 6). To identify yeast genes that play a role in sensitivity to cisplatin, Ishida et al. started with a simple genetic approach. By using the transposon mutagenesis approach of Snyder and coworkers (7), they screened yeast loss-of-function mutants for cisplatin resistance. An advantage of the transposon mutagenesis approach is that in general only one gene is mutated per cell and the mutation is usually a complete loss of function. In addition, the mutated gene is marked by the transposon, greatly simplifying identification of the mutated gene. This approach is similar to that used in recent studies that have employed a set of yeast strains carrying deletions in all ORFs encoding nonessential genes. The deletion set has been used to characterize, on a genome scale, genes required for repair of DNA damage caused by a variety of different DNAdamaging agents (8–10). Both approaches allow efficient identification of all loss-offunction mutations that result in a specific phenotype. The strain that generated the highest level of cisplatin resistance carried a mutation in the MAC1 gene, a transcription factor that regulates the catalase genes, as well as genes required for the uptake of iron and copper (11). Each of the known genes regulated by MAC1 were sequentially deleted, and it was observed that deletion of the gene encoding the highaffinity copper transporter CTR1 nearly recapitulated the cisplatin resistance that was observed in mac1-deficient cells. This result indicated that the resistance seen in mac1 mutants was principally caused by a failure to express CTR1. Interestingly, deletion of a second yeast high-affinity copper transporter CTR3 produced only a minor effect on cisplatin sensitivity. Ishida et al. carefully exclude the possibility that cisplatin resistance caused by ctr1 arises from indirect effects, rather than the ability of CTRp to transport cisplatin into cells. For example, because mutation of ctr1 results in an impairment of copper uptake, activities of enzymes, such as superoxide dismutase, that rely on copper or iron can be impaired. A series of deletion strains carrying defects in genes such as SOD1 (superoxide dismutase), FET3 (a protein encoding a ferro-O2oxidoreductase that is part of the highaffinity iron transport system), LYS7 (a copper chaperone for superoxide dismutase that is important for protection from oxidative stress and which also confers an auxotrophy for lysine), and others were examined, but none of the single mutants resulted in significant enhancement of cisplatin resistance. The final key point in the demonstration that CTR1p plays an important role in cisplatin influx was that ctr1 results in a reduction of cisplatin levels in yeast cells, but does not