IntroductionThe 90% survival rate of patients with testicular cancer highlights the effectiveness of the widely used chemotherapy drug cisplatin,cis-diamminedichloro-platinum(ii).1Although cisplatin and later-generation Pt drugs carboplatin and oxaliplatin (seeChart 1) are also used against other cancers, several drawbacks limit the survival rate observed in testicular cancer. In many cases, treatment is diminished by side effects of nephrotoxicity and neurotoxicity, as well as inherent and acquired resistance of many cancer types to this class of drug. Understanding the molecular basis for resistance is critical to discovering new treatment regimens in order to expand the effectiveness of these drugs.Clinically important Pt drugs.Cisplatin is administered as a formulation that includes 154 mM NaCl that suppresses hydrolysis of the chloride ligands from platinum. The high chloride ion concentration in the blood (∼105 mM) presumably allowscis-[Pt(NH3)2Cl2] to remain neutral until in enters the cytosol. Here it encounters a 4 mM chloride ion concentration which facilitates hydrolysis of the chloro ligands to form aquated [Pt(NH3)2(OH2)Cl]+, [Pt(NH3)2(OH)Cl], [Pt(NH3)2(OH2)2]2+, and [Pt(NH3)2(OH)2] species. The aquo and hydroxo forms are more reactive towards nucleophiles than the original chloro species, and upon entering the nucleus they bind to N7 positions on adjacent guanine bases of DNA. These intrastrand DNA cross-links are the primary biological targets that lead to cell death.1Any side reaction that diverts thecis-Pt(NH3)22+core from forming these toxic lesions on DNA is a potential pathway for drug inactivation, increased side effects, or resistance. Such pathways include inactivation of the Pt compounds by sulfur-containing molecules like glutathione, increased repair of the cell-killing Pt–DNA adducts, and decreased drug accumulation.2Of these mechanisms, the decreased accumulation of Pt drugs is the most common defect found in resistant cells and therefore demands critical attention in order to maximize the effectiveness of this well proven class of drugs across a wider range of cancer types.Observations that cisplatin uptake is not saturable, does not have a pH optimum, and is not inhibited by structural analogs lead to the long-standing hypothesis that cisplatin passively diffuses across cell membranes.1While passive diffusion remains a viable mechanism for Pt drug entry, current estimates suggest that only half of Pt drug uptake isviapassive diffusion, while the remainder enters by other transport mechanisms that are only beginning to be discovered and appreciated.3,4One of these is the cell-surface copper transport protein Ctr1.5Increasing and compelling evidence is revealing that cellular processes associated with Cu homeostasis are modulating Pt accumulation, particularly in resistant cells.6A connection between Cu and Pt trafficking was suggested by observations of bidirectional cross-resistance of Pt drugs and Cu in a variety of cell lines.5,7Cells selected for resistance to high levels of Cu were found to be resistant to Pt drugs; likewise, highly Pt-resistant cell lines were found to be moderately resistant to Cu.8The cytotoxic properties of these two metals are quite different, implying that their similar phenotype derives from impairment in shared uptake or efflux mechanisms. Work in yeast and mammalian cells identified the shared uptake mechanism as the transmembrane copper transporter Ctr1,9and both ATP7A and ATP7B, two structurally similarP-type ATPases that mediate Cu efflux from mammalian cells, have been implicated in removing Pt drugs from cells.5,7,10,11Yeast cells in which the CTR1 gene is deleted show increased resistance to cisplatin that correlates with reduced uptake of cisplatin and reduced formation of the critical Pt–DNA adducts compared with control cells.9,12Cisplatin has been shown to induce internalization and degradation of hCtr1 as well as impede the cellular uptake of both carboplatin and oxaliplatin.13,14These initial reports clearly establish a biological link between Ctr1 and Pt drugs; however, how cisplatin interacts with Ctr1 and how the two molecules modulate each other’s trafficking and function is unclear. Recent work from the Howell lab using murine embryonic fibroblasts in which both CTR1 alleles were deleted indicates that the copper transporter controls cellular accumulation of cisplatin, carboplatin, and oxaliplatin. Subsequently, Howellet al.observed that oxaliplatin at higher concentrations may use a different cellular entry mechanism.15In addition to these conventional mononuclear Pt drugs, an experimental polynuclear Pt compound BBR3464 has also been demonstrated to utilize hCtr1 to gain cellular access.16Studies of a variety of human cell lines in which hCtr1 is over-expressed reveal that simply increasing the level of hCtr1 does not always correlate with an increase in cell death by Pt drugs. For example, human ovarian carcinoma cells engineered to over-express hCtr1 accumulate more Pt than their counterparts, but this over-accumulation does not lead to increased Pt–DNA adducts or cisplatin sensitivity.7This implies that not all Pt drugs brought inviaa Ctr1-mediated pathway reach their DNA target. Not all cisplatin-resistant cell lines show reduction in endogenous Ctr1 expression levels compared with cisplatin-sensitive counterparts.17Similarly, at least one Pt-resistant cell line, a cervix squamous cell carcinoma, does not accumulate increased levels of cisplatin when it is engineered to over-express hCtr1.18However, because it is not clear that over-expressed Ctr1 functions normally in human cells, it is not at all obvious that its forced over-expression should be expected to lead to an increase in Pt accumulation.15More conflicting results describe the trafficking of Ctr1 in the presence of Pt drugs. In human ovarian carcinoma cells that endogenously or exogenously express hCtr1, cisplatin causes rapid disappearance of the transporter from the plasma membrane, suggesting that cisplatin down-regulates its own transporter in these cells.19On the other hand, Ctr1 expressed in human embryonic kidney cells is stabilized at the plasma membrane as a multimeric complex, and is not endocytosed or degraded upon Pt treatment.20These seemingly conflicting results expose the need to more fully understand the factors governing Pt drug access to cells in general and Pt–Ctr1 interactions in particular.Human hCtr1 contains three transmembrane regions that situate the amino terminus on the extracellular side with the carboxyl terminus facing the cytosol.21–24The protein likely exists as a homotrimer in its active form.21,25–27The unique feature of the amino-terminal region of Ctr1, which is the first gate of entry for cellular copper, is the presence of methionine-rich domains arranged as MXXM or MXM motifs containing 3–5 methionine residues per “Mets” motif. Yeast yCtr1 has 8 Mets motifs, with a total of 30 methionine residues in the ∼140-residue extracellular domain, whereas human hCtr1 has two Mets motifs in a 65-residue extracellular region.The extracellular N-terminal region of Ctr1 is critical for Ctr1-mediated Pt drug entry into cells. Pt drug accumulation studies with hCtr1 deletion mutants show that full-length and C-terminally truncated protein both transport Pt drugs, whereas an N-terminal deletion mutant fails to do so.17,28The N-terminal Mets motifs were also found to be responsible for stabilizing multimeric hCtr1 at the plasma membrane in HEK cells expressing hCtr1.20Mutagenesis of the eight methionines that make up the two Mets motifs of hCtr1 to alanines results in protein that appropriately traffics to the plasma membrane but does not trimerize upon cisplatin exposure, suggesting that the stable trimers observed in the parent protein result from cisplatin cross linking adjacent proteinsviamethionine binding. Further evidence for this model is provided by the fact that trimer formation is reversed upon exposure to Pt chelating compounds.20A study that used fluorescent fusion proteins to monitor molecular dynamics of yCtr1 found that Cu(i) induced FRET but that cisplatin, which accumulated in a Ctr1-dependent manner, did not.28This result highlights the fact that differences in the chemistry of copper and platinum translate into differences in metal-induced protein conformational changes and mechanisms of metal transport by Ctr1.Several lines of evidence point to Cu(i) as the active redox state for Ctr1,21,26,29–31and we have previously shown that isolated Mets domains are capable of binding Cu(i) selectively over Cu(ii).32Additionally, yCtr1 is unable to transport Zn(ii) or other divalent metal ions but readily takes in Ag(i) as a surrogate for Cu(i).27Given the fact that Ctr1 does not transport divalent metal ions and that it seems to be ideally suited for transporting soft, kinetically labile, monovalent cations, it would actually be quite surprising that this protein transports intact Pt(ii) coordination compounds. Apart from the fact that both Cu(i) and Pt(ii) prefer soft, sulfur-rich coordination environments, their coordination chemistry otherwise is quite different. Whereas Cu(i) can adopt linear, trigonal, or tetrahedral coordination geometries, Pt(ii) is rigorously square planar. In addition, the ligands on Pt(ii) are less likely to be kinetically labile, although inner-sphere ligand exchange reactions can occur, with their likelihood depending on thetranseffect of the ligands.The affinity of Pt(ii) for sulfur-containing ligands has provoked a long-standing question of how Pt drugs reach their DNA targets in spite of significant competition for binding to S-containing amino acids and proteins.33,34Because neuro- and nephrotoxicity associated with Pt drug treatment has been attributed to S-containing proteins, and because intracellular S-containing compounds like glutathione may act as intracellular drug reservoirs,33numerous studies have been done to describe the interactions of simple amino acids like cysteine, methionine, and glutathione with various Pt(ii) compounds.35–42These studies have revealed that the thioether of methionine can displace chloro or aqua ligands of cisplatin. Thioether sulfur has a strongtransinfluence, therefore methionine coordination to square-planarcis-diaminePt(ii) core can result in loss of the amine ligandtransto the incoming thioether. Many model studies have used stable tridentate chelates of diethylenetriamine (dien) in order to suppress ligand loss and focus exclusively on the Pt–S bonding interaction.43–49However, the potential replacement of the pendant ligands on Pt drugs will have important repercussions on the mechanism of action of Ctr1-transported Pt, since thecis-diamminePt(ii) core is the active metal complex that binds to DNA to affect its cytotoxic action. Therefore we focus here exclusively on the interactions of hCtr1 peptides with the clinically important drugs themselves. Methionine–platinum adducts are not unique to human Ctr1 species as work by Arnesanoet al. have shown that in the presence of a methionine-rich sequence of yCtr1, cisplatin is stripped of its ligands and binds in a 1 : 1 ratio.50,51Certain Pt(ii) complexes selectively hydrolyze peptide amide bonds by first anchoring onto Met or His side chains.52–54Cisplatin is included in this family of inorganic hydrolases, and has been shown to cleave the second amide bond upstream from an anchoring Met in relatively simple acetylated di- and tripeptides.55–57These model studies strongly encourage the examination of peptides containing multiple Met residues such as those found in hCtr1 to determine if hydrolysis is a possible mechanism of action.The differences in the coordination chemistry of Cu(i) and Pt(ii) raise two fundamental questions related to Ctr1-mediated Pt drug uptake: (1) What happens to Pt drugs upon interacting with the extracellular domains of Ctr1? Our hypothesis is that methionine residues in the Mets domains induce ligand exchange reactions that change the molecular speciation of the original drug, which has important ramifications for its overall efficacy. (2) How does the interaction of the protein with Pt drugs change the integrity of the protein itself? Given the proclivity of Pt compounds to hydrolyze amide bonds, it is possible that Pt drugs may degrade hCtr1. To gain insight into these questions, we examined the reaction products of model Mets peptides derived from human hCtr1 with Pt drugs by combined liquid-chromatography–mass spectrometry (LC–MS).