The Role of Human Copper Transporter 1, hCTR1, in Copper and Cisplatin Entry in Mammalian Cells

Copper is a trace element that is important to drive a wide array of biochemical processes that are essential for life. Copper plays a major role in cellular respiration, iron metabolism, and skin and hair pigmentation to name a few. Elevated levels of copper are toxic and disruptions of copper homeostasis are seen in such human disorders as Wilson’s and Menkes disease. In order to maintain copper homeostasis, efficient mechanisms have evolved to facilitate copper import, copper export and cellular distribution of the metal. Copper uptake is regulated by the CTR (copper transporter) family of proteins, which are highly conserved. High affinity copper uptake is facilitated by CTR1 and CTR3 in Saccharomyces cerevisiae and by hCTR1 in humans. Cisplatin (cDDP) is an anti-cancer drug used in a number of malignancies including testicular, ovarian, cervical, bladder, lung, head, and neck cancers. Its use is limited by the development of resistance, often rationalized via effects on cellular uptake. It has been claimed that hCTR1, the human high affinity copper transporter, is the major entry pathway for cDDP and related drugs via a mechanism that mimics copper. We compared the uptake rates of copper with cDDP (and several analogs) into HEK293 cells over-expressing wild-type or mutant hCTR1, mouse embryonic fibroblasts (mefs) that do or do not express CTR1, and human ovarian tumor cells, sensitive or resistant to cDDP. We have also compared the effects of extracellular copper, which causes regulatory endocytosis of hCTR1, to those of cDDP. We confirm the correlation between higher hCTR1 levels and higher Pt-drug uptake in tumor cells sensitive to the drug. However, we show that hCTR1 is not the major entry route of platinum-drugs and that the copper transporter is not internalized in response to extracellular drug. Our data suggest the major entry pathway for platinum-drugs is not saturable at relevant concentrations and probably not protein-mediated. Clinical trials have been initiated that depend upon regulating membrane levels of hCTR1. If reduced drug uptake is a major factor in resistance, hCTR1 is unlikely to be a productive target in attempts to enhance efficacy, although the proteins involved in copper homeostasis may play a role. We also investigated three hCTR1-independent copper uptake systems. A chloride-dependent anion system was identified in intestinal and kidney cells. This anion uptake system is located on the apical membrane in polarized cells thus being appropriately localized to faciltate copper uptake from the diet. We studied HEK 293 cells overexpressing DMT1, the divalent metal transporter. Metal accumulation analysis showed that DMT1 transports both Fe and Mn, but neither Cu (I) nor Cu (II). CTR2 is highly homologous to CTR1 and has been suggested to facilitate low-affinity copper uptake or perhaps play a role in intracellular copper transport. We show that over-expresssed hCTR2 is partially localized at the plasma membrane in HEK 293 cells as shown by cell surface biotinylation and sucrose gradient fractionations. Measurement of 64Cu uptake rates into HEK cells overexpressing hCTR2 show that although expressed at the surface, hCTR2 transports copper very poorly, if at all.