Copper transporter 2 regulates endocytosis and controls tumor growth and sensitivity to cisplatin in vivo.

Copper transporter 2 (CTR2) is one of the four copper transporters in mammalian cells that influence the cellular pharmacology of cisplatin and carboplatin. CTR2 was knocked down using a short hairpin RNA interference. Robust expression of CTR2 was observed in parental tumors grown in vivo, whereas no staining was found in the tumors formed from cells in which CTR2 had been knocked down. Knockdown of CTR2 reduced growth rate by 5.8-fold, increased the frequency of apoptotic cells, and decreased the vascular density, but it did not change copper content. Knockdown of CTR2 increased the tumor accumulation of cis-diamminedichloroplatinum(II) [cisplatin (cDDP)] by 9.1-fold and greatly increased its therapeutic efficacy. Because altered endocytosis has been implicated in cDDP resistance, uptake of dextran was used to quantify the rate of macropinocytosis. Knockdown of CTR2 increased dextran uptake 2.5-fold without reducing exocytosis. Inhibition of macropinocytosis with either amiloride or wortmannin blocked the increase in macropinocytosis mediated by CTR2 knockdown. Stimulation of macropinocytosis by platelet-derived growth factor coordinately increased dextran and cDDP uptake. Knockdown of CTR2 was associated with activation of the Rac1 and cdc42 GTPases that control macropinocytosis but not activation of the phosphoinositide-3 kinase pathway. We conclude that CTR2 is required for optimal tumor growth and that it is an unusually strong regulator of cisplatin accumulation and cytotoxicity. CTR2 regulates the transport of cDDP in part through control of the rate of macropinocytosis via activation of Rac1 and cdc42. Selective knockdown of CTR2 in tumors offers a strategy for enhancing the efficacy of cDDP.

Copper transporters and the cellular pharmacology of the platinum-containing cancer drugs.

Multiple lines of evidence indicate that the platinum-containing cancer drugs enter cells, are distributed to various subcellular compartments, and are exported from cells via transporters that evolved to manage copper homeostasis. The cytotoxicity of the platinum drugs is directly related to how much drug enters the cell, and almost all cells that have acquired resistance to the platinum drugs exhibit reduced drug accumulation. The major copper influx transporter, copper transporter 1 (CTR1), has now been shown to control the tumor cell accumulation and cytotoxic effect of cisplatin, carboplatin, and oxaliplatin. There is a good correlation between change in CTR1 expression and acquired cisplatin resistance among ovarian cancer cell lines, and genetic knockout of CTR1 renders cells resistant to cisplatin in vivo. The expression of CTR1 is regulated at the transcriptional level by copper via Sp1 and at the post-translational level by the proteosome. Copper and cisplatin both trigger the down-regulation of CTR1 via a process that involves ubiquitination and proteosomal degradation and requires the copper chaperone antioxidant protein 1 (ATOX1). The cisplatin-induced degradation of CTR1 can be blocked with the proteosome inhibitor bortezomib, and this increases the cellular uptake and the cytotoxicity of cisplatin in a synergistic manner. Copper and platinum(II) have similar sulfur binding characteristics, and the presence of stacked rings of methionines and cysteines in the CTR1 trimer suggest a mechanism by which CTR1 selectively transports copper and the platinum-containing drugs via sequential transchelation reactions similar to the manner in which copper is passed from ATOX1 to the copper efflux transporters.

The role of the methionines and histidines in the transmembrane domain of mammalian copper transporter 1 in the cellular accumulation of cisplatin.

Mammalian copper transporter 1 (CTR1) is a high-affinity copper influx transporter that also mediates the uptake of platinum-containing chemotherapeutic agents including cisplatin (cDDP). Methionines 150, 154, and histidine 139 have been proposed to form a series of stacked rings in the pore formed by the CTR1 homotrimer, each of which is required for maximal copper transport. To examine the mechanism by which hCTR1 also transports cDDP, variant forms of hCTR1 in which methionines 150 and 154 were converted to isoleucines or in which histidine 139 was converted to alanine were re-expressed in cells in which both alleles of CTR1 had been knocked out. Each of these conversions disabled copper transport and increased cellular resistance to the cytotoxic effect of copper. In contrast, conversion of the methionines increased the uptake and cytotoxicity of cDDP well above that attained with wild-type hCTR1. Conversion of His139 to alanine did not impair cDDP uptake and actually enhanced cytotoxicity. Thus, although Met150 and Met154 facilitate the movement of copper through the pore, they serve to obstruct the passage of cDDP. None of the modifications altered the ability of cDDP to trigger the degradation of hCTR1, indicating that cDDP must interact with hCTR1 at other sites as well. Although both copper and cDDP may rely on a series of transchelation reactions to pass through the hCTR1 trimeric complex, the details of the molecular interactions must be different, which provides a potential basis for selective pharmacological modulation of copper versus cDDP cytotoxicity.

Regulation of copper transporter 2 expression by copper and cisplatin in human ovarian carcinoma cells.

Down-regulation of copper transporter 1 (CTR1) reduces uptake and sensitivity, whereas down-regulation of CTR2 enhances both. Cisplatin (DDP) triggers the rapid degradation of CTR1 and thus limits its own accumulation. We sought to determine the effect of DDP and copper on the expression of CTR2. Changes in CTR1 and CTR2 mRNA and protein levels in human ovarian carcinoma 2008 cells and ATOX1(+/+) and ATOX1(-/-) mouse embryo fibroblasts in response to exposure to DDP and copper were measured by quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and deconvolution microscopy. DDP triggered rapid degradation of CTR1 in 2008 human ovarian cancer cells. However, it increased the expression of CTR2 mRNA and protein levels. Expression of CTR2 was heavily modulated by changes in intracellular copper concentration; copper depletion produced rapid disappearance of CTR2, whereas excess copper increased the level of CTR2 protein. This increase was associated with an increase in CTR2 mRNA and prolongation of the CTR2 half-life. Consistent with prior observations that short hairpin RNA interference-mediated knockdown of CTR2 enhanced DDP uptake and tumor cell kill, reduction of CTR2 by copper starvation also enhanced DDP uptake and cytotoxicity. Comparison of the ability of copper and DDP to modulate the expression of CTR1 in ATOX1(+/+) and ATOX1(-/-) indicated that ATOX1 participates in the regulation of CTR2 expression. Unlike CTR1, the expression of CTR2 is increased rather than decreased by DDP. Therefore, these two copper transporters have opposite effects on DDP sensitivity. CTR2 expression is regulated by copper availability via the copper-dependent regulator ATOX1.

The role of the mammalian copper transporter 1 in the cellular accumulation of platinum-based drugs.

The mammalian copper transporter 1 (CTR1) is responsible for the uptake of copper from the extracellular space. In this study, we used an isogenic pair of CTR1(+/+) and CTR1(-/-) mouse embryo fibroblasts to examine the contribution of CTR1 to the influx of cisplatin (DDP), carboplatin (CBDCA), oxaliplatin (L-OHP), and transplatin. Exposure to DDP triggered the rapid degradation of CTR1, suggesting that its contribution to influx was likely to be on the initial phase of drug entry. Loss of CTR1 decreased the initial binding of DDP to cells and reduced influx measured over the first 5 min of drug exposure by 81%. Loss of CTR1 almost completely eliminated the initial influx of CBDCA and reduced the initial uptake of L-OHP by 68% but had no effect on the influx of transplatin. Loss of CTR1 rendered cells resistant to even high concentrations of DDP when measured in vitro, and re-expression of CTR1 in the CTR1(-/-) cells restored both DDP uptake and cytotoxicity. The growth of CTR1(-/-) tumor xenografts in which CTR1 levels were restored by infection with a lentivirus expressing wild-type CTR1 was reduced by a single maximum tolerated dose of DDP in vivo, whereas the CTR1(-/-) xenografts failed to respond at all. We conclude that CTR1 mediates the initial influx of DDP, CBDCA, and L-OHP and is a major determinant of responsiveness to DDP both in vitro and in vivo.

Transport of cisplatin by the copper efflux transporter ATP7B.

ATP7B is a P-type ATPase that mediates the efflux of copper. Recent studies have demonstrated that ATP7B regulates the cellular efflux of cisplatin (DDP) and controls sensitivity to the cytotoxic effects of this drug. To determine whether DDP is a substrate for ATP7B, DDP transport was assayed in vesicles isolated from Sf9 cells infected with a baculovirus that expressed either the wild-type ATP7B or a mutant ATP7B that was unable to transport copper as a result of conversion of the transmembrane metal binding CPC motif to CPA. Only the wild-type ATP7B-expressing vesicles exhibited copper-dependent ATPase activity, copper-induced acyl-phosphate formation, and ATP-dependent transport of copper. The amount of DDP that became bound was higher for vesicles expressing either type of ATP7B than for those not expressing either form of ATP7B, but only the vesicles expressing wild-type ATP7B mediated ATP-dependent accumulation of the drug. At pH 4.6, the vesicles expressing the wild-type ATP7B exhibited ATP-dependent accumulation of DDP with an apparent K(m) of 1.2 +/- 0.5 (S.E.M.) muM and V(max) of 0.03 +/- 0.002 (S.E.M.) nmol/mg of protein/min. DDP also induced the acyl-phosphorylation of ATP7B but at a much slower rate than copper. Copper and DDP each inhibited the ATP-dependent transport of the other. These results establish that DDP is a substrate for ATP7B but is transported at a much slower rate than copper.

Role of copper transporters in the uptake and efflux of platinum containing drugs.

Cellular mechanisms for the uptake, intracellular distribution and efflux of the platinum (Pt) containing compounds cisplatin (DDP), carboplatin (CBDCA) and oxaliplatin (LOHP) are unknown. Current data suggest that specialized transporters/carriers mediate the transport of Pt drugs across the cellular membranes. Specific roles for the copper (Cu) transporters CTR1, ATP7A and ATP7B have been demonstrated during recent years. The finding that in cultured cells and tumor samples a correlation can be found between the expression of Cu transporters and the degree of the acquired resistance to Pt drug suggests that the Cu transporters are important constituents of the program that regulates sensitivity to Pt drugs. A model is presented that describes the function of Cu transporters in the regulation of Pt drug uptake and efflux.

Intracellular localization and trafficking of fluorescein-labeled cisplatin in human ovarian carcinoma cells.

PURPOSE: We sought to identify the subcellular compartments in which cisplatin [cis-diamminedichloroplatinum (DDP)] accumulates in human ovarian carcinoma cells and define its export pathways. EXPERIMENTAL DESIGN: Deconvoluting digital microscopy was used to identify the subcellular location of fluorescein-labeled DDP (F-DDP) in 2008 ovarian carcinoma cells stained with organelle-specific markers. Drugs that block vesicle movement were used to map the traffic pattern. RESULTS: F-DDP accumulated in vesicles and were not detectable in the cytoplasm. F-DDP accumulated in the Golgi, in vesicles belonging to the secretory export pathway, and in lysosomes but not in early endosomes. F-DDP extensively colocalized with vesicles expressing the copper efflux protein, ATP7A, whose expression modulates the cellular pharmacology of DDP. Inhibition of vesicle trafficking with brefeldin A, wortmannin, or H89 increased the F-DDP content of vesicles associated with the pre-Golgi compartments and blocked the loading of F-DDP into vesicles of the secretory pathway. The importance of the secretory pathway was confirmed by showing that wortmannin and H89 increased whole cell accumulation of native DDP. CONCLUSIONS: F-DDP is extensively sequestered into vesicular structures of the lysosomal, Golgi, and secretory compartments. Much of the distribution to other compartments occurs via vesicle trafficking. F-DDP detection in the vesicles of the secretory pathway is consistent with a major role for this pathway in the efflux of F-DDP and DDP from the cell.

Acquisition of resistance to cisplatin is accompanied by changes in the cellular pharmacology of copper.

Impaired uptake of cisplatin (DDP) consistently accompanies the acquisition of resistance to the platinum drugs. The pathways by which DDP enters or exits from cells remain poorly defined. Using three pairs of human ovarian carcinoma cell lines, each consisting of a sensitive parental line and a stably DDP-resistant subline derived by in vitro selection, resistance to DDP was found to be accompanied by cross-resistance to Cu. Accumulation of DDP in the resistant sublines ranged from 38 to 67% of that in the parental line at 1 h, and DNA adduct formation varied from 10 to 38% of that in the sensitive cells. The DDP-resistant cells had 22-56% lower basal levels of copper, and the copper levels were only 27-46% of those observed in the sensitive parental lines after a 24-h exposure to medium supplemented with copper. The initial influx rate for DDP in the three resistant cell lines ranged from 23 to 55% of that in the sensitive cells of each pair; the initial influx rate for copper in the resistant cells varied from 56 to 75% of control. Studies performed using one pair of cell lines demonstrated that for both copper and DDP the initial efflux rate was lower, whereas the terminal efflux rate was higher in the resistant cells. On Western blot analysis all three resistant lines exhibited increased expression of one or the other of the two copper export pumps (ATP7A or ATP7B) with no change in the HAH1 chaperone. We conclude that the acquisition of DDP resistance in ovarian carcinoma is accompanied by alterations in the cellular pharmacology of DDP that are paralleled by similar changes in the uptake and efflux of copper. These results are consistent with the concept that DDP enters and exits from the cell via transporters that normally mediate copper homeostasis.

Abnormal lysosomal trafficking and enhanced exosomal export of cisplatin in drug-resistant human ovarian carcinoma cells.

Previous work has shown that cisplatin (CDDP) becomes concentrated in lysosomes, and that acquired resistance to CDDP is associated with abnormalities of protein trafficking and secretion. The lysosomal compartment in CDDP-sensitive 2008 human ovarian carcinoma cells was compared with that in CDDP-resistant 2008/C13*5.25 subline using deconvoluting imaging and specific dyes and antibodies. The lysosomal compartment in CDDP-resistant cells was reduced to just 40% of that in the parental CDDP-sensitive cells (P<0.002). This was accompanied by a reduced expression of the lysosome-associated proteins 1 and 2 (LAMP1 and LAMP2) as determined by both microscopy and Western blot analysis. The CDDP-resistant cells released more protein as exosomes and Western blot analysis revealed that these exosomes contained substantially more LAMP1 than those released by the CDDP-sensitive cells. Following loading of the whole cell with CDDP, the exosomes released from 2008/C13*5.25 cells contained 2.6-fold more platinum than those released from sensitive cells. Enhanced exosomal export was accompanied by higher exosomal levels of the putative CDDP export transporters MRP2, ATP7A, and ATP7B. Expression profiling identified significant increases in the expression of several genes whose products function in membrane fusion and vesicle trafficking. This study shows that the lysosomal compartment of human ovarian carcinoma cells selected for stable resistance to CDDP is markedly reduced in size, and that these cells abnormally sort some lysosomal proteins and the putative CDDP transporters into an exosomal pathway that also exports CDDP.

Copper transporters regulate the cellular pharmacology and sensitivity to Pt drugs.

Recent studies have demonstrated that the major Cu influx transporter CTR1 regulates tumor cell uptake of cisplatin (DDP), carboplatin (CBDCA) and oxaliplatin (L-OHP), and that the two Cu efflux transporters ATP7A and ATP7B regulate the efflux of these drugs. Evidence for the concept that these platinum (Pt) drugs enter cells and are distributed to various subcellular compartments via transporters that have evolved to manage Cu homeostasis includes the demonstration of: (1) bidirectional cross-resistance between cells selected for resistance to either the Pt drugs or Cu; (2) parallel changes in the transport of Pt and Cu drugs in resistant cells; (3) altered cytotoxic sensitivity and Pt drug accumulation in cells transfected with Cu transporters; and (4) altered expression of Cu transporters in Pt drug-resistant tumors. Appreciation of the role of the Cu transporters in the development of resistance to DDP, CBDCA, and L-OHP offers novel insights into strategies for preventing or reversing resistance to this very important family of anticancer drugs.

Increase in expression of the copper transporter ATP7A during platinum drug-based treatment is associated with poor survival in ovarian cancer patients.

PURPOSE: The Cu efflux transporter ATP7A is overexpressed in some cisplatin-resistant ovarian carcinoma cell lines. We examined the expression of ATP7A in the major normal human organs and in several types of human malignancies and sought to determine whether ATP7A expression changed during treatment of ovarian carcinomas with Pt-containing regimens. EXPERIMENTAL DESIGN: ATP7A expression was quantified by immunohistochemical staining using microarrays containing normal and malignant tissues, and standard sections of 54 paired tumor samples obtained from ovarian carcinoma patients before and after at least two cycles of platinum-based therapy. RESULTS: ATP7A was expressed in normal endometrium, prostate, testis, and kidney but was not detected in the other major organs. ATP7A was expressed in some of the most common human malignancies, including prostate (7 of 7), breast (10 of 10), lung (8 of 8), colon (5 of 8), and ovary (6 of 7), as well as in a wide variety of other types of malignancy. ATP7A staining was detected in 28 of 54 ovarian carcinomas before treatment. Patients with increased ATP7A expression after treatment (18 of 54) exhibited poorer actuarial survival (P<0.0057 by log-rank test). Expression of ATP7A either before or after treatment was not associated with other clinical factors. CONCLUSIONS: Although ATP7A is not detectable in most normal tissues it is expressed in a considerable fraction of many common tumor types. Enrichment of the tumor for ATP7A-expressing cells during platinum drug-based treatment of ovarian cancers is associated with poor survival. These findings are in agreement with results of in vitro studies from this laboratory demonstrating that increased expression of ATP7A renders cells resistant to cisplatin and carboplatin.

Confocal microscopic analysis of the interaction between cisplatin and the copper transporter ATP7B in human ovarian carcinoma cells.

Some cisplatin (DDP)-resistant cells overexpress the copper export transporter ATP7B, and cells molecularly engineered to overexpress ATP7B are resistant to DDP. The interaction of Cu with ATP7B normally triggers its relocalization from the perinuclear region to more peripheral vesicles. To investigate the interaction of DDP with ATP7B, we examined the effect of DDP on the subcellular localization of ATP7B using human ovarian carcinoma cells expressing a cyan fluorescent protein (ECFP)-tagged ATP7B (2008/ECFP-ATP7B). ATP7B expression was confirmed in 2008/ECFP-ATP7B cells by Western blotting, and its functionality was documented by showing that it rendered the cells 1.9-fold resistant to CuSO(4) and 4.1-fold resistant to DDP and also reduced the accumulation of both drugs. There was greater sequestration of Pt into intracellular vesicles in the 2008/ECFP-ATP7B cells than in the 2008/ECFP cells. Confocal digital microscopy revealed that ECFP-ATP7B localized in the perinuclear region in absence of drug exposure and that both Cu and DDP triggered relocalization to more peripheral vesicular structures. A fluorescein-labeled form of DDP that retained cytotoxicity and was subject to the same mechanisms of resistance as DDP colocalized with ECFP-ATP7B in the 2008/ECFP-ATP7B cells, whereas the same fluorochrome lacking the DDP moiety did not. These results provide evidence that DDP directly interacts with ATP7B to trigger its relocalization and that ATP7B mediates resistance to DDP by sequestering it into vesicles of the secretory pathway for export from the cell.

Increased expression of the copper efflux transporter ATP7A mediates resistance to cisplatin, carboplatin, and oxaliplatin in ovarian cancer cells.

PURPOSE: The goal of this study was to determine the effect of small changes in ATP7A expression on the pharmacodynamics of cisplatin, carboplatin, and oxaliplatin in human ovarian carcinoma cells. EXPERIMENTAL DESIGN: Drug sensitivity and cellular pharmacology parameters were determined in human 2008 ovarian carcinoma cells and a subline transfected with an ATP7A-expression vector ATP7A (2008/MNK). Drug sensitivity was determined by clonogenic assay, platinum (Pt) levels were measured by inductively coupled plasma mass spectroscopy, copper (Cu) accumulation was quantified with (64)Cu, and the subcellular distribution of ATP7A was assessed by confocal digital microscopy. RESULTS: The 1.5-fold higher expression of ATP7A in the 2008/MNK cells was sufficient to alter Cu cellular pharmacokinetics but not confer Cu resistance. In contrast, it was sufficient to render the 2008/MNK cells resistant to cisplatin, carboplatin, and oxaliplatin. Resistance was associated with increased rather than decreased whole-cell Pt drug accumulation and increased sequestration of Pt into the vesicular fraction. Cu triggered relocalization of ATP7A away from the perinuclear region, whereas at equitoxic concentrations the Pt drugs did not. CONCLUSIONS: A small increase in ATP7A expression produced resistance to all three of the clinically available Pt drugs. Whereas increased expression of ATP7A reduced Cu accumulation, it did not reduce accumulation of the Pt drugs. Under conditions where Cu triggered ATP7A relocalization, the Pt drugs did not. Thus, although ATP7A is an important determinant of sensitivity to the Pt drugs, there are substantial differences between Cu and the Pt drugs with respect to how they interact with ATP7A and the mechanism by which ATP7A protects the cell.

Cross-resistance to cisplatin in cells with acquired resistance to copper.

PURPOSE: Cells selected for resistance to cisplatin (DDP) demonstrate cross-resistance to copper (Cu) suggesting one or more common mechanisms of cellular defense. We sought to determine whether cells selected for resistance to Cu are cross-resistant to DDP. MATERIALS AND METHODS: Parental HuH7 human hepatoma cells and the CuR27 Cu-resistant subline were compared for sensitivity to Cu and DDP by clonogenic assay, and with respect to drug uptake and efflux by measuring cellular Cu and Pt content. RESULTS: CuR27 cells were found to be 1.8-fold resistant to Cu and 8.6-fold cross-resistant to DDP. Changes in the cellular pharmacokinetics of Cu in the CuR27 cells were paralleled by changes in the kinetics of DDP. The accumulations of Cu and DDP measured at 1 min were, respectively, 36% and 26% of those in the parental HuH7 cells. The initial rate of efflux from the CuR27 cells was 6.2-fold faster for Cu and 2.5-fold faster for DDP than from the HuH7 cells. Cu reduced the accumulation of DDP in the HuH7 cells in a concentration-dependent manner and vice versa. DDP also reduced the efflux of Cu. Western blot analysis demonstrated that expression of the Cu exporter ATP7B was increased 3.9-fold in the CuR27 cells. CONCLUSIONS: In this model system, cross-resistance between Cu and DDP was bidirectional and accompanied by parallel changes in the cellular pharmacokinetics of both compounds. The results are consistent with the idea that transporters and chaperones that normally mediate Cu homeostasis also directly or indirectly modulate the accumulation of DDP.

The role of copper transporters in the development of resistance to Pt drugs.

Recent studies in yeast, mouse and human cells suggest that the conserved metal binding transporters of the Cu homeostasis pathway can mediate resistance to Pt drugs in cancer cells. This review summarizes the data available from these studies. The observation that cells selected for resistance to Cu or the Pt drugs display bidirectional cross-resistance, parallel defects in the transport of Cu and the Pt drugs and altered expression of Cu transporters is consistent with the concept that the Cu homeostasis proteins regulate sensitivity to the Pt drugs by influencing their uptake, efflux and intracellular distribution. This model is supported by the finding that when mammalian and yeast cells are genetically engineered to express altered levels of the Cu transporters they exhibit altered sensitivity to Pt drugs and are defective in intracellular Pt accumulation due to altered uptake and/or efflux rates. Negative associations between the expression of ATP7A and ATP7B and the outcome of Pt therapy further support the significance of the Cu homeostasis proteins as both markers of and contributors to Pt resistance.

Molecular modulation of the copper and cisplatin transport function of CTR1 and its interaction with IRS-4.

The copper influx transporter CTR1 is also a major influx transporter for cisplatin (cDDP) in tumor cells. It influences the cytotoxicity of cDDP both in vivo and in vitro. Whereas Cu triggers internalization of CTR1 from the plasma membrane, cDDP does not. To investigate the mechanisms of these effects, myc-tagged forms of wild type hCTR1 and variants in which Y103 was converted to alanine, C189 was converted to serine, or the K178/K179 dilysine motif was converted to alanines were re-expressed in mouse embryo cells in which both alleles of CTR1 had been knocked out and also in HEK293T cells. The Y103A mutation and to a lesser extent the C189S mutation reduced internalization of CTR1 induced by Cu while the K178A/K179A had little effect. Both Y103 and C189 were required for Cu and cDDP transport whereas the K178/K179 motif was not. While Y103 lies in an YXXM motif that, when phosphorylated, is a potential docking site for phosphatidylinositol 3-kinase and other proteins involved in endocytosis, Western blot analysis of immunoprecipitated myc-CTR1, and proteomic analysis of peptides derived from CTR1, failed to identify any basal or Cu-induced phosphorylation. However, proteomic analysis did identify an interaction of CTR1 with IRS-4 and this was confirmed by co-immunoprecipitation from HEK cells expressing either FLAG-CTR1 or myc-CTR1. The interaction was greater in the Y103A-expressing cells. We conclude that Y103 is required for the internalization of hCTR1 in response to Cu, that this occurs by a mechanism other than phosphorylation and that mutation of Y103 modulates the interaction with IRS-4.

The role of metal binding and phosphorylation domains in the regulation of cisplatin-induced trafficking of ATP7B.

The copper (Cu) exporter ATP7B mediates cellular resistance to cisplatin (cDDP) by increasing drug efflux. ATP7B binds and sequesters cDDP in into secretory vesicles. Upon cDDP exposure ATP7B traffics from the trans-Golgi network (TGN) to the periphery of the cell in a manner that requires the cysteine residues in its metal binding domains (MBD). To elucidate the role of the various domains of ATP7B in its cDDP-induced trafficking we expressed a series of mCherry-tagged variants of ATP7B in HEK293T cells and analyzed their subcellular localization in basal media and after a 1 h exposure to 30 µM cDDP. The wild type ATP7B and a variant in which the cysteines in the CXXC motifs of MBD 1-5 were converted to serines trafficked out of the trans-Golgi (TGN) when exposed to cDDP. Conversion of the cysteines in all 6 of the CXXC motifs to serines, or in only the sixth MBD, rendered ATP7B incapable of trafficking on exposure to cDDP. Truncation of MBD1-5 or MBD1-6 resulted in the loss of TGN localization. Addition of the first 63 amino acids of ATP7B to these variants restored TGN localization to a great extent and enabled the MBD1-5 variant to undergo cDDP-induced trafficking. A variant of ATP7B in which the aspartate 1027 residue in the phosphorylation domain was converted to glutamine localized to the TGN but was incapable of cDDP-induced trafficking. These results demonstrate that the CXXC motif in the sixth MBD and the catalytic activity of ATP7B are required for cDDP-induced trafficking as they are for Cu-induced redistribution of ATP7B; this provides further evidence that cDDP mimics Cu with respect to the molecular mechanisms by they control the subcellular distribution of ATP7B.

The CXXC motifs in the metal binding domains are required for ATP7B to mediate resistance to cisplatin.

The copper (Cu) exporter ATP7B mediates resistance to cisplatin (cDDP) but details of the mechanism are unknown. We explored the role of the CXXC motifs in the metal binding domains (MBDs) of ATP7B by investigating binding of cDDP to the sixth metal binding domain (MBD6) or a variant in which the CXXC motif was converted to SXXS. Platinum measurement showed that cDDP bound to wild type MBD6 but not to the SXXS variant. Wild type ATP7B rendered ovarian 2008 cells resistant to cDDP. In 2008 and in HEK293T cells, wild type ATP7B trafficked from TGN to peripheral locations in response to Cu or cDDP. A variant in which the CXXC motifs in all 6 MBDs were converted to SXXS localized correctly to the TGN but failed to traffic when exposed to either Cu or cDDP. Deletion of either the first 5 MBDs or all 6 MBDs resulted in failure to localize to the TGN. Neither the SXXS variant nor the deletion variant was able to mediate resistance to cDDP. We conclude that cDDP binds to the CXXC motifs of ATP7B and that this interaction is essential to the trafficking of ATP7B and to its ability to mediate resistance to cDDP.

The role of the N-terminus of mammalian copper transporter 1 in the cellular accumulation of cisplatin.

The mammalian copper transporter 1 (CTR1) is responsible for the uptake of copper (Cu) from the extracellular space, and has been shown to play a major role in the initial accumulation of platinum-based drugs. In this study we re-expressed wild type and structural variants of hCTR1 in mouse embryo fibroblasts in which both alleles of mCTR1 had been knocked out (CTR1(-/-)) to examine the role of the N-terminal extracellular domain of hCTR1 in the accumulation of cisplatin (cDDP). Deletion of either the first 45 amino acids or just the (40)MXXM(45) motif in the N-terminal domain did not alter subcellular distribution or the amount of protein in the plasma membrane but it eliminated the ability of hCTR1 to mediate the uptake of Cu. In contrast it only partially reduced cDDP transport capacity. Neither of these structural changes prevented cDDP from triggering the rapid degradation of hCTR1. However, they did alter the potency of the cDDP that achieved cell entry, possibly reflecting the fact that hCTR1 may mediate the transport of cDDP both through the pore it forms in the plasma membrane and via endocytosis. We conclude that cDDP interacts with hCTR1 both at (40)MXXM(45) and at sites outside the N-terminal domain that produce the conformational changes that trigger degradation.