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At least 19 recordsLinked to original sources
PubMed2002

Biochemical characterization and subcellular localization of human copper transporter 1 (hCTR1).

The human copper transporter 1 gene (hCTR1) was previously identified by functional complementation in ctr1-deficient yeast. Overexpression of hCTR1 in wild-type yeast leads to increased sensitivity to copper toxicity, and mice with a homozygous disruption at the Ctr1 locus die early during embryogenesis. It is proposed that hCTR1 is responsible for high-affinity copper uptake into human cells, but the underlying molecular mechanisms are unknown. To begin to investigate the biochemical characteristics of hCTR1, a polyclonal antiserum was raised against recombinant hCTR1-fusion peptides. Biosynthetic studies using this antiserum revealed that hCTR1 was synthesized as a precursor protein of 28 kDa containing N-linked oligosaccharides, and is then converted to a mature protein of approx. 35 kDa, which is ubiquitously expressed. Immunofluorescence studies showed that subcellular hCTR1 localization differed markedly between cell types. In some cell lines, hCTR1 was located predominantly in an intracellular vesicular perinuclear compartment, and in others hCTR1 was located predominantly at the plasma membrane. In contrast with the copper export P-type ATPases mutated in Wilson disease and Menkes disease, the localization of hCTR1 was not influenced by copper concentrations. Inhibition of endocytosis by methyl-beta-cyclodextrin caused a partial redistribution of hCTR1 to the cell surface of HeLa cells. Taken together, the results in this study suggest a cell-specific control of copper uptake, which involves subcellular localization of the hCTR1 protein.

Base Sequence↗
PubMed2006

The internalization and degradation of human copper transporter 1 following cisplatin exposure.

The human copper transporter 1 (hCTR1), the major transporter responsible for copper influx, mediates one component of the cellular accumulation of cisplatin (DDP). Both copper and DDP cause rapid down-regulation of hCTR1 expression in human ovarian carcinoma cells. In this study, we investigated the mechanism of this effect using digital deconvolution microscopy and Western blot analysis of cells stained with antibodies directed at both ends of the protein. Treatment of 2008 cells with DDP in combination with inhibitors of various endosomal pathways (amiloride, cytochalasin D, nystatin, and methyl-beta-cyclodextrin) showed that hCTR1 degradation was blocked by amiloride and cytochalasin D, indicating that hCTR1 was internalized primarily by macropinocytosis. Expression of transdominant-negative forms of dynamin I and Rac showed that loss of hCTR1 was not dependent on pathways regulated by either of these proteins. DDP-induced loss of hCTR1 was blocked by the proteasome inhibitors lactacystin, proteasome inhibitor 1, and MG132. This study confirms that DDP triggers the rapid loss of hCTR1 from ovarian carcinoma cells at clinically relevant concentrations. The results indicate that DDP-induced loss of hCTR1 involves internalization from the plasma membrane by macropinocytosis followed by proteasomal degradation. Because hCTR1 is a major determinant of early DDP uptake, prevention of its degradation offers a potential approach to enhancing tumor sensitivity.

Amiloride↗
PubMed2004

Cloning, expression pattern and essentiality of the high-affinity copper transporter 1 (ctr1) gene in zebrafish.

The high-affinity copper transporter 1 (Ctr1) is a highly conserved transmembrane protein that mediates the internalization of copper ions from the extracellular medium. In this study, we have isolated the zebrafish ctr1 gene. The zebrafish ctr1 cDNA encodes a protein with 69% identity to the human orthologue and shows conservation of specific amino acid residues involved in copper transport. We find only a single ctr1 gene in the zebrafish genome which maps to linkage group 5. The genomic structure of the zebrafish gene shows that it consists of five exons and that exon-intron boundaries are absolutely conserved with the mammalian ctr1 genes. Expression in embryos was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and by in situ hybridization. Zebrafish ctr1 is maternally loaded, and transcripts can be detected throughout development and in adult fish. Distribution of ctr1 message appears ubiquitous during early stages becoming restricted to the brain and ventral tissues by 24 h post fertilization (hpf). Beginning at 3 days post fertilization (dpf), expression is found mainly in the developing intestine. Specific knockdown of ctr1 by antisense morpholino oligonucleotides (MOs) causes early larval lethality. Defects include cell death in tissues where ctr1 is most heavily expressed, a finding similar to that described for a mouse knockout of mCtr1. Despite the existence of at least one other copper transport mechanism in the fish, our studies show that zebrafish ctr1 is an essential gene for development.

Amino Acid Sequence↗
PubMedDate not supplied

Mouse extrahepatic hepatoma detected on MicroPET using copper (II)-64 chloride uptake mediated by endogenous mouse copper transporter 1.

PURPOSE: This study was conducted to assess the positron-emitting copper (II)-64 chloride ((64)CuCl(2)) as a probe for imaging mouse extrahepatic hepatoma expressing mouse copper transporter 1 (mCtr1) with positron emission tomography (PET). PROCEDURES: Following the intravenous administration of (64)CuCl(2), athymic mice bearing extrahepatic hepatoma grafts were subjected to whole-body static PET imaging with a Concorde microPET R4 tomograph. Upon completion of the imaging study, immunohistochemistry (IHC) study of mCtr1 was performed with postmortem tissues. RESULTS: The mouse extrahepatic hepatoma grafts were well visualized on static microPET images. Quantitative analysis demonstrated that the tracer concentration in the hepatoma was significantly higher than those in the soft tissue of the right shoulder opposite to the tumor site and the brain (p < 0.001). mCtr1 immunoreactivity in the hepatoma graft was approximately 70% of that in liver, whereas (64)CuCl(2) concentration in the graft was approximately 11% of the liver concentration. CONCLUSIONS: The extrahepatic mouse hepatoma grafts may be visualized by Cu-64 PET, taking advantage of the (64)CuCl(2) uptake mediated by the functional endogenous mCtr1.

Animals↗
PubMed2003

The N-terminus of the human copper transporter 1 (hCTR1) is localized extracellularly, and interacts with itself.

We have used indirect immunofluorescense studies and glycosylation-site insertion and deletion mapping to characterize the topology of human copper transporter 1 (hCTR1), the putative human high-affinity copper-import protein. Both approaches indicated that hCTR1 contains three transmembrane domains and that the N-terminus of hCTR1, which contains several putative copper-binding sites, is localized extracellularly, whereas the C-terminus is exposed to the cytosol. Based on previous observations that CTR1 proteins form high-molecular-mass complexes, we investigated directly whether CTR1 proteins interact with themselves. Yeast two-hybrid studies showed that interaction of yeast, mouse, rat and human CTR1 occurs at the sites of their N-terminal domains, and is not dependent on the copper concentration in the growth media. Analysis of deletion constructs indicated that multiple regions in the N-terminus are essential for this self-interaction. In contrast, the N-terminal tail of the presumed low-affinity copper transporter, hCTR2, does not interact with itself. Taken together, these results suggest that CTR1 spans the membrane at least six times, permitting formation of a channel, which is consistent with its proposed role as a copper transporter.

Amino Acid Sequence↗
PubMed2006

Expression of the human copper influx transporter 1 in normal and malignant human tissues.

The major copper influx transporter, copper transporter 1 (hCTR1), controls the cellular accumulation of cisplatin in mammalian cells. The goal of this study was to determine the pattern of hCTR1 expression in normal and malignant human tissues. Tissue arrays were stained with an antibody specific for hCTR1 using standard immunohistochemical techniques. Particularly strong staining was noted in the alpha cells of the pancreatic islets, enteroendocrine cells of the gastric mucosa and bronchioles, C cells of the thyroid, and a subset of cells in the anterior pituitary. Frequency and intensity of hCTR1 staining in malignant tissues reflected the levels found in their normal tissue counterparts. For example, neither normal prostate nor prostate cancers expressed hCTR1, whereas it was commonly expressed in both normal colonic epithelium and in colon carcinomas. Strong staining was observed in a limited number of cases of carcinoid tumors, Ewing's sarcoma, and undifferentiated carcinomas. Although all tissues require copper, expression of hCTR1 was highly variable among normal tissues and among the major human malignancies, with the highest levels found in enteroendocrine cells. No hCTR1 expression was found in several common types of cancer, suggesting that hCTR1 expression is not commonly enhanced by transformation.

Biomarkers, Tumor↗
PubMed2004

The copper influx transporter human copper transport protein 1 regulates the uptake of cisplatin in human ovarian carcinoma cells.

Cells selected for resistance to cisplatin are often cross-resistant to copper and vice versa, and the major copper influx transporter copper transport protein 1 (CTR1) has been shown to regulate the uptake of cisplatin, carboplatin, and oxaliplatin in yeast. To further define the role of hCTR1 in human tumor cells, the ovarian carcinoma cell line A2780 was molecularly engineered to increase expression of hCTR1 by a factor of 20-fold. Enhanced expression of hCTR1 in the A2780/hCTR1 cells was associated with a 6.5-fold increase in basal steady-state copper content and a 13.7-fold increase in initial copper influx, demonstrating that the exogenously expressed hCTR1 was functional in altering copper homeostasis. The A2780/hCTR1 cells accumulated 46% more platinum after a 1-h exposure to 2 microM cisplatin, and 55% more after a 24 h exposure, than the control A2780/empty vector cells. The initial uptake of cisplatin was 81% higher in the A2780/hCTR1 cells when measured at 5 min. Thus, increased expression of hCTR1 had a substantially larger effect on the cellular pharmacology of copper than cisplatin. Interestingly, the increased uptake of copper and cisplatin was accompanied by only a marginal increase in sensitivity to the cytotoxic effect of copper and cisplatin, and there was no increase in the extent of cisplatin-DNA adduct formation. Thus, although increased expression of hCTR1 mediates greater cellular accumulation of copper and cisplatin, hCTR1 delivers these compounds into intracellular compartments from which they do not have ready access to their key cytotoxic targets.

Antineoplastic Agents↗
PubMed2006

PET of human prostate cancer xenografts in mice with increased uptake of 64CuCl2.

UNLABELLED: Our objective was to determine whether human prostate cancer xenografts in mice can be localized by PET using 64CuCl2 as a probe (64Cu PET). METHODS: Athymic mice bearing human prostate cancer xenografts were subjected to 64Cu PET, followed by quantitative analysis of the tracer concentrations and immunohistochemistry study of human copper transporter 1 expression in the tumor tissues. RESULTS: Human prostate cancer xenografts expressing high levels of human copper transporter 1 were well visualized on the PET images obtained 24 h after injection but not on the images obtained 1 h after injection. PET quantitative analysis demonstrated a high concentration of 64CuCl2 in the tumors in comparison to that in the left shoulder regions (percentage injected dose per gram of tissue: 3.6 +/- 1.3 and 0.6 +/- 0.3, respectively; P = 0.004), at 24 h after injection. CONCLUSION: The data from this study suggested that locally recurrent prostate cancer might be localized with 64Cu PET using 64CuCl2 as a probe.

Animals↗
PubMed2004

Cellular pharmacology of cisplatin in relation to the expression of human copper transporter CTR1 in different pairs of cisplatin-sensitive and -resistant cells.

The molecular mechanism of cisplatin uptake remains poorly defined and impaired drug accumulation may be implicated in the acquisition of resistance to cisplatin. Thus, we used cell lines of different tumor types (ovarian carcinoma A2780 and IGROV-1, osteosarcoma U2-OS, cervix squamous cell carcinoma A431) and stable cisplatin-resistant sublines, exhibiting variable levels of resistance (between 2.5 and 18.4), to investigate the mechanisms of cellular accumulation of cisplatin. Among the resistant lines we found that reduced cisplatin uptake was a common feature and ranged between 23 and 76%. In an attempt to examine the role of human copper transporter 1 (CTR1) in cisplatin accumulation by human cells, we selected the well characterized A431 cell line and the resistant variant A431/Pt. As compared with A431/Pt cells, A431/Pt transfectants overexpressing CTR1 (3.4-fold) exhibited increased uptake of copper, thereby supporting the expression of a functional transporter. However, no changes in cisplatin uptake and cellular sensitivity to drug were observed. Also overexpression of CTR1 in A431 cells did not produce modulation of cisplatin accumulation. An analysis of the expression of other factors that could affect drug accumulation indicated that A431/Pt cells displayed increased expression of ATPase, Cu(2+) transporting, alfa polypeptide. In conclusion, our results indicate that the overexpression of a functional CTR1 in a human cell line characterized by impaired cisplatin uptake fails (a) to restore cellular drug accumulation to the level of the parental cell line and (b) to modulate cisplatin sensitivity. Our data are consistent with the interpretation that the defects in cellular accumulation by resistant cells are not mediated by expression of CTR1, that plays a marginal role, if any, in cisplatin transport.

Adenosine Triphosphatases↗
PubMed2005

Stable plasma membrane levels of hCTR1 mediate cellular copper uptake.

The human copper transporter 1 (hCtr1), when heterologously overexpressed in insect cells, mediates saturable Cu uptake. In mammalian expression systems, a rapid Cu-dependent internalization of hCtr1 has been reported in cells that overexpress epitope-tagged hCtr1 when exposed to Cu in the external medium. This finding led to the suggestion that such internalization may be a step in the hCtr1 transmembrane Cu transport mechanism. We have demonstrated that preincubation in Cu-containing media of sf9 cells stably expressing hCtr1 has no effect on the initial rate of Cu transport. Furthermore, Western blot analyses of fractionated sf9 cell membranes show no evidence of a regulatory Cu-dependent internalization from the plasma membrane. In similar studies on human embryonic kidney (HEK) 293 cells, we showed that incubation with Cu does not alter the initial rate of Cu uptake mediated by endogenous levels of hCtr1 compared with untreated cells. Confirmation that hCtr1 mediates this transport is provided by specific small interfering RNA-dependent decreases in hCtr1 protein levels and in Cu transport rates. Western blot analysis and confocal microscopy of human embryonic kidney 293 cells showed that the majority of hCtr1 protein is localized at the plasma membrane and no significant internalization is detected upon Cu treatment. We concluded that internalization of hCtr1 is not a required step in the transport pathway; we suggest that oligomeric hCtr1 acts as a conventional transporter providing a permeation pathway for Cu through the membrane and that internalization of endogenous hCtr1 in response to elevated extracellular Cu levels does not play a significant regulatory role in Cu homeostasis.

Animals↗
PubMed2005

Differential profiles of copper-induced ROS generation in human neuroblastoma and astrocytoma cells.

To determine neuronal and glial responses to copper (Cu) elevation in the CNS, human neuroblastoma and astrocytoma cells were used to compare their responses to Cu in terms of reactive oxygen species (ROS) generation and expression of enzymes responsible for anti-oxidation. Astrocytoma cells, not neuroblastoma cells, were responsive to Cu and Cu elevation was associated with ROS generation. Intracellular Cu levels as determined by inductively coupled plasma-mass spectrometry (ICP-MS), and expression levels of copper-transporting ATPase (ATP7A) and human copper transporter 1 (hCtr1) as detected by quantitative reverse transcription-polymerase chain reaction (RT-PCR), were comparable in both cell lines. Differences in Cu-induced ROS between two cell lines paralleled superoxide dismutase (SOD)-catalase expression as detected by Western blot analysis. Copper,zinc-SOD (Cu,Zn-SOD) and catalase protein levels were upregulated by Cu in neuroblastoma cells while Cu,Zn-SOD was down-regulated by Cu and catalase level was not changed in astrocytoma cells. Manganese-SOD (Mn-SOD) was not responsive to Cu in either cell line. Furthermore, 78-kDa glucose-regulated protein aggregation and upregulation were observed in Cu-treated astrocytoma cells, but not neuroblastoma cells. These data suggest that neurons use the SOD-catalase system to scavenge Cu-induced ROS while glia rely on the endoplasmic reticulum stress response to compensate for the reduction of ROS scavenging capacity.

Adenosine Triphosphatases↗
PubMed2005

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.

Adenosine Triphosphatases↗
PubMed2002

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.

Adenosine Triphosphatases↗
PubMed2004

Functional and molecular responses of suckling rat pups and human intestinal Caco-2 cells to copper treatment.

Ctr1 and Atp7A are copper (Cu) transporters that may play a role in the regulation of intestinal Cu absorption; however, intestinal regulation of these transporters by Cu in vivo has not been well defined. In this study, we hypothesized that Cu supplementation would alter the expression of intestine Ctr1 and Atp7A in vivo and further documented effects of Cu exposure on Cu transport, Ctr1 and Atp7A levels and localization in enterocyte-like Caco-2 cells. Suckling rat pups were supplemented with Cu (0 and 25 microg Cu/day) for 10 days and small intestine Cu concentration, Ctr1, Atp7A and metallothionein (MT) gene expression were measured by Northern blot analysis. Caco-2 cells were treated with basal medium, or medium supplemented with 3 and 94 microM CuSO4 and 67Cu transport, Ctr1 and Atp7A levels and localization were determined. In rat pups, Cu supplementation increased intestinal Cu, Ctr1 and MT gene expression; however, Atp7A gene expression was not significantly affected. Caco-2 cells treated with 94 microM Cu had lower cellular Cu uptake and export compared to untreated cells. While Ctr1 and Atp7A gene and protein levels were unaffected, confocal microscopy indicated that Ctr1 was endocytosed and co-localized with transferrin in Cu treated cells. This study demonstrates the functional response of intestinal cells to Cu treatment and suggests that both Ctr1 and Atp7A may regulate Cu absorption.

Absorption↗
PubMed2002

Correction of the copper transport defect of Menkes patient fibroblasts by expression of two forms of the sheep Wilson ATPase.

The Wilson disease (WD) protein (ATP7B) is a copper-transporting P-type ATPase that is responsible for the efflux of hepatic copper into the bile, a process that is essential for copper homeostasis in mammals. Compared with other mammals, sheep have a variant copper phenotype and do not efficiently excrete copper via the bile, often resulting in excessive copper accumulation in the liver. To investigate the function of sheep ATP7B and its potential role in the copper-accumulation phenotype, cDNAs encoding the two forms of ovine ATP7B were transfected into immortalised fibroblast cell lines derived from a Menkes disease patient and a normal control. Both forms of ATP7B were able to correct the copper-retention phenotype of the Menkes cell line, demonstrating each to be functional copper-transporting molecules and suggesting that the accumulation of copper in the sheep liver is not due to a defect in the copper transport function of either form of sATP7B.

Adenosine Triphosphatases↗
PubMed2006

Mammary gland copper transport is stimulated by prolactin through alterations in Ctr1 and Atp7A localization.

Milk copper (Cu) concentration declines and directly reflects the stage of lactation. Three Cu-specific transporters (Ctr1, Atp7A, Atp7B) have been identified in the mammary gland; however, the integrated role they play in milk Cu secretion is not understood. Whereas the regulation of milk composition by the lactogenic hormone prolactin (PRL) has been documented, the specific contribution of PRL to this process is largely unknown. Using the lactating rat as a model, we determined that the normal decline in milk Cu concentration parallels declining Cu availability to the mammary gland and is associated with decreased Atp7B protein levels. Mammary gland Cu transport was highest during early lactation and was stimulated by suckling and hyperprolactinemia, which was associated with Ctr1 and Atp7A localization at the plasma membrane. Using cultured mammary epithelial cells (HC11), we demonstrated that Ctr1 stains in association with intracellular vesicles that partially colocalize with transferrin receptor (recycling endosome marker). Atp7A was primarily colocalized with mannose 6-phosphate receptor (M6PR; late endosome marker), whereas Atp7B was partially colocalized with protein disulfide isomerase (endoplasmic reticulum marker), TGN38 (trans-Golgi network marker) and M6PR. Prolactin stimulated Cu transport as a result of increased Ctr1 and Atp7A abundance at the plasma membrane. Although the molecular mechanisms responsible for these posttranslational changes are not understood, transient changes in prolactin signaling play a role in the regulation of mammary gland Cu secretion during lactation.

Adenosine Triphosphatases↗