Copper is an essential metal nutrient, yet copper overload is toxic. Here, we report that human copper transporter (hCtr) 1 plays an important role in the maintenance of copper homeostasis by demonstrating that expression of hCtr1 mRNA was up-regulated under copper-depleted conditions and down-regulated under copper-replete conditions. Overexpression of full-length hCtr1 by transfection with a recombinant hCtr1 cDNA clone reduced endogenous hCtr1 mRNA levels, whereas overexpression of N terminus-deleted hCtr1 did not change endogenous hCtr1 mRNA levels, suggesting that increased functional hCtr1 transporter, which leads to increased intracellular copper content, down-regulates the endogenous hCtr1 mRNA. A luciferase assay using reporter constructs containing the hCtr1 promoter sequences revealed that three Sp1 binding sites are involved in the basal and copper concentration-dependent regulation of hCtr1 expression. Modulation of Sp1 levels affected the expression of hCtr1. We further demonstrated that the zinc-finger domain of Sp1 functions as a sensor of copper that regulates hCtr1 up and down in response to copper concentration variations. Our results demonstrate that mammalian copper homeostasis is maintained at the hCtr1 mRNA level, which is regulated by the Sp1 transcription factor.

The human high-affinity copper transporter (hCtr1) plays an important role in the regulation of intracellular copper homeostasis. hCtr1 is involved in the transport of platinum-based antitumor agents such as cisplatin (CDDP); however, the mechanisms that regulate hCtr1-mediated transport of these agents have not been well elucidated. We compared the mechanisms of hCtr1-mediated transport of copper and CDDP. We found that replacements of several methionine residues that are essential for hCtr1-mediated copper transport conferred a dominant-negative effect on the endogenous hCtr1's function, resulting in reduced rates of Cu(I) and CDDP transport and increased resistance to the toxicities of copper and CDDP treatments. Kinetic constant analyses revealed that although these mutations reduced maximal transport rates (V max ) for Cu(I) and CDDP, reduction of K m only for Cu(I) but not for CDDP was observed. Mutation in Gly167, which is located in the third transmembrane domain and is involved in helix packing of hCtr1, also conferred dominant-negative property of Cu(I) transport but not of CDDP transport. Deleting the N-terminal 45 amino acids that contain two methionine-rich motifs resulted in cytoplasmic localization of the hCtr1 and abolished the dominant-negative function of these mutants. Nonetheless, these mutations did not affect the capacities of hCtr1 oligomerization induced by copper or CDDP, suggesting a distinct structural requirement between metal transport and oligomerization. Finally, we also observed that expressing the dominant-negative hCtr1 mutants up-regulates endogenous hCtr1 mRNA expression, consistent with our previous report that intracellular copper homeostasis and homeostatic levels of hCtr1 mRNA are mutually regulated.

Platinum (Pt)-based antitumor agents are widely used in cancer chemotherapy. Drug resistance is a major obstacle to the successful use of these agents because once drug resistance develops, other effective treatment options are limited. Recently, we have conducted a clinical trial using a copper (Cu)-lowering agent to overcome Pt drug resistance in ovarian cancer patients and the preliminary results are encouraging. In supporting this clinical study, using three pairs of cisplatin (cDDP)-resistant cell lines and two ovarian cancer cell lines derived from patients who had failed in Pt-based chemotherapy, we demonstrated that cDDP resistance associated with reduced expression of the high affinity copper transporter (hCtr1) which is also a cDDP transporter, can be preferentially re-sensitized by copper-lowering agents due to enhanced hCtr1 expression, as compared with their drug-sensitive counterparts. Such a preferential induction of hCtr1 expression in cDDP-resistant variants by Cu chelation can be explained by the mammalian Cu homeostasis regulatory mechanism. Enhanced cell-killing efficacy by a Cu-lowering agent was also observed in animal xenografts bearing cDDP-resistant cells. Finally, by analyzing a public gene expression dataset, we found that ovarian cancer patients with elevated levels of hCtr1 in their tumors, but not ATP7A and ATP7B, had more favorable outcomes after Pt-drug treatment than those expressing low hCtr1 levels. This study reveals the mechanistic basis for using Cu chelation to overcome cDDP resistance in clinical investigations.

Previous studies have demonstrated that treating cultured cells with cisplatin (CDDP) up-regulated the expression of glutathione (GSH) and its de novo rate-limiting enzyme glutamate-cysteine ligase (GCL), which consists of a catalytic (GCLC) and a modifier (GCLM) subunit. It has also been shown that many CDDPresistant cell lines exhibit high levels of GCLC/GCLM and GSH. Because the GSH system is the major intracellular regulator of redox conditions that serve as an important detoxification cytoprotector, these results have been taken into consideration that elevated levels of GCL/GSH are responsible for the CDDP resistance. In contrast to this context, we demonstrated here that overexpression of GSH by transfection with an expression plasmid containing the GCLC cDNA conferred sensitization to CDDP through up-regulation of human copper transporter (hCtr) 1, which is also a transporter for CDDP. Depleting GSH levels in these transfected cells reversed CDDP sensitivity with concomitant reduction of hCtr1 expression. Although rates of copper transport were also up-regulated in the transfected cells, these cells exhibited biochemical signature of copper deficiency, suggesting that GSH functions as an intracellular copper-chelator and that overexpression of GSH can alter copper metabolism. More importantly, our results reveal a new role of GSH in the regulation of CDDP sensitivity. Overproduction of GSH depletes the bioavailable copper pool, leading to upregulation of hCtr1 and sensitization of CDDP transport and cell killing. These findings also have important implications in that modulation of the intracellular copper pool may be a novel strategy for improving chemotherapeutic efficacy of platinumbased antitumor agents.

Copper is an essential micronutrient for cell growth but is toxic in excess. Copper transporter (Ctr1) plays an important role in regulating adequate copper levels in mammalian cells. We have shown previously that expression of the human high-affinity copper transporter (hCtr1) was transcriptionally up-regulated under copper-depleted conditions and downregulated under replete conditions; moreover, elevated hCtr1 levels suppress hCtr1 expression. Specificity protein 1 (Sp1) regulates expression of hCtr1 under copper-stressed conditions. In this study, we made the following important observations: 1) Sp1 expression is down-regulated under copper-replete conditions but up-regulated under copperdepleted conditions. These up-and down-regulations of Sp1 in turn regulate hCtr1 expression to control copper homeostasis. 2) Copper-regulated Sp1 expression involved Sp1 binding to its own promoter as demonstrated by the chromatin immunoprecipitation assay; therefore, Sp1 is also transcriptionally self-regulated via hCtr1/copper intermediation. 3) Both zinc finger and glutamine-rich transactivation domains of Sp1 are involved in the Sp1-mediated hCtr1 and Sp1 regulation by copper stresses. 4) Although Sp3 expression is also regulated by copper availability, Sp3 does not regulate hCtr1 homeostasis. Collectively, our results demonstrated that mammalian cells use Sp1 oscillation in response to copper availability to regulate copper homeostasis through hCtr1 expression in a tripartite inter-regulatory relationship. These findings have important implications in mammalian copper physiology regulation.

The expression of retinoic acid receptor B 2 (RAR-B 2 ) is frequently lost in various cancers and their premalignant lesions. However, the restoration of RAR-B 2 expression inhibits tumor cell growth and suppresses cancer development. To understand the molecular mechanisms responsible for this RAR-B 2 -mediated antitumor activity, we did restriction fragment differential display-PCR and cloned a novel retinoid receptor-induced gene 1 (RRIG1), which is differentially expressed in RAR-B 2 -positive and RAR-B 2 -negative tumor cells. RRIG1 cDNA contains 2,851 bp and encodes a protein with 276 amino acids; the gene is localized at chromosome 9q34. Expressed in a broad range of normal tissues, RRIG1 is also lost in various cancer specimens. RRIG1 mediates the effect of RAR-B 2 on cell growth and gene expression (e.g., extracellular signal-regulated kinase 1/2 and cyclooxygenase-2). The RRIG1 protein is expressed in the cell membrane and binds to and inhibits the activity of a small GTPase RhoA. Whereas induction of RRIG1 expression inhibits RhoA activation and f-actin formation and consequently reduces colony formation, invasion, and proliferation of esophageal cancer cells, antisense RRIG1 increases RhoA activity and f-actin formation and thus induces the colony formation, invasion, and proliferation of these cells. Our findings therefore show a novel molecular pathway involving RAR-B 2 regulation of RRIG1 expression and RRIG1-RhoA interaction. An understanding of this pathway may translate into better control of human cancer. (Cancer Res 2006; 66(14): 7111-8)

Histone-induced thrombotic thrombocytopenic purpura in adamts13-/zebrafish depends on von Willebrand factor

Platinum-based antitumor agents have been the mainstay in cancer chemotherapy for many human malignancies. Drug resistance is an important obstacle to achieving the maximal therapeutic efficacy of these drugs. Understanding how platinum drugs enter cells is of great importance in improving therapeutic efficacy. It has been demonstrated that human high-affinity copper transporter 1 (hCtr1) is involved in transporting cisplatin into cells to elicit cytotoxic effects, although other mechanisms may exist. In this communication, we demonstrate that cisplatin transcriptionally induces the expression of hCtr1 in time- and concentration-dependent manners. Cisplatin functions as a competitor for hCtr1-mediated copper transport, resulting in reduced cellular copper levels and leading to upregulated expression of Sp1, which is a positive regulator for hCtr1 expression. Thus, regulation of hCtr1 expression by cisplatin is an integral part of the copper homeostasis regulation system. We also demonstrate that Ag(I) and Zn(II), which are known to suppress hCtr1-mediated copper transport, can also induce hCtr1/Sp1 expression. In contrast, Cd(II), another inhibitor of copper transport, downregulates hCtr1 expression by suppressing Sp1 expression. Collectively, our results demonstrate diverse mechanisms of regulating copper metabolism by these heavy metals.