Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals.
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Cisplatin is a chemotherapeutic drug used to treat a variety of cancers. Both intrinsic and acquired resistance to cisplatin, as well as toxicity, limit its effectiveness. Molecular mechanisms that underlie cisplatin resistance are poorly understood. Here we demonstrate that deletion of the yeast CTR1 gene, which encodes a high-affinity copper transporter, results in increased cisplatin resistance and reduced intracellular accumulation of cisplatin. Copper, which causes degradation and internalization of Ctr1 protein (Ctr1p), enhances survival of wild-type yeast cells exposed to cisplatin and reduces cellular accumulation of the drug. Cisplatin also causes degradation and delocalization of Ctr1p and interferes with copper uptake in wild-type yeast cells. Mouse cell lines lacking one or both mouse Ctr1 (mCtr1) alleles exhibit increased cisplatin resistance and decreased cisplatin accumulation in parallel with mCtr1 gene dosage. We propose that cisplatin uptake is mediated by the copper transporter Ctr1p in yeast and mammals. The link between Ctr1p and cisplatin transport may explain some cases of cisplatin resistance in humans and suggests ways of modulating sensitivity and toxicity to this important anticancer drug. (+info)
Correction of the copper transport defect of Menkes patient fibroblasts by expression of two forms of the sheep Wilson ATPase.
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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. (+info)
Delineation of hypoxia in canine myocardium using PET and copper(II)-diacetyl-bis(N(4)-methylthiosemicarbazone).
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Copper(II)-diacetyl-bis(N(4)-methylthiosemicarbazone) (copper-ATSM) is a hypoxia-avid tracer for the selective identification of hypoxic tissue. Using canine models of hypoxic myocardium, we report our findings on *Cu-ATSM PET (*Cu is defined as either (60)Cu, (61)Cu, or (64)Cu) for the delineation of ischemic and hypoxic myocardium. METHODS: In protocol I, myocardial hypoxia was induced by global hypoxia (n = 3). In protocol II, myocardial ischemia was generated by occlusion of the left anterior descending coronary artery (n = 9). In protocol III, coronary artery stenosis was induced by a stenosis in the left anterior descending coronary artery (n = 4). PET dynamic data were acquired immediately after tracer injection. Tracer retention kinetics were analyzed using either monoexponential analysis (1/k(mono)) or a simple 2-compartment model (1/k(4)). RESULTS: In protocol I, tracer retention in hypoxic myocardium was 2-fold greater than in normal myocardium, despite a 7-fold increase in blood flow (normal, 0.70 +/- 0.42 mL.min(-1).g(-1); hypoxic, 4.94 +/- 3.00 mL.min(-1).g(-1) [P < 0.005]). In protocol II, approximately 3 h after occlusion, retention of *Cu-ATSM within 20 min was greater in ischemic regions (myocardial blood flow, 0.28 +/- 0.26 mL.min(-1).g(-1)) than in normal tissue (myocardial blood flow, 0.52 +/- 0.19 mL.min(-1).g(-1)) (1/k(mono), 40.72 +/- 39.0 min vs. 26.69 +/- 22.29 min [P < 0.05]; 1/k(4), 6.85 +/- 4.90 min vs. 3.51 +/- 1.97 min [P < 0.05]). In selected dogs, tracer retention decreased at 24 h, suggesting the development of necrosis with no subsequent retention of *Cu-ATSM. In protocol III, dobutamine infusion after stenosis placement resulted in increased tracer retention consistent with hypoxia in the damaged regions. CONCLUSION: *Cu-ATSM PET has shown quantitative selective uptake in hypoxic myocardium within 20 min of tracer administration in 3 canine models of hypoxia. (+info)
Prion infection impairs copper binding of cultured cells.
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The molecular mechanism of neurodegeneration in transmissible spongiform encephalopathies (TSEs) remains unclear. Using radioactive copper ((64)Cu) at physiological concentration, we showed that prion infected cells display a marked reduction in copper binding. The level of full-length prion protein known to bind the metal ion was not modified in infected cells, but a fraction of this protein was not releasable from the membrane by phosphatidylinositol-specific phospholipase C. Our results suggest that prion infection modulates copper content at a cellular level and that modification of copper homeostasis plays a determinant role in the neuropathology of TSE. (+info)
In vivo evaluation of pretargeted 64Cu for tumor imaging and therapy.
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Pretargeting involves administration of a tumor-targeting monoclonal antibody (mAb) covalently linked to a molecule having a high-affinity binding site for a rapidly distributed radiolabeled effector molecule. The aim of this study was to compare pretargeting to a conventionally labeled antibody for tumor targeting of the intermediate-lived radionuclide (64)Cu, which has shown promise for PET imaging and radioimmunotherapy of cancer. METHODS: DOTA-biotin (where DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid) and the intact immunoconjugate DOTA-NR-LU-10 were labeled to high specific activities with (64)Cu, and the serum stabilities and target binding capabilities of each agent were assayed in vitro. Nude mice bearing SW1222 human colorectal carcinoma xenografts were administered (64)Cu-DOTA-biotin, with and without pretreatment with the mAb-streptavidin conjugate NR-LU-10/SA and the synthetic clearing agent Biotin-GalNAc(16), or injected with (64)Cu-DOTA-NR-LU-10. Biodistributions of both agents were obtained from 5 min to 48 h after injection. RESULTS: Both (64)Cu-DOTA-biotin and (64)Cu-DOTA-NR-LU-10 were 100% stable in serum in vitro. (64)Cu-DOTA-biotin exhibited >98% specific binding to immobilized streptavidin, whereas the immunoreactivity of (64)Cu-DOTA-NR-LU-10 averaged nearly 80%. Biodistributions in SW1222-bearing mice showed that NR-LU-10/SA-pretargeted (64)Cu-DOTA-biotin attained a peak tumor uptake of 18.9 percentage injected dose per gram (%ID/g) at 1 h, with concomitant rapid disappearance from blood and renal excretion. In the absence of pretargeting, (64)Cu-DOTA-biotin had very similar biodistribution and clearance properties, except with extremely low nonspecific tumor uptake. In contrast, (64)Cu-DOTA-NR-LU-10 reached 80.3 %ID/g in tumor tissue, after 48 h, whereas blood clearance was considerably slower than pretargeted (64)Cu-DOTA-biotin. Comparison of the time-activity curves for tumor uptake and blood clearance of pretargeted (64)Cu and the (64)Cu-labeled antibody revealed that the maximum tumor accumulations of radioactivity were similar for each agent, 17.9 percentage injected activity per gram (%IA/g) and 20.7 %IA/g, respectively. However, the tumor-to-blood ratio of areas under the curves was 14 times higher for pretargeted (64)Cu-DOTA-biotin because of the substantial increase in blood clearance of the small effector molecule. CONCLUSION: The extremely rapid tumor uptake and blood clearance of pretargeted (64)Cu-DOTA-biotin should afford markedly superior PET imaging contrast and therapeutic efficacy, compared with conventionally labeled (64)Cu-DOTA-NR-LU-10. Further comparison of the therapeutic efficacy, toxicity, and dosimetry of these 2 agents is warranted. (+info)
Effects of age and sex on copper absorption, biological half-life, and status in humans.
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Healthy, free-living men and women aged 20-83 y (n = 127) were studied to determine the effects of age and sex on copper absorption, biological half-life (BH), and status. Copper absorption was greater in women (71%) than in men (64%) aged 20-59 y (P = 0.02), but did not differ in men and women aged 60-83 y. BH of 67Cu ranged from 13 to 33 d and differed between men and women aged 20-59 y (P = 0.006), but not between men and women aged 60-83 y. Plasma copper, enzymatic ceruloplasmin (Cp), and immunoreactive (RID) Cp were significantly higher in women than in men (P < 0.005), but superoxide dismutase (SOD) and in vitro 67Cu uptake by red blood cells did not differ. Plasma copper, RID Cp, and cytochrome oxidase in platelets and mononuclear cells were significantly affected by age (P < 0.005). Oral contraceptives elevated plasma copper, enzymatic Cp, and SOD activity but not copper absorption and BH in women aged 20-39 y. Copper intake from self-selected diets was 0.9-1.2 mg/d for women and 1.2-1.3 mg/d for men, but net copper absorption (micrograms Cu.kg body wt-1.d-1) did not differ. Thus, dietary copper intake requirements may differ between men and women. (+info)
Enhancing targeted radiotherapy by copper(II)diacetyl- bis(N4-methylthiosemicarbazone) using 2-deoxy-D-glucose.
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Most cancer deaths are a consequence of resistance to conventional chemotherapy and radiation therapy. This may be attributable to unique phenotypic characteristics of solid tumors. We have exploited two well-described characteristics of solid tumors commonly associated with treatment failure, high glucose use and hypoxia, to design a unique therapy based on the selective accumulation of two cytotoxic compounds, 2-deoxyglucose (2-DG) and copper(II)diacetyl-bis(N(4)-methylthiosemicarbazone) ((64)Cu-ATSM). (64)Cu-ATSM localizes to hypoxic regions of tumors and has been used for administering a high local dose of radiation therapy after uptake by cells. 2-DG, a glucose analog, selectively accumulates in cancer cells and interferes with energy metabolism, resulting in cancer cell death. 2-DG has been shown to potentiate the cytotoxic effect of ionizing radiation and certain chemotherapeutic agents. We have tested the effect of 2-DG on tumor response when combined with (64)Cu-ATSM in a mouse breast tumor model using the highly aggressive mouse mammary carcinoma cell line EMT-6. 2-DG administered up to 2 mg/g of body weight daily resulted in no weight loss or systemic symptoms. EMT-6 mammary tumors had similar uptake of [(18)F]fluoro-2-deoxyglucose before and after 2 weeks of 2-DG treatment as determined by microPET imaging, indicating that resistance to 2-DG uptake does not develop. Pretreatment of tumor-bearing mice with 2-DG resulted in increased uptake of (64)Cu-ATSM by tumors compared with nontreated mice. This effect was not observed with the nonhypoxia-specific agent copper(II)pyruvaldehyde-bis(N(4)-methylthiosemicarbazone. When 2-DG was combined with a single dose of (64)Cu-ATSM (2 mCi), tumor growth was inhibited approximately 60% compared with untreated mice, and animals survived approximately 50% longer than untreated mice or animals treated with each agent alone (32 versus 20 days). The maximum effect on tumor growth and survival was observed when 2-DG was administered daily for the lifetime of the mouse. Our results indicate that 2-DG potentiates the effect of (64)Cu-ATSM on tumoricidal activity and animal survival. We hypothesize that 2-DG alters the metabolic state of the cell, leading to increased uptake of (64)Cu-ATSM by the tumor. This would result in a higher local dose of radiotherapy. The continued presence of 2-DG would then prevent the repair of damaged cells, leading to inhibition of tumor growth. Our data indicate that the strategy of combining tumor-specific cytotoxic agents that function by differing mechanisms can result in an effective, selective, tumor-specific cell death with minimal effect on the host. (+info)
Subcellular localization of radiolabeled somatostatin analogues: implications for targeted radiotherapy of cancer.
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Copper-64 (T(1/2) = 12.7 h; beta(+), 17.4%; beta(-), 39%) has been used both in positron emission tomography imaging and in radiotherapy. Copper-64 radiopharmaceuticals have shown tumor growth inhibition with a relatively low radiation dose in animal models; however, the mechanism of cytotoxicity has not been fully elucidated. These studies incorporate the use of somatostatin receptor-positive AR42J rat pancreatic tumor cells in vitro to understand the cell killing mechanism of (64)Cu by focusing on subcellular distribution of the somatostatin analogues (64)Cu-labeled 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid-octreotide ((64)Cu-TETA-OC) and (111)In-labeled diethylenetriaminepentaacetic acid-octreotide ((111)In-DTPA-OC). Cell uptake and organelle isolation studies were conducted on (64)Cu-TETA-OC and (111)In-DTPA-OC. Nuclear localization of (64)Cu and (111)In from (64)Cu-TETA-OC and (111)In-DTPA-OC, respectively, increased over time, with 19.5 +/- 1.4% and 6.0 +/- 1.0% in the cell nucleus at 24 h, respectively. In pulse-chase experiments, in which (64)Cu-TETA-OC was incubated with AR42J cells for 4 h, it was found that the nuclear localization of (64)Cu increased significantly over the next 20 h (from 9.8 +/- 1.0% to 26.3 +/- 5.4%). In a control pulse-chase experiment, levels of (64)Cu from [(64)Cu]cupric acetate decreased from 4 to 24 h postadministration (20.6 +/- 8.7 to 5.4 +/- 1.9), suggesting that the redistribution mechanism, or the kinetics of (64)Cu from (64)Cu-TETA-OC is different from that for (64)Cu from [(64)Cu]cupric acetate. The amount of (64)Cu from (64)Cu-TETA-OC also increased in the mitochondria over time, with 21.1 +/- 3.6% in the mitochondria at 24 h postadministration. These results suggest that localization of substantial quantities of (64)Cu to the cell nucleus and mitochondria may contribute to cell killing with (64)Cu radiopharmaceuticals. (+info)