Expression Of CTR1 Research Articles

High fructose feeding induces copper deficiency in Sprague–Dawley rats: A novel mechanism for obesity related fatty liver

Dietary copper deficiency is associated with a variety of manifestations of the metabolic syndrome, including hyperlipidemia and fatty liver. Fructose feeding has been reported to exacerbate complications of copper deficiency. In this study, we investigated whether copper deficiency plays a role in fructose-induced fatty liver and explored the potential underlying mechanism(s). Male weanling Sprague-Dawley rats were fed either an adequate copper or a marginally copper deficient diet for 4 weeks. Deionized water or deionized water containing 30% fructose (w/v) was also given ad lib. Copper and iron status, hepatic injury and steatosis, and duodenum copper transporter-1 (Ctr-1) were assessed. Fructose feeding further impaired copper status and led to iron overload. Liver injury and fat accumulation were significantly induced in marginal copper deficient rats exposed to fructose as evidenced by robustly increased plasma aspartate aminotransferase (AST) and hepatic triglyceride. Hepatic carnitine palmitoyl-CoA transferase I (CPT I) expression was significantly inhibited, whereas hepatic fatty acid synthase (FAS) was markedly up-regulated in marginal copper deficient rats fed with fructose. Hepatic antioxidant defense system was suppressed and lipid peroxidation was increased by marginal copper deficiency and fructose feeding. Moreover, duodenum Ctr-1 expression was significantly increased by marginal copper deficiency, whereas this increase was abrogated by fructose feeding. Our data suggest that high fructose-induced nonalcoholic fatty liver disease (NAFLD) may be due, in part, to inadequate dietary copper. Impaired duodenum Ctr-1 expression seen in fructose feeding may lead to decreased copper absorption, and subsequent copper deficiency.

Prognostic value of the copper transporters, CTR1 and CTR2, in patients with ovarian carcinoma receiving platinum-based chemotherapy

Objective CTR1 and CTR2 are copper transporters that have been associated with platinum sensitivity in several human cancers. We investigated the prognostic significance of CTR1 and CTR2 in women with ovarian carcinoma. Materials and methods We evaluated the expression of CTR1 and CTR2 using real-time PCR in 40 women with ovarian carcinoma (IIb = 2, IIIb = 2, IIIc = 30, IV = 6). We compared the expression of CTR1 and CTR2 with participants' clinicopathological findings. Results We found lower expression of CTR1 and CTR2 mRNA in ovarian cancer cells against normal ovarian tissue with statistically significant differences ( p = 0.018 and 0.011, respectively). High CTR1 expression was a prognostic factor for improved survival after adjusting for age, tumor grade, stage, residual tumor, and CTR2 mRNA expression (HR, 0.35; 95% CI, 0.15–0.84). However, CTR2 expression did not exhibit any prognostic significance. Of the 20 women with elevated CTR1 expression, 17 (85%) were sensitive to platinum-based chemotherapy. Of the 7 women with low CTR1 expression and high CTR2 expression, 6 (85.7%) were resistant to platinum-based chemotherapy and had the shortest progression-free survival of all women in our study sample. Conclusion In our sample of 40 women with ovarian carcinoma, high CTR1 expression was significantly associated with sensitivity to platinum-based chemotherapy and longer progression-free survival. Conversely, low CTR1 expression and high CTR2 expression were significantly associated with resistance to platinum-based chemotherapy and the shortest survival.

Physiological response to a metal-contaminated invertebrate diet in zebrafish: Importance of metal speciation and regulation of metal transport pathways

Dietary metal uptake in fish is determined by metal bioavailability in prey and the metal requirements of the fish. In this study zebrafish were fed the intertidal polychaete worm Nereis diversicolor (3% wet weight day −1) collected from Ag, Cd and Cu-impacted Restronguet Creek (RC) or a reference site, Blackwater estuary (BW), for 21 days. On days 0, 7, 14 and 21 fish were fed a single meal of RC or BW N. diversicolor labeled with 110mAg or 109Cd for measurements of metal assimilation efficiency (AE). Zebrafish intestines were also taken for mRNA expression analysis of copper transporter 1 ( ctr1), divalent metal transporter 1 ( dmt1) and metallothionein 2 ( mt2). No significant difference was observed in the AE of 109Cd in metal naïve fish at day 0 between RC and BW worms, 11.8 ± 2.1 and 15.3 ± 2.8%, respectively. However, AE of 110mAg was significantly greater in fish fed worms from BW compared to RC, 5 ± 1.2% and 1.6 ± 0.5%, respectively at day 0. Fractionation analysis of radiolabeled metal partitioned in N. diversicolor from RC revealed a greater proportion of Ag (40 ± 1.1%) in a fraction containing protein and organelle bound metal, associated with high trophic availability, compared to BW polychaetes (24 ± 2.5%). Lower AE of 110mAg from RC polychaetes is therefore unlikely due to speciation of 110mAg in N. diversicolor from RC, but to the high concentration of Cu, a potential Ag antagonist. Exposure to RC polychaetes significantly increased the AE of 110mAg (6.2 ± 1%), but not 109Cd, from RC worms, after 21 days. AE of 110mAg and 109Cd was unaffected by pre-exposure to BW. Elevated concentration of intestinal Cu and increased expression of ctr1, dmt1 and mt2 after 14 days exposure in fish fed worms from RC suggest altered Cu handling strategy of these fish which may increase AE of Ag via shared Ag and Cu transport pathways. These data suggest metal exposure history of invertebrates may affect metal bioavailability to fish, and fish may alter intestinal uptake physiology during chronic dietary exposure with implications for the assimilation and toxicity of dietary metals.

Roles of copper chaperone for superoxide dismutase 1 and metallothionein in copper homeostasis

Copper chaperone for SOD1 (CCS) specifically delivers copper (Cu) to copper, zinc superoxide dismutase (SOD1) in cytoplasm of mammalian cells. In the present study, small interfering RNA (siRNA) targeting CCS was introduced into metallothionein-knockout mouse fibroblasts (MT-KO cells) and their wild type cells (MT-WT cells) to reveal the interactive role of CCS with other Cu-regulating proteins, in particular, MT. CCS knockdown significantly decreased Ctr1, a Cu influx transporter, mRNA expression. On the other hand, Atp7a, a Cu efflux transporter, mRNA expression was increased 3.0 and 2.5 times higher than those of the control in MT-WT and MT-KO cells. These responses of Cu-regulating genes to the CCS knockdown reflected the presence of excess Cu in the cells. To evaluate the Atp7a function in the Cu-replete cells, siRNA of Atp7a and the other Cu transporter, Atp7b were introduced into MT-WT and MT-KO cells. The Atp7a knockdown significantly increased the intracellular Cu concentration, whereas the Atp7b knockdown had no affect. Although two MT isoforms were induced by the CCS knockdown in MT-WT cells, the expression and activity of SOD1 were maintained in both MT-WT and MT-KO cells even when CCS protein expression was reduced to 0.30-0.35 of control. This suggests that the amount of CCS protein exceeds that required to supply Cu to SOD1 in the cells. Further, the CCS knockdown induces Cu accumulation in cells, however, the Cu accumulation is ameliorated by the MT induction, the decrease of Ctr1 expression and the increase of Atp7a expression to maintain Cu homeostasis.

Cadmium regulates copper homoeostasis by inhibiting the activity of Mac1, a transcriptional activator of the copper regulon, in Saccharomyces cerevisiae

Cadmium is a toxic metal and the mechanism of its toxicity has been studied in various model systems from bacteria to mammals. We employed Saccharomyces cerevisiae as a model system to study cadmium toxicity at the molecular level because it has been used to identify the molecular mechanisms of toxicity found in higher organisms. cDNA microarray and Northern blot analyses revealed that cadmium salts inhibited the expression of genes related to copper metabolism. Western blotting, Northern blotting and chromatin immunoprecipitation experiments indicated that CTR1 expression was inhibited at the transcriptional level through direct inhibition of the Mac1 transcriptional activator. The decreased expression of CTR1 results in cellular copper deficiency and inhibition of Fet3 activity, which eventually impairs iron uptake. In this way, cadmium exhibits a negative effect on both iron and copper homoeostasis.

The Drosophila Copper Transporter Ctr1C Functions in Male Fertility

Living organisms have evolved intricate systems to harvest trace elements from the environment, to control their intracellular levels, and to ensure adequate delivery to the various organs and cellular compartments. Copper is one of these trace elements. It is at the same time essential for life but also highly toxic, not least because it facilitates the generation of reactive oxygen species. In mammals, copper uptake in the intestine and copper delivery into other organs are mediated by the copper importer Ctr1. Drosophila has three Ctr1 homologs: Ctr1A, Ctr1B, and Ctr1C. Earlier work has shown that Ctr1A is an essential gene that is ubiquitously expressed throughout development, whereas Ctr1B is responsible for efficient copper uptake in the intestine. Here, we characterize the function of Ctr1C and show that it functions as a copper importer in the male germline, specifically in maturing spermatocytes and mature sperm. We further demonstrate that loss of Ctr1C in a Ctr1B mutant background results in progressive loss of male fertility that can be rescued by copper supplementation to the food. These findings hint at a link between copper and male fertility, which might also explain the high Ctr1 expression in mature mammalian spermatozoa. In both mammals and Drosophila, the X chromosome is known to be inactivated in the male germline. In accordance with such a scenario, we provide evidence that in Drosophila, the autosomal Ctr1C gene originated as a retrogene copy of the X-linked Ctr1A, thus maintaining copper delivery during male spermatogenesis.

Dietary iron affects mRNA expression of porcine copper transporters

Weanling male pigs (21 days of age) were fed diets deficient (20 mg Fe/kg; L‐Fe), adequate (120 mg Fe/kg; A‐Fe) or high (520 mg Fe/kg; H‐Fe) in iron (Fe) for 32 days to evaluate the effects of dietary Fe level on copper (Cu) transporters and chaperones. Increasing dietary Fe increased Fe in proximal duodenum and liver. Liver Cu was lower in A‐Fe than in L‐Fe and H‐Fe pigs and did not differ among pigs fed L‐Fe and H‐Fe diets. Expression of liver copper transporter 1 (Ctr1) was higher in L‐Fe vs. A‐Fe pigs. Liver Atp7b was lower in both L‐Fe and H‐Fe pigs vs. A‐Fe pigs. Ceruloplasmin (Cp) expression in liver was up‐regulated in L‐Fe and H‐Fe pigs vs. A‐Fe pigs. Cp was also increased in pigs fed L‐Fe vs. H‐Fe diets. Feeding a L‐Fe diet tended to decrease Commd1 compared to feeding an A‐Fe diet. Liver cytochrome c oxidase assembly protein 17 (Cox17), antioxidant 1 (Atox1), copper chaperone for Cu/Zn superoxide dismutase (CCS), Cu/Zn superoxide dismutase (Sod1), and metallothionein 1a (Mt1a) were unaffected by dietary Fe. Duodenal Cu concentration and expression of duodenal Ctr1, Atox1, Cox17, CCS, Sod1, Atp7a, or Mt1a were not affected by dietary Fe. These data indicate that dietary Fe affects cellular Cu transporters involved in import and export of Cu to a greater extent in the liver than in duodenum. Reduced hepatic expression of Atp7b in both L‐Fe and H‐Fe pigs may explain the increased liver Cu seen in these pigs relative to A‐Fe.Grant Funding Source: Micronutrients

Differential expression of ATP7A, ATP7B and CTR1 in adult rat dorsal root ganglion tissue.

BackgroundATP7A, ATP7B and CTR1 are metal transporting proteins that control the cellular disposition of copper and platinum drugs, but their expression in dorsal root ganglion (DRG) tissue and their role in platinum-induced neurotoxicity are unknown. To investigate the DRG expression of ATP7A, ATP7B and CTR1, lumbar DRG and reference tissues were collected for real time quantitative PCR, RT-PCR, immunohistochemistry and Western blot analysis from healthy control adult rats or from animals treated with intraperitoneal oxaliplatin (1.85 mg/kg) or drug vehicle twice weekly for 8 weeks.ResultsIn DRG tissue from healthy control animals, ATP7A mRNA was clearly detectable at levels similar to those found in the brain and spinal cord, and intense ATP7A immunoreactivity was localised to the cytoplasm of cell bodies of smaller DRG neurons without staining of satellite cells, nerve fibres or co-localisation with phosphorylated heavy neurofilament subunit (pNF-H). High levels of CTR1 mRNA were detected in all tissues from healthy control animals, and strong CTR1 immunoreactivity was associated with plasma membranes and vesicular cytoplasmic structures of the cell bodies of larger-sized DRG neurons without co-localization with ATP7A. DRG neurons with strong expression of ATP7A or CTR1 had distinct cell body size profiles with minimal overlap between them. Oxaliplatin treatment did not alter the size profile of strongly ATP7A-immunoreactive neurons but significantly reduced the size profile of strongly CTR1-immunoreactive neurons. ATP7B mRNA was barely detectable, and no specific immunoreactivity for ATP7B was found, in DRG tissue from healthy control animals.ConclusionsIn conclusion, adult rat DRG tissue exhibits a specific pattern of expression of copper transporters with distinct subsets of peripheral sensory neurons intensely expressing either ATP7A or CTR1, but not both or ATP7B. The neuron subtype-specific and largely non-overlapping distribution of ATP7A and CTR1 within rat DRG tissue may be required to support the potentially differing cuproenzyme requirements of distinct subsets of sensory neurons, and could influence the transport and neurotoxicity of oxaliplatin.

ATP7B detoxifies silver in ciliated airway epithelial cells

Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B −/− mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag +/Cu + transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

Two Chlamydomonas CTR Copper Transporters with a Novel Cys-Met Motif Are Localized to the Plasma Membrane and Function in Copper Assimilation

Inducible high-affinity copper uptake is key to copper homeostasis in Chlamydomonas reinhardtii. We generated cDNAs and updated gene models for four genes, CTR1, CTR2, CTR3, and COPT1, encoding CTR-type copper transporters in Chlamydomonas. The expression of CTR1, CTR2, and CTR3 increases in copper deficient cells and in response to hypoxia or Ni(2+) supplementation; this response depends on the transcriptional activator CRR1. A copper response element was identified by mutational analysis of the 5' upstream region of CTR1. Functional analyses identify CTR1 and CTR2 as the assimilatory transporters of Chlamydomonas based on localization to the plasma membrane and ability to rescue a Saccharomyces cerevisiae mutant defective in high-affinity copper transport. The Chlamydomonas CTRs contain a novel Cys-Met motif (CxxMxxMxxC-x(5/6)-C), which occurs also in homologous proteins in other green algae, amoebae, and pathogenic fungi. CTR3 appears to have arisen by duplication of CTR2, but CTR3 lacks the characteristic transmembrane domains found in the transporters, suggesting that it may be a soluble protein. Thus, Chlamydomonas CTR genes encode a distinct subset of the classical CTR family of Cu(I) transporters and represent new targets of CRR1-dependent signaling.

Deletion of hepatic Ctr1 reveals its function in copper acquisition and compensatory mechanisms for copper homeostasis

Copper is a vital trace element required for normal growth and development of many organisms. To determine the roles for copper transporter 1 (Ctr1) in hepatic copper metabolism and the contribution of the liver to systemic copper homeostasis, we have generated and characterized mice in which Ctr1 is deleted specifically in the liver. These mice express less than 10% residual Ctr1 protein in the liver and exhibit a small but significant growth retardation, which disappears with age. Hepatic copper concentrations and the activities of copper-requiring enzymes are reduced; however, mild copper deficiency relative to Ctr1 protein deficit indicates compensatory mechanisms for copper metabolism. Copper concentrations of other organs did not alter despite the defect in hepatic copper uptake. Whereas biliary copper excretion is reduced, urinary copper concentration in these mice is higher than that of control mice. Our data indicate that Ctr1 plays a critical role in copper acquisition in the liver, and, when Ctr1 expression is compromised, compensatory mechanisms facilitate copper uptake and/or retention in the liver and excretion of copper via urine.

Self-renewal and chemotherapy resistance of p75NTR positive cells in esophageal squamous cell carcinomas

Backgroundp75NTR has been used to isolate esophageal and corneal epithelial stem cells. In the present study, we investigated the expression of p75NTR in esophageal squamous cell carcinoma (ESCC) and explored the biological properties of p75NTR+ cells.Methodsp75NTR expression in ESCC was assessed by immunohistochemistry. p75NTR+ and p75NTR- cells of 4 ESCC cell lines were separated by fluorescence-activated cell sorting. Differentially expressed genes between p75NTR+ and p75NTR- cells were determined by real-time quantitative reverse transcription-PCR. Sphere formation assay, DDP sensitivity assay, 64copper accumulation assay and tumorigenicity analysis were performed to determine the capacity of self-renewal, chemotherapy resistance and tumorigenicity of p75NTR+ cells.ResultsIn ESCC specimens, p75NTR was found mainly confined to immature cells and absent in cells undergoing terminal differentiation. The percentage of p75NTR+ cells was 1.6%–3.7% in Eca109 and 3 newly established ESCC cell lines. The expression of Bmi-1, which is associated with self-renewal of stem cells, was significantly higher in p75NTR+ cells. p63, a marker identified in keratinocyte stem cells, was confined mainly to p75NTR+ cells. The expression of CTR1, which is associated with cisplatin (DDP)-resistance, was significantly decreased in p75NTR+ cells. Expression levels of differentiation markers, such as involucrin, cytokeratin 13, β1-integrin and β4-integrin, were lower in p75NTR+ cells. In addition, p75NTR+ cells generated both p75NTR+ and p75NTR- cells, and formed nonadherent spherical clusters in serum-free medium supplemented with growth factors. Furthermore, p75NTR+ cells were found to be more resistant to DDP and exhibited lower 64copper accumulation than p75NTR- cells.ConclusionOur results demonstrated that p75NTR+ cells possess some characteristics of CSCs, namely, self-renewal and chemotherapy resistance. Chemotherapy resistance of p75NTR+ cells may probably be attributable to decreased expression of CTR1.

Transcriptional Activation in Yeast in Response to Copper Deficiency Involves Copper-Zinc Superoxide Dismutase

Copper is an essential trace element, yet excess copper can lead to membrane damage, protein oxidation, and DNA cleavage. To balance the need for copper with the necessity to prevent accumulation to toxic levels, cells have evolved sophisticated mechanisms to regulate copper acquisition, distribution, and storage. In Saccharomyces cerevisiae, transcriptional responses to copper deficiency are mediated by the copper-responsive transcription factor Mac1. Although Mac1 activates the transcription of genes involved in high affinity copper uptake during periods of deficiency, little is known about the mechanisms by which Mac1 senses or responds to reduced copper availability. Here we show that the copper-dependent enzyme Sod1 (Cu,Zn-superoxide dismutase) and its intracellular copper chaperone Ccs1 function in the activation of Mac1 in response to an external copper deficiency. Genetic ablation of either CCS1 or SOD1 results in a severe defect in the ability of yeast cells to activate the transcription of Mac1 target genes. The catalytic activity of Sod1 is essential for Mac1 activation and promotes a regulated increase in binding of Mac1 to copper response elements in the promoter regions of genomic Mac1 target genes. Although there is precedent for additional roles of Sod1 beyond protection of the cell from oxygen radicals, the involvement of this protein in copper-responsive transcriptional regulation has not previously been observed. Given the presence of both Sod1 and copper-responsive transcription factors in higher eukaryotes, these studies may yield important insights into how copper deficiency is sensed and appropriate cellular responses are coordinated.

Characterization and Identification of Hepatic mRNA Related to Copper Metabolism and Homeostasis in Cattle

Copper is an essential trace mineral required for growth and development. Copper homeostasis within the cell is mediated by the expression of the Cu transporter protein (CTR1), ATPase7A (ATP7A), ATPase7B (ATP7B), Cox17, and Cu chaperone for Cu-Zn superoxide dismutase (CCS) which helps to regulate Cu uptake, export, and intracellular compartmentalization in non-ruminants. Copper also serves as a cofactor of antioxidant, superoxide dismutase1 (SOD1). Liver tissue from eighteen Holstein bull calves (average BW 201 +/- 58.5 kg, 7.3 +/- 1.9 months) from a previous experiment were utilized to characterize and identify hepatic mRNA related to Cu metabolism and homeostasis in cattle. Hepatic Cu concentration was determined via flame atomic absorption, and total RNA was extracted using TRI reagent and purified using RNeasy. Hepatic Cu concentrations ranged from 86 to 801 mg of Cu/kg DM. Real-time polymerase chain reaction analysis revealed that CTR1, ATP7A, and ATP7B mRNA expressions were negatively correlated with hepatic Cu concentration, while CCS (P = 0.0887) and SOD1 had a tendency (P = 0.0733) to be negatively correlated to hepatic Cu concentration. These data indicate that higher than normal hepatic Cu concentration downregulates gene expression of CTR1, ATP7A, ATP7B, and Cox17, which are involved in bovine liver copper homeostasis.

Gefitinib Maintenance in Stage III Non–Small-Cell Lung Cancer

■■■ TOTHEEDITOR:Kellyetal 1 recentlyreportedinJournalofClinical Oncology that gefitinib maintenance after chemoradiotherapy and consolidation chemotherapy was associated with a significant decreaseinoverallsurvival(butonlyaslightdecreaseinprogression-free survival)comparedwithplaceboinstageIIInon‐small-celllungcancer(NSCLC).Theyofferpotentialexplanationsforthis,asdoKeedyet al 2 in an accompanying editorial. We would like to suggest an additional possible reason for this outcome. The flattening of the NSCLC dose-response curve at higher chemotherapy doses suggests that the most important factor in chemotherapy resistance is deficiency of factors required for efficacy. 3 Platinum-resistant cell lines may have decreased expression of a range of transporters that are important for cellular uptake of chemotherapy and nutrients. 4 In patients with a variety of resistant tumor types, we assessed tumor expression (by immunohistochemistry) of the copper transporter CTR1, which is also important in platinum uptake. CTR1 expression was significantly lower in tumors of patients who had received either chemotherapy or targeted therapies withintheprevious3monthsthanintumorsofpatientswithalonger interval off therapy. 5 The correlation with time from last chemotherapy or targeted therapy was stronger than the correlation with time from last cytotoxic therapy alone. This leads us to hypothesize that either chemotherapy or targeted therapy may result in a broad reduction in membrane transporters and that this, in turn, may generate broad cross resistance. This could, in part, explain why adding tyrosine kinase inhibitors to chemotherapy did not improve outcome in chemotherapy-naive advanced NSCLC patients 6 and might also predict that monoclonal antibodies (that do not require uptake across membranes) might add to chemotherapy even if small molecules did not. If correct, this would also predict that most other currentlyavailablesmallmoleculestargetingtumorcellswilladdlittle

Copper transporter 1, metallothionein and glutathione reductase genes are differentially expressed in tissues of sea bream ( Sparus aurata) after exposure to dietary or waterborne copper

The high affinity copper transporter 1 (Ctr1), metallothionein (MT) and glutathione reductase (GR) are essential for copper uptake, sequestration and defense respectively. Following rearing on a normal commercial diet (12.6 ± 0.2 mg kg − 1 Cu), sea bream were fed an experimental control diet lacking mineral mix (7.7 ± 0.3 mg kg − 1 Cu), an experimental diet enhanced with Cu (135 ± 4 mg kg − 1 Cu) or an experimental diet (7.7 ± 0.3 mg kg − 1 Cu) whilst exposed to Cu in water (0.294 ± 0.013 mg L − 1 ). Fish were sampled at 0, 15 and 30 days after exposures. Fish fed the Cu-enhanced experimental diet showed lower levels of expression of Ctr1 in the intestine and liver compared to fish fed control experimental diets, whilst Ctr1 expression in the gill and kidney was unaffected by excess dietary Cu exposure. Waterborne-Cu exposure increased Ctr1 mRNA levels in the intestine and the kidney compared to experimental controls. Excess dietary Cu exposure had no effect on levels of metallothionein (MT) mRNA, and the only effect of dietary excess Cu on glutathione reductase (GR) mRNA was a decrease in the intestine. Both MT mRNA and GR were increased in the liver and gill after waterborne-Cu exposure, compared to levels in fish fed experimental control low Cu diets. Thus, Ctr1, MT and GR mRNA expression in response to excess Cu is dependent on the route of exposure. Furthermore, the tissue expression profile of sea bream Ctr1 is consistent with the known physiology of copper exposure in fish and indicates a role both in essential copper uptake and in avoidance of excess dietary and waterborne copper influx.

THE EFFECT OF CISPLATIN ON THE COPPER TRANSPORTERS AND SPERMATOGENESIS IN THE TESTIS

Cisplatin (cis-diamminedichloroplatinum(II)) is a chemotherapeutic agent that efficiently eliminates tumor cells, especially in testicular germ cell tumors. Unfortunately, cisplatin also can cause a loss of normal germ cells from the testis. It is believed that long-term infertility that this agent may cause is due to the loss and/or dysfunction of stem germ cells. Previous studies of our lab showed that sub-chronic cisplatin exposure of C57/Bl/6J mice increased the incidence of germ cell apoptosis and caused a long-term failure of spermatogenesis. Spermatogonia, arising from stem germ cells, underwent apoptosis and were unable to re-populate the testis of cisplatin-exposed mice. These data suggest that either direct damage by cisplatin occurs to stem germ cells or that damage occurs to Sertoli cells that then cannot create a supportive microenvironment to support the development and differentiation of the various germ cell subtypes. Here we report that exposure of the Sertoli cell lines TM4 or ASC-17D to cisplatin (100 μM) show a significant decrease of mRNA expression of copper efflux transporter ATP7B within 6 hours of treatment. However, no differences in mRNA expression of influx transporter Ctr1 were observed after cisplatin treatment. By ELISA, no changes in inhibin B or transferrin in protein levels were measured in media of TM4 or ASC-17D cells after cisplatin treatment. After long-term exposure of 2.5 mg/kg cisplatin, partial loss of influx transporter function in Ctr1+/- mice resulted in 4% reduction of testis/body weight ratio compared with nontreatment control, whereas C57/Bl/6J wild-type mice showed a significant decrease of testis/body weight ratio after cisplatin treatment (reduced 22%). In addition, Ctr1+/- mice treated with cisplatin showed no significant change of TUNEL-positive germ cell numbers, indicating that the reduction in cisplatin uptake correlated with the sensitivity of cisplatin-induced injury. These observations demonstrate the copper transporters play important roles in cisplatin-stimulated testicular injury and spermatogenesis recovery. (poster)

Survey of the expression of genes for ethylene synthesis and perception during maturation and ripening of ‘Sunrise’ and ‘Golden Delicious’ apple fruit

Ethylene is important in the ripening of apple fruit and different cultivars of apple show very different ripening patterns. The patterns of expression of the currently known genes for ethylene synthesis and perception in apple were examined for the summer apple, ‘Sunrise’ (SR), and the later season ‘Golden Delicious’ (GD). Comparisons were made during the last 4 weeks of maturation on the tree and after post-harvest ripening using reverse transcriptase quantitative PCR. Increases in gene expression for ACC synthase 1 ( ACS1) and ACC Oxidase 1 ( ACO1) were 1000- and 10,000-fold, respectively, for both cultivars confirming the importance of these genes in the climacteric ethylene burst. ACS1 levels remained below detection level until the ripening stage. Additional apple genes for ACS and ACO did not appear to contribute to this ripening-associated ethylene. ACS3 gene expression in both cultivars increased 100-fold during maturation and reached near maximum levels a full week before commercial harvest. Unique DNA sequences for apple ethylene receptors ETR2 and ETR5 and ethylene control element CTR1 were determined. Expression of four ethylene receptors ( ETR1, 2, 5 and ERS1) and two control elements, CTR1 and EIN2, were evaluated. Only small changes (less than 5-fold) were observed for these perception elements. Splice variants of CTR1 did not appear to be differentially expressed in these tissues. Genes for ACO3 and ERS1 (in GD) responded in a fashion consistent with feedback inhibition of ethylene production in ripening tissue. Differences between the cultivars included a more gradual increase for ACO1 in GD, an increase in ERS1 for GD upon ripening that was not seen in SR, and a larger decrease in expression of CTR1 for GD compared with SR.

Relevance of drug uptake and efflux for cisplatin sensitivity of tumor cells

Platinum sensitivity and platinum resistance may involve altered activity of transport proteins. In order to assess the role of drug uptake and efflux in this phenomenon, we compared the expression of three copper transporters, intracellular platinum accumulation, DNA platination and cytotoxicity of cisplatin in two cisplatin-sensitive and -resistant tumor cell line pairs (ovarian A2780/A2780cis and cervical HeLa/HeLaCK cells). Gene expression of importer CTR1, and ATP7A and ATP7B efflux transporters (with and without cisplatin treatment) was investigated using quantitative real-time PCR and platinum concentrations were determined by flameless atomic absorption spectrometry.After incubation with cisplatin, DNA platination was significantly lower in the resistant variants compared to the respective sensitive cell lines, whereas no obvious difference in DNA repair was found. Accordingly, the resistant variants exhibited lower intracellular platinum concentrations than their respective parental cells (2.5- and 2.9-fold lower in A2780cis and HeLaCK cells, respectively). No differences in efflux were observed. Resistant cells expressed lower levels of CTR1 (1.5–1.8-fold) than their sensitive counterparts. Expression differences of ATP7A and ATP7B between resistant and sensitive cells were cell type-specific.The results highlight the relevance of CTR1 for cisplatin sensitivity as there is a clear relationship between lower CTR1 expression, intracellular concentration, DNA platination and cytotoxicity of cisplatin in both resistant cell lines. Our data provide the basis for a quantitative understanding of alterations in uptake and efflux processes leading to cisplatin resistance and might hence facilitate the development of ex vivo assays that can predict cisplatin sensitivity in tumor specimens of patients.

Copper transporter (Ctr1) expression in mouse tissue is impacted by age and dietary copper

Copper transporter (Ctr1) expression in mouse tissue is impacted by age and dietary copper