Accuracy and Discomfort of Different Types of Intranasal Specimen Collection Methods for Molecular Influenza Testing in Emergency Department Patients.

STUDY OBJECTIVE: While development is under way of accurate, point-of-care molecular tests for influenza infection, the optimal specimen type for molecular tests remains unclear. Compared with standard nasopharyngeal swab specimens, less invasive nasal swab and midturbinate swab specimens may cause less patient discomfort and be more suitable for routine emergency department (ED) testing, although possibly at the expense of diagnostic accuracy. We compare both the accuracy of a polymerase chain reaction molecular influenza test and discomfort between these 3 intranasal specimen types. METHODS: A convenience sample of adult and pediatric patients with influenza-like illness and presenting to 2 Northern California EDs and 2 EDs in Santiago, Chile, was prospectively enrolled during the 2015 to 2016 influenza season. Research nurses collected nasopharyngeal swab, midturbinate swab, and nasal swab specimens from each subject and assessed discomfort on a validated 6-point scale. Specimens were tested for influenza A and B by real-time polymerase chain reaction at reference laboratories. Outcome measures were comparison of test performance between nasal swab and midturbinate swab, when compared with a reference standard nasopharyngeal swab; and comparison of discomfort between all 3 specimen types. RESULTS: Four hundred eighty-four subjects were enrolled, and all 3 swabs were obtained for each subject; 14% were children. The prevalence of influenza (A or B) was 30.0% (95% confidence interval [CI] 26.0% to 34.8%). The sensitivity for detecting influenza was 98% (95% CI 94.25% to 99.65%) with the midturbinate swab versus 84.4% (95% CI 77.5% to 89.8%) with the nasal swab, difference 13.6% (95% CI 8.2% to 19.3%). Specificity was 98.5% (95% CI 96.6% to 99.5%) with the midturbinate swab versus 99.1% (95% CI 97.4% to 99.8%) with the nasal swab, difference -0.6% (95% CI -1.8% to 0.6%). Swab discomfort levels correlated with the depth of the swab type. Median discomfort scores for the nasal swab, midturbinate swab, and nasopharyngeal swab were 0, 1, and 3, respectively; the median differences were nasopharyngeal swab-midturbinate swab 2 (95% CI 1 to 2), nasopharyngeal swab-nasal swab 3 (95% CI 2 to 3), and midturbinate swab-nasal swab 1 (95% CI 1 to 2). CONCLUSION: Compared with the reference standard nasopharyngeal swab specimen, midturbinate swab specimens provided a significantly more comfortable sampling experience, with only a small sacrifice in sensitivity for influenza detection. Nasal swab specimens were significantly less sensitive than midturbinate swab. Our results suggest the midturbinate swab is the sampling method of choice for molecular influenza testing in ED patients.

Imaging guidance with C-arm CT: prospective evaluation of its impact on patient radiation exposure during transhepatic arterial chemoembolization.

PURPOSE: To prospectively evaluate the impact of C-arm CT on radiation exposure to hepatocellular carcinoma (HCC) patients treated by chemoembolization. MATERIALS AND METHODS: Patients with HCC (N = 87) underwent digital subtraction angiography (DSA; control group) or combined C-arm CT/DSA (test group) for chemoembolization. Dose-area product (DAP) and cumulative dose (CD) were measured for guidance and treatment verification. Contrast agent volume and C-arm CT utility were also measured. RESULTS: The marginal DAP increase in the test group was offset by a substantial (50%) decrease in CD from DSA. Use of C-arm CT allowed reduction of DAP and CD from DSA imaging (P = .007 and P = .017). Experienced operators were more efficient in substituting C-arm CT for DSA, resulting in a negligible increase (7.5%) in total DAP for guidance, compared with an increase of 34% for all operators (P = .03). For treatment verification, DAP from C-arm CT exceeded that from DSA, approaching that of conventional CT. The test group used less contrast medium (P = .001), and C-arm CT provided critical or supplemental information in 20% and 17% of patients, respectively. CONCLUSIONS: Routine use of C-arm CT can increase stochastic risk (DAP) but decrease deterministic risk (CD) from DSA. However, the increase in DAP is operator-dependent, thus, with experience, it can be reduced to under 10%. C-arm CT provides information not provided by DSA in 33% of patients, while decreasing the use of iodinated contrast medium. As with all radiation-emitting modalities, C-arm CT should be used judiciously.

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.

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.

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.

Evaluations of HIV type 1 rev gene diversity and functional domains following perinatal transmission.

The human immunodeficiency virus type 1 (HIV-1) rev exons 1 and 2 sequences were analyzed from six mother-infant pairs following perinatal transmission. The rev open reading frame was maintained with a frequency of 93.96% in six mother-infant pairs' sequences. There was a low degree of viral heterogeneity and estimates of genetic diversity in mother-infant pairs' rev sequences. However, the distances of rev sequences between epidemiologically unlinked individuals were greater than in epidemiologically linked mother-infant pairs. Furthermore, phylogenetic parameters revealed that the epidemiologically linked mother-infant pairs were closer evolutionarily to each other as compared with epidemiologically unlinked mother-infant pairs. Both mothers and infants were under positive selection pressure as determined by the ratios of nonsynonymous to synonymous substitutions. The functional domains required for Rev activity, including nuclear export of RNA, RNA binding domain, and nuclear import signals, were conserved in all mother-infant pairs' sequences. The conservation of functional domains of rev and a low degree of heterogeneity following vertical transmission are consistent with an indispensable role of rev in the HIV-1 life cycle.

Cisplatin rapidly down-regulates its own influx transporter hCTR1 in cultured human ovarian carcinoma cells.

PURPOSE: Cisplatin (DDP)-resistant cells commonly exhibit reduced drug accumulation. Previous studies have shown that the major copper (Cu) influx transporter CTR1 controls the uptake of DDP in yeast and mammalian cells. The goal of this study was to examine the effect of Cu and DDP on the level and subcellular localization of hCTR1 protein in human ovarian carcinoma cells. EXPERIMENTAL DESIGN: Cultured human ovarian carcinoma A2780 cells were exposed to DDP and Cu, and the effect on hCTR1 was determined using Western blot analysis and confocal digital deconvolution microscopy. RESULTS: Loss of hCTR1 was triggered by DDP exposure in a concentration and time-dependent manner. Exposure to 0.5 micromol/L DDP for 5 minutes reduced hCTR1 levels and exposure to DDP concentrations > or =2 micromol/L caused almost complete disappearance. The loss of hCTR1 was observed within 1 minute of the start of exposure to 2 micromol/L DDP. Treatment of cells with 100 micromol/L Cu for 5 minutes produced a smaller effect. Pretreatment of cells with 2 micromol/L DDP for 5 minutes resulted in a 50% decrease in 64Cu uptake, demonstrating that the DDP-induced loss of hCTR1 detected by Western blot analysis and imaging was functionally significant. CONCLUSIONS: DDP down-regulated the amount of its major influx transporter in cultured human ovarian carcinoma cells in a concentration- and time-dependent manner. The effect was observed at DDP concentrations within the range found in the plasma of patients being treated with DDP, and it occurred very quickly relative to the half-life of the drug.

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.

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.

The chemoattractant chemerin suppresses melanoma by recruiting natural killer cell antitumor defenses.

Infiltration of specialized immune cells regulates the growth and survival of neoplasia. Here, in a survey of public whole genome expression datasets we found that the gene for chemerin, a widely expressed endogenous chemoattractant protein, is down-regulated in melanoma as well as other human tumors. Moreover, high chemerin messenger RNA expression in tumors correlated with improved outcome in human melanoma. In experiments using the B16 transplantable mouse melanoma, tumor-expressed chemerin inhibited in vivo tumor growth without altering in vitro proliferation. Growth inhibition was associated with an altered profile of tumor-infiltrating cells with an increase in natural killer (NK) cells and a relative reduction in myeloid-derived suppressor cells and putative immune inhibitory plasmacytoid dendritic cells. Tumor inhibition required host expression of CMKLR1 (chemokine-like receptor 1), the chemoattractant receptor for chemerin, and was abrogated by NK cell depletion. Intratumoral injection of chemerin also inhibited tumor growth, suggesting the potential for therapeutic application. These results show that chemerin, whether expressed by tumor cells or within the tumor environment, can recruit host immune defenses that inhibit tumorigenesis and suggest that down-regulation of chemerin may be an important mechanism of tumor immune evasion.

Allosteric inhibition of lysyl oxidase-like-2 impedes the development of a pathologic microenvironment.

We have identified a new role for the matrix enzyme lysyl oxidase-like-2 (LOXL2) in the creation and maintenance of the pathologic microenvironment of cancer and fibrotic disease. Our analysis of biopsies from human tumors and fibrotic lung and liver tissues revealed an increase in LOXL2 in disease-associated stroma and limited expression in healthy tissues. Targeting LOXL2 with an inhibitory monoclonal antibody (AB0023) was efficacious in both primary and metastatic xenograft models of cancer, as well as in liver and lung fibrosis models. Inhibition of LOXL2 resulted in a marked reduction in activated fibroblasts, desmoplasia and endothelial cells, decreased production of growth factors and cytokines and decreased transforming growth factor-beta (TGF-beta) pathway signaling. AB0023 outperformed the small-molecule lysyl oxidase inhibitor beta-aminoproprionitrile. The efficacy and safety of LOXL2-specific AB0023 represents a new therapeutic approach with broad applicability in oncologic and fibrotic diseases.

Contribution of the major copper influx transporter CTR1 to the cellular accumulation of cisplatin, carboplatin, and oxaliplatin.

The goal of this study was to determine the ability of the major copper influx transporter CTR1 to mediate the cellular accumulation of cisplatin (DDP), carboplatin (CBDCA), and oxaliplatin (L-OHP). Wild-type murine embryonic fibroblasts (CTR1+/+) and a subline in which both alleles of CTR1 were deleted (CTR1-/-) were tested for their ability to accumulate platinum when exposed to increasing concentrations of DDP, CBDCA, or L-OHP for 1 h. They were also tested for their sensitivity to the growth-inhibitory effect of each drug. Platinum content was measured by ion-coupled plasmon mass spectroscopy. The experimental model was validated by measuring copper accumulation and cytotoxicity. CTR1-/- cells accumulated only 5.7% as much copper as CTR1+/+ cells during a 1-h exposure to 2 microM copper. When exposed to DDP, CBDCA, or L-OHP at 2 microM, accumulation in the CTR1-/- cells was only 35 to 36% of that in the CTR1+/+ cells. When tested at a 5-fold higher concentration, this deficit remained for DDP and CBDCA, but accumulation of L-OHP was no longer CTR1-dependent. There was an association between the effect of loss of CTR1 function on uptake of the platinum drugs and their cytotoxicity. The CTR1-/- cells were 3.2-fold resistant to DDP, 2.0-fold resistant to CBDCA, but only 1.7-fold resistant to L-OHP. Thus, whereas CTR1 controls the cellular accumulation of all three drugs at low concentrations, accumulation of L-OHP is not dependent on CTR1 at higher concentrations. We conclude that L-OHP is a substrate for some other cellular entry mechanism, a feature consistent with its different clinical spectrum of activity.

The copper transporter CTR1 regulates cisplatin uptake in Saccharomyces cerevisiae.

Resistance to cisplatin (DDP) is often accompanied by impaired accumulation in mammalian cells. The mechanism of impaired DDP accumulation is unknown, but copper uptake is diminished as well. We investigated the ability of the copper transporter CTR1 to control the accumulation of DDP in Saccharomyces cerevisiae. Parallel studies of copper and DDP cellular pharmacokinetics were carried out using an isogenic pair of wild-type CTR1 and ctr1 knockout S. cerevisiae strains. Both copper and platinum accumulation increased linearly as a function of time and drug concentration in the parental cells. Deletion of CTR1 resulted in a 16-fold reduction in the uptake of copper and an 8-fold reduction in the uptake of DDP measured at 1 h. The CTR1-deficient cells accumulated 2.3-fold (p < 0.05) less platinum in their DNA and were 1.9-fold more resistant to the cytotoxic effect of DDP than the CTR1-replete cells. The kinetics of cellular copper accumulation were similar to those of DDP. Based on measurements of accumulation at 1 h, the K(m) for copper influx was 128.8 microM, and the V(max) was 169.5 ng/mg of protein/min; for DDP, the K(m) was 140.2 microM and the V(max) was 76.9 ng/mg of protein/min. DDP blocked the uptake of copper into the parental cells but not ctr1-deficient cells. CTR1-deficient cells also demonstrated impaired accumulation of the DDP analogs carboplatin, oxaliplatin, and ZD0473 [cis-amminedichloro(2-methylpyridine) platinum (II)]. These results indicate that CTR1 function markedly influences the uptake of all of the clinically used platinum-containing drugs and suggest that this copper transporter may also transport DDP.

The copper export pump ATP7B modulates the cellular pharmacology of carboplatin in ovarian carcinoma cells.

Human tumor cells lines with acquired resistance to cisplatin (DDP) and carboplatin (CBDCA) are often cross-resistant to copper and vice versa, and some DDP-resistant cells overexpress the copper export pump ATP7B. We sought to demonstrate that ATP7B directly mediates resistance to DDP and CBDCA by stably transfecting human carcinoma cells with a vector designed to express ATP7B. Increased expression of ATP7B rendered all three cell lines tested more resistant to a 1-h exposure to DDP (1.6-2.6-fold), CBDCA (1.5-1.6-fold), and copper (1.2-1.4-fold). The effect of ATP7B on the cellular pharmacology of 64Cu and [14C]CBDCA was investigated in more detail using one cell pair (2008 cells transfected with an empty vector or an ATP7B-expressing vector). In the 2008/ATP7B subline, steady-state copper levels were decreased under both basal and copper-supplemented conditions, as was steady-state CBDCA content upon exposure to 50 microM [14C]CBDCA. Over the first 5 min, the average rate of accumulation of copper and CBCDA in the 2008/ATP7B cells was reduced by 37 and 61%, respectively. Efflux was more rapid from 2008/ATP7B cells for both copper and CBDCA. Two-compartment modeling indicated that the second phase of efflux was increased by a factor of 3.9-fold for CBCDA and to an even greater extent for copper. We conclude that expression of ATP7B regulates sensitivity to CBDCA as well as to DDP and copper and that a transporter that normally mediates copper homeostasis modulates the cellular pharmacology of CBDCA.

Modulation of the cellular pharmacology of cisplatin and its analogs by the copper exporters ATP7A and ATP7B.

The copper efflux transporters ATP7A and ATP7B sequester intracellular copper into the vesicular secretory pathway for export from the cell. The influence of these transporters on the pharmacodynamics of cisplatin, carboplatin, and oxaliplatin was investigated using human Menkes' disease fibroblasts (Me32a) that do not express either transporter and sublines molecularly engineered to express either ATP7A (MeMNK) or ATP7B (MeWND). Cellular copper levels were significantly higher in the Me32a cells than in the MeMNK and MeWND sublines. These transporter-proficient sublines were resistant to the cytotoxic effect of copper, cisplatin, and carboplatin but were hypersensitive to oxaliplatin. Whole-cell accumulation of platinum after a 24-h exposure was significantly increased in the MeMNK and MeWND cells for all three platinum drugs, but this was accompanied by an increase in the amount of platinum reaching the DNA only for oxaliplatin. Vesicles isolated from MeMNK cells contained more platinum after exposure to cisplatin and carboplatin, whereas the platinum content of vesicles from MeWND cells was increased after exposure to all three drugs. Although copper triggered relocalization of ATP7A from the perinuclear region to more peripheral locations, the platinum drugs did not. These results demonstrate that both ATP7A and ATP7B modulate the pharmacodynamics of all three clinically used platinum drugs. The data are consistent with the hypothesis that these copper exporters sequester the platinum drugs into subcellular compartments, limiting their cytotoxicity, similar to their effect on copper. However, in this model system, although copper is readily exported after vesicular sequestration, the platinum drugs are not.

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.