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Experimental Therapeutics |
Department of Medicine and the Cancer Center, University of California, San Diego, La Jolla, California 92093-0058 [K. K., A. K., R. S., A. H., G. S., M. M., S. B. H.]; Skyepharma, Inc., San Diego, California 92121 [M. R.]; and University of California, San Francisco, San Francisco, California 94143 [Y-M. K.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Biochemical studies have not succeeded in conclusively identifying the basis of resistance in any type of cell selected with DDP, but they have defined several mechanisms that can contribute to resistance. The effectiveness of cell killing is a function of how much drug gets into the cell, how much of it enters the nucleus and actually reacts with DNA, how tolerant the cell is of lesions in its DNA, and how effectively it removes these adducts (5) . Intracellular detoxification of DDP through mechanisms that involve binding to thiols may contribute to resistance (reviewed in Ref. 6 ). Both defects in the ability of the cell to recognize adducts in DNA (reviewed in Ref. 7 ) and enhanced repair of and tolerance to adducts (8) have been identified as contributing to resistance in some cell types. However, impaired uptake of DDP is the single most consistently identified feature of cells with acquired DDP resistance both in vitro and in vivo (reviews Refs. 5 , 9, 10, 11, 12, 13, 14, 15, 16 ).
The mechanism of impaired DDP accumulation is unknown, and in fact, the mechanism by which DDP enters or exits from cells remains poorly defined. DDP enters cells relatively slowly compared with most anticancer agents, and earlier evidence suggested that at least one component of DDP uptake is mediated by a transport mechanism or channel (17, 18, 19, 20) . DDP efflux is characterized by an initial very rapid phase followed by long terminal half-life (21 , 22) . An increased rate of efflux has been reported in some DDP-resistant cell lines (23) and in cells that overexpress glutathione GS-X pump (24 , 25) . However, the lack of a convenient isotopically labeled form of DDP and technical limitations on the measurement of very small amounts of platinum have impeded further progress in defining the DDP export mechanisms.
On the basis of our observation that cells selected for DDP resistance are cross-resistant to copper and that cells selected for resistance to copper are cross-resistant to DDP (26) , we are proposing the novel unifying concept that DDP enters the cell, is distributed to subcellular compartments, and is exported from the cell using transporters and chaperones that normally mediate copper homeostasis. The body of prior data on the cellular pharmacology of DDP is consistent with this hypothesis. We report here studies of the cellular pharmacology of copper and DDP in three pairs of isogenic DDP-sensitive and -resistant ovarian carcinoma cell lines that provide additional support for this hypothesis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines.
Three human ovarian carcinoma cell lines (A2780, 2008, and IGROV-1) and their DDP-resistant cell lines (A2780/CP, 2008/C13*5.25, and IGROV-1/CP) were maintained at 37°C in a humidified incubator containing 5% CO2 in RPMI 1640 supplemented with 10% (A2780, A2780/CP, IGROV-1, and IGROV-1/CP) or 5% (2008 and 2008/C13*5.25) fetal bovine serum. The resistant cell lines were not grown in DDP as their phenotype was stable.
Cisplatin Whole Cell Uptake and Accumulation in DNA.
Cells were grown to 80% confluence in 150-cm2 flasks and were then incubated in fresh medium containing 0–200 µM DDP for 1 h. The cells were then trypsinized, washed three times with PBS, and then scraped free in 1 ml of PBS and transferred to a centrifuge tube. The flask was rinsed a second time with an additional 1 ml of PBS, and this was also transferred to the same centrifuge tube. The cells were pelleted by centrifugation, the supernatant removed, and the pellets stored at -20°C until ready for analysis. A Wizard Genomic DNA Purification Kit (Promega, Madison, WI) was used for isolation of DNA. Aliquots of the DNA were digested in 1 M HCl at 75°C for 2 h, and the hydrolysate was used for the quantitation of platinum by flameless atomic absorption spectrophotometry (Perkin-Elmer Model 2380). Relative differences in DNA platination were determined by comparing the slopes plot of platinum content versus DDP concentration, and the values reported are the result of three independent experiments each performed with triplicate cultures.
Measurements of the initial uptake rates of DDP and copper were made using 100-mm tissue culture plates seeded with 106 cells each and incubated in medium until they were 75–80% confluent. Five plates were used for each data point. For uptake experiments, the medium was replaced by 10 ml of fresh medium containing various concentrations of CuSO4 or DDP, and the cells were incubated at 37°C for various periods of time. Measurements of efflux rates were made by exposing the cells to 400 µM DDP or CuSO4 for 10 min, after which the cells were rinsed and incubated in drug-free medium at 37°C. At the requisite time point for both types of experiments, cultures were quickly rinsed three times with ice-cold PBS, and cells were harvested into 10 ml of ice-cold PBS using a rubber policeman. After centrifugation at 3000 rpm for 10 min, the cells were resuspended in PBS, an aliquot was used for protein assay, and the remainder was digested in 70% nitric acid. Cell lysates were heated for 2 h at 75°C, diluted to 5% nitric acid, and assayed for platinum and copper content using a model 3000DV Perkin-Elmer inductively coupled plasma optical emission spectroscope from the Analytical Facility at the Scripps Institute of Oceanography.
Colony Formation Assays.
Colony assays were performed using triplicate cultures of 200 cells/60-mm plate grown in 3 ml of medium. The cells were allowed to attach, exposed for 1 h to different concentrations of DDP or CuSO4, and then incubated in fresh medium until visible colonies had formed (10–14 days). The dishes were rinsed twice with PBS, fixed with 100% methanol, and stained with a 0.5% crystal violet solution. A ChemiImager 400 instrument (Alpha Innotech, San Leandro, CA) was used for counting colonies of >50 cells.
Pharmacokinetic Analysis.
Cells were loaded by exposure to Cu or DDP for 10 min, and aliquots were harvested by a rapid sampling technique at various time points over the ensuing 2 h. Six independent cultures were sampled at each time point. Mean data were fitted using a 2 compartment pharmacokinetic model assuming a first order disposition process using WinNonlin, Professional 3.1 (Pharsight Corp, CA).
DNA Sequencing.
RNA was extracted from the cell lines using Trizol (Life Technologies, Inc.), and cDNA was generated using the SuperScript Preamplication system (Life Technologies, Inc.) following manufacturer’s protocol. CTR1 was amplified by PCR in the GeneAmp PCR system 9700 (Applied Biosystems) using the primers 5'-CACGTCGAGCCGGGTAGAAG-3' and 5'-TGGAGCAGGAATCACGTCTTC-3'. The DNA product was sequenced with the primer 5'-GTGACGGGTTAAGATTCGGAGAG-3'.
Real-Time PCR.
Ten µg of total RNA, extracted with Trizol reagent (Life Technologies, Inc.), were treated with DNase and converted to cDNA using random hexamer primers with the SuperScript First-Strand Synthesis System (Invitrogen). A Perkin-Elmer ABI Prism 7700 and Sequence Detection System software was used for RT-PCR and primer design, respectively. Triplicate PCR amplifications of 10 ng of the cDNA were performed using the Taqman Master Mix provided by Perkin-Elmer. Fold change in RNA abundance was calculated using the Standard Curve Method for quantification (ABI Prism 7700 SDS User Bulletin No. 2 P/N 4303859 Rev. A).5
Gene Bank sequence no. U83460 was used for CTR1 primer design. The CTR1 forward primer was 5'-AGGACTCAAGATAGCCCGAGAGA-3', the reverse primer 5'-CCTGGGACAGGCATGGAA-3', and the probe was 5'-CTGCGTAAGTCACAAGTCAGCATTCGCTACA-3'.
Western Blot Analysis.
The protein samples were heated before electrophoresis and were subjected to 4–15% SDS-PAGE. Transfer to nitrocellulose membranes (Bio-Rad Co.) was performed electrophoretically for 30 min at 200 volts (constant voltage) using a Transblot SD apparatus (Bio-Rad Co.). The membrane was blocked with 5% skimmed milk in buffer C [0.35 M NaCl, 10 mM Tris-HCl (pH 8.0), and 0.05% Tween 20] for 1 h at room temperature and then incubated overnight at 4°C with 4000-fold diluted polyclonal antibody against ATP7A, 3000-fold diluted polyclonal antibody against ATP7B, or 1000-fold diluted polyclonal antibody against HAH1, respectively. These antibodies were obtained from Dr. Jonathan D. Gitlin (Washington University School of Medicine, St. Louis, MO). The membrane was washed three times with buffer C and then incubated for 1 h with 1000-fold or 500-fold- diluted horseradish peroxidase-conjugated antirabbit IgG (Amersham, Buckinghamshire, United Kingdom) for detection of ATP7B or HAH1, respectively. For detection of ATP7A, 500-fold diluted horseradish peroxidase-conjugated antisheep IgG (Santa Cruz Biotechnology, Santa Cruz, CA) was used. The nitrocellulose membrane was rinsed three times for 5 min with buffer C and then evenly coated using the enhanced chemiluminescence Western blotting detection system (Amersham) for 1 min. The membrane was immediately exposed to Fuji medical X-ray film (RX-U; Fujifilm, Kanagawa, Japan) in a film cassette at room temperature for various periods.
Statistics.
All data were analyzed by use of a two-sided paired Student’s t test with the assumption of unequal variance.
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RESULTS |
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presents the IC50 for each cell line determined using a clonogenic assay and a 1 h exposure to DDP or CuSO4. As measured by IC50s, the A2780/CP, 2008/C13*5.25, and IGROV-1/CP sublines were 8.1-, 5.7-, and 7.5-fold resistant to DDP, respectively, relative to the parental cell lines from which they were derived. These DDP-resistant sublines were 2.1-, 1.5-, and 2.0-fold cross-resistant to copper. All three DDP-resistant sublines were even more cross-resistant to copper when tested using continuous exposure to DDP and copper throughout the period of colony formation as well (27)
. Using continuous exposure, the A2780/CP cells were 6.3-fold resistant to DDP and 4.1-fold cross-resistant to CuSO4. Likewise, the 2008/C13*5.25 cells were 10.4-fold resistant to DDP and 1.9-fold cross-resistant to CuSO4, whereas the IGROV-1/CP cells were 7.6-fold resistant to DDP and 2.4-fold cross-resistant to CuSO4. We have previously reported that human hepatoma cell lines selected for resistance to copper are cross-resistant to DDP (27)
. The results of the current study establish that the cross-resistance operates in both directions and that cell lines selected for resistance to DDP also demonstrate substantial degrees of cross-resistance to copper. It is noteworthy that in each case, the magnitude of the resistance to DDP was greater than that to copper and that there was an excellent correlation between resistance to the two agents (r = 0.99).
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presents plots of the cellular accumulation of platinum at the end of a 1-h exposure to DDP as a function of drug concentration. The platinum accumulation rate was constant with time over the first hour of uptake in all cells (data not shown). The amount of platinum in the cell at 1 h increased linearly with DDP concentration in both the sensitive and resistant members of all three pairs of cell lines. Table 2
presents the slopes of the increase in pmol Pt/mg cell protein as a function of DDP concentration. In all three resistant sublines, there was a substantial impairment of platinum accumulation. Mean uptake in the resistant sublines was 67% of that in the parental line for A2780/CP, 41% for 2008/C13*5.25, and 38% for IGROV-1/CP. There was no significant association between IC50 and whole cell platinum uptake for either the DDP-sensitive or -resistant cells.
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, as for whole cell uptake, platinum accumulation in DNA increased linearly as a function of DDP concentration. Table 3
presents the slopes of the increase in pg Pt/µg DNA as a function of DDP concentration. Accumulation of platinum in the DNA was substantially lower in the DDP-resistant sublines than in the parental cells. For the A2780/CP subline, accumulation was only 38% of that in the parental cells, whereas for the 2008/C13*5.25 cells it was 10% and for the IGROV-1/CP cells it was 29%. There was no significant association between IC50 and DNA platinum accumulation, nor between whole cell uptake and the extent of DNA platination for either the DDP-sensitive or -resistant cells.
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indicate that when grown in standard tissue culture medium the DDP-resistant cells did indeed have 22–56% lower basal levels of copper consistent with either a reduction in influx or an increase in efflux rate.
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. The copper content was reduced in all three DDP-resistant lines to levels ranging from 27 to 46% of those observed in the sensitive parental lines.
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. There was a marked reduction in the initial influx rate for DDP in all three resistant cell lines; the rate ranged from 23 to 55% of that observed in the sensitive cells of each pair. Despite the limited number of pairs of cell lines examined, there was an excellent correlation between the reduction in initial influx rate and the decrease in the slope of the whole cell accumulation at 1 h as a function of concentration (r = 0.954). The reduction in initial DDP uptake was accompanied by a decrease in the initial uptake rate of copper in all three DDP-resistant lines. The initial rate of copper uptake was 75% of control in the A2780/CP line, 61% of control in the 2008/C13*5.25 line, and 56% of control in the IGROV-1/CP cell line.
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, and the calculated half-lives are presented in Table 7
. Interestingly, for both copper and DDP, the initial half-life was longer in the resistant cells, reflecting reduced initial efflux of these compounds. The terminal half-life was shorter in the resistant cells for both copper and DDP, reflecting an apparent increase in the rate of late efflux. On the basis of the goodness of fit, there is substantial confidence that the initial efflux rate was lower in the resistance cells; however, because of the more limited number of time points sampled, there is less confidence that the difference in terminal efflux is real. The data suggest that in the 2008/C13*5.25 cells both a defect in initial influx and an enhanced terminal efflux appear to contribute to the lower steady-state copper levels and the impaired net accumulation of DDP and copper. In the resistant cells, the initial half-life was increased by 2.37-fold for DDP and 2.39-fold for copper. It is noteworthy that the magnitude of the change was virtually identical for both compounds.
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Expression of ATP7A, ATP7B, HAH1, and CTR1.
The level of expression of the copper homeostasis proteins for which antibodies are currently available (ATP7A, ATP7B, and HAH1) was examined by Western blot analysis. HAH1 is the major copper chaperone that delivers copper to either ATP7A or ATP7B. In the absence of an antibody to CTR1, RT-PCR was used to estimate relative mRNA levels. Fig. 4
shows that A2780/CP and 2008/C13*5.25 cell lines overexpressed ATP7A, whereas the IGROV-1/CP cells exhibited increased expression of ATP7B. None of the DDP-resistant cell lines demonstrated detectable differences in the level of HAH1. Thus, all three of the DDP-resistant cells had increased expression of one or the other, but not both, of the known copper export pumps. The ratio of the level of CTR1 mRNA in the DDP-resistant A2780/CP, 2008/C13*5.25, and IGROV-1/CP lines relative to their DDP-sensitive parental lines was 0.6 ± 0.2 (SE), 1.2 ± 0.4 (SE) and 1.0 ± 0.2 (SE), respectively. Thus, the DDP-resistant phenotype was not consistently accompanied by a change in CTR1 message level.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows a schematic diagram of the current understanding of copper transport pathways in mammalian cells; these pathways have been highly conserved during evolution and are very similar to homologous pathways in Saccharomyces cerevisiae (reviews Refs. 29
, 30
). Copper delivered by plasma ceruloplasmin is reduced by a cell surface reductase before being passively transported across the plasma membrane in an energy-independent manner by CTR1 (31
, 32)
. CTR1 hands the copper to one of three different chaperones, each of which serves to deliver copper to a specific target in the cell. COX17 delivers copper to cytochrome c oxidase in the mitochondria (33)
; Ccs1 delivers copper to superoxide dismutase (34)
, and HAH1 delivers copper to a P-type ATPase transporter (ATP7A or ATP7B). ATP7A and ATP7B function to sequester copper into the trans-Golgi network where it is loaded onto copper-dependent enzymes in the secretory pathway for export from the cell (35)
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The initial efflux rate from the DDP-resistant 2008/C13*5.25 cells was reduced for both copper and DDP in a strikingly parallel manner. This is consistent with the fact that copper is first sequestered from the cytoplasm into the trans-Golgi apparatus and only subsequently exported from the cell. Thus, if the increased level of ATP7A in the 2008/C13*5.25 cells actually accounts for the altered export, it appears to have the effect of rendering less of the intracellular copper and DDP available for rapid efflux while at the same time increasing total export capability.
Additional evidence suggesting a linkage between copper and DDP homeostasis is provided by the finding that each one of the DDP-resistant cell lines overexpressed one or the other of the two variant forms of the major copper export pump. Overexpression of ATP7B has previously been reported to render human KB cells resistant to copper and DDP (36) , and this finding has recently been confirmed for ovarian carcinoma cells in this laboratory (37) . Although there is currently no information as to whether molecular engineering of human cells to overexpress ATP7A also confers resistance to copper and DDP, ATP7A and ATP7B are highly homologous and provide the same export function for copper in different normal tissues in the body. The finding of ATP7A overexpression in two of three DDP-resistant cell lines mandates additional investigation of the ability of DDP to serve as a substrate for this exporter as well.
Because CTR1 is the major copper influx transporter in mammalian cells, the impaired initial influx of copper observed in the DDP-resistant cells suggests either down-regulation or disability of this plasma membrane protein. Because no mutations were detected in any of the exons of CTR1 in the DDP-resistant cells, the implication is that these cells express fewer CTR1 molecules on their surface or that some other protein that operates in conjunction with CTR1 is altered. RT-PCR analysis demonstrated a reduction in CTR1 mRNA in the A2780/CP cells relative to that in the A2780 cells, but no change in the other two DDP-resistant cell lines was observed. However, the relationship between CTR1 mRNA and protein level has not been established, and in the absence of a method for quantifying CTR1 protein, the significance of the change in the A2780/CP levels remains to be determined.
It is of interest that, in both the cell lines used in the current studies and in our studies of cells selected for resistance to copper (27) , relatively small changes in sensitivity to the cytotoxic effect of copper were accompanied by substantially larger changes in sensitivity to DDP. This finding is consistent with a prior study of the A2780 and A2780/CP20 cell lines as well (38) . The molecular basis for this is not apparent but could be explained if the efficiency of transport of copper by CTR1 was greater than for DDP or if intracellular DDP is more available to the export mechanism. It is also noteworthy that in the current studies there was not a clear association between the whole cell uptake of DDP and the extent of DNA adduct formation. This lack of association was also recently observed in a more extensive study of the cellular pharmacology of oxaliplatin (39) . This suggests that having gained access to the cytoplasm there remain significant barriers to successful adduct formation and that detoxification mechanisms capable of preventing adduct formation are variably activated in different DDP-resistant sublines.
Because of the relatively slow influx of copper and DDP, measurements of initial influx and efflux rates had to be made using cells exposed to relatively high concentrations of drug that were likely substantially above the Km for the copper transport processes. It will be of interest to examine influx and efflux at lower drug concentrations as well. This is of particular importance because of the fact that under physiological conditions the concentration of free copper in the cell is <10-18 M (40) . The unique redox chemistry of copper allows oxidation and reduction under physiological conditions, permitting it to serve as a redox cofactor for a variety of processes (41) . However, this same characteristic allows production of reactive oxygen species, and copper can also be toxic to the cell. The complex system of copper transporters and chaperones that has evolved serves the dual role of both protecting copper(I) during its uptake and distribution to copper-requiring enzymes and preventing copper toxicity (42 , 43) . The central feature of this system is that copper(I) is chelated by a unique set of metal binding sequences into a protective pocket in each of the transporters and chaperones and that one protein hands the copper to the next through an intimate protein-protein interaction such that copper is virtually never free in the cell (40 , 44, 45, 46) . If indeed DDP uses the copper transporters and chaperones for uptake, distribution and efflux, the kinetic behavior of DDP may be different when examined at lower and more clinically relevant concentrations at which the transport and chaperone function may not be saturated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by NIH Grant CA78648. This work was conducted, in part, by the Clayton Foundation for Research: California Division. R. S. and S. B. H. are Clayton Foundation investigators. 
2 Presented, in part, at the 2002 meeting of the American Association of Cancer Research.
3 To whom requests for reprints should be addressed, at Department of Medicine 0058, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0058. Phone: (858) 822-1110; Fax: (858) 822-1111; E-mail: showell{at}ucsd.edu
4 The abbreviations used are: DDP, cisplatin; RT-PCR, reverse transcription-PCR; CTR1, copper transporter 1.
5 www2.perkin-elmer.com/ab/techsupp/7700.html.
Received 6/10/02. Accepted 9/18/02.
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R. Jensen and P. M. Glazer Cell-interdependent cisplatin killing by Ku/DNA-dependent protein kinase signaling transduced through gap junctions PNAS, April 20, 2004; 101(16): 6134 - 6139. [Abstract] [Full Text] [PDF]
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G. D. Kruh Lustrous Insights into Cisplatin Accumulation: Copper Transporters Clin. Cancer Res., December 1, 2003; 9(16): 5807 - 5809. [Full Text] [PDF]
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G. Samimi, N. M. Varki, S. Wilczynski, R. Safaei, D. S. Alberts, and S. B. Howell Increase in Expression of the Copper Transporter ATP7A during Platinum Drug-Based Treatment Is Associated with Poor Survival in Ovarian Cancer Patients Clin. Cancer Res., December 1, 2003; 9(16): 5853 - 5859. [Abstract] [Full Text] [PDF]
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K. Katano, R. Safaei, G. Samimi, A. Holzer, M. Rochdi, and S. B. Howell The Copper Export Pump ATP7B Modulates the Cellular Pharmacology of Carboplatin in Ovarian Carcinoma Cells Mol. Pharmacol., August 1, 2003; 64(2): 466 - 473. [Abstract] [Full Text] [PDF]
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, parental cell line; 
, DDP-resistant subline. A, A2780 and A2780/CP cells; B, 2008 and 2008/C13*5.25 cells; C, IGROV-1 and IGROV-1/CP cells. Each data point represents the mean of three independent experiments each performed with triplicate cultures; vertical bars: ± SE.
