Functional Nucleoside Transporters Are Required for Gemcitabine Influx and Manifestation of Toxicity in Cancer Cell Lines1

[CANCER RESEARCH 58. 4349-4357. October I. I998| Functional Nucleoside Transporters Are Required for Gemcitabine Influx and Manifestation of Toxicity in Cancer Cell Lines1

John R. Mackey2, Ra.jam S. Mani, Milada Seiner, Delores Mowles, James D. Young, Judith A. Belt, Charles R. Crawford, and Carol E. Cass Department of Oncology, University- of Alberta, unti Cross Cancer Institute, Edmonton, Alberili, T6G 1Z2 Canuda (J. R. M., R. S. M., M. S., D. M., C. E. C.Â¡;Department of Physiology, University of Alberiti, Edmonton, T6G 172 Canada Â¡J.D. Y.I; and Department of Molecular Pharmacology. St. Jude Children's Research Hospital. Memphis. Tennessee 38105 Â¡].A. B.. C. K. CJ

ABSTRACT furanoxylcytosine), cladribine (chlorodeoxyadenosine). and fludara- Gemcitabine (2',2'-difluorodeoxycytidine) is a novel pyrimidine nucle- bine (fluoroarabinosyladenine)], gemcitabine has demonstrated sig nificant activity in Phase II and Phase III clinical trials in patients with oside drug with clinical efficacy in several common epithelial cancers. We advanced breast (1,2), bladder (3), ovarian (4), non-small cell lung (5, have proposed that gemcitabine requires nucleoside transporter (NT) 6), pancreatic (7, 8), and head and neck cancers (9). The mecha- proteins to permeate the plasma membrane and to exhibit pharmacolog ical activity. In humans, there are seven reported distinct NT activities nism(s) accounting for the unique and broad spectrum of gemcitabine varying in substrate specificity, sodium dependence, and sensitivity to activity has not been fully determined. Although gemcitabine and inhibition by nitrobenzylthioinosine (NBMPR) and dipyridamole. To de cytarabine both require phosphorylation to produce the corresponding termine which NTs are required for gemcitabine-dependent growth inhi toxic triphosphates, dFdCTP and ara-CTP, the cellular pharmacology bition, cultures from a panel of 12 cell lines with defined plasma mem of gemcitabine differs from that of cytarabine, with relatively pro brane NT activities were incubated with different concentrations of longed intracellular accumulation of gemcitabine triphosphate (10). gemcitabine. Cell proliferation was assessed by the sulforhodamine B dFdCTP can be incorporated into DNA, which is believed to be the assay and cell enumeration to identify the concentrations of gemcitabine that inhibited cell replication by 50% (IC50s). NT activity was a prereq main mechanism of cytotoxicity (11, 12). Gemcitabine is a substrate uisite for growth inhibition in vitro because: (a) the nucleoside transport- for deoxycytidine deaminase, producing the noncytotoxic compound 2',2'-defluorodeoxyuridine (12). deficient cells were highly resistant to gemcitabine; and (In treatment of cells that exhibited only equilibrative NT activity with NBMPR or dipyr For unknown reasons, some human tumors exhibit de novo gem idamole increased resistance to gemcitabine by 39- to 1800-fold. These citabine resistance [as shown by low response rates in clinical trials of data suggested that the type of NT activities possessed by a cell may be an patients with advanced colon cancers (13)], whereas other initially important determinant of its sensitivity to gemcitabine and that NT sensitive cancers may develop resistance during therapy. The in vivo deficiency may confer significant gemcitabine resistance. We analyzed the uptake kinetics of [3H]gemcitabine by each of five human NT activities in mechanisms of resistance to gemcitabine therapy are not defined, cell lines that exhibited a single NT activity in isolation; transient trans- although several in vitro mechanisms of gemcitabine resistance have fection of the cDNAs encoding the human concentrative NT proteins been identified. Gemcitabine resistance was induced by exposing (hCNTl and hCNT2) was used to study the rii and rif activities, respec A2780 human ovarian carcinoma cells to stepwise increases in gem tively. The efficiency of gemcitabine uptake varied markedly among the citabine concentration. The identified mechanism of resistance was a cell lines with single NTs: es = dt > ei > db Â»> df. The transport deficiency in deoxycytidine kinase, the enzyme necessary for the ability of [3H]gemcitabine was demonstrated by reconstitution of the activation of gemcitabine to gemcitabine monophosphate (14). Trans- human es NT in proteoliposomes, confirming that gemcitabine permeation fection of CEM human leukemia cells with a vector that forced is a protein-mediated process. expression of cytidine deaminase conferred a 2.4-fold increase in gemcitabine resistance (15). Gemcitabine efficacy was decreased in INTRODUCTION fibrosarcoma cells induced by transfection to produce large quantities of heat shock protein 70 (16). Resistance to gemcitabine was not Gemcitabine (2',2'-difluorodeoxycytidine, dFdC,3 Gemzar) is a associated with increased P-glycoprotein expression and was not seen novel pyrimidine nucleoside anticancer drug with demonstrated clin in cell lines resistant to alkylating agents and cisplatinum (17). Plasma ical activity in common epithelial cancers. Unlike other nucleoside membrane transport has been studied in Chinese hamster ovary cells, anticancer drugs used in clinical practice [cytarabine ( l -ÃŸ-D-arabino- where membrane transport did not appear to be a major factor con tributing to the more potent cytotoxicity of gemcitabine when com Received 3/10/98: accepted 8/18/98. pared with cytarabine (10). The costs of publication of this article were defrayed in part by the payment of page The biochemical targets for gemcitabine are intracellular, and the charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. obligatory first step in cytotoxicity is permeation across the plasma ' Supported by a Canadian Cancer Society operating grant from the National Cancer Institute of Canada and a Pilot Project and New Investigator's Award from the Alberta membrane. Because gemcitabine, like other anticancer nucleosides, is Cancer Board. C. E. C. is a Terry Fox Cancer Research Scientist of the National Cancer hydrophilic and would not be expected to readily permeate plasma Institute of Canada, and J. D. Y. is a Heritage Medical Scientist of the Alberta Heritage membranes by passive diffusion, its cellular uptake requires the pres Foundation for Medical Research. Isolation of the L12IO/DNC3 and L1210/DU-5 cell ence of specialized plasma membrane NT proteins (18). Seven distinct lines was also supported by National Cancer Institute (USA) Research Grant CA55056. Cancer Center Support Grant CA21765. and the American Lebanese Syrian Associated NT activities have been demonstrated in human cells. Nucleoside Charities of St. Jude Children's Research Hospital (to J. A. B. and C. R. C.). transport occurs by both sodium-independent, bidirectional equilibra 2 To whom requests for reprints should be addressed, at Cross Cancer Institute. 11560 tive (ENT) and sodium-dependent, inwardly directed concentrative University Avenue, Edmonton, Alberta, T6G 1Z2 Canada. 1The abbreviations used are: dFdC. gemcitabine; CEM. CCRF-CEM; NT, nucleoside (CNT) processes: es (broad permeant selectivity); ei (broad selectiv transporter: ENT. equilibrative nucleoside transporter; CNT, concentrative nucleoside ity), cir, cib (broad permeant selectivity); cif (purine-nucleoside se transporter; NBMPR. nitrobenzylmercaptopurine ribonucleoside (nitrobenzylthioinosine); ei, equilibrative NBMPR-insensitive: es, equilibrative NBMPR-sensitive: cif, concentra lectivity); csg (concentrative, NBMPR-insensitive, guanosine selec tive. insensitive to NBMPR and formycin B-selective; cit, concentrative, insensitive to tivity); and ci (concentrative, NBMPR-insensitive, unknown NBMPR and thymidine selective: cib, concentrative. insensitive to NBMPR and broadly selective; OCTG. octylglucopyranoside; h. human; NBTGR. nitrobenzylmercaptoguanine selectivity). cDNAs encoding four human NTs have recently been ribonucleoside (nitrobenzylthioguanosine). cloned and functionally expressed: hENTl, which displays es activity 4349

(19); HENT2, which displays ei activity (20, 21); hCNTl, which selected using the de novo uridylate synthesis inhibitor Brequinar in the displays cit activity (22); and hSPNT, or in our terminology, hCNT2, presence of the salvageable nucleoside uridine. The LI210/DU-5 cell line was which displays cit activity (23).4 The equilibrative NTs appear to be cloned from this selection and had a single copy of rCNTl inserted into its widely distributed in different cell types and tissues, whereas the genome (33). K562 is a human erythroid leukemia cell line that possesses es and ei transport activities (27). CEM is a human T-lymphoblast cell line with concentrative NTs appear to be limited to specialized cells such as intestinal and renal epithelia. liver, and leukemic cells. It is likely that es transport activity originally derived from a patient with acute lymphocytic leukemia (34). The NT-defective ARAC-8C cell line is a clonal derivative of different human tissues and cell lines vary widely in NT activities, due a hypoxanthine-guanine phosphoribosyltransferase-deficient CEM cell line to variations in the number and type of NT proteins expressed on the originally isolated by mutagen exposure and selection for resistance to 8 /UM plasma membrane (24). cytarabine. For the present investigations, the absence of hypoxanthine-gua To determine whether gemcitabine resistance could be correlated nine phosphoribosyltransferase. a purine-metabolizing enzyme, was not perti with nucleoside transport capabilities, we examined the gemcitabine nent to the metabolism or cytotoxicity of gemcitabine (35). CaCo-2 is a human sensitivity of 12 murine and human cell lines with defined nucleoside colon carcinoma cell line that possesses es, ei, and cib activities (24). HeLa is transport characteristics. To study the effect of pharmacological ma a human cervical carcinoma cell line that possesses es and ei activities (26). nipulation of nucleoside transport on gemcitabine-dependent inhibi BeWo is a human choriocarcinoma cell line that possesses es and ei activities tion of proliferation, the transport inhibitors NBMPR and dipyri- (36, 37). damole were used to specifically inhibit, respectively, es NT activity Chemosensitivity Testing. The chemosensitivity of suspension cell lines was assessed using the sulforhodamine B assay, essentially as described or both es and ei NT activities. To further define the transportability of gemcitabine, we analyzed the uptake kinetics of [^HJgemcitabine previously (38. 39). Cells were seeded in multiples of eight wells in 96-well cell culture plates, with â€”¿20,000cells/well, then exposed to drug. Drug by five human nucleoside transporters acting in cell lines exhibiting a exposures were performed in growth media with 10% horse serum for murine single NT activity in isolation. To confirm the mediated character of cell lines and 10% fetal bovine serum for human cell lines. Gemcitabine was | 'H]gemcitabine permeation in a noncellular system, the human es NT added to final concentrations that ranged from I0~4-10~9 M. Cells were was solubilized from membranes of cultured human BeWo chorio- enumerated with the sulforhodamine B assay at the time of drug addition and carcinoma cells and functionally reconstituted in proteoliposomes, at 48 h after drug addition. All adherent cell lines were plated in triplicate 24 h which were used to study ['HJgemcitabine uptake. before drug exposure, then similarly assessed for growth inhibition by trypsinization and cell enumeration with an electronic cell counter after 72 h of drug exposure. Chemosensitivity was expressed as the concentration of gem MATERIALS AND METHODS citabine that inhibited cell proliferation by 50% (IC50), as determined by Materials. ['H]Gemcitabine was a kind gift of Eli Lilly. Inc. (Indianapolis. nonlinear regression analysis using GraphPad Prism 2.01 (GraphPad Software, Indiana). |G-'H|NBMPR (22.5 Ci/mmol) and [5,6-'H]uridine (40 Ci/mmol) San Diego. CA). All experiments were performed independently on two occasions, and mean IC5(>values are reported. were purchased from Moravek Biochemicals. Inc. (Brea. CA). Radiolabeled nucleosides were purified by high-pressure liquid chromatography using wa- Generation of Expression Constructs. For expression in HeLa cells, the full coding sequences of hCNTl (22) and hCNT24 were removed from the ter-methanol gradients on a C,8 reverse phase column; repurification of I'Hlgemcitabine was essential because some preparations contained 'H deg original cloning vector pGEM3Z by Afrlll/Xhal digestion. Subsequently, the 5' radation producÃs. ['''CICholesteryl oleate (0.1 mCi/ml) was obtained from overhang was blunt-ended with the Klenow fragment and ligated into pCD- NAI/Amp at the EcoRV site. The 3' ends of hCNTl and hCNT2 were ligated Amersham Canada. Ltd. (Oakville. Ontario, Canada). Asolectin (soybean into the vector at the Xbal site. The structures of pCDNAI/Amp-hCNTl and phospholipids) was purchased from Associated Concentrates (Woodside. NY) pCDNAI/Amp-hCNT2 were confirmed by restriction mapping and sequence and stored under N:. Other phospholipids and cholesterol were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). OCTG, adenosine, uridine, analysis using an ABI Prism 310 Genetic Analyser (Norwalk, CT). NBMPR. and dipyridamole were purchased from Sigma Chemical Co., and Transient Expression of hCNTl and hCNT2 in HeLa Cells. The trans Sephadex G-50 (fine and medium) was from Pharmacia, Canada. Dilazep port characteristics of recombinant hCNTl (cit activity) and hCNT2 (cif K/V,/V'-bis|3-(3,4.5-trimethoxy-ben/oyloxy)-propyl] homopiperazine] was a activity) were studied using a method described previously (40) for recombi nant rCNTl produced in COS-1 cells. HeLa cells were plated (5.0 X 10" per gift from F. Hoffman La Roche and Co. (Basel. Switzerland). All other 100-mm plate) and grown to 50% confluency in RPMI with 10% calf serum. chemicals were of analytical grade and commercially available. Transfection was performed by a modification of the DEAE-dextran method Cell Culture. Murine cell lines were grown in RPMI and 10% horse serum, and human cell lines were cultured in RPMI 1640 and 10% fetal bovine serum (40) in which cells in DMEM and 10% NuSerum with 200 Â¡IMchloroquine (5 as described elsewhere (25-27). Experiments were initiated with cells in ml) were exposed to 100 Â¿ilof DEAE-dextran DNA mixture, using 5 /j,g of DNA per plate. Cells were incubated at 37Â°Cin 5% CO, for 2 h, then 2 ml of exponential growth phase. S49 mouse lymphoma cells possess only es nucle oside transport activity, which can be completely inhibited by either NBMPR DMSO 10% solution were added. Plates were incubated for 2 min at room (1 /Â¿M)or dipyridamol (10 /IM: Ref. 28). AE1 is a NT-deficient S49 mutant temperature, washed three times with PBS. and placed in growth media for 24 h. Cells were then trypsinized, pooled, and replated on 60-mm plates to (29). LI 210 murine leukemia cells have three NT activities: es, ei, andc//(30). B23.1 is double mutant derived from the parental L1210 line by mutagen eliminate any differences in transfection efficiencies among the individual exposure and selection with 7 /xM cytarabine. erythro-9-[3-(2-hydroxynony- cultures. Cells were used for uptake assays 72 h after transfection. Transfection l)]adenine. and NBMPR; it possesses only es transport activity (25). MA = efficiencies (determined by counting stained and unstained cells in cultures that had been independently transfected with ÃŸ-galactosidase) ranged from 27.1 is a mutant L1210 line that possesses only ci/activity and was isolated by 15% to 30%. Cells were treated with 10 /J.Mdilazep to inhibit endogenous es- mutagen exposure and selection with tubercidin and cytarabine (31). LI210/ DNC3 is a transport-deficient cell line isolated from L1210/B23.1 after mu- and e/-mediated NT activity prior to uptake assays. [3H]Gemcitabine and l'11irridine Cellular Uptake Assays. Nucleoside lagenesis and selection with 0.25 JU.Mtubercidin plus 1.0 Â¡UM1-ÃŸ-D-arabino- uptake assays were conducted at room temperature under zero-trans conditions furanoxylcytosine and lacks all three of the nucleoside transport activities found in wild-type LI210 cells. L12IO/DU-5 is a stable cell line expressing in either sodium-containing transport buffer (20 mM Tris/HCl. 3 mM K,HPO4. recombinant rat CNT1 (32) in the transport-incompetent background of LI 210/ 1 mM MgCl,-6H,O. 2 mM CaCU, 5 mM glucose, and 130 mM NaCl, pH 7.4; 300 Â± 15 mOsm) or sodium-free transport buffer (20 mM Tris/HCL, 3 mM DNC3. rCNTl was subcloned into the mammalian expression vector pcDNA3 and electroporated into LI210/DNC3 cells. Transport-competent cells were K2HPO4. 1 mM MgCl:-6H,O. 2 mM CaCU, 5 mM glucose, and 130 mM N-methyl-D-glucamine/HCl, pH 7.4; 300 Â± 15 mOsm). Cells were washed

4 M. W. L. Ril/el. S. Y. M. Yao. A. M. L. Ng, J. R. Mackey, C. E. Cass. and J. D. once with the appropriate transport buffer and then processed immediately, or in some experiments, incubated with NBMPR or dilazep at room temperature Young. Molecular cloning, functional expression and chromosomal localization of a cDNA encoding a human Na*/nucleoside cotransporter (hCNT2) selective for purine for 30 min before the uptake assay. For suspension cell lines (27), each sample nucleosides and uridine. suhmitled for purification. was processed individually, and permeant fluxes were terminated using the 4350

"inhibitor-oil" stop method. Dilazep dihydrochloride was used at 200 /UMas homogenizer (Brinkman Instruments. Westbury, NY), and resuspended in the stopping reagent. Uptake at time "zero" was determined by the addition of 9.25% sucrose with removal of nuclei by centrifugation (2 min. 900 X g). cold (4Â°C)dilazep to the assay mixtures, followed by the addition of cells and Plasma membranes were then separated on a 15-45% (w/w) sucrose gradient immediate centrifugaron (16,000 X #). Cell-associated radioactivity was and stored at -80Â°C in 15% (v/v) DMSO. The fractions collected at 25% determined by liquid scintillation counting using a xylene-detergent scintillant. sucrose were used for reconstitution experiments because they were enriched For adherent cell lines. ~ 106 cells were plated on 60-mm plates 48 h before in plasma membranes when assayed for 5'-nucleotidase (EC 3.1.3.5) activity transport studies. Uptake intervals were started by adding 1.5 ml of transport (Sigma Diagnostics 5'-nucleotidase assay kit). buffer containing 'H-labeled permeant and inhibitors. Uptake intervals were Membranes were thawed and washed three times in ice-cold reconstitution ended by rapidly aspirating the permeant solution and immersing the culture buffer (pH 7.4) composed of 100 mM KC1. 10 mM Tris. 0.1 niM MgCU and 0.1 dishes in 1.5 I of ice-cold transport buffer containing dilazep. The dishes were mM CaCU. Membranes were then combined with 1% OCTG and 0.15% drained, and the cells were solubilized in 1 ml of 5% (v/v) Triton X-100 and asolectin in reconstitution buffer and incubated on ice for l h with frequent combined with 5.0 ml of Ecoute scintillant for radioactivity measurements. mixing. The insoluble material was removed by centrifugation (60 min, Uptake at time zero was determined by incubating cells for 10 min at 4Â°Cwith 100.000 X g. 4Â°C).and the supernatants were retained on ice until use. The transport buffer that contained 10 JU.Mdilazep and. immediately thereafter, for membrane protein concentrations were monitored using the modified Bradford Â£3 s with ice-cold transport buffer that contained 3H-labeled permeant and assay (42) and adjusted to â€”¿200Â¿igof protein/ml. either 10 /UMNBMPR or 10 /J.Mdilazep. For kinetic studies with transfected Proteoliposome Reconstitution Procedure and 3H-Labeled Nucleoside HeLa cells, ['H]uridine uptake was measured both in sodium-containing and Uptake Assays. The reconstitution technique is described in detail elsewhere sodium-free transport buffer in cells transfected with the vector alone or that (43, 44). Solubilized BeWo membranes (2 ml) were supplemented with a contained the appropriate cDNA constructs. Initial rates of uptake were derived sonicated preparation of lipids (0.3 ml) consisting of phosphatidylcholine by linear regression analysis of time courses of uptake of radioactive nucleo- (bovine brain), cholesterol, phosphatidylethanolamine (egg), and phosphati- sides. Kinetic parameters were estimated from nonlinear regression analysis dylserine (bovine brain) in molar ratios of 33:33:26:8, respectively, plus a trace using GraphPad Prism 2.01. and the resulting Km and Vmail values were (10s dpm/ml) of [l4C]cholesteryl oleate. The detergent was removed by averaged. filtration on Sephadex G-50. and the resulting proteoliposomes were pooled, Isolation and Soin bili/al ion of Be Wo Membranes. BeWo cells were frozen in ethanol and dry ice. and stored at -80Â°C for up to 1 month. chosen as a source of es NT due to their unusually high NT activity and high Proteoliposomes were thawed at room temperature, centrifuged (40.000 X g, numbers of high-affinity NBMPR-binding sites (26). Cells (5 X IO6 per flask) 20 min), resuspended in ~ 1 ml of reconstitution buffer, sonicated for 15 s in were grown in 15 Corning disposable tissue culture flasks and. although a cylindrical tank sonicator (Laboratory Supplies Company Inc.. Hicksville. actively proliferating, the harvest yielded 8 x 10S-I X IO9 cells. Plasma NY), and held on ice until use. membranes were prepared as described previously (41 ). In brief, washed cells The uptake assays were initiated by the rapid addition of 100 Â¿ilof were swollen by suspension in 1 mM ZnCU, fragmented using a Polytron proteoliposome suspension (~5 fig protein, with or without inhibitors) to 25 /Â¿I

Table 1 Gemcittlhine i'heniosensiÃivity in cell lines wilh tiffined NT Ãl(7/w'//Ã'.v Cultures were exposed to graded concentrations of gemcitabine (10 -10 M)for 48 or 72 h. as described in "Materials and Methods." Cells were enumerated, and chemosensitivity was expressed as the concentration of gemcitabine that inhibited cell replication by 50% (IC5(,). All studies were performed Iwice in independent experimenls. and each IC50 is reported.

Nucleoside transport activities

lineS49AEIL12IOB23.1DU-5MA27.1DNC-3K562K562Cell eiMurine es cif(fiM)0.0130.0091.61.0+ cib IC50 gemcitabineresistance1.0118l.o131.773018001.0>18001.0164267>32001.01.1391.01.11.3 +S49 lymphoma

mutantMurineNT-deficient

+L1210 leukemia 4- 0.00870.00550.1140.070+

+L1210rCNTlmutant

transfectantL1210stable 0.0150.009+

mutantLI210 6.04.3150.0570.049>IOO>IOO0.0320.0305.84.48.77.9>100>1000.0360.0200.0390.0231.40.8+

mutantErylhroidNT-deficient

+Erythroid leukemia +

+dipyridamoleCEMCEM leukemiaLymphoblastic

+Lymphoblastic leukemia

+NBMPRCEM leukemiaLymphoblastic

-1-dipyridamoleCEM-ARAC8CHeLaHeLaleukemiaNT-deficient

mutantCervicalCEM

+Cervical cancer +

+NBMPRHeLa +Cervical cancer

+dipyridamoleCaCo-2CaCo-2 cancerColon

+Coloncancer + 0.00370.0025+

+NBMPRCaCo-2 +Coloncancer 0.00390.0029+

+ dipyridamoleOrigin cancercil 0.00410.0035Relative

4351

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1998 American Association for Cancer Research. GEMCITABINE CYTOTOXICITY AND NUCLEOSIDE TRANSPORT of reconstitution buffer with 20 /IM "H-labeled permeant. After specific incu 10.0-, bation times, 100 ÃŸ\of the reaction mixtures were layered on ice-cold Seph- adex minicolumns ( 1-ml syringes fitted with a polyethylene filter), equilibrated in reconstitution buffer containing 10 /Â¿Mdila/.ep and NBMPR. and centrifuged (45 s. 7(X) x #). The effluents were collected in preweighed tubes from which portions were removed for the determination of protein. 3H. and I4C contents. Estimates of/ero-time uptake values were obtained by measuring the uptake of 'H at =2 s in the presence of ice-cold solutions containing 10 itiM adenosine. 10 /IM dipyridamole, and 10 /IM NBTGR. Protein was measured using BSA as the standard with appropriate corrections for detergent interfer ence (42. 45).

RESULTS Fig. 3. Initial rates of uptake of ['Hlgemcitabine by nucleoside-transport competent Growth Inhibition Studies. We have compared the ability of (CEM) and incompetent (CEM-AraC-8Cl cells. Cells were exposed to 10 fiM ['H|gcm- gemcitabine to inhibit proliferation of murine and human cancer cell citahine for the lime periods indicated, permeant fluxes were terminated using the lines exhibiting a spectrum of NT activities. In the experiments inhibitor-oil slop method, and cell-associated radioactivity was determined (see "Materials and Methods"). Each point represents the mean of experiments performed in triplicate; summari/.ed in Table 1, cells were exposed to graded concentrations bars, SE (not shown where smaller than data points). A single representative experiment of four is shown. â€¢¿.CEMcells: O, AraC-C8 cells.

*- of gemcitabine: (a) for 48 h (suspension cultures) and quantitated WC using the sultbrhodamine B assay (38); or (b) for 72 h (adherent ^â€”D 0)C 1X cultures) and quantitated by trypsinization and electronic enumera 0Â«1,> !1b-Q-o-o7 tion. Chemosensitivity was expressed as the concentration of gemcit abine that inhibited cell proliferation by 50% (IC50). and results are 50-25-0-1IJ summarized in Table 1. We first compared chemosensitivity in pairs

\â€¢ / 1â€” v of cell lines that were either transport competent (parental line) or *Jo1Â£10075- transport incompetent (mutant line) in experiments similar to the one shown in Fig. 1 for CEM, a human T-lymphoblast cell line with ex transport activity (34). and ARAC-8C. a NT-incompetent clonal de -15 -13 -11 -9 -7 -5 rivative that was isolated by mutagen exposure and selection for resistance to 8 /Â¿Mcytarabine(35). The ARAC-8C cell line was highly gemcitabine resistant with an IC50 exceeding 100 /AMcompared with Log gemcitabine an IC5(Iof 0.031 /AMfor the CEM line (Fig. 1). The other transport- concentration (M) incompetent cell lines produced genetically by mutation (AE1, DNC-3) were also significantly less sensitive to gemcitabine than the Fig. I. A comparison of the anliptulifcralive action of gemcitabine against nucleoside transport-incompetent (CEM-AraC-8C) and nucleoside transport-competent (CEM) cells. matched parental cell lines (S49, LI 210). The IC5,,s of gemcitabine in Cells were exposed lo a range of gemcitabine concentrations for 48 h and then quantitated by the sulf'orhodaniinc B assay. Each point represents the mean of eight replicates; bars. the transport-incompetent cell lines, when related to the IC5()s in parental cell lines, increased by 118-fold to greater than 3200-fold. SE (not shown where smaller than the size of data points). A single representative experiment is shown. O. CEM cells; â€¢¿AraC-C8 cells. The high levels of resistance seen in transport-incompetent cells indicated that uptake of gemcitabine in the absence of functional NTs in the plasma membrane was inefficient and suggested that diffusional uptake of gemcitabine through the plasma membrane was capable of producing growth-inhibitory concentrations inside cells only in the = 0) u presence of large transmembrane concentration gradients. To determine the contributions of the two bidirectional equilibra IÂ§ tive NT activities to the antiproliferative action of gemcitabine, we o next examined the growth-inhibitory effects of gemcitabine in three . _ human cancer cell lines (K562, CEM, and HeLa) in the presence and 0) Â£ absence of pharmacological inhibitors of es and ei (Table I ). At the concentrations used in these studies, NBMPR (100 nM) selectively inhibits ev-mediated transport, whereas dipyridamole (10 /Â¿M)inhibits both ex- and ii-mediated transport. Although NBMPR exposures conferred 164-fold resistance to gemcitabine in CEM cells, which exhibit es NT activity in isolation, NBMPR was not capable of protecting HeLa cells, which exhibit both es and ei NT activities (26). In contrast, dipyridamole exposures conferred 39-fold resistance in HeLa cells, indicating that Â«'-mediated uptake alone was sufficient to Log gemcitabine concentration (M) achieve intracellular concentrations of gemcitabine that inhibited pro Fig. 2. The effects of inhibitors of equilibrative nucleoside transport on (he antipro- liferation. Dipyridamole increased gemcitabine resistance by 39- to liferative action of gemcitabine against CaCo-2 human colon cancer cells. Cells were plated in triplicate for 24 h and then exposed to graded concentrations of gemcitabine for > 1800-fold in cell lines with only equilibrative NT activities (K562, 72 h. lrypsini/ed. and enumerated. Each point represents the mean; burs. SE (not shown CEM, and HeLa), demonstrating that pharmacological inhibition of where smaller than data points). â€¢¿.gemcitahinealone; Q. gemcitabine after preincuhation with I(K) nM NBMPR; O. gemcitabine after preincubation with 10 Â¿IMdipyridamole. A NT activities can significantly modulate the antiproliferative action of single representative experiment is shown. gemcitabine. 4352

Table 2 Kinetic parameters of Â¡"Hfgemcitabine transport in cell lines with defined NT adivines

Cells (Iransfected and/or inhibitor treated as indicated) were exposed to graded concentrations of [ HJgemcitabine. and initial uptake rates were determined. Assays were conducted at room temperature using short gemcitabine exposures terminated by inhibitor/oil stop methods (CEM cells) or inhibilor/cold stop methods (all other cell types). Kinetic parameters were estimated from nonlinear regression analysis. Each value represents the mean Â±SE of values from independent experiments conducted in triplicale.

of independent cells)17.0 TransportereseicitcifdbCelltreatmentCEMHeLa line and (MM)329 experiments43323 Â±91832 Â±4.34.2 Â±5.9215 NBMPRRecombinant+ 100 HM Â±20418.3 Â±1.30.94 Â±2022.8 cellsRecombinanthCNTl in HeLa 7.2No Â± Â±0.22NA" Â±9.7NA hCNT2 in HeLa cells measurable transport CaCo2 colon cancer cells + dilazepKm Rate insufficient for accurate kineticsVniax(pmol/s/IO6Rate insufficient for accurate kineticsÂ«JVmM20.1NANo. ' NA, not assessable.

In addition to the equilibrative NTs described above, mammalian activity has been observed in intestinal enterocytes, brush-border cells possess sodium-coupled inwardly directed concentrative NT membrane vesicles of several species, and rabbit choroid plexus (18). activities (18). To determine the contributions of the concentrative Because cit activity has not yet been demonstrated in cultured cell NTs to gemcitabine activity, we examined gemcitabine-induced lines, we used L1210/DU-5, a stable transfectant that produces re- growth inhibition in cell lines that exhibited a single concentrative NT combinant rat CNT1 (32) in the transport-incompetent background of activity (cit, cif, or cib; Table 1). L1210/DNC3. L1210/DU-5 cells exhibit sodium-dependent transport The cit NT exhibits sodium dependence, selectivity for pyrimidine- of thymidine that is inhibited by the pyrimidine nucleosides uridine nucleosides, and insensitivity to inhibition by NBMPR, and cit NT and cytidine but not by the purine nucleosides inosine or guanosine (33). Gemcitabine inhibited proliferation of DU-5 cells (IC5() 0.092 /J.M) in contrast to the parental, transport-incompetent DNC-3 cells (IC5() 13 /LIM),suggesting that the cit transporter mediated gemcitabine 20, influx. The cif NT exhibits sodium dependence, selectivity for purine- -8 10-1 nucleosides, and uridine, and insensitivity to inhibition by NBMPR. o and Â«/NT activity has been observed in mouse splenocytes, macro Q. phages, rat macrophages, and hepatocytes and in several cultured cell types, including mouse L1210 leukemia cells (18). The L1210/MA27 1000 2000 3000 cell line, which exhibits only cif NT activity, was 730-fold less (MM) sensitive to gemcitabine (IC5() 5.2 /J.M)than the parental L1210 cell line (IC50 0.0071 /J.M),suggesting that gemcitabine is a poor substrate B for the a/NT. The cib NT has the ability to transport a wide range of both purine and pyrimidine nucleosides and has been described in the HL-60 "=3 o o cultured human promyelocytic leukemia cell line after induction of differentiation (46) and in the CaCo-2 colon carcinoma cell line (24). In the experiment of Fig. 2, we examined gemcitabine inhibition of 1000 2000 3000 growth in CaCo-2 cells that were treated with dipyridamole to block (MM) endogenous es and ei activities. There were no differences in the responses to gemcitabine between untreated and inhibitor-treated cells. It has been shown previously that dipyridamole enhanced the toxicity of 9-ÃŸ-D-arabinofuranosyladenine against the L1210/C2 cell 1.0, line (47), which has es, ei, and Â«'/'transport activities (30). Although Â«41 the initial rate of drug uptake was reduced by dipyridamole, there was -â€”o> 0.5. _ o o an intracellular accumulation of drug above the steady-state intracel- E lular concentration because of the elimination of drug efflux via the 0.0 0 25 Â¿0 /5 100 125 bidirectional equilibrative transporters. That dipyridamole treatment of CaCo-2 cells did not increase gemcitabine sensitivity suggested (uM) that release of gemcitabine back into the growth medium via the Fig. 4. Kinetic analysis of initial uptake of ['HJgemcitabine by human es, ei, and cit NT activities. Cells were exposed to graded concentrations of ['HJgemcitabine. and the initial bidirectional equilibrative NTs was not a major component of net rates of update were determined (see "Materials and Methods"). Each point represents the gemcitabine retention. mean of the rate derived from by linear analysis of experiments performed in triplicate; [3H]Gemcitabine and [3H]Uridine Cellular Uptake Assays. To burs, SE (not shown where smaller that data points). A single representative experiment maximize the potential clinical relevance of this work, gemcitabine is shown for each transporter. Nonlinear regression analysis was used to analyze uptake kinetics. A, kinetics of Â«-mediated ['HJgemcitabine uptake in CEM cells. Initial rates of transport by each of the human NTs was examined individually, either uptake were determined as described in "Materials and Methods." Analysis of initial in transport-competent cell lines or in human cell lines transiently [3H]gemcitabine uptake kinetics revealed a Km of 410 yM and a Vmaxof 16.1 pmol/s/10 6 cells. B, kinetics of f/-medialed ['HJgemcitabine uptake in HeLa cells. Initial rates of transfected with cloned human NT cDNAs. The transport-incompe uptake were determined as described in "Materials and Methods." and endogenous es tent CEM-AraC-8C cell line was studied first to establish whether activity was inhibited by 100 nM NBMPR. Analysis of initial ['HJgemcitabine uptake kinetics reveals a Afmof 737 JIM and a Vmâ€žof 1.99 pmol/s/10 6 cells. C, kinetics of gemcitabine was capable of entry into cells in the absence of a (â€¢Â«-mediated['HJgemcitabine uptake in HeLa cells transiently transfected with hCNTl. functional nucleoside transporter (i.e., by diffusion). No significant HeLa cells were transiently transfected as described in "Materials and Methods." After [-'H]gemcitabine uptake occurred (Fig. 3), because uptake rates did 72 h cells were preincubated with 10 JIM dilaz.ep to inhibit endogenous equilibrative NT activity. Analysis of initial [â€¢'HJgemcitabineuptake kinetics reveals a Km of 31.8 JAMand not significantly vary from zero. The parental cell line CEM, from a ymÂ«of 0.82 pmol/s/106 cells. which CEM-AraC8C was derived, possesses only ex activity, with 4353

HeLa cells transfected with the hCNTl vector. Gemcitabine was a 10.0, good permeant for recombinant hCNTl, with a Km of 18.3 Â±7.2 /J.M and Vmaxof 0.94 Â±0.22 pmol/s/106 cells (Table 2; Fig. 4C). Con versely, transfection of the hCNT2 cDNA into HeLa cells had no 8 7.5- effect on uptake of [3H]gemcitabine, although the expected [3H]uri- "o dine uptake was observed (Fig. 5fi). This result was consistent with 5.0- the observed gemcitabine resistance in L1210/MA27 cells, which exhibit only the purine-nucleoside selective cif NT. Analysis of de-dependent transport of gemcitabine was difficult. A human cDNA encoding a sodium-dependent NT with broad permeant selectivity has not yet been reported, and the CaCo-2 cell line exhibits 12 10 relatively low levels of endogenous cib activity in addition to ex and time (s) ei activities (24). Uptake of [3H]gemcitabine was examined (not shown) in CaCo-2 cells in the presence of 10 Â¡Ã•Mdilazepto block es B and ei activities. Gemcitabine entry was mediated by the cib trans porter, but uptake rates were too low for kinetic analysis. The dem 10.0, onstration of Â«'Â¿-mediatedgemcitabine uptake explains our earlier Â£ observation (Fig. 2) of high-level gemcitabine activity in CaCo-2 o 7.5. Â«D despite the presence of inhibitors of equilibrative NT. O Transport efficiency can be expressed as A"m/Vmax,where low 5.0. values indicate efficient transporter-mediated permeation. The values ! 2.5. of Table 2 indicate that the es (Arm/Vmax,20.1) and dt (KJVmâ€žâ€ž22.8) Q. NT activities were the most efficient mediators of gemcitabine influx, 0.0. followed by the ei (Km/Vmax, 215) NT activity. The Â«/NT did not 0 4 Ã¨ 10 12 mediate permeation of gemcitabine across plasma membranes. time (s) Because uridine is accepted as a permeant by all of the reported Fig. 5. Initial rales of uptake of | 'HJgemcitabinc and |'H)uridine in HeLa cells human NTs, it is considered to be a "universal permeant" and was transiently Iransfeclcd with hCNTl (i-ii NT) or HCNT2 (Â«yNT). Cells were transiently chosen as a reference nucleoside with which to compare gemcitabine transfected with vector alone (controls) or that contained either hCNTl M) or hCNT2 (ÃŸ) cDNAs as described in "Materials and Methods." After 72 h. cells were preincubated with uptake rates. To allow an assessment of efficiency of gemcitabine IO /IM dila/cp to inhibit endogenous equilibrarne NT activity. Cells were then exposed to either K) JIM [ 'H|genicitabine or [lH)uridine for the indicated time periods, and cell- uptake among the human NTs at the therapeutically and physiologi associated radioactivity was determined (see "Materials and Methods"). Each point cally relevant concentration of 10 /J.M,uptake rates of both gemcit represents the mean of experiments performed in triplicate; Aurs. SE (not shown where abine and uridine were determined for each of the human NTs (Table smaller than data points). A single representative experiment is shown for each recom binant transporter. A. |'H Â¡undineuptake (â€¢)or [ 'Hjgenicilabinc uptake (Q) in transfected 3). Relative uptake rates (gemcitabine/uridine) were 0.12 for es, 0.26 cell with hCNTl cDNA; |'H]uridinc uptake (O) or ['H]gemcitabine uptake <â€¢>incells for ei, 0.10 for cit. and 0.58 for cib (there was no gemcitabine uptake transfected with vector alone. R, | lH)uridine uptake (â€¢)or ['Hjgemcitabine uptake (O) in for cif). Thus, although gemcitabine was transported by four of the cells transfected with hCNT2 cDNA. Values for uptake of ['Hjuridine and ['Hlgemcit- five NTs, the physiological substrate uridine was transported by all abine by cells transfected with vector alone were not significantly different from zero and are thus not shown. five with much greater efficiency. In the HeLa cell line, known to possess both es and ei activities (26), the relative contributions to initial ['HJgemcitabine uptake at a concentration of 10 /J.Mwere 72.4 -3.3 X IO5 NBMPR-binding sites/cell (48). Uptake of ['HJgemcit- and 27.6%, respectively (Table 4), indicating that the major compo abine by CEM cells was rapid, with kinetic analyses of initial uptake nent of gemcitabine influx was mediated by the es NT. rates demonstrating a Km of 329 Â±91 /Â¿Mand Vmaxof 17.0 Â±4.3 [â€¢'HJGemcitabineand l'il|l ridine Uptake by Proteoliposomes pmol/s/10(' cells (mean Â±SE of four independent experiments per Prepared from Detergent-solubilized Membrane Fractions En formed in triplicate: Table 2: Fig. 4/1). riched in NBMPR-binding Activity. The representative experiment Human cells that exhibit ei NT activity in isolation have not been shown in Fig. 6 examined the initial rates of uptake of 20 H.M described. However, HeLa cells possess es and ei without concentra- [3H]gemcitabine by proteoliposomes prepared from BeWo cells by tive NT activity. By selectively inhibiting HeLa es activity with NBMPR I(X)nM, the NBMPR-insensitive endogenous ei activity was studied in isolation. Uptake of [3H]gemcitabine was linear over 10 s, Table 3 Comparison of initial rules of uptake of [' HÃ¬geineÃ¬tahÃ¬neanil/" Hlitritline bv human mtcleositlt' transporters and kinetic analysis revealed a Km of 832 Â±204 /Â¿Mand Vmaxof Cells (transfected or inhibitor treated as indicated) were exposed to either 10 /AM 4.3 Â±1.3 pmol/s/106 cells (mean Â±SE of three independent exper ["Hjgemcitabine or [ H|uridine. Initial uptake rates (transport) were determined at room iments performed in triplicate; Table 2; Fig. 4fi). temperature using short gemcitabine exposures terminated by inhibitor/oil stop methods (CEM cells) or inhibitor/cold stop methods (other cell types). Each transport value Although human cell lines that exhibit at or d/NT activities have represents the mean Â±SE of values from two to four independent experiments, each not been described, the recent isolation of cDNAs encoding human conducted in triplicate. NTs with either cil (22) or cif activities4 has provided an approach to kinetic analysis of these NTs in intact cells. We studied gemcitabine NTtypees transport transport typeCEM (pmol/s/IO6cells)0.109 (pmol/s/106cells)0.894 transport by recombinant cit or cif after transient transfection of HeLa cells, respectively, with hCNTl or hCNT2 cDNA. Transport studies Â±0.049 Â±0.145 ei HeLa + NBMPR 0.034 Â±0.008 0.131 Â±0.022 0.26 were performed in the presence of 10 /AMdilazep to inhibit endoge citcif Recombinant hCNTl 53No0.337 Â±0.1 3.25 Â±1.480.76 0.100 nous equilibralive NT activities normally present in HeLa cells. The in HeLa Recomhinant hCNT2 measurable uptake Â±0.09 results of Fig. 5/4 demonstrated: (a) the lack of mediated uptake of 'H-labeled nucleoside uptake in HeLa cells transfected with vector in HeLa cibCell CaCo-2 + dilazepGemcilabine0.064 Â±0.01Uridine 0.11 Â±0.01Ratio"0.120.58 alone: and (b) uptake of both ['Hjgemcitabine and [3H]uridine in ' Gemcitabine:uridine transport rates.

4354

sucrose-density centrifugation and examined in the presence and 1.00, absence of a mixture of inhibitors (adenosine, NBTGR, and dilazep) oÃes- and e/'-mediated transport. We have shown previously (41) that = a> 0.75. proteoliposomes so prepared exhibit only es NT activity, despite the presence of both es and ei activities in BeWo crude membranes. The || jS Â» 0.50. value from four separate experiments for the uninhibited initial rate of Ã¼i E o gemcitabine uptake was 20 Â±2 pmol/mg protein/s, and preincubation oÃE 025- of proteoliposome with the mixture of transport inhibitors decreased gÃ¬ this rate to 13 Â±2 pmol/mg protein/s, or 65% of total uptake. The mediated component of [3H]gemcitabine uptake, which was obtained 0.00.O 2"5 5"0 7*5 100 125 150 by subtracting the inhibited value from the total value, was 7 Â±2 pmol/mg protein/s. In a separate study conducted similarly, the initial time (s) rate of uptake of 20 JU.M[3H]uridine (six separate experiments) was Fig. 6. Time course of ['HJgemcitahine uptake by proteoliposomes prepared from 45.0 Â±5.0 pmol/mg proteins/s (41). The ratio (0.15) of [3H]gemcit- detergenl-solubilized membrane fractions from BeWo cells. Proteoliposomes were pre abine to [3H]uridine uptake rates by the reconstituted BeWo es NT pared from OCTG-solubilizcd membrane fractions that were isolated by density-gradient centrifugation in 25% sucrose as described in "Materials and Methods." The proteolipo was very similar to that (0.12) for the native es NT of CEM cells. somes were incubated with 20 (ÃŒM['H|gemcitabine for the indicated times in the presence These results support the use of functional reconstitution of individual (O. non-mediated uptake) and absence (â€¢.total uptake) of 10 nut adenosine. 10 (ÃŒM NBTGR. and 10 fiM dipyridamolc. Values for mediated uptake (â€¢)were calculated from human NT proteins as an appropriate means of studying human NT the difference between total and nonmedialed uptake. Assays were conducted in duplicate activities in isolation; this is the first report of gemcitabine uptake and plots from one of four separate experiments are shown. mediated by a reconstituted NT. plasma membranes was a relatively minor component of gemcitabine DISCUSSION uptake. By studying gemcitabine-dependent inhibition of proliferation in a panel of murine and human cell lines with defined NT activities, Although resistance to gemcitabine because of decreased drug we were able to infer that gemcitabine uptake was mediated by the es, influx has not been reported previously, there is evidence that nucle- ei, cit, and cib NTs but not by the cif NT. The es, ei, and cib NTs oside-transport deficiency may be a mechanism of resistance to anti- exhibit broad permeant selectivity, accepting both purine and pyrim- cancer nucleosides both in vitro and in vivo. Decreased es NT activity idine nucleosides, whereas the cit and cif NTs accept uridine and correlated with in vivo cytarabine resistance in human acute myelog- exhibit selectivity, respectively, for pyrimidine and purine nucleosides enous leukemia (49). Resistance to cytarabine therapy has also been (18). shown to be associated with decreased es activity in freshly isolated To confirm the transportability of gemcitabine suggested by the human leukemia cells (50, 51). Estimates of the numbers of es growth-inhibition studies, we performed detailed kinetic studies of transporters have been inferred from quantitative analysis of cellular [3H]gemcitabine uptake in human cells. Initial rates of uptake were NBMPR binding. Decreases in NBMPR-binding sites have been measured in cells in which NT activity was absent or in which only a observed during treatment of leukemia; in a patient with T-cell acute single NT activity was present (either naturally or by transient trans- lymphoblastic leukemia, the leukemic blasts examined at relapse fection with the relevant cDNA). The results of the transport studies exhibited a 75% reduction in NBMPR binding when compared with were entirely concordant with those of the growth-inhibition studies. blasts examined before initiation of cytarabine treatment (52). Murine Diffusion was not a measurable component of gemcitabine cellular erythroleukemia cells that were selected in vitro for resistance to influx, and gemcitabine uptake was mediated by several NT activities. cytotoxic purine nucleosides exhibited decreased levels of the es However, the NT activities exhibited by the various cell lines varied transporter, as indicated by a reduction in NBMPR-binding sites (53). markedly in their apparent efficiencies of gemcitabine permeation: es Similarly, CEM T lymphoblasts exhibited a 75% decease in 2',3'- = cit > ei > cib Â»> cif. Because the absolute numbers of dideoxyctidine sensitivity when transmembrane nucleoside transport functional transporters (which probably differ among the various cell activity was decreased by 80% (35). types) could not be determined, it was not possible to directly compare The mechanisms of gemcitabine permeation have not been defined. the intrinsic transportability of gemcitabine by the different NTs. The present study demonstrated that transport-incompetent cells, pro Uridine is accepted as a permeant by all of the known human NTs duced either by mutation or by pharmacological inhibition of NT and was therefore used as a standard with which to compare NT- activity, were resistant to gemcitabine inhibition, and in some cell mediated gemcitabine uptake. Initial uptake rates for a concentration lines, the lack of mediated uptake conferred greater than 1000-fold (10 /XM)that is therapeutically relevant for gemcitabine and physio resistance to the antiproliferative action of gemcitabine. These obser logically relevant for uridine were determined for the five NT activ vations suggested that passive diffusion of gemcitabine through ities (see Table 3). The ratios of gemcitabine:uridine uptake rate varied among the NTs, and gemcitabine rates were consistently less Table 4 /" H'Â¡Cemcitabine initial uptake rates b\ HeLtt fells in the presence and than uridine rates for the es, ei, cit, and cib NTs. The cif NT, which absence of inhibitors of nucleoside transpon mediates transport of uridine and purine nucleosides, including HeLa cells were exposed to 10 ^.M [3H]gemcitabine in the presence or absence of 100 cladribine (54), did not mediate gemcitabine influx. nm NBMPR or 10 JIM dilazep. Initial uptake rales were determined at room temperature using short gemcitabine exposures terminated by inhibitor/cold stop methods. The final Because the equilibrative NTs are bidirectional, net gemcitabine values represent the mean Â±SE from four independen! experiments, each conducted in uptake would be expected to represent the combined contributions of triplicate. Uptake rates (pmol/s/106 cells) plus SE are recorded. NT-mediated influx and efflux. Therefore, the antiproliferative action Uptake of gemcitabine might be potentiated by inhibitors of equilibrative (no inhibitors)0.504 (NBMPR)0.110 (dilazep)000 transport, if a concentrative NT was also present. This possibility was explored in the CaCo-2 cell line, which possesses es, ei, and cib; 0.214 0.078 0.331 0.045 however, dipyridamole exposure did not potentiate gemcitabine cy- 0.387 0.161 00 totoxicity, suggesting that drug efflux by the equilibrative NTs was 0.359 Â±0.060Uptake 0.099 Â±0.024Uptake not a major component of net gemcitabine retention. 4355

The pharmacokinetic properties of gemcitabine suggest that effi Ahratt. R. P.. Bezwoda. W. R.. Falkson. G.. Goedhals. L.. Hacking. D.. and Rugg. T. A. Efficacy and safety profile of gemcitabine in non-small-cell lung cancer: a phase cient uptake of gemcitabine may be required for anticancer efficacy in H study. J. Clin. Oncol., 12: 1535-1540. 1994. vivo. Gemcitabine is typically administered to humans as an i.V. bolus Casper. E. S.. Green. M. R.. Kelsen. D. P.. Heelan. R. T.. Brown. T. D.. Flombaum. infusion lasting 30 min, in doses ranging from 800 to 1200 mg/m2. C. D.. Trochanowski. B.. and Tarassoff. P. G. Phase II trial of gemcitabine (2,2'- difluorodeoxycytidine) in patients with adenocarcinoma of the pancreas.Invest. New The most common dosage schedule is weekly administration on the Drugs. 12: 29-34, 1994. first, eighth, and fifteenth day of a 28-day cycle. Peak serum gemcit Moore. M. Activity of gemcitabine in patients with advanced pancreatic carcinoma. abine concentrations are dose dependent and generally do not exceed Cancer (Phila.). 78: 633-638. 1996. 50 /IM (55). However, after bolus injections, enzyme-mediated deami- Catimel. G.. Vermorken. J. B.. Clavel. M., de Mulder. P.. Judson. I.. Sessa, C., Piccarl. M., Bruntsch. U.. Verweij. J.. Wanders, J.. Franklin. H.. and Kay. S. B. A nation causes a rapid decrease in plasma concentrations, and within phase II study of gemcitabine (LY 188011) in patients with advanced squamous cell 2 h, the plasma concentration of the parent drug falls below quanti carcinoma of the head and neck. Ann. Oncol.. 5: 543-547. 1994. 10. Hcinemann. V.. Hertel. L. W.. Grindey. G. B.. and Plunkett. W. Comparison of the fiable levels (56). These observations suggest that cells in vivo may be cellular pharmacokinelics and toxicity of 2',2'-difluorodeoxycytidine and l-ÃŸ-i> exposed to significant gemcitabine concentrations for only short pe arabinofuranosylcytosine. Cancer Res.. 48: 4024-4031. 1988. Ruiz van Haperen. V. W.. Veerman. G., Vermorken. J. B.. and Peters, G. J. 2',2'- riods and raise the possibility that inefficient cellular uptake may be a Difluoro-deoxycytidine (gemcitabine) incorporation into RNA and DNA of tumour mechanism underlying the observed resistance to gemcitabine in some cell lines. Biochem. Pharmacol.. 46: 762-766. 1993. solid tumors. Plunkett. W.. Huang. P.. Xu. Y. Z.. Heinemann. V.. Grunewald. R.. and Gandhi. V. Gemcitabine: metabolism, mechanisms of action, and self-potentiation. Semin. On Although gemcitabine was shown to be a substrate for four NTs, the col., 22: 3-10. 1995. major mediators of gemcitabine uptake in human tissues are probably Fink. U.. Molle. B., and Daschner. H. Phase II study of gemcitabine in metastatie the equilibrative NTs, because human cil activity has, thus far, only colorectal cancer. Proc. Am. Soc. Clin. Oncol.. Il: A507. 1992. been demonstrated in kidney, liver, and intestinal epithelium (22, 57), Ruiz van Haperen, V. W., Veerman. G.. Eriksson, Sâ€žBoven.E., Stegmann, A. P., Hermsen. M.. Vermorken. J. B.. Pinedo. H. M., and Peters. G. J. Development and and human cib activity has only been demonstrated in human myeloid molecular characterization of a 2'.2'-difluorodcoxycytidine-rcsistant variant of the leukemic cell lines, freshly isolated myeloblasts (46), and the colon human ovarian carcinoma cell line A2780. Cancer Res.. 54: 4138-4143. 1994. cancer cell line used in this report, CaCo-2 (24). By implication, 15. Neff, T.. and Blau. C. A. Forced expression of cytidine deaminase confers resistance to l-/3-r>-arabinofuranosylcytosine and gemcitabine. Exp. Hemalol.. 24: 1340-1346. deficiency of equilibrative NT in tumor cells may be a mechanism of 1996. gemcitabine resistance in vivo. Given that both deoxycytidine kinase 16. Sliutz. G.. Karlseder. J.. Tempfer. C.. Orel. L.. Holzer. G., and Simon. M. M. Drug deficiency and NT deficiency confer high-level resistance to gemcit resistance against gemcitabine and topotecan mediated by constitutive hsp70 over- expression in vitro: implication of qucrcetin as sensitiser in chemotherapy. Br. J. abine cytotoxicity in vitro, either mechanism could be the basis of the Cancer, 74: 172-177. 1996. acquired or de novo resistance to gemcitabine observed in clinical Waud. W. R., Gilbert. K. S.. Grindey. G. B., and Worzalla. J. F. Lack of in vim crossresistancewith gemcitabine against drug-resistant murine P388 leukemias. Can trials. cer Chemother. Pharmacol.. 38: 178-180. 1996. In summary, we have demonstrated that functional NTs are re 18. Cass. C. E. Nucleoside transport. In: N. H. Georgopapadakou (ed.). Drug Transport quired for manifestation of gemcitabine toxicity in vitro. Conversely, in Antimicrobial Therapy and Anticancer Therapy, pp. 403-451. New York: Marcel a deficiency in nucleoside-transport activity was capable of conferring Dekker. 1995. 19. Griffiths. M.. Beaumont. N., Yao, S. Y., Sundaram. M.. Boumah. C. E.. Davies. A.. high-level resistance to the toxicity of gemcitabine. We have per Kwong. F. Y.. Coe, I., Cass, C. E., Young, J. D.. and Baldwin. S. A. Cloning of a formed detailed kinetic studies of gemcitabine uptake in human cell human nucleosidc transporter implicated in the cellular uptake of adenosine and chemolherapeutic drugs. Nat. Med.. 3: 89-93. 1997. lines exhibiting a range of NT activities and have demonstrated 20. Crawford. C. R.. Palei. D. H.. Naeve. C.. and Belt. J. A. Cloning of the human marked variability in the capacity of the different NTs to mediate equilibrative. nitrobenzylmercaptopurine riboside (NBMPR I-insensitive nucleoside transporter ei by functional expression in a transport-deficient cell line. J. Biol. gemcitabine influx. Finally, we have demonstrated mediated uptake of Chem.. 273: 5288-5293. 1998. gemcitabine by the human o NT reconstituted in proteoliposomes. Griffiths. M.. Yao, S. Y. M., Abidi. F.. Phillips, S. E. V.. Cass. C. E.. Young. J. D.. Although the potential for clinical application of these observations is and Baldwin. S. A. Molecular cloning and characterization of a nilroben/ylthioi- nosine-insensitive (ei) equilibrative nucleoside transporter from human placenta. high, efforts to rationally modulate the antitumor and normal tissue Biochem. J.. 328: 739-743. 1997. toxicities of gemcitabine must await a clearer understanding of the Ritzel. M. W. L.. Yao. S. Y. M.. Huang. M. Y.. Elliott. J. F., Cass.C. E., and Young. tumor and tissue distribution of the NT proteins. The recent cloning of J. D. Molecular cloning and functional expression of cDNAs encoding a human cDNAs encoding the four major human NTs ( 19-23)4 has led to new Na*-nucleoside cotransporter (hCNTl). Am. J. Physiol.. 272: C707-C7I4. 1997. Wang. J.. Su, S-F.. Dresser. M. J.. Schnaner. M. E.. Washington. C. B., and molecular and immunological probes to study NT distribution which, Giacomini. K. M. Na ' -dependent purine nucleoside transporter from human kidney: in turn, may lead to transport-based strategies capable of improving cloning and functional characterization. Am. J. Physiol.. 27.?; F1058-F1065. 1997. Belt. J. A.. Marina. N. M.. Phelps. D. A., and Crawford. C. R. Nucleoside transport the therapeutic indices of anticancer nucleosides. in normal and neoplastic cells. Adv. Enzyme Regul.. 33: 235-252. 1993. Vijayalakshmi. D.. Dagnino. L.. Belt, J. A.. Gali. W. P., Cass. C. E.. and Palerson, A. R. L1210/B23.1 cells expressequilibrative. inhibitor-sensitive nucleoside transport ACKNOWLEDGMENTS activity and lack two parental nucleoside transport activities. J. Biol. Chem.. 267: 16951-16956. 1992. We acknowledge Dr. Kathryn Graham and Thack Lang for the provision of Boumah. C. E.. Hogue. D. L.. and Cass, C. E. Expression of high levels of nitroben- zyllhioinosine-scnsitive nucleoside transport in cultured human choriocarcinoma expression constructs used in this work. (BeWo) cells. Biochem. J.. 288: 987-996. 1992. Boleti. H.. Coe. I. R.. Baldwin. S. A.. Young. J. D.. and Cass, C. E. Molecular identification of the equilibrative NBMPR-sensilive (es) nucteoside transporter and REFERENCES demonstration of an equilihrative NBMPR- insensilive (ei) transport activity in 1. Blackslcin. M.. Vogel, C. L., and Amhiner. R. Phase 11study of gemcitabine in human erythroleukemia (K562) cells. Neuropharmacology. 36: 1167-1179. 1997. patients with mctastalic breast eancer. Proc. Am. Soc. Clin. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1998 American Association for Cancer Research. Functional Nucleoside Transporters Are Required for Gemcitabine Influx and Manifestation of Toxicity in Cancer Cell Lines

John R. Mackey, Rajam S. Mani, Milada Selner, et al.

Cancer Res 1998;58:4349-4357.

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