© 1999 by Oxford University Press
Journal of the National Cancer Institute, Vol. 91, No. 21, 1876-1881,
November 3, 1999
© 1999 Oxford University Press
REPORTS |
Gemcitabine Transport in Xenopus Oocytes Expressing Recombinant Plasma Membrane Mammalian Nucleoside Transporters
Affiliations of authors: J. R. Mackey, C. E. Cass, Department of Oncology, University of Alberta, Canada, and Cross Cancer Institute, Edmonton, AB; S. Y. M. Yao, K. M. Smith, E. Karpinski, J. D. Young, Department of Physiology, University of Alberta; S. A. Baldwin, School of Biochemistry and Molecular Biology, University of Leeds, U.K.
Correspondence to: John R. Mackey, M.D., 11560 University Ave., Edmonton, AB T6G 1Z2, Canada (e-mail: johnmack{at}cancerboard.ab.ca).
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionNotesReferences |
|---|
BACKGROUND: Gemcitabine, a pyrimidine analogue of deoxycytidine, is an anticancer nucleoside drug that requires functional plasma membrane nucleoside transporter proteins to reach its intracellular targets and cause cytotoxicity. Because of technical difficulties inherent in studying nucleoside transport in human cells, we rigorously defined gemcitabine membrane transportability by producing each of the available human (h) and rat (r) recombinant nucleoside transporters (NTs) individually in Xenopus laevis oocytes. METHODS: Oocytes were microinjected with in vitro-transcribed RNAs derived from complementary DNAs encoding (C = concentrative) rCNT1, rCNT2, hCNT1, hCNT2, (E = equilibrative) rENT1, rENT2, hENT1, and hENT2. Uptake of [3H]gemcitabine and [14C] uridine was measured 3 days after microinjection to determine kinetic constants. We also used the two-electrode, voltage-clamp technique to investigate the electrophysiology of hCNT1-mediated gemcitabine transport. RESULTS: Gemcitabine was transported by most of the tested proteins (the exceptions being the purine-selective rCNT2 and hCNT2), with the greatest uptake occurring in oocytes producing recombinant rCNT1 and hCNT1. Influxes of gemcitabine mediated by hCNT1, hENT1, and hENT2 were saturable and conformed to Michaelis-Menten kinetics with apparent Km values of 24, 160, and 740 µM, respectively. Gemcitabine had a limited ability to cross the lipid bilayer of oocyte membranes by simple diffusion. External application of gemcitabine to oocytes producing recombinant hCNT1 induced an inward current, which demonstrated that hCNT1 functions as a Na+/nucleoside co-transport protein and confirmed the transporter's ability to transport gemcitabine. CONCLUSIONS: Mammalian nucleoside transporters vary widely in their affinity and capacity to transport gemcitabine. Variation in the tumor and tissue distribution of plasma membrane nucleoside transporter proteins may contribute to the solid tumor activities and schedule-dependent toxic effects of gemcitabine.
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionNotesReferences |
|---|
Gemcitabine (2',2'-difluorodeoxycytidine, dFdC; Gemzar®) is a pyrimidine analogue of deoxycytidine in which two fluorine atoms are present at the 2' position of the deoxyribose ring. Gemcitabine is unique among nucleoside drugs because it is active against epithelial cancers, including non-small-cell lung, breast, bladder, ovarian, and head and neck cancers (1). After intravenous administration, gemcitabine permeates the plasma membrane and is converted to 2',2'-difluorodeoxycytidine monophosphate (dFdCMP) by deoxycytidine kinase, and the latter is subsequently phosphorylated to the cytotoxic 5'-diphosphate and 5'-triphosphate derivatives by pyrimidine monophosphate and diphosphate kinases (2). 2',2'-difluorodeoxycytidine diphosphate inhibits ribonucleotide reductase, while 2',2'- difluorodeoxycytidine triphosphate is incorporated into DNA and RNA (3). Gemcitabine exhibits the properties of self-potention and masked chain termination. Among other mechanisms of self-potentiation, gemcitabine triphosphate inhibits deoxycytidine monophosphate deaminase, thereby decreasing triphosphate catabolism (2). In masked chain termination, an additional physiologic nucleotide is incorporated prior to inhibition of DNA polymerase, which conceals the incorporated gemcitabine nucleotide from exonuclease activity (4).
Because the molecular targets of gemcitabine are intracellular, permeation through the plasma membrane is the obligatory first step in cytotoxicity. Physiologic and therapeutic nucleosides are generally hydrophilic and require plasma membrane nucleoside transporter proteins for efficient cellular entry [reviewed in (5-7)]. Four major functionally distinct nucleoside transporter processes, each of which has been defined in molecular terms through isolation and functional expression of complementary DNAs (cDNAs) encoding the transporter proteins, have been described in human cells (8-13). These nucleoside transporters belong to two previously unrecognized families of membrane proteins and function either as equilibrative, bidirectional transporters (the equilibrative nucleoside transporter [ENT] family) or as concentrative, sodium/nucleoside cotransporters (the concentrative nucleoside transporter [CNT] family). The ENT proteins accept both pyrimidine and purine nucleosides as permeants but differ in their sensitivity to inhibition by nitrobenzylthioinosine (NBMPR): Human ENT1 (hENT1, an equilibrative sensitive, es-type transporter) is inhibited by nanomolar concentrations of NBMPR, whereas hENT2 (an equilibrative insensitive, ei-type transporter) is unaffected by low concentrations (<1 µM) of NBMPR. One of the CNT proteins (hCNT1, a concentrative NBMPR-insensitive thymidine selective or cit-type transporter) is selective for pyrimidine nucleosides but also transports adenosine, albeit inefficiently. The other protein (hCNT2, a concentrative insensitive and formycin B-selective or cif-type transporter) is selective for purine nucleosides and uridine. The tissue and tumor distribution of the nucleoside transporters is not fully defined, but hENT1 is present in most human cells, while concentrative nucleoside transporters have been identified in liver (13), kidney (12-14), intestine (13,15,16), choroid plexus (17), and some cancer cell lines (18-21). Rat homologues of each of these proteins have also been identified and are designated rENT1, rENT2, rCNT1, and rCNT2 (22-25).
In cytotoxicity experiments performed in vitro(26), a deficiency in plasma membrane nucleoside transport, produced either pharmacologically or genetically, conferred two- to three-log protection from gemcitabine growth inhibition in human and murine cancer cell lines. By investigating gemcitabine cytotoxicity in a panel of murine and human cell lines with defined nucleoside transporter activities, we concluded that gemcitabine uptake was apparently mediated by hENT1, hENT2, and hCNT1 but not by hCNT2. Studies with radiolabeled gemcitabine that were conducted in the same panel of cultured human cell lines with single nucleoside transport activities confirmed mediated uptake by the hENT1, hENT2, and hCNT1 transporters. Gemcitabine was most efficiently transported by hENT1 and hCNT1 but at rates approximately 10-fold lower than those of uridine. Plasma membrane diffusion of gemcitabine was much slower than mediated transport.
Because of the technical difficulties inherent in studying nucleoside transport in human cells (the presence of multiple endogenous nucleoside transport activities, variable transfection efficiencies when studying recombinant transporters, and characteristically rapid uptake rates requiring inhibitor-oil stop techniques), we have undertaken a definitive study of radiolabeled gemcitabine transportability by producing each of the available human and rat nucleoside transporter cDNAs individually in oocytes from the amphibian Xenopus laevis. In addition, by the use of whole-cell, two-electrode, voltage-clamp electrophysiology studies, we have studied hCNT1-mediated transport of uridine and gemcitabine without the requirement for radiolabeled permeants.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionNotesReferences |
|---|
CNT and ENT cDNAs
cDNAs encoding rCNT1 (GenBank accession No. U10279), rCNT2 (GenBank accession No. U66723), hCNT1 (GenBank accession No. U62966), hCNT2 (GenBank accession No. AF036109), rENT1 (GenBank accession No. AF015304), rENT2 (GenBank accession No. AF015305), hENT1 (GenBank accession No. U81375), and hENT2 (GenBank accession No. AF029358) in the plasmid expression vectors pGEM-3Z (Promega Corp., Madison, WI; rCNT1), pGEM-T (Promega Corp.; rCNT2, rENT1, and rENT2), or pBluescript II KS(+) (Stratagene, La Jolla, CA; hCNT1, hCNT2, hENT1, and hENT2) were obtained as described previously (8,9,11,13, 23-25).
Expression of cDNAs Encoding Recombinant Transporters inXenopus Oocytes
Linearized plasmids were transcribed with T3 polymerase (hENT1 and hENT2), T7 polymerase (rCNT1, rCNT2, and hCNT1), or SP6 polymerase (rENT1 and rENT2) in the presence of the m7GpppG cap by use of the MEGAscript (Ambion, Austin, TX) transcription system. The remaining template was removed by digestion with deoxyribonuclease 1. Defolliculated oocytes (23) were microinjected with either 10 nL of water containing 10 ng RNA transcript or 10 nL of water alone. Xenopus care was in accordance with institutional guidelines.
Radioisotope Flux Studies
Uptake of gemcitabine and uridine was measured 3 days after microinjection by use of high-performance liquid chromatography-repurified [3H]gemcitabine (Eli Lilly and Co., Indianapolis, IN) or [14C]uridine (Amersham Life Science Inc., Piscataway, NJ) at concentrations of 2 µCi/mL and 1 µCi/mL, respectively. Flux measurements were performed at room temperature (20 °C) on groups of 10-12 oocytes in medium (0.2 mL) containing the following: 100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl, and 10 mM HEPES (pH 7.5). At the end of the incubation period, extracellular label was removed by six rapid washes in ice-cold medium. Individual oocytes were dissolved in 5% (wt/vol) sodium dodecyl sulfate for quantitation of oocyte-associated 3H or 14C by liquid scintillation counting (LS 6000 IC; Beckman Instruments, Inc., Fullerton, CA). Incubation periods for kinetic studies were selected to be within the initial linear phase of uptake curves to approximate zero-trans conditions and to measure initial rates of transport.
Results are presented as means with 95% confidence intervals (CIs) for 10-12 individual oocytes. Kinetic constants (apparent Km and Vmax) were determined by nonlinear regression analysis (ENZFITTER; Elsevier-Biosoft, Furguson, MO). Each experiment was performed at least twice on different batches of oocytes.
Measurements of hENT1-Induced Sodium Currents
Oocyte membrane currents were measured by use of a CA-1B oocyte clamp (Dagan Corp.,
Minneapolis, MN) in the whole-cell, two-electrode, voltage clamp mode. The microelectrodes
were filled with 3 M KCl and had resistances that ranged from 1 to 2.5 M
(mega
ohm). The CA-1B was interfaced to a computer via a Digidata 1200B A/D converter and
controlled by Axoscope software (Axon Instruments, Foster City, CA). Current signals were
filtered at 20 Hz (four-pole Bessel filter) and sampled at a sampling interval of 50 msec. For data
presentation, the current signals were further filtered at 0.5 Hz by use of pCLAMP software
(Axon Instruments). All electrophysiologic experiments were performed at room temperature.
The oocytes were penetrated and the membrane potential was observed for 15 minutes. If the
membrane potential was unstable or less than -30 mV, these oocytes were not used. For
measurements of hCNT1-generated currents, the oocyte membrane potential was clamped at
-50 mV. Oocytes were then perfused with medium of the same composition used for
radioisotope flux studies. For transport measurements, the medium was changed to one
containing substrate, either gemcitabine (100 µM) or uridine (100 µM). In experiments examining Na+ dependence, sodium in the medium was
replaced with equimolar choline.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionNotesReferences |
|---|
In Fig. 1,
we show a representative recombinant
expression experiment in Xenopus oocytes that measured the
uptake of gemcitabine or uridine (10 µM, 20 °C,
10-minute flux) in cells injected with water alone (control) or with
water that contained transcripts for either rCNT1, rCNT2, hCNT1, hCNT2,
eENT1, rENT2, hENT1, or hENT2. Gemcitabine, at the clinically
achievable concentration of 10 µM, was a permeant of six
of the eight recombinant transporters tested, with the greatest uptake
occurring in oocytes that produced recombinant rCNT1 and hCNT1.
Gemcitabine was not a permeant of either rCNT2 or hCNT2, transporters
that have been previously shown to be selective for purine nucleosides.
|
Subsequent experiments (Fig. 2)
investigated the concentration
dependence of gemcitabine transport by each of the four recombinant human transporters. Initial
rates of transport (influx) were determined by use of an incubation period of 1 minute for hCNT1,
2 minutes for hENT2, 5 minutes for hENT1, and 60 minutes for hCNT2. Under these conditions,
intracellular gemcitabine levels at the end of the incubation period were typically less than
15% of starting extracellular concentration, and uptake time courses were linear. To
facilitate comparison between the different recombinant transporters, influx values were calculated
as pmol/oocyte - minute-1. Kinetic parameters derived from the gemcitabine
transport data in Fig. 2 are shown in Table 1
together with previously determined apparent Km and Vmax values for uridine influx.
|
|
Influxes of gemcitabine mediated by hCNT1, hENT1, and hENT2 were saturable and conformed to Michaelis-Menten kinetics with apparent Km values of 24, 160, and 740 µM, respectively. Except for recombinant hENT2, which exhibited a relatively low affinity for gemcitabine, the Km values of the other recombinant transporters for gemcitabine influx were similar to the apparent Km values of these transporters for uridine influx (45, 240, and 200 µM, respectively, for hCNT1, hENT1, and hENT2) (8,9,11,13). In the case of hENT2 (uridine Km 200 µM), the recombinant transporter's relatively low affinity for gemcitabine (Km value of 740 mM) was compensated by a higher Vmax value, so that the Vmax : Km ratio for hENT2-mediated gemcitabine transport was 0.059 compared with 0.032 for uridine transport (Table 1
). Corresponding Vmax : Km ratios for gemcitabine and uridine influx by the
other recombinant transporters were in good agreement with the 10-µM-uptake
data shown in Fig. 1. In contrast to the other recombinant transporters, no
significant mediated transport of gemcitabine was detected for hCNT2, even at high permeant
concentrations (data not shown). Influx of gemcitabine in water-injected oocytes, like that in
hCNT2-transcript-injected cells, was approximately linear over the concentration ranges studied
(10 µM-5 mM, suggesting a lack of endogenous-mediated transport of
gemcitabine in oocytes. At a concentration of 1 mM, the magnitude of the gemcitabine
flux (0.091 pmol/oocyte - minute-1 [95% CI =
0.042-0.14]) was similar to that determined previously for uridine (0.102 pmol/oocyte -
minute-1 [95% CI = 0.010-0.104]) (24). Therefore, like uridine, gemcitabine has a limited ability to cross the lipid bilayer of
cell membranes by simple diffusion.
Finally, we used whole-cell recording by the two-electrode, voltage-clamp technique to
investigate the electrophysiology of hCNT1-mediated gemcitabine and uridine transport. In this
technique, one microelectrode clamps the oocyte membrane to a predetermined potential
(-50 mV in the case of the present experiments), while a second microelectrode delivers
current to maintain that potential. The current (in nano-Amperes [nA]) needed to hold
the membrane at the predetermined potential (-50 mV) is the measured parameter. As
shown for the representative experiment in Fig. 3, external application of
gemcitabine (100 µM) to oocytes producing recombinant hCNT1 induced an
inward current that returned to baseline on removal of the drug. The gemcitabine-induced current
was not seen in control, water-injected oocytes and was abolished when extracellular Na+ was replaced by choline. In three separate experiments with different oocytes, the range of
Na+ currents induced by 100 µM gemcitabine was 10-15 nA
compared with 55-68 nA for 100 µM uridine. This 3.7-fold difference in
gemcitabine-induced and uridine-induced Na+ currents was consistent with the
estimates for fluxes of 100 µM gemcitabine and uridine calculated from the hCNT1
kinetic parameters in Table 1 (4.7 and 17.9 pmol/oocyte - minute-1 for 100 µM gemcitabine and uridine, respectively). As was the
case for gemcitabine, the uridine current was not seen in control, water-injected oocytes and was
abolished when Na+ was replaced by choline. These studies demonstrated that
hCNT1 functions as a Na+/nucleoside co-transport protein and confirmed the
recombinant transporter's ability to transport gemcitabine.
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionNotesReferences |
|---|
Our results demonstrated that gemcitabine, like each of the physiologic nucleosides, diffuses only slowly through plasma membranes and requires protein-mediated transport for efficient cell entry. Mediated transport of gemcitabine by recombinant hCNT1, hENT1, and hENT2 was saturable and conformed to Michaelis-Menten kinetics with apparent Km values of 24, 160, and 740 µM, respectively. These characteristics were similar to those observed previously (26) in human cell lines that produced only a single nucleoside transporter in isolation, in which we also found that diffusional uptake of gemcitabine was slow and that the influx of gemcitabine mediated by recombinant hCNT1, hENT1, and hENT2 was saturable with apparent Km values of 18 , 330, and 830 µM, respectively.
Although there have been few prior reports of differences in permeant selectivity or kinetics between ENT1- and ENT2-mediated transport processes, we report here marked differences in kinetics of gemcitabine transport by recombinant hENT1 and hENT2 in Xenopus oocytes. hENT1 transports gemcitabine with high affinity and low capacity, while hENT2 transports gemcitabine with low affinity and high capacity. These differences may, in part, explain why bolus infusion of gemcitabine has clinical efficacy against epithelial cancers, while it causes only modest degrees of myelotoxicity. Hematopoietic progenitor cells are believed to possess predominantly hENT1 nucleoside transport capabilities based on the effectiveness of low concentrations of NBMPR in protecting such cells from the toxicity of cytotoxic nucleosides (27) and the enhancement of antifolate cytotoxicity to hematopoietic progenitor cells by nanomolar concentrations of NBMPR, which prevents nucleoside transporter-mediated thymidine uptake, thereby preventing thymidine rescue from inhibition of de novo synthesis of nucleotide precursors (28). In contrast, human epithelial cancer cell lines, including HeLa (26), MCF-7, and MDA-MB-435S (unpublished data), have substantial rates of hENT2-mediated gemcitabine uptake. These observations, taken together, suggest that human epithelial cancers may also possess hENT2 activity and might thereby take up gemcitabine more efficiently than hematopoietic precursors at the high (>50 µM) peak plasma concentrations achieved by 30-minute bolus gemcitabine (29).
Variation in the tissue and tumor distribution of the nucleoside transporters may also explain, in part, the different schedule-dependent toxic effects of bolus-infusion gemcitabine and prolonged-infusion gemcitabine. Gemcitabine is routinely administered on days 1, 8, and 15 of each 4-week cycle. When given as a 30-minute bolus infusion at doses of approximately 1200 mg/m2, peak plasma levels exceeding 50 µM are achieved after 15 minutes (29,30); however, on completion of the infusion, gemcitabine is rapidly eliminated from the serum, with a half-life of 8 minutes (29). This gemcitabine administration schedule produces mild and noncumulative myelotoxicity and minimal hepatotoxicity. However, when given by weekly continuous infusion at doses of 10 mg/m2 per minute for 120-280 minutes, median steady-state serum concentrations approach 25 µM, and cumulative myelotoxicity is dose limiting (31). Similarly, prolonging the duration of infusion to 1 hour of an otherwise standard gemcitabine dose causes hepatotoxicity as shown by elevated serum transaminases (32), possibly because of hCNT1-mediated accumulation of gemcitabine in hepatocytes. We have recently demonstrated the presence of hCNT1 transcript in human liver (13), and the rat homologue rCNT1 (33) has been identified functionally and molecularly in rat liver (34).
Although the accumulation of gemcitabine triphosphate by peripheral blood mononuclear cells and leukemic blasts is saturated by gemcitabine dose rates of 10 mg/m2 per minute in large part due to saturation of deoxycytidine kinase (35,36), the relative importance of plasma membrane transport and metabolism is not known for other tissues or for solid tumors. Molecular and immunologic probes will be required to define the tissue and tumor distribution of the human nucleoside transporters and may help guide clinical trials exploring rational scheduling and dosage regimens for the use of gemcitabine in cancer therapy (37).
In addition to using radioisotope flux measurements, we have investigated gemcitabine transportability by the two-electrode, voltage-clamp technique, a method that has not been used previously for the study of native or recombinant nucleoside transporters. These experiments provided a direct demonstration of the sodium dependence of hCNT1-mediated transport of gemcitabine and uridine. By confirming the protein-mediated nature of gemcitabine uptake by recombinant hCNT1 expressed in Xenopus oocytes, we have demonstrated that it is possible to use this technique to screen potential sodium-dependent permeants without the requirement for radiolabeling and to determine the relative efficiency of uptake among physiologic and therapeutic nucleosides. This result validates the utility of the Xenopus expression system as an appropriate method for kinetic determination of nucleoside drug transport by human nucleoside transport processes.
|
NOTES |
|---|
Supported by an Alberta Cancer Board New Investigators Award, the National Cancer Institute of Canada (NCIC), with funds from the Terry Fox Foundation, and by the Alberta Cancer Board and the Alberta Heritage Foundation for Medical Research (AHFMR). C. E. Cass is a Terry Fox Cancer Research Scientist of the NCIC. J. D. Young is a Heritage Medical Scientist of the AHFMR.
We thank Eli Lilly and Co. for the gift of gemcitabine and 3H-gemcitabine.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionNotesReferences |
|---|
1 Allegra CJ, Grem JL. Antimetabolites. In: DeVita VT Jr, editor. Cancer: principles and practice of oncology. Philadelphia (PA): Lippincott-Raven; 1997. p. 490-8.
2
Heinemann V, Xu YZ, Chubb S, Sen A, Hertel LW, Grindey GB,
et al. Cellular elimination of 2',2'-difluorodeoxycytidine 5'-triphosphate: a
mechanism of self-potentiation. Cancer Res 1992;52:533-9.
3 Baker CH, Banzon J, Bollinger JM, Stubbe J, Samano V, Robins MJ, et al. 2'-Deoxy-2'-methylenecytidine and 2'-deoxy-2',2'-difluorocytidine 5'-diphosphates: potent mechanism-based inhibitors of ribonucleotide reductase. J Med Chem 1991;34:1879-84.[CrossRef][Web of Science][Medline]
4 Plunkett W, Huang P, Xu YZ, Heinemann V, Grunewald R, Gandhi V. Gemcitabine: metabolism, mechanisms of action, and self-potentiation. Semin Oncol 1995;22(Suppl 11):3-10.[Web of Science][Medline]
5 Mackey JR, Baldwin SA, Young JD, Cass CE. The role of nucleoside transport in anticancer drug resistance. Drug Resistance Updates 1998;1:310-24.[CrossRef][Medline]
6 Griffith DA, Jarvis SM. Nucleoside and nucleobase transport systems of mammalian cells. Biochim Biophys Acta 1996;1286:153-81.[Medline]
7 Wang J, Schaner ME, Thomassen S, Su SF, Piquette-Miller M, Giacomini KM. Functional and molecular characteristics of Na(+)-dependent nucleoside transporters. Pharm Res 1997;14:1524-32.[CrossRef][Web of Science][Medline]
8 Griffiths M, Beaumont N, Yao SY, Sundaram M, Boumah CE, Davies A, et al. Cloning of a human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs. Nat Med 1997;3:89-93.[CrossRef][Web of Science][Medline]cancerlit;97140266
9
Ritzel MW, Yao SY, Huang MY, Elliott JF, Cass CE, Young JD.
Molecular cloning and functional expression of cDNAs encoding a human Na+-nucleoside cotransporter (hCNT1). Am J Physiol 1997;272:C707-14.
10
Crawford CR, Patel DH, Naeve C, Belt JA. Cloning of the
human equilibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive nucleoside
transporter ei by functional expression in a transport-deficient cell line. J Biol Chem 1998;273:5288-93.
11 Griffiths M, Yao SY, Abidi F, Phillips SE, Cass CE, Young JD, et al. Molecular cloning and characterization of a nitrobenzylthioinosine-insensitive (ei) equilibrative nucleoside transporter from human placenta. Biochem J 1997;328:739-43.
12 Wang J, Su SF, Dresser MJ, Schnaner ME, Washington CB, Giacomini KM. Na(+)-dependent purine nucleoside transporter from human kidney: cloning and functional characterization. Am J Physiol 1997;273:F1058-65.
13 Ritzel MW, Yao SY, Ng AM, Mackey JR, Cass CE, Young JD. Molecular cloning, functional expression and chromosomal localization of a cDNA encoding a human Na+/nucleoside cotransporter (hCNT2) selective for purine nucleosides and uridine. Mol Membr Biol 1999;15:203-11.
14 Gutierrez MM, Brett CM, Ott RJ, Hui AC, Giacomini KM. Nucleoside transport in brush border membrane vesicles from human kidney. Biochim Biophys Acta 1992;1105:1-9.[Medline]
15
Patil SD, Unadkat JD. Sodium-dependent nucleoside transport in
the human intestinal brush-border membrane. Am J Physiol 1997;272:G1314-20.
16 Chandrasena G, Giltay R, Patil SD, Bakken A, Unadkat JD. Functional expression of human intestinal Na+-dependent and Na+-independent nucleoside transporters in Xenopus laevis oocytes. Biochem Pharmacol 1997;53:1909-18.[CrossRef][Web of Science][Medline]
17 Washington CB, Giacomini KM, Brett CM. Nucleoside transport in isolated human and rhesus choroid plexus tissue slices. Pharm Res 1998;15:1145-7.[CrossRef][Web of Science][Medline]
18 Roovers KI, Meckling-Gill KA. Characterization of equilibrative and concentrative Na+-dependent (cif) nucleoside transport in acute promyelocytic leukemia NB4 cells. J Cell Physiol 1996;166:593-600.[CrossRef][Web of Science][Medline]cancerlit;96177178
19
Flanagan SA, Meckling-Gill KA. Characterization of a novel Na+-dependent, guanosine-specific, nitrobenzylthioinosine-sensitive transporter in
acute promyelocytic leukemia cells. J Biol Chem 1997;272:18026-32.
20 Belt JA, Harper EH, Byl JA, Noel LD. Sodium-dependent nucleoside transport inhuman myeloid leukemic cell lines and freshly isolated myeloblasts [abstract]. Proc Am Assoc Cancer Res 1992;33:20.
21 Belt JA, Marina NM, Phelps DA, Crawford CR. Nucleoside transport in normal and neoplastic cells. Adv Enzyme Regul 1993;33:235-52.[CrossRef][Web of Science][Medline]cancerlit;93362560
22
Che M, Ortiz DF, Arias IM. Primary structure and functional
expression of a cDNA encoding the bile canalicular, purine-specific Na(+)-nucleoside
cotransporter. J Biol Chem 1995;270:13596-9.
23
Huang QQ, Yao SY, Ritzel MW, Paterson AR, Cass CE, Young
JD. Cloning and functional expression of a complementary DNA encoding a mammalian
nucleoside transport protein. J Biol Chem 1994;269:17757-60.
24 Yao SY, Ng AM, Ritzel MW, Cass CE, Young JD. Transport of adenosine by recombinant purine- and pyrimidine-selective sodium/nucleoside cotransporters from rat jejunum expressed in Xenopus laevis oocytes. Mol Pharmacol 1996;50:1529-35.[Abstract]
25
Yao SY, Ng AM, Muzyka WR, Griffiths M, Cass CE, Baldwin
SA, et al. Molecular cloning and functional characterization of nitrobenzylthioinosine
(NBMPR)-sensitive (es) and NBMPR-insensitive (ei) equilibrative nucleoside transporter proteins
(rENT1 and rENT2) from rat tissues. J Biol Chem 1997;272:28423-30.
26
Mackey JR, Mani RS, Selner M, Mowles D, Young JD, Belt JA,
et al. Functional nucleoside transporters are required for gemcitabine influx and manifestation of
toxicity in cancer cell lines. Cancer Res 1998;58:4349-57.
27
Cass CE, King KM, Montano JT, Janowska-Wieczorek A. A
comparison of the abilities of nitrobenzylthioinosine, dilazep, and dipyridamole to protect human
hematopoietic cells from 7-deazaadenosine (tubercidin). Cancer Res 1992;52:5879-86.
28
Allay JA, Spencer HT, Wilkinson SL, Belt JA, Blakley RL,
Sorrentino BP. Sensitization of hematopoietic stem and progenitor cells to trimetrexate using
nucleoside transport inhibitors. Blood 1997;90:3546-54.
29 Abbruzzese JL, Grunewald R, Weeks EA, Gravel D, Adams T, Nowak B, et al. A phase I clinical, plasma, and cellular pharmacology study of gemcitabine. J Clin Oncol 1991;9:491-8.[Abstract]cancerlit;91154854
30 Storniolo AM, Allerheiligen SR, Pearce HL. Preclinical, pharmacologic, and phase I studies of gemcitabine. Semin Oncol 1997;24(Suppl 7):S7-2-S7-7.
31
Touroutoglou N, Gravel D, Raber MN, Plunkett W, Abbruzzese
JL. Clinical results of a pharmacodynamically-based strategy for higher dosing of gemcitabine in
patients with solid tumors. Ann Oncol 1998;9:1003-8.
32 Pollera CF, Ceribelli A, Crecco M, Oliva C, Calabresi F. Prolonged infusion gemcitabine: a clinical phase I study at low- (300 mg/m2) and high-dose (875 mg/m2) levels. Invest New Drugs 1997;15:115-21.[CrossRef][Web of Science][Medline]cancerlit;97363998
33 Fang X, Parkinson FE, Mowles DA, Young JD, Cass CE. Functional characterization of a recombinant sodium-dependent nucleoside transporter with selectivity for pyrimidine nucleosides (cNT1rat) by transient expression in cultured mammalian cells. Biochem J 1996;317:457-65.cancerlit;96313797
34 Felipe A, Ferrer-Martinez A, Casado FJ, Pastor-Anglada M. Expression of sodium-dependent purine nucleoside carrier (SPNT) mRNA correlates with nucleoside transport activity in rat liver. Biochem Biophys Res Commun 1997;233:572-5.[CrossRef][Web of Science][Medline]
35 Grunewald R, Abbruzzese JL, Tarassoff P, Plunket W. Saturation of 2',2'-difluorodeoxycytidine 5'-triphosphate accumulation by mononuclear cells during a phase I trial of gemcitabine. Cancer Chemother Pharmacol 1991;27:258-62.[CrossRef][Web of Science][Medline]cancerlit;91152814
36
Grunewald R, Kantarjian H, Keating MJ, Abbruzzese J,
Tarassoff P, Plunkett W. Pharmacologically directed design of the dose rate and schedule of
2',2'-difluorodeoxycytidine (Gemcitabine) administration in leukemia. Cancer Res 1990;50:6823-6.
37 Tempero M, Plunkett W, Ruiz van Haperen V, Hainsworth J, Hochster H, Lenzi R, et al. Randomized phase II trial of dose intense gemcitabine by standard influsion versus fixed dose rate in metastatic pancreatic adenocarcinoma [abstract]. Proc ASCO 1999;18:273.
Manuscript received February 23, 1999; revised August 18, 1999; accepted September 3, 1999.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  R. Marechal, J. R. Mackey, R. Lai, P. Demetter, M. Peeters, M. Polus, C. E. Cass, J. Young, I. Salmon, J. Deviere, et al. Human Equilibrative Nucleoside Transporter 1 and Human Concentrative Nucleoside Transporter 3 Predict Survival after Adjuvant Gemcitabine Therapy in Resected Pancreatic Adenocarcinoma Clin. Cancer Res., April 15, 2009; 15(8): 2913 - 2919. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-H. Tan, S.-C. Lee, B.-C. Goh, and J. Wong Pharmacogenetics in Breast Cancer Therapy Clin. Cancer Res., December 15, 2008; 14(24): 8027 - 8041. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Veltkamp, D. Pluim, O. van Tellingen, J. H. Beijnen, and J. H. M. Schellens Extensive Metabolism and Hepatic Accumulation of Gemcitabine After Multiple Oral and Intravenous Administration in Mice Drug Metab. Dispos., August 1, 2008; 36(8): 1606 - 1615. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Veltkamp, D. Pluim, M. A.J. van Eijndhoven, M. J. Bolijn, F. H.G. Ong, R. Govindarajan, J. D. Unadkat, J. H. Beijnen, and J. H.M. Schellens New insights into the pharmacology and cytotoxicity of gemcitabine and 2',2'-difluorodeoxyuridine Mol. Cancer Ther., August 1, 2008; 7(8): 2415 - 2425. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Veltkamp, R. S. Jansen, S. Callies, D. Pluim, C. M. Visseren-Grul, H. Rosing, S. Kloeker-Rhoades, V. A.M. Andre, J. H. Beijnen, C. A. Slapak, et al. Oral Administration of Gemcitabine in Patients with Refractory Tumors: A Clinical and Pharmacologic Study Clin. Cancer Res., June 1, 2008; 14(11): 3477 - 3486. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Fowler, J. A. Brown, K. A. Johnson, and Z. Suo Kinetic Investigation of the Inhibitory Effect of Gemcitabine on DNA Polymerization Catalyzed by Human Mitochondrial DNA Polymerase J. Biol. Chem., May 30, 2008; 283(22): 15339 - 15348. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Santini, G. Perrone, B. Vincenzi, R. Lai, C. Cass, R. Alloni, C. Rabitti, A. Antinori, F. Vecchio, S. Morini, et al. Human equilibrative nucleoside transporter 1 (hENT1) protein is associated with short survival in resected ampullary cancer Ann. Onc., April 1, 2008; 19(4): 724 - 728. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Veltkamp, J. H. Beijnen, and J. H.M. Schellens Prolonged Versus Standard Gemcitabine Infusion: Translation of Molecular Pharmacology to New Treatment Strategy Oncologist, March 1, 2008; 13(3): 261 - 276. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Sugiyama, N. Kaniwa, S.-R. Kim, R. Kikura-Hanajiri, R. Hasegawa, K. Maekawa, Y. Saito, S. Ozawa, J.-i. Sawada, N. Kamatani, et al. Pharmacokinetics of Gemcitabine in Japanese Cancer Patients: The Impact of a Cytidine Deaminase Polymorphism J. Clin. Oncol., January 1, 2007; 25(1): 32 - 42. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Pauwels, A. E.C. Korst, F. Lardon, and J. B. Vermorken Combined Modality Therapy of Gemcitabine and Radiation Oncologist, January 1, 2005; 10(1): 34 - 51. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Spratlin, R. Sangha, D. Glubrecht, L. Dabbagh, J. D. Young, C. Dumontet, C. Cass, R. Lai, and J. R. Mackey The Absence of Human Equilibrative Nucleoside Transporter 1 Is Associated with Reduced Survival in Patients With Gemcitabine-Treated Pancreas Adenocarcinoma Clin. Cancer Res., October 15, 2004; 10(20): 6956 - 6961. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Smith, A. M. L. Ng, S. Y. M. Yao, K. A. Labedz, E. E. Knaus, L. I. Wiebe, C. E. Cass, S. A. Baldwin, X.-Z. Chen, E. Karpinski, et al. Electrophysiological characterization of a recombinant human Na+-coupled nucleoside transporter (hCNT1) produced in Xenopus oocytes J. Physiol., August 1, 2004; 558(3): 807 - 823. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Duflot, B. Riera, S. Fernandez-Veledo, V. Casado, R. I. Norman, F. J. Casado, C. Lluis, R. Franco, and M. Pastor-Anglada ATP-Sensitive K+ Channels Regulate the Concentrative Adenosine Transporter CNT2 following Activation by A1 Adenosine Receptors Mol. Cell. Biol., April 1, 2004; 24(7): 2710 - 2719. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. M. Larrayoz, F. J. Casado, M. Pastor-Anglada, and M. P. Lostao Electrophysiological Characterization of the Human Na+/Nucleoside Cotransporter 1 (hCNT1) and Role of Adenosine on hCNT1 Function J. Biol. Chem., March 5, 2004; 279(10): 8999 - 9007. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Zhang, F. Visser, M. F. Vickers, T. Lang, M. J. Robins, L. P.C. Nielsen, I. Nowak, S. A. Baldwin, J. D. Young, and C. E. Cass Uridine Binding Motifs of Human Concentrative Nucleoside Transporters 1 and 3 Produced in Saccharomyces cerevisiae Mol. Pharmacol., December 1, 2003; 64(6): 1512 - 1520. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Garcia-Manteiga, M. Molina-Arcas, F. J. Casado, A. Mazo, and M. Pastor-Anglada Nucleoside Transporter Profiles in Human Pancreatic Cancer Cells: Role of hCNT1 in 2',2'-Difluorodeoxycytidine- Induced Cytotoxicity Clin. Cancer Res., October 15, 2003; 9(13): 5000 - 5008. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Huang, Y. Wang, M. Collins, J. J. Gu, B. S. Mitchell, and L. M. Graves Inhibition of Nucleoside Transport by p38 MAPK Inhibitors J. Biol. Chem., August 2, 2002; 277(32): 28364 - 28367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Y. M. Yao, A. M. L. Ng, M. F. Vickers, M. Sundaram, C. E. Cass, S. A. Baldwin, and J. D. Young Functional and Molecular Characterization of Nucleobase Transport by Recombinant Human and Rat Equilibrative Nucleoside Transporters 1 and 2. CHIMERIC CONSTRUCTS REVEAL A ROLE FOR THE ENT2 HELIX 5-6 REGION IN NUCLEOBASE TRANSLOCATION J. Biol. Chem., July 5, 2002; 277(28): 24938 - 24948. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Mackey, L. L. Jennings, M. L. Clarke, C. L. Santos, L. Dabbagh, M. Vsianska, S. L. Koski, R. W. Coupland, S. A. Baldwin, J. D. Young, et al. Immunohistochemical Variation of Human Equilibrative Nucleoside Transporter 1 Protein in Primary Breast Cancers Clin. Cancer Res., January 1, 2002; 8(1): 110 - 116. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Gourdeau, M. L. Clarke, F. Ouellet, D. Mowles, M. Selner, A. Richard, N. Lee, J. R. Mackey, J. D. Young, J. Jolivet, et al. Mechanisms of Uptake and Resistance to Troxacitabine, a Novel Deoxycytidine Nucleoside Analogue, in Human Leukemic and Solid Tumor Cell Lines Cancer Res., October 1, 2001; 61(19): 7217 - 7224. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. W. Blackstock, H. Lightfoot, L. D. Case, J. E. Tepper, S. K. Mukherji, B. S. Mitchell, S. G. Swarts, and S. M. Hess Tumor Uptake and Elimination of 2',2'-Difluoro-2'-deoxycytidine (Gemcitabine) after Deoxycytidine Kinase Gene Transfer: Correlation with in Vivo Tumor Response Clin. Cancer Res., October 1, 2001; 7(10): 3263 - 3268. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. F. Mata, J. M. Garcia-Manteiga, M. P. Lostao, S. Fernandez-Veledo, E. Guillen-Gomez, I. M. Larrayoz, J. Lloberas, F. J. Casado, and M. Pastor-Anglada Role of the Human Concentrative Nucleoside Transporter (hCNT1) In the Cytotoxic Action of 5[Prime]-Deoxy-5-fluorouridine, an Active Intermediate Metabolite of Capecitabine, a Novel Oral Anticancer Drug Mol. Pharmacol., June 1, 2001; 59(6): 1542 - 1548. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Dresser, K. M. Gerstin, A. T. Gray, D. D. F. Loo, and K. M. Giacomini Electrophysiological Analysis of the Substrate Selectivity of a Sodium-Coupled Nucleoside Transporter (rCNT1) Expressed in Xenopus laevis Oocytes Drug Metab. Dispos., September 1, 2000; 28(9): 1135 - 1140. [Abstract] [Full Text]
|
|
|
|
|
|
M. W. L. Ritzel, A. M. L. Ng, S. Y. M. Yao, K. Graham, S. K. Loewen, K. M. Smith, R. G. Ritzel, D. A. Mowles, P. Carpenter, X.-Z. Chen, et al. Molecular Identification and Characterization of Novel Human and Mouse Concentrative Na+-Nucleoside Cotransporter Proteins (hCNT3 and mCNT3) Broadly Selective for Purine and Pyrimidine Nucleosides (System cib) J. Biol. Chem., January 19, 2001; 276(4): 2914 - 2927. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Soler, R. Valdes, J. Garcia-Manteiga, J. Xaus, M. Comalada, F. J. Casado, M. Modolell, B. Nicholson, C. MacLeod, A. Felipe, et al. Lipopolysaccharide-induced Apoptosis of Macrophages Determines the Up-regulation of Concentrative Nucleoside Transporters Cnt1 and Cnt2 through Tumor Necrosis Factor-alpha -dependent and -independent Mechanisms J. Biol. Chem., August 3, 2001; 276(32): 30043 - 30049. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||