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).
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ABSTRACT |
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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.
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RESULTS |
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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.
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DISCUSSION |
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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.
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NOTES |
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We thank Eli Lilly and Co. for the gift of gemcitabine and 3H-gemcitabine.
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Manuscript received February 23, 1999; revised August 18, 1999; accepted September 3, 1999.
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