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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 [[sup.3]H]gemcitabine and [[sup.14]C] 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 [K.sub.m] values of 24, 160, and 740 [micro]M/, 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.sup.+]/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. [J Natl Cancer Inst 1999;91:1876-81]

3′-Deoxy-3′-fluorothymidine (FLT) is a positron emission tomography (PET) tracer used to identify proliferating tumor cells. The purpose of this study was to characterize FLT transport by human nucleoside transporters (hNTs) and to determine the role of hNTs for FLT uptake in various human cancer cell lines. FLT binding to hNTs was monitored by the inhibitory effects of FLT on [3H]uridine uptake in yeast cells producing recombinant hNT proteins. hCNT1 displayed the lowest FLT Kivalue for inhibition of [3H]uridine uptake, followed by hCNT3, hENT2, hENT1, and hCNT2. [3H]FLT was efficiently transported in Xenopus laevisoocytes individually producing hENT1, hENT2, hCNT1, or hCNT3. [3H]FLT uptake in MCF-7, A549, U251, A498, MIA PaCa-2, and Capan-2 cells was inhibited at least 50% by the hENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside (NBMPR). According to results of real-time polymerase chain reactions, hENT1 and hENT2 had the most abundant hNT transcripts in all cell lines. Cell lines also underwent 1) [3H]NBMPR equilibrium binding assays with or without 5-S-{2-(1-[(fluorescein-5-yl)thioureido]hexanamido)ethyl}-6-N-(4-nitrobenzyl)-5-thioadenosine, a membrane-impermeable NBMPR analog, to determine plasma membrane hENT1 levels, and 2) dose-response NBMPR inhibition of [3H]FLT uptake. MCF-7, A549, and Capan-2 cells displayed NBMPR IC50values that were smaller or equal to NBMPR Kdvalues, suggesting that 50% inhibition of hENT1 reduced [3H]FLT uptake by at least 50%. A strong correlation between extracellular NBMPR binding sites/cell and [3H]FLT uptake was observed for all cell lines except MIA PaCa-2. These data suggest that plasma membrane hNTs (especially hENT1) are important determinants of cellular FLT uptake.

hCNT1 and hCNT2 mediate concentrative (Na+-linked) cellular uptake of nucleosides and nucleoside drugs by human cells and tissues. The two proteins (650 and 658 residues, 71 kDa) are 72% identical in sequence and contain 13 putative transmembrane helices (TMs). When produced in Xenopusoocytes, recombinant hCNT1 is selective for pyrimidine nucleosides (system cit), whereas hCNT2 is selective for purine nucleosides (system cif). Both transport uridine. We have used (i) chimeric constructs between hCNT1 and hCNT2, (ii) sequence comparisons with a newly identified broad specificity concentrative nucleoside transporter (system cib) from Eptatretus stouti, the Pacific hagfish (hfCNT), and (iii) site-directed mutagenesis of hCNT1 to identify two sets of adjacent residues in TMs 7 and 8 of hCNT1 (Ser319/Gln320and Ser353/Leu354) that, when converted to the corresponding residues in hCNT2 (Gly313/Met314and Thr347/Val348), changed the specificity of the transporter from citto cif. Mutation of Ser319in TM 7 of hCNT1 to Gly enabled transport of purine nucleosides, whereas concurrent mutation of Gln320to Met (which had no effect on its own) augmented this transport. The additional mutation of Ser353to Thr in TM 8 converted hCNT1/S319G/Q320M, from cibto cif, but with relatively low adenosine transport activity. Additional mutation of Leu354to Val (which had no effect on its own) increased the adenosine transport capability of hCNT1/S319G/Q320M/S353T, producing a full cif-type transporter phenotype. On its own, the S353T mutation converted hCNT1 into a transporter with novel uridine-selective transport properties. Helix modeling of hCNT1 placed Ser319(TM 7) and Ser353(TM 8) within the putative substrate translocation channel, whereas Gln320(TM 7) and Leu354(TM 8) may exert their effects through altered helix packing.