Electrogenic uptake of nucleosides and nucleoside-derived drugs by the human nucleoside transporter 1 (hCNT1) expressed in Xenopus laevis oocytes.

The concentrative pyrimidine-preferring nucleoside transporter 1 (hCNT1), cloned from human fetal liver, was expressed in Xenopus laevis oocytes. Using the two-electrode voltage-clamp technique, it is shown that translocation of nucleosides by this transporter generates sodium inward currents. Membrane hyperpolarization (from -50 to -150 mV) did not affect the K(0.5) for uridine, although it increased the transport current approximately 3-fold. Gemcitabine (a pyrimidine nucleoside-derived drug) but not fludarabine (a purine nucleoside-derived drug) induced currents in oocytes expressing the hCNT1 transporter. The K(0.5) value for gemcitabine at -50 mV membrane potential was lower than that for natural substrates, although this drug induced a lower current than uridine and cytidine, thus suggesting that the affinity binding of the drug transporter is high but that translocation occurs more slowly. The analysis of the currents generated by the hCNT1-mediated transport of nucleoside-derived drugs used in anticancer and antiviral therapies will be useful in the characterization of the pharmacological profile of this family of drug transporters and will allow rapid screening for uptake of newly developed nucleoside-derived drugs.

Nucleoside transporters in absorptive epithelia.

There are two families of nucleoside transporters, concentrative (termed CNTs) and equilibrative (called ENTs). The members of both families mediate the transmembrane transport of natural nucleosides and some drugs whose structure is based on nucleosides. CNT transporters show a high affinity for their natural substrates (with Km values in the low micromolar range) and are substrate selective. In contrast, ENT transporters show lower affinity and are more permissive regarding the substrates they accept. Both types of transporters are tightly regulated in all cell types studied so far, both by endocrine and growth factors and by substrate availability. The degree of cell differentiation and the proliferation status of a cell also affect the pattern of expressed transporters. Although the presence of both types of transporters in the cells of absortive epithelia suggested the possibility of a transepithelial flux of nucleosides, their exact localization in the different plasma membrane domains of epithelial cells had not been demonstrated until recently. Concentrative transporters are found in the apical membrane while equlibrative transporters are located in the basolateral membrane, thus strengthening the hypothesis of a transepithelial flux of nucleosides.

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.

We attempt to identify the plasma membrane transporter involved in the uptake of 5'-deoxy-5-fluorouridine (5'-DFUR), an intermediate metabolite of capecitabine. This novel oral fluoropyrimidine is used in cancer treatments and is a direct precursor of the cytostatic agent 5'-fluorouracil. We also examine the role of the transporter in 5'-DFUR cytotoxicity. The human concentrative nucleoside transporter (hCNT1) was cloned from human fetal liver and expressed in Xenopus laevis oocytes. The two-electrode voltage-clamp technique was used to demonstrate that 5'-DFUR, but not capecitabine or 5'-FU, is an hCNT1 substrate. Then, hCNT1 was heterologously expressed in the mammalian cell line Chinese hamster ovary-K1. Functional expression was demonstrated by monitoring transport of radiolabeled substrates and by using a monospecific polyclonal antibody generated against the transporter. hCNT1-expressing cells were more sensitive to 5'-DFUR than vector-transfected or wild-type cells. The sensitivity of the three cell types to other agents such as cisplatin or 5'-FU was identical. In conclusion, this study shows that 1) the pharmacological profile of a nucleoside transporter can be determined by an electrophysiological approach; 2) the hCNT1 transporter is involved in 5'-DFUR uptake; and 3) hCNT1 expression may increase cell sensitivity to 5'-DFUR treatment. This study also reports for the first time the generation of an antibody against hCNT1, which may be useful in the elucidation of the relationship between hCNT1 expression and tumor response to capecitabine treatment.