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4'-Thio-beta-D-arabinofuranosyl cytosine (TaraC) is in phase I development for treatment of cancer. In human equilibrative nucleoside transporter (hENT) 1-containing CEM cells, initial rates of uptake (10 microM; picomoles per microliter of cell water per second) of [3H]TaraC and [3H]1-beta-D-arabinofuranosyl cytosine (araC) were low (0.007 +/- 003 and 0.034 +/- 0.003, respectively) compared with that of [3H]uridine (0.317 +/- 0.048), a highactivity hENT1 permeant. In hENT1- and hENT2-containing HeLa cells, initial rates of uptake (10 microM; picomoles per cell per second) of [3H]TaraC, [3H]araC, and [3H]deoxycytidine were low (0.30 +/- 0.003, 0.42 +/- 0.03, and 0.51 +/- 0.11, respectively) and mediated primarily by hENT1 (approximately 74, approximately 65, and approximately 61%, respectively). In HeLa cells with recombinant human concentrative nucleoside transporter (hCNT) 1 or hCNT3 and pharmacologically blocked hENT1 and hENT2, transport of 10 microM[3H]TaraC and [3H]araC was not detected. The apparent affinities of recombinant transporters (produced in yeast) for a panel of cytosine-containing nucleosides yielded results that were consistent with the observed low-permeant activities of TaraC and araC for hENT1/2 and negligible permeant activities for hCNT1/2/3. During prolonged drug exposures of CEM cells with hENT1 activity, araC was more cytotoxic than TaraC, whereas coexposures with nitrobenzylthioinosine (to pharmacologically block hENT1) yielded identical cytotoxicities for araC and TaraC. The introduction by gene transfer of hENT2 and hCNT1 activities, respectively, into nucleoside transport-defective CEM cells increased sensitivity to both drugs moderately and slightly. These results demonstrated that nucleoside transport capacity (primarily via hENT1, to a lesser extent by hENT2 and possibly by hCNT1) is a determinant of pharmacological activity of both drugs.

Human concentrative nucleoside transporters 1, 2, and 3 (hCNT1, hCNT2, and hCNT3) exhibit different functional characteristics, and a better understanding of their permeant selectivities is critical for development of nucleoside analog drugs with optimal pharmacokinetic properties. In this study, the sensitivity of a high-throughput yeast expression system used previously for hCNT1 and hCNT3 was improved and used to characterize determinants for interaction of uridine (Urd) with hCNT2. The observed changes of binding energy between hCNT2 and different Urd analogs suggested that it interacts with C(3′)-OH, C(5′)-OH, and N(3)-H of Urd. The C(2′) and C(5) regions of Urd played minor but significant roles for Urd-hCNT2 binding, possibly through Van der Waals interactions. Because the yeast assay only provided information about potential transportability, the permeant selectivities of recombinant hCNT1, hCNT2, and hCNT3 produced in Xenopus laevis oocytes were investigated using a two-electrode voltage clamp assay. hCNT1-mediated transport was sensitive to modifications of the N(3), C(3′), and C(5′) positions of Urd. hCNT2 showed some tolerance for transporting Urd analogs with C(2′) or C(5) modifications, little tolerance for N(3) modifications, and no tolerance for any modifications at C(3′) or C(5′) of Urd. Although hCNT3 was sensitive to C(3′) modifications, it transported a broad range of variously substituted Urd analogs. The transportability profiles identified in this study, which reflected the binding profiles well, should prove useful in the development of anticancer and antiviral therapies with nucleoside drugs that are permeants of members of the hCNT protein family.

Human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) differ functionally in that hENT2 generally displays lower affinity for its nucleoside permeants and is less sensitive to inhibition by the coronary vasodilators dilazep and dipyridamole. In previous work, we demonstrated that mutation of residues 33 (Met versus Ile) of hENT1 and hENT2 altered sensitivity to dilazep and dipyridamole and that the hENT2 mutant (I33M) displayed a K(m) value for uridine that was lower than that of hENT2 and similar to that of hENT1 (J Biol Chem 277:395-401, 2002). In this study, we report results of an in-depth investigation of the role of residue 33 in hENT2. We found that hENT2-I33M displayed decreased K(m) values for both pyrimidine and purine nucleosides and increased V(max) values for purine nucleosides. Cys or Ser at position 33 had similar effects on the kinetic parameters of hENT2 as Met, indicating that hydrophobic (Met and Cys) or hydrogen-bonding energy (Ser) contributed to permeant binding by these residues. hENT2-I33M and I33C displayed increased sensitivities to dipyridamole compared with wild-type hENT2, hENT2-I33A, and hENT2-I33S, suggesting interaction of the sulfur atom of Met and Cys with aromatic moieties on dipyridamole. hENT2-I33C was inhibited by the membrane-impermeant sulfhydryl reactive reagent p-chloromercuribenzyl sulfonate, and uridine, adenosine, and dipyridamole protected against inhibition. Our results indicated that residue 33 resides in an extracellular domain as predicted by the current hENT2 topology model and suggested that it is a functionally important component of both the permeant and dipyridamole binding sites.

Benzamide riboside (BR) and tiazofurin (TR) are converted to analogs of NAD that inhibit IMP dehydrogenase (IMPDH), resulting in cellular depletion of GTP and dGTP and inhibition of proliferation. The current work was undertaken to identify the human nucleoside transporters involved in cellular uptake of BR and TR and to evaluate their role in cytotoxicity. Transportability was examined in Xenopus laevis oocytes and Saccharomyces cerevisiae that produced individual recombinant human concentrative nucleoside transporter (CNT) and equilibrative nucleoside transporter (ENT) types (hENT1, hENT2, hCNT1, hCNT2, or hCNT3). TR was a better permeant than BR with a rank order of transportability in oocytes of hCNT3hENT1hENT2hCNT2hCNT1. The concentration dependence of inhibition of [(3)H]uridine transport in S. cerevisiae by TR exhibited lower K(i) values than BR: hCNT3 (5.4 versus 226 microM), hENT2 (16 versus 271 microM), hENT1 (57 versus 168 microM), and hCNT1 (221 versus 220 microM). In cytotoxicity experiments, BR was more cytotoxic than TR to cells that were either nucleoside transport-defective or -competent, and transport-competent cells were more sensitive to both drugs. Exposure to nitrobenzylmercaptopurine ribonucleoside conferred resistance to BR and TR cytotoxicity to hENT1-containing CEM cells, thereby demonstrating the importance of transport capacity for manifestation of cytoxicity. A breast cancer cell line with mutant p53 exhibited 9-fold higher sensitivity to BR than the otherwise similar cell line with wild-type p53, suggesting that cells with mutant p53 may be potential targets for IMPDH inhibitors. Further studies are warranted to determine whether this finding can be generalized to other cell types.

Pharmacologically important drugs were examined as potential inhibitors or permeants of human concentrative nucleoside transporters 1 (hCNT1)- and 2 (hCNT2)-producing stable transfectants by assessing their abilities to inhibit uridine transport. hCNT1 exhibited high affinities for uridine analogs (5-fluorouridine, 2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, and 5-fluoro-5'-deoxyuridine) with K(i) values of 22 to 33 microM, whereas hCNT2 exhibited moderate affinities for 5-fluoro-2'-deoxyuridine, high affinities for 2'-deoxyuridine and 5-fluorouridine, and low affinity for 5-fluoro-5'-deoxyuridine. The uridine analogs were transported at 2-fold higher rates (at 10 microM) by hCNT1 than by hCNT2. Enantiomeric configuration and the 3'-hydroxyl group of the ribose ring were important determinants for interaction with hCNTs, whereas the 2'-hydroxyl group was less important. Both transporters bound N(6)-(p-aminobenzyl)adenosine with affinities similar to those of adenosine (K(i) = 28-39 microM). Other adenosine receptor ligands, including caffeine, bound better to hCNT1 than to hCNT2 (K(i) = 46 versus 103 microM, respectively), whereas 2-chloroadenosine bound better to hCNT2 than to hCNT1 (K(i) = 37 and 101 microM, respectively). There was a greater than 3-fold difference in binding affinities between hCNT1 and hCNT2 for nicotine (K(i) = 63 versus 227 microM). However, direct measurements of nicotine and caffeine uptake rates (10 microM) failed to demonstrate mediated uptake by either transporter. Although hCNT1 bound several adenosine analogs relatively well, it did not transport 2-chloro-2'-deoxyadenosine (cladribine) or 2-fluoro-9-beta-d-arabinofuranosyladenine (fludarabine), whereas hCNT2 transported both, albeit with low activities. The results indicated that although hCNT1 and hCNT2 possess some overlap in transport of several uridine and adenosine analogs, they also exhibit distinct differences in capacity to interact with some adenosine receptor ligands, adenosine-based drugs, and nicotine.

An extensive series of structural analogs of uridine that differed in substituents in the sugar and/or base moieties were subjected to inhibitor-sensitivity assays in a yeast expression system to define uridine structural determinants for inhibitors of human concentrative nucleoside transporters 1 and 3 (hCNT1 and hCNT3). The production of recombinant hCNT1 and hCNT3 in a nucleoside-transporter deficient strain of yeast was confirmed by immunoblotting, and uridine transport parameters (Km, Vmax) were determined by defining the concentration dependence of initial rates of uptake of [3H]uridine by intact yeast. The Ki values of uridine analogs were obtained from inhibitory-effect curves and converted to binding energies. hCNT1 and hCNT3 recognized uridine through distinguishable binding motifs. hCNT1 was sensitive to modifications at C(3), less sensitive at C(5') or N(3), and much less sensitive at C(2'). hCNT3 was sensitive to modifications at C(3'), but much less sensitive at N(3), C(5') or C(2'). The changes of binding energy between transporter proteins and different uridine analogs suggested that hCNT1 formed hydrogen bonds (H-bonds) with C(3')-OH, C(5')-OH, or N(3)-H of uridine, but not with C(2')-OH, whereas hCNT3 formed H-bonds to C(3')-OH, but not to N(3)-H, C(5')-OH, and C(2')-OH. Both transporters barely tolerated modifications at C(3') or inversion of configurations at C(2')orC(3'). The binding profiles identified in this study can be used to predict the potential transportability of nucleoside analogs, including anticancer or antiviral nucleoside drugs, by hCNT1 and hCNT3.

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 [(3)H]uridine uptake in yeast cells producing recombinant hNT proteins. hCNT1 displayed the lowest FLT K(i) value for inhibition of [(3)H]uridine uptake, followed by hCNT3, hENT2, hENT1, and hCNT2. [(3)H]FLT was efficiently transported in Xenopus laevis oocytes individually producing hENT1, hENT2, hCNT1, or hCNT3. [(3)H]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) [(3)H]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 [(3)H]FLT uptake. MCF-7, A549, and Capan-2 cells displayed NBMPR IC(50) values that were smaller or equal to NBMPR K(d) values, suggesting that 50% inhibition of hENT1 reduced [(3)H]FLT uptake by at least 50%. A strong correlation between extracellular NBMPR binding sites/cell and [(3)H]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.

2-Chloro-9-(2'-deoxy-2'-fluoro-beta-d-arabinofuranosyl)adenine (Cl-F-ara-A, clofarabine), a purine nucleoside analog with structural similarity to 2-chloro-2'-deoxyadenosine (Cl-dAdo, cladribine) and 9-beta-d-arabinofuranosyl-2-fluoroadenine (F-ara-A, fludarabine), has activity in adult and pediatric leukemias. Mediated transport of the purine nucleoside analogs is believed to occur through the action of two structurally unrelated protein families, the equilibrative nucleoside transporters (ENTs) and the concentrative nucleoside transporters (CNTs). The current work assessed the transportability of Cl-F-ara-A, Cl-dAdo, and F-ara-A in cultured human leukemic CEM cells that were either nucleoside transport-defective or possessed individual human nucleoside transporter types and in Xenopus laevis oocytes and Saccharomyces cerevisiae yeast that produced individual recombinant human nucleoside transporter types. Cells producing hENT1 or hCNT3 exhibited the highest uptake of Cl-F-ara-A, whereas nucleoside transport-deficient cells and cells producing hCNT1 lacked uptake altogether. When Cl-F-ara-A transport rates by hENT1 were compared with those of Cl-dAdo and F-ara-A, Cl-dAdo had the highest efficiency of transport, although Cl-F-ara-A showed the greatest accumulation during 5-min exposures. In cytotoxicity studies with the CEM lines, Cl-F-ara-A was more cytotoxic to cells producing hENT1 than to the nucleoside transport-deficient cells. The efficiency of Cl-F-ara-A transport by oocytes with recombinant transporters was hCNT3 > hENT2 > hENT1 > hCNT2; no transport was observed with hCNT1. Affinity studies with recombinant transporters produced in yeast showed that hENT1, hENT2, and hCNT3 all had higher affinities for Cl-F-ara-A than for either Cl-dAdo or F-ara-A. These results suggest that the nature and activity of the plasma membrane proteins capable of inward transport of nucleosides are important determinants of Cl-F-ara-A activity in human cells.

CEM-ARAC leukemia cells with resistance to cytarabine were shown to lack equilibrative transporter (hENT1) expression and activity. Stable transfer of hCNT2 cDNA into CEM-ARAC enabled Na(+)-dependent transport of purine and pyrimidine nucleoside analogs and provided a unique in vitro model for studying hCNT2. Analysis of [(3)H]uridine inhibitory activity by test substances in hCNT2 transfectant ARAC/D2 revealed structural requirements for interaction with hCNT2: 1) ribosyl and 2'-deoxyribosyl nucleosides were better inhibitors than 3'-deoxyribosyl, 2',3'-dideoxyribosyl or arabinosyl nucleosides; 2) uridine analogs with halogens at position 5 were better inhibitors than 5-methyluridine or thymidine; 3) 2-chloroadenosine was a better inhibitor than 2-chloro-2'-deoxyadenosine (cladribine); and 4) cytosine-containing nucleosides, 7-deazaadenosine and nucleobases were not inhibitors. Quantification of inhibitory capacity yielded K(i) values of 34-50 microM (5-halogenated uridine analogs, 2'-deoxyuridine), 82 microM (5-fluoro-2'-deoxyuridine), 197-246 microM (5-methyluridine < 5-bromo-2'-deoxyuridine < 5-iodo-2'-deoxyuridine), and 411 microM (5-fluoro-5'-deoxyuridine, capecitabine metabolite). Comparisons of hCNT2-mediated transport rates indicated halogenated uridine analogs were transported more rapidly than halogenated adenosine analogs, even though hCNT2 exhibited preference for physiologic purine nucleosides over uridine. Kinetics of hCNT2-mediated transport of 5-fluorouridine and uridine were similar (K(m) values, 43-46 microM). The impact of hCNT2-mediated transport on chemosensitivity was assessed by comparing antiproliferative activity of nucleoside analogs against hCNT2-containing cells with transport-defective, drug-resistant cells. Chemosensitivity was restored partially for cladribine, completely for 5-fluorouridine and 5-fluoro-2'-deoxyuridine, whereas there was little effect on chemosensitivity for fludarabine, 7-deazaadenosine, or cytarabine. These studies, which demonstrated hCNT2 uptake of halogenated uridine analogs, suggested that hCNT2 is an important determinant of cytotoxicity of this class of compounds in vivo.