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 (K m , V max ) were determined by defining the concentration dependence of initial rates of uptake of [ 3 H]uridine by intact yeast. The K i values of uridine analogs were obtained from inhibitory-effect curves and converted to binding energies.

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 ϩ -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 2Ј-deoxyribosyl nucleosides were better inhibitors than 3Ј-deoxyribosyl, 2Ј,3Ј-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-2Ј-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 M (5-halogenated uridine analogs, 2Ј-deoxyuridine), 82 M (5-fluoro-2Ј-deoxyuridine), 197-246 M (5-methyluridine Ͻ 5-bromo-2Ј-deoxyuridine Ͻ 5-iodo-2Ј-deoxyuridine), and 411 M (5-fluoro-5Ј-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 M). The impact of hCNT2-mediated transport on chemosensitivity was assessed by comparing antiproliferative activity of nucleoside analogs against hCNT2-containing cells with transport-defective, drugresistant cells. Chemosensitivity was restored partially for cladribine, completely for 5-fluorouridine and 5-fluoro-2Ј-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.

Resistance to fludarabine is observed in the clinic, and molecular predictive assays for benefit from chemotherapy are required. Our objective was to determine if expression of nucleoside transport and metabolism genes was associated with response to fludarabine therapy in patients with chronic lymphocytic leukemia (CLL).