Acquisition of human concentrative nucleoside transporter 2 (hCNT2) activity by gene transfer confers sensitivity to fluoropyrimidine nucleosides in drug-resistant leukemia cells

CEM-ARAC leukemia cells with resistance to cytarabine were shown to lack eq uilibrative transporter (hENT1) expression and activity. Stable transfer of hCNT2 cDNA into CEM-ARAC enabled Na+-dependent transport of purine and pyr imidine nucleoside analogs and provided a unique in vitro model for studyin g hCNT2. Analysis of [H-3]uridine inhibitory activity by test substances in hCNT2 transfectant ARAC/D2 revealed structural requirements for interactio n with hCNT2: 1) ribosyl and 2'-deoxyribosyl nucleosides were better inhibi tors 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 t han 2-chloro-2'-deoxyadenosine (cladribine); and 4) cytosine-containing nuc leosides, 7-deazaadenosine and nucleobases were not inhibitors. Quantificat ion of inhibitory capacity yielded K-i values of 34-50 muM (5-halogenated u ridine analogs, 2'-deoxyuridine), 82 muM (5-fluoro-2'-deoxyuridine), 197-24 6 muM (5-methyluridine < 5-bromo-2'-deoxyuridine < 5-iodo-2'-deoxyuridine), and 411 muM (5-fluoro-5'-deoxyuridine, capecitabine metabolite). Compariso ns of hCNT2-mediated transport rates indicated halogenated uridine analogs were transported more rapidly than halogenated adenosine analogs, even thou gh hCNT2 exhibited preference for physiologic purine nucleosides over uridi ne. Kinetics of hCNT2-mediated transport of 5-fluorouridine and uridine wer e similar (K-m values, 43-46 muM). The impact of hCNT2-mediated transport o n 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 cladribi ne, completely for 5-fluorouridine and 5-fluoro-2'-deoxyuridine, whereas th ere was little effect on chemosensitivity for fludarabine, 7-deazaadenosine , or cytarabine. These studies, which demonstrated hCNT2 uptake of halogena ted uridine analogs, suggested that hCNT2 is an important determinant of cy totoxicity of this class of compounds in vivo.