Acquisition of Human Concentrative Nucleoside Transporter 2 (hCNT2) Activity by Gene Transfer Confers Sensitivity to Fluoropyrimidine Nucleosides in Drug-Resistant Leukemia Cells

Abstract

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.

Functional characterization of a H+/nucleoside co‐transporter (CaCNT) from Candida albicans, a fungal member of the concentrative nucleoside transporter (CNT) family of membrane proteins

“…With the exception of 5-fluorouridine, the measured fluxes were smaller than those for uridine and inosine, and similar to those reported previously for hCNT2-mediated transport of ddI, 5-fluorouridine, fludarabine and cladribine in oocytes and transfected mammalian cells (Ritzel et al, 1998;Lang et al, 2001). CaCNT was, therefore, relatively tolerant of substitutions at the 2 (5-fluoro-2 -deoxyuridine, cladribine, fludarabine) and 3 (cordycepin, ddI) positions of the sugar, with significant import at micromolar concentrations.…”

Nucleoside anticancer drugs: the role of nucleoside transporters in resistance to cancer chemotherapy

“…The importance of 3 0 -and 5 0 -hydroxyl groups of the ribosyl moiety of uridine for permeant recognition by hCNT2 was demonstrated in the stably transfected CEM-ARAC (transport deficient clone) leukemic cell line (Lang et al, 2001). The purine nucleoside selectivity of hCNT2 is demonstrated by its lower K m for adenosine (8 mm) than for uridine (40 mm).…”

“…hCNT1 and hCNT2 differ in permeant selectivity in that hCNT1 transports zidovudine and zalcitabine whereas hCNT2 transports didanosine. The importance of 3 0 -and 5 0 -hydroxyl groups of the ribosyl moiety of uridine for permeant recognition by hCNT2 was demonstrated in a stably transfected CEM-ARAC (transport-deficient clone) leukemic cell line (Lang et al, 2001). Removal of the hydroxyl group at the 2 0 -position did not affect the interaction of uridine with hCNT2.…”

Nucleoside transporters in chronic lymphocytic leukaemia

“…A similar specificity profile seems to apply to cladribine. 41,46,47 Which nucleoside transporters do CLL cells express?…”

“…43 Similarly, the acquisition of hCNT2 function by gene transfer into a drugresistant T-lymphoblast cell line devoid of nucleoside transporter activity increased sensitivity to halogenated uridine analogues. 79 The role of CNT isoforms in nucleoside-derived drug anticancer therapy remains unclear, but it is likely that the two nucleoside influx pathways contribute to drug cytotoxicity.…”