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Vol. 60, Issue 5, 1143-1152, November 2001
Canadian Institute of Health Research Membrane Protein Research
Group (T.T.L., M.S., J.D.Y., C.E.C.), Departments of Oncology
(T.T.L., M.S., C.E.C.) and Physiology (J.D.Y.), University of Alberta;
and the Cross Cancer Institute (T.T.L., M.S., C.E.C.), Edmonton,
Alberta, Canada
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
[3H]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 Ki values of 34-50 µM (5-halogenated
uridine analogs, 2'-deoxyuridine), 82 µM (5-fluoro-2'-deoxyuridine),
197-246 µM (5-methyluridine < 5-bromo-2'-deoxyuridine < 5-iodo-2'-deoxyuridine), and 411 µM (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 (Km values, 43-46 µM). 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.
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