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Role of Human Nucleoside Transporters in the Cellular Uptake of Two Inhibitors of IMP Dehydrogenase, Tiazofurin and Benzamide Riboside

Membrane Protein Research Group, Departments of Biochemistry (C.E.C.), Oncology (V.L.D., F.V., J.Z., D.M., C.E.C.) and Physiology (A.M.L.N., J.D.Y.), University of Alberta, Edmonton, Alberta, Canada; Cross Cancer Institute, Edmonton, Alberta, Canada (V.L.D., F.V., J.Z., D.M. C.E.C.); and the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine and Richard Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana (H.N.J.)

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 hCNT3 >> hENT1 > hENT2 > hCNT2 >> hCNT1. The concentration dependence of inhibition of [³H]uridine transport in S. cerevisiae by TR exhibited lower K_i values than BR: hCNT3 (5.4 versus 226 µM), hENT2 (16 versus 271 µM), hENT1 (57 versus 168 µM), and hCNT1 (221 versus 220 µM). 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.

Address correspondence to: Dr. Carol E. Cass, Department of Oncology, Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta T6G 1Z2 Canada. E-mail: carol.cass{at}cancerboard.ab.ca

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