Received for publication June 30, 2004.
Revised October 7, 2004.
Accepted for publication October 12, 2004.

Role of human nucleoside transporters in the cellular uptake of inhibitors of IMP dehydrogenase, tiazofurin and benzamide riboside

Abstract

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 X. laevis oocytes and S. cerevisiae that produced individual recombinant human nucleoside transporter 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 vs. 226 µM), hENT2 (16 vs. 271 µM), hENT1 (57 vs. 168 µM) and hCNT1 (221 vs. 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 (NBMPR) 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 nine-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 if this finding can be generalized to other cell types.

Key words: Nucleoside/Nucleotide, Nucleoside/Nucleotide derivatives