Molecular Pharmacology, Vol 13, 1014-1023, Copyright © 1977 by the American Society for Pharmacology and Experimental Therapeutics

Inhibition of Uridine Uptake in HeLa Cells by Nitrobenzylthioinosine and Related Compounds

¹ Cancer Research Unit (McEachern Laboratory), and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Rates of uridine uptake by logarithmically proliferating monolayer cultures of HeLa cells at 20° were constant for intervals of several minutes, and rate saturability was evident. Nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport, abolished saturable uptake of uridine, revealing a nonsaturable component of uptake that apparently was due to simple diffusion. Under conditions of the uptake assay, the principal metabolite of uridine was UTP. NBMPR had no effect on uridine kinase in HeLa cell extracts, or on the relative proportions of uridine anabolites in intact cells. These results indicate that NBMPR inhibited mediated passage of uridine into the cell. The inhibition of uridine transport by NBMPR was characterized by unchanged V_max values, with increases in apparent K_m values, whether the inhibitor and permeant were added simultaneously in the uptake assay or the cells had previously been treated with inhibitor. To assess the structural requirements for inhibitory activity, compounds related structurally to NBMPR were tested for their ability to inhibit uridine uptake; of these, the most effective were the 2'-deoxyribosyl and arabinosyl analogues of NBMPR and the S⁶-nitrobenzyl derivatives of 6-thioguanosine, 2'-deoxy-6-thioguanosine, and 6-selenoguanosine.

Note:
ACKNOWLEDGMENTS For the provision of nucleoside derivatives we are grateful to Drs. M. J. Robins, L. B. Townsend, K. C. Tsou, J. P. Miller, R. A. Long, H. J. Schaeffer, and Drug Research and Development, National Cancer Institute, Bethesda, Md. The latter agency generously provided 6-thioinosine and 6-thioguanosine for the synthesis of S⁶ derivatives. We acknowledge competent assistance by S. C. Kim, J. R. Schwarzkopf, L. R. Babb, and J. H. Paran.