MODULATION OF THE METABOLISM OF BETA-L-(-)-2',3'-DIDEOXY-3'-THIACYTIDINE BY THYMIDINE, FIUDARABINE, AND NITROBENZYLTHIOINOSINE

beta-L-(-)-2',3'-Dideoxy-3'-thiacytidine (3TC) is a cytosine nucleosid e analog that potently inhibits the replication of human and duck hepa titis B viruses and human immunodeficiency virus through the activity of its 5'-triphosphate ester metabolite. The present study examined th e intracellular decay of 3TC 5'-phosphates and tested strategies for m odulating the cellular content of those nucleotides in primary culture s of duck hepatocytes and in human hepatoma 2.2.15 cells and CCRF-CEM T lymphoblasts. Inhibition by deoxycytidine of the 5'-phosphorylation of 3TC in duck hepatocytes confirmed that, as in mammalian cells, deox ycytidine kinase catalyzed 3TC activation. The 5'-mono, 5'-di-, and 5' -triphosphates of 3TC underwent monoexponential elimination from duck hepatocytes and 2.2.15 cells (half-lives, 3.6 to 8.0 h). Thymidine and fludarabine, which are agents that enhance the activity of deoxycytid ine kinase, were tested in strategies for increasing the cellular cont ent of 3TC 5'-phosphates. Coordinate treatment of cells with 3TC and t hymidine (50 mu m)) increased the content of 3TC 5'-monophosphate in d uck hepatocytes and the content of 3TC 5'-di- and 5'-triphosphates in 2.2.15 cells, but enhancement of 3TC 5'-phosphate levels in CCRF-CERI cells required a higher thymidine concentration (100 mu M). Fludarabin e (5 mu M) did not affect the contents of 3TC 5'-di- and 5'-triphospha tes in duck hepatocytes, but modestly increased the contents of those nucleotides in 2.2.15 cells and CCRF-CERI cells, Nitrobenzylthioninosi ne (NBMPR), an inhibitor of the es facilitated diffusion nucleoside tr ansporter, reduced the level of entry of 3TC into 2.2.15 cells and abo lished inward fluxes of thymidine, adenosine, and deoxycytidine. In 2. 2.15 cells and CCRF-CEM cells, NBMPR reduced the formation of 3TC 5'-d i- and 5'-triphosphates and reversed the thymidine- and fludarabine-in duced increases in the formation of those nucleotides, NBMPR protected against the cytotoxicity of 3TC in CCRF-CEM cells, whereas thymidine potentiated that toxicity, apparently by enhancing the formation of 3T C 5'-triphosphate. Taken together, these results indicate that deoxycy tidine kinase and the es nucleoside transporter are targets for manipu lation of the metabolism and activity of STC.