Modulatory Activity of 2′,2′-Difluorodeoxycytidine on the Phosphorylation and Cytotoxicity of Arabinosyl Nucleosides

This investigation analyzed the metabolism of 2′,2′-difluorodeoxycy-tidine (dFdC) in K562 human leukemia cells and evaluated it as a biochemical modulator for the phosphorylation of several arabinosyl nucleosides. The rate of accumulation of dFdC triphosphate was linear up to 3 h and maximal during incubation with 10 mm dFdC (92 pM/h). Deoxynucleotides analyzed at this time showed a decrease in dCTP, dATP, and dGTP levels, indicating an inhibitory role of dFdC nucleotides in ribonucleotide reduction. We evaluated the hypothesis that dFdC-mediated deoxyribonucleoside triphosphate perturbation enhances the phosphorylation of substrates that use deoxycytidine kinase or deoxy-guanosine kinase, because these enzymes are inhibited by dCTP or dGTP, respectively. When the activity of these nucleoside kinases was rate limiting to triphosphate formation, the accumulation of triphosphates of deoxycytidine, l-β-D-arabinofuranosylcytosine, and 1-β-D-arabinofura-nosylguanine was potentiated in cells pretreated with dFdC. In contrast, the phosphorylation of 9-β-D-arabinofuranosyladenine was not affected, since it is mainly phosphorylated by adenosine kinase, which is not influenced by deoxyribonucleoside triphosphates. Treatment of cells with dFdC followed by l-β-D-arabinofuranosylcytosine resulted in greater cytotoxicity than sum effects of each drug alone. The data indicate that an enhanced cytotoxicity could be obtained by administering dFdC as a modulator followed by 1-β-D-arabinofuranosylcytosine or 1-β-D-arabi-nofuranosylguanine in optimal sequence, suggesting that these results should be considered in the design of combination clinical protocols.