Inhibition of ribonucleotide reduction in CCRF-CEM cells by 2',2'-difluorodeoxycytidine

The new deoxycytidine analogue 2',2'-difluorodeoxycytidine (dFdC) is a specific inhibitor of DNA synthesis that has marked cytotoxicity and therapeutic activity. A 2-hr incubation with 0.1-10 μM dFdC decreased cellular viability 78-97%. This treatment reduced deoxynucleoside triphosphate pools, similar to the action of the ribonucleotide reductase inhibitor hydroxyurea. The most pronounced decrease occurred in the dCTP pool, quantitatively followed by the decrease of dATP, dGTP, and dTTP. In contrast, inhibition of DNA synthesis by arabinosylcytosine did not affect the dCTP level, whereas dATP, dGTP, and dTTP pools increased, but less than 2-fold. The incorporation of [5-³H]cytidine into the dCTP pool, a measure of ribonucleotide reductase activity in whole cells, was reduced to 3% of controls by 0.1 μM dFdC, but to only 40% by 0.1 μM ara-C. Each drug decreased incorporation of [5-³H]cytidine into DNA to a similar extent (>94%), suggesting limitation by a reaction proximal to this step. The cellular concentration of dFdC 5'-diphosphate was 0.3 μM at 50% inhibition of the in situ activity of ribonucleotide reductase. Direct assays of partially purified ribonucleoside diphosphate reductase (EC 1.17.4.1) demonstrated 50% inhibition by 4 μM dFdC 5'-diphosphate; dFdC 5'-triphosphate was much less inhibitory. We conclude that dFdC 5'-diphosphate acts as an inhibitor of ribonucleoside diphosphate reductase.