The role of DNA synthesis inhibition in the cytotoxicity of 2′,2′-difluoro-2′-deoxycytidine

Purpose. Cytotoxicity from the anticancer drug 2',2'-difluoro-2'-deoxycytidine (dFdCyd) has been correlated with its incorporation into DNA. However, cytotoxicity may also result from inhibition of DNA synthesis, due to either (1) dFdCyd diphosphate-mediated inhibition of ribonucleotide reductase, or (2) direct inhibition of DNA polymerases by the 5'-triphosphate of dFdCyd (dFdCTP). To elucidate the role of DNA synthesis inhibition in the cytotoxicity of dFdCyd, we compared dFdCyd to hydroxyurea (HU), a ribonucleotide reductase inhibitor, and aphidicolin, an inhibitor of DNA polymerases, in the U251 and D54 human glioblastoma cell lines. Methods. Sensitivity to dFdCyd, HU, and aphidicolin were determined using a colony formation assay. The effects of these drugs on DNA synthesis were measured by dual parameter flow cytometry, while the effects on nucleotide pool levels were analyzed by high-performance liquid chromatography. Results. HU and aphidicolin elicited substantially less cytotoxicity than the multi-log killing with dFdCyd. When used at equitoxic concentrations (24-h IC50 values), dFdCyd and HU decreased purine dNTP pools primarily, but dFdCyd was less effective than HU. dFdCyd had decreased dATP by about 80% after 4-12 h, and required 8-24 h to decrease DNA synthesis by 50%. In contrast, HU rapidly depleted dATP by >98% within 2 h, which resulted in >90% inhibition of DNA synthesis. Aphidicolin at a concentration similar to its K-i values for DNA polymerases (1 muM) decreased DNA synthesis by >70% within 2 h. However, this decreased cell survival by only 10% (U251 cells) and 40% (D54 cells). Conclusions. These results demonstrate that HU and aphidicolin produced a more rapid and profound inhibition of DNA synthesis than dFdCyd, but resulted in significantly less cytotoxicity. This suggests that inhibition of DNA synthesis accounted for less than one log of the multi-log cytotoxicity observed with dFdCyd, whereas incorporation of dFdCTP into DNA is a more lethal event.