Consequences of 2',2'-difluorodeoxycytidine (gemcitabine) on replicative DNA synthesis in intact HL-60 cells

The technique of pH-step alkaline elution was used to assess the effects of gemcitabine (dFdC) on replicative DNA synthesis in intact HL-60 human myeloid leukemia cells. Although gemcitabine did cause profound inhibition of DNA chain elongation, it was progressively incorporated through nascent DNA replication intermediates of increasing size into genomic DNA. Hence, in the intact cell, it is not a chain terminator, at least not in the absolute sense of the term. In comparison to cytosine arabinoside (ara-C), the progression of incorporated gemcitabine from small nascent DNA fragments to genomic-length DNA was less complete. Furthermore, at equitoxic exposures, less gemcitabine was incorporated into DNA than ara-C. Studies of the effects of gemcitabine on ribonucleotide reduction in HL-60 cells revealed that dGTP pools, but not dCTP pools, were reduced by a 3-hour exposure to 40 nmol/L gemcitabine (the concentration that causes 50% lethality). This reduction was transient, and recovery of dGTP pool size was accomplished within 16 hours. These studies indicate that the effects of gemcitabine on inhibiting replicative DNA chain elongation comprise an important component of the cytotoxicity of the drug.