The success of
gemcitabine (2',2'-difluorodeoxycytidine; dFdC) resulted in new interest in its
purine congeners. Based on the structure-activity relationship studies of catabolism and anabolism,
2',2'-difluorodeoxyguanosine (dFdG) emerged as a lead candidate among the difluoropurine analogs. The cytotoxicity, metabolism, and actions of dFdG on
DNA synthesis were studied in the human
leukemia lymphoblastoid line CCRF-CEM. The IC50 values of dFdG after a 72-hour continuous incubation were 0.01, 0.03, and 0.28 mumol/L for CCRF-CEM, K562, and HL-60 cells, respectively. A cell line deficient in dCyd
kinase was equally sensitive to dFdG, suggesting that, in contrast to dFdC, dFdG may be activated by other deoxynucleoside
kinase(s). Consistent with these data, coincubation with dGuo spared the dFdG-mediated toxicity; however, up to 500 mumol/L dCyd failed to reverse the toxicity of dFdG. These observations indicated that dGuo
kinase, which phosphorylates arabinosylguanine, also appears to play a major role in activating dFdG. CCRF-CEM cells incubated with varying concentrations of [3H]dFdG accumulated dFdGTP in a dose-dependent manner; a 3-hour incubation with 1 mmol/L dFdG resulted in more than 600 mumol/L intracellular dFdGTP. This is in contrast to the
gemcitabine triphosphate accumulation, which is saturated
at 10 to 20 mumol/L of exogenous dFdC. dFdG metabolites affected
ribonucleotide reductase, resulting in a lowering of the
dCTP pool; this is in agreement with the effect of dFdC on dNTP pools in
leukemia cell lines. The major effect of dFdG on macromolecular synthesis was inhibition of
DNA synthesis.
DNA primer extension over a defined template revealed that dFdGTP was a good substrate for
DNA polymerase alpha and incorporated opposite C sites of the template. Unlike arabinosyl analogs, but similar to
gemcitabine triphosphate, dFdGTP incorporation caused
DNA polymerase to pause after one normal deoxynucleotide was incorporated beyond the analog. The unique activation requirements of dFdG, its novel mode of inhibition of
DNA synthesis, and its potent toxicity to human
leukemia cells make it a promising new
antimetabolite.