Difluorodeoxyguanosine: cytotoxicity, metabolism, and actions on DNA synthesis in human leukemia cells.
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.
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D001087:Arabinonucleosides / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D006224:Cricetinae; D004261:DNA Replication / Q000187:drug effects*; D004273:DNA, Neoplasm / Q000187:drug effects*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology / Q000633:toxicity; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007938:Leukemia / Q000188:drug therapy*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D012334:RNA, Neoplasm / Q000096:biosynthesis / Q000187:drug effects; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured / Q000187:drug effects; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D001087:Arabinonucleosides; D004273:DNA, Neoplasm; D009711:Nucleotides; D012334:RNA, Neoplasm; D003841:Deoxycytidine; C096208:LY 223592; C028771:9-arabinofuranosylguanine; D017853:Phosphotransferases (Alcohol Group Acceptor); D003849:Deoxyguanosine; D000093542:Gemcitabine