Cytotoxicity, metabolism, and mechanisms of action of 2',2'-difluorodeoxyguanosine in Chinese hamster ovary cells.
The emerging clinical success of gemcitabine (2',2'-difluorodeoxycytidine) stimulated interest in the synthesis and evaluation of purine congeners. The cytotoxicity, metabolism, and mechanisms of action of the lead candidate, 2',2'-difluorodeoxyguanosine (dFdGuo), were studied in Chinese hamster ovary cells. Unlike the natural nucleoside deoxyguanosine (dGuo), dFdGuo was not a substrate for purine nucleoside phosphorylase. Wild-type Chinese hamster ovary cells and a mutant line deficient in deoxycytidine (dCyd) kinase were similarly affected by dFdGuo (50% inhibitory concentration, 7.5 and 6.5 microM, respectively), suggesting that unlike gemcitabine, dCyd kinase was not responsible for activation of dFdGuo. This was further confirmed by separation of nucleoside kinases (adenosine kinase, dGuo kinase, and dCyd kinase) of Chinese hamster ovary cells on DEAE-cellulose column chromatography. The kinase activity that phosphorylated dGuo also converted dFdGuo to its monophosphate, suggesting that dGuo kinase activated dFdGuo. Consistent with this result, coincubation with dGuo spared the dFdGuo-mediated toxicity; however, addition of up to 10 mM dCyd did not reverse the toxicity of dFdGuo. Intracellularly, dFdGuo was phosphorylated to its mono-, di-, and triphosphates; dFdGuo triphosphate (dFdGTP) was the major metabolite and accumulated to 45 microM after a 6-h incubation with 30 microM dFdGuo. The elimination of dFdGTP was monophasic with a t1/2 of about 6 h. Deoxynucleotides were decreased in cells incubated with dFdGuo, suggesting that ribonucleotide reductase was inhibited. dATP, which decreased 78% after a 4-h incubation with 30 microM dFdGuo, was most affected. dFdGuo was a potent inhibitor of DNA synthesis. Extension of a DNA primer over a defined template in the presence of dFdGTP revealed that dFdGTP was a good substrate for incorporation opposite C sites of the template by DNA polymerase alpha. dFdGTP incorporation caused DNA polymerase alpha to pause after the polymerization of one additional deoxynucleotide. This pattern of inhibition, which is shared by gemcitabine, distinguishes 2',2'-difluoronucleosides from arabinosylnucleosides which halt primer extension at the incorporation site. dGTP competed effectively with dFdGTP for incorporation by DNA polymerase alpha. The unique activation requirements and patterns of inhibition of DNA synthesis distinguish this promising new antimetabolite from other nucleoside analogues.
Gandhi
V
V
Department of Clinical Investigation, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Mineishi
S
S
Huang
P
P
Chapman
A J
AJ
Yang
Y
Y
Chen
F
F
Nowak
B
B
Chubb
S
S
Hertel
L W
LW
Plunkett
W
W
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D002455:Cell Division / Q000187:drug effects; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011683:Purine-Nucleoside Phosphorylase / Q000378:metabolism*; D012313:RNA / Q000096:biosynthesis*; D013379:Substrate Specificity; D000093542:Gemcitabine
D003841:Deoxycytidine; D012313:RNA; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D011683:Purine-Nucleoside Phosphorylase; D000093542:Gemcitabine