Cytotoxicity, Metabolism, and Mechanisms of Action of 2’,2’-Difluorodeoxyguanosine in Chinese Hamster Ovary Cells

Varsha Gandhi, Shin Mineishi, Peng Huang, Amy J. Chapman, Yandan Yang, Feng Chen, Billie Nowak, Sherri Chubb, Larry W. Hertel, William Plunkett

Research output: Contribution to journalArticle

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Abstract

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 um, 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 um after a 6-h incubation with 30 um dFdGuo. The elimination of dFdGTP was monophasic with a tvx 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 um 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 a. dFdGTP incorporation caused DNA polymerase a to pause after the polymerization of one additional de-oxynucleotide. 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 a. The unique activation requirements and patterns of inhibition of DNA synthesis distinguish this promising new antimetabolite from other nucleoside analogues.

Original languageEnglish (US)
Pages (from-to)1517-1524
Number of pages8
JournalCancer Research
Volume55
Issue number7
StatePublished - Apr 1995

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Cricetulus
gemcitabine
Ovary
Deoxycytidine Kinase
deoxyguanosine kinase
Deoxyguanosine
DNA-Directed DNA Polymerase
nucleoside phosphotransferase
Nucleosides
Adenosine Kinase
Purine-Nucleoside Phosphorylase
Nucleic Acid Synthesis Inhibitors
Ribonucleotide Reductases
Antimetabolites
DEAE-Cellulose Chromatography
Deoxycytidine
DNA Primers
Diphosphates
triphosphoric acid
Polymerization

All Science Journal Classification (ASJC) codes

  • Oncology
  • Cancer Research

Cite this

Gandhi, V., Mineishi, S., Huang, P., Chapman, A. J., Yang, Y., Chen, F., ... Plunkett, W. (1995). Cytotoxicity, Metabolism, and Mechanisms of Action of 2’,2’-Difluorodeoxyguanosine in Chinese Hamster Ovary Cells. Cancer Research, 55(7), 1517-1524.
Gandhi, Varsha ; Mineishi, Shin ; Huang, Peng ; Chapman, Amy J. ; Yang, Yandan ; Chen, Feng ; Nowak, Billie ; Chubb, Sherri ; Hertel, Larry W. ; Plunkett, William. / Cytotoxicity, Metabolism, and Mechanisms of Action of 2’,2’-Difluorodeoxyguanosine in Chinese Hamster Ovary Cells. In: Cancer Research. 1995 ; Vol. 55, No. 7. pp. 1517-1524.
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title = "Cytotoxicity, Metabolism, and Mechanisms of Action of 2’,2’-Difluorodeoxyguanosine in Chinese Hamster Ovary Cells",
abstract = "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 um, 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 um after a 6-h incubation with 30 um dFdGuo. The elimination of dFdGTP was monophasic with a tvx 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 um 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 a. dFdGTP incorporation caused DNA polymerase a to pause after the polymerization of one additional de-oxynucleotide. 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 a. The unique activation requirements and patterns of inhibition of DNA synthesis distinguish this promising new antimetabolite from other nucleoside analogues.",
author = "Varsha Gandhi and Shin Mineishi and Peng Huang and Chapman, {Amy J.} and Yandan Yang and Feng Chen and Billie Nowak and Sherri Chubb and Hertel, {Larry W.} and William Plunkett",
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Gandhi, V, Mineishi, S, Huang, P, Chapman, AJ, Yang, Y, Chen, F, Nowak, B, Chubb, S, Hertel, LW & Plunkett, W 1995, 'Cytotoxicity, Metabolism, and Mechanisms of Action of 2’,2’-Difluorodeoxyguanosine in Chinese Hamster Ovary Cells', Cancer Research, vol. 55, no. 7, pp. 1517-1524.

Cytotoxicity, Metabolism, and Mechanisms of Action of 2’,2’-Difluorodeoxyguanosine in Chinese Hamster Ovary Cells. / Gandhi, Varsha; Mineishi, Shin; Huang, Peng; Chapman, Amy J.; Yang, Yandan; Chen, Feng; Nowak, Billie; Chubb, Sherri; Hertel, Larry W.; Plunkett, William.

In: Cancer Research, Vol. 55, No. 7, 04.1995, p. 1517-1524.

Research output: Contribution to journalArticle

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T1 - Cytotoxicity, Metabolism, and Mechanisms of Action of 2’,2’-Difluorodeoxyguanosine in Chinese Hamster Ovary Cells

AU - Gandhi, Varsha

AU - Mineishi, Shin

AU - Huang, Peng

AU - Chapman, Amy J.

AU - Yang, Yandan

AU - Chen, Feng

AU - Nowak, Billie

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N2 - 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 um, 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 um after a 6-h incubation with 30 um dFdGuo. The elimination of dFdGTP was monophasic with a tvx 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 um 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 a. dFdGTP incorporation caused DNA polymerase a to pause after the polymerization of one additional de-oxynucleotide. 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 a. The unique activation requirements and patterns of inhibition of DNA synthesis distinguish this promising new antimetabolite from other nucleoside analogues.

AB - 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 um, 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 um after a 6-h incubation with 30 um dFdGuo. The elimination of dFdGTP was monophasic with a tvx 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 um 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 a. dFdGTP incorporation caused DNA polymerase a to pause after the polymerization of one additional de-oxynucleotide. 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 a. The unique activation requirements and patterns of inhibition of DNA synthesis distinguish this promising new antimetabolite from other nucleoside analogues.

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