Metabolic channeling of 5-fluoro-2'-deoxycytidine utilizing inhibitors of its deamination in cell culture

D. A. Boothman, T. V. Briggle, S. Greer

Research output: Contribution to journalArticle

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Abstract

The metabolism of 5-fluoro-2'-deoxycytidine (FdC) with and without tetrahydrouridine (H4U) or 2'-deoxytetrahydrouridine (dH4U) was examined in log phase HEp-2 cells using HPLC and TLC methods which quantified: (a) the incorporation of FdC-related antimetabolites into RNA and DNA and (b) pool size levels of FdC-related antimetabolites. [3H]-FdC administered to log phase HEp-2 cells at a concentration of 0.01 μM for 24 hr resulted in the incorporation of 5.22 x 10-8 mol of FdC/mol of DNA phosphate, a 0.021% substitution of FdC for dC. Coadministration of 1.0 mM H4U or dH4U resulted in 2- and 25-fold increases in the incorporation of FdC, respectively. No detectable incorporation of 5-fluoro-2'-deoxyuridine (FdU) into HEp-2 DNA resulted (detection limit, approximately 5 fmol). In contrast, treatment of HEp-2 cells with 0.1 μM FdU resulted in the incorporation of 1.83 x 10-9 mol of FdU (74.7 fmol detected)/mol of DNA phosphate. A linear incorporation of FdC into the DNA of HEp-2 cells was found with increasing concentrations of FdC and 1.0 mM dH4U. 0.1 μM FdC resulted in the incorporation of 2.39 x 10-6 of FUMP/mol of cytoplasmic RNA phosphate and 2.23 x 10-5 mol of FUMP/mol of nuclear RNA phosphate. Similarly, HEp-2 cells treated with 0.1 μM FdU resulted in the incorporation of 1.10 x 10-5 of FUMP/mol of nuclear RNA phosphate and 9.44 x 10-7 mol of FUMP/mol of cytoplasmic RNA phosphate. In contrast, no detectable FUMP incorporation into either nuclear or cytoplasmic RNAs of HEp-2 cells resulted when H4U or dH4U was coadministered with 0.1 μM FdC. Pool size analyses of log phase HEp-2 cells following a 30-min exposure to FdU or FdC with and without H4U or dH4U were also performed; 0.1 μM FdC treatment resulted in the formation of 169 fmol of FUMP/1.0 x 106 viable HEp-2 cells. Treatment with 0.1 μM FdU produced 253 fmol of FUMP/1.0 x 106 viable HEp-2 cells. In contrast, no detectable FUMP pools were formed when H4U or dH4U was coadministered with 0.1 μM FdC (detection limit, approximately 5 fmol). Pool levels of FdUMP, the inhibitor of thymidylate synthetase, were also assayed; 36.9 fmol of FdUMP/1.0 x 106 viable HEp-2 cells were detected upon administration of 0.1 μM FdC. Coadministration of 1.0 mM H4U with 0.1 μM FdC increased FdUMP pools 2.3-fold, while 1.0 mM dH4U resulted in a 3.2-fold decrease; 0.1 μM FdU resulted in the formation of 55.2 fmol of FdUMP/1.0 x 106 viable HEp-2 cells, 34.3% less than that formed when 1.0 mM H4U was coadministered with 0.1 μM FdC. These studies demonstrate that the coadministration of H4U effectively directs the metabolism of FdC in neoplastic cells through the deoxycytidine kinase-deoxycytidylate deaminase pathway to the formation of FdUMP without the incorporation of FUMP into RNA or the formation of RNA-level antimetabolite pools (i.e., FUra, FUrd, or FUMP); this metabolic restriction was not found to be due to the inhibition of thymidine phosphorylase by H4U.

Original languageEnglish
Pages (from-to)584-594
Number of pages11
JournalMolecular Pharmacology
Volume27
Issue number5
StatePublished - Jan 1 1985
Externally publishedYes

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Deamination
Cell Culture Techniques
Fluorodeoxyuridylate
Phosphates
RNA
Antimetabolites
DNA
5-fluoro-2'-deoxycytidine
Nuclear RNA
Tetrahydrouridine
Limit of Detection
DCMP Deaminase
Deoxycytidine Kinase
Thymidine Phosphorylase
Thymidylate Synthase

ASJC Scopus subject areas

  • Pharmacology

Cite this

Metabolic channeling of 5-fluoro-2'-deoxycytidine utilizing inhibitors of its deamination in cell culture. / Boothman, D. A.; Briggle, T. V.; Greer, S.

In: Molecular Pharmacology, Vol. 27, No. 5, 01.01.1985, p. 584-594.

Research output: Contribution to journalArticle

Boothman, D. A. ; Briggle, T. V. ; Greer, S. / Metabolic channeling of 5-fluoro-2'-deoxycytidine utilizing inhibitors of its deamination in cell culture. In: Molecular Pharmacology. 1985 ; Vol. 27, No. 5. pp. 584-594.
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abstract = "The metabolism of 5-fluoro-2'-deoxycytidine (FdC) with and without tetrahydrouridine (H4U) or 2'-deoxytetrahydrouridine (dH4U) was examined in log phase HEp-2 cells using HPLC and TLC methods which quantified: (a) the incorporation of FdC-related antimetabolites into RNA and DNA and (b) pool size levels of FdC-related antimetabolites. [3H]-FdC administered to log phase HEp-2 cells at a concentration of 0.01 μM for 24 hr resulted in the incorporation of 5.22 x 10-8 mol of FdC/mol of DNA phosphate, a 0.021{\%} substitution of FdC for dC. Coadministration of 1.0 mM H4U or dH4U resulted in 2- and 25-fold increases in the incorporation of FdC, respectively. No detectable incorporation of 5-fluoro-2'-deoxyuridine (FdU) into HEp-2 DNA resulted (detection limit, approximately 5 fmol). In contrast, treatment of HEp-2 cells with 0.1 μM FdU resulted in the incorporation of 1.83 x 10-9 mol of FdU (74.7 fmol detected)/mol of DNA phosphate. A linear incorporation of FdC into the DNA of HEp-2 cells was found with increasing concentrations of FdC and 1.0 mM dH4U. 0.1 μM FdC resulted in the incorporation of 2.39 x 10-6 of FUMP/mol of cytoplasmic RNA phosphate and 2.23 x 10-5 mol of FUMP/mol of nuclear RNA phosphate. Similarly, HEp-2 cells treated with 0.1 μM FdU resulted in the incorporation of 1.10 x 10-5 of FUMP/mol of nuclear RNA phosphate and 9.44 x 10-7 mol of FUMP/mol of cytoplasmic RNA phosphate. In contrast, no detectable FUMP incorporation into either nuclear or cytoplasmic RNAs of HEp-2 cells resulted when H4U or dH4U was coadministered with 0.1 μM FdC. Pool size analyses of log phase HEp-2 cells following a 30-min exposure to FdU or FdC with and without H4U or dH4U were also performed; 0.1 μM FdC treatment resulted in the formation of 169 fmol of FUMP/1.0 x 106 viable HEp-2 cells. Treatment with 0.1 μM FdU produced 253 fmol of FUMP/1.0 x 106 viable HEp-2 cells. In contrast, no detectable FUMP pools were formed when H4U or dH4U was coadministered with 0.1 μM FdC (detection limit, approximately 5 fmol). Pool levels of FdUMP, the inhibitor of thymidylate synthetase, were also assayed; 36.9 fmol of FdUMP/1.0 x 106 viable HEp-2 cells were detected upon administration of 0.1 μM FdC. Coadministration of 1.0 mM H4U with 0.1 μM FdC increased FdUMP pools 2.3-fold, while 1.0 mM dH4U resulted in a 3.2-fold decrease; 0.1 μM FdU resulted in the formation of 55.2 fmol of FdUMP/1.0 x 106 viable HEp-2 cells, 34.3{\%} less than that formed when 1.0 mM H4U was coadministered with 0.1 μM FdC. These studies demonstrate that the coadministration of H4U effectively directs the metabolism of FdC in neoplastic cells through the deoxycytidine kinase-deoxycytidylate deaminase pathway to the formation of FdUMP without the incorporation of FUMP into RNA or the formation of RNA-level antimetabolite pools (i.e., FUra, FUrd, or FUMP); this metabolic restriction was not found to be due to the inhibition of thymidine phosphorylase by H4U.",
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T1 - Metabolic channeling of 5-fluoro-2'-deoxycytidine utilizing inhibitors of its deamination in cell culture

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N2 - The metabolism of 5-fluoro-2'-deoxycytidine (FdC) with and without tetrahydrouridine (H4U) or 2'-deoxytetrahydrouridine (dH4U) was examined in log phase HEp-2 cells using HPLC and TLC methods which quantified: (a) the incorporation of FdC-related antimetabolites into RNA and DNA and (b) pool size levels of FdC-related antimetabolites. [3H]-FdC administered to log phase HEp-2 cells at a concentration of 0.01 μM for 24 hr resulted in the incorporation of 5.22 x 10-8 mol of FdC/mol of DNA phosphate, a 0.021% substitution of FdC for dC. Coadministration of 1.0 mM H4U or dH4U resulted in 2- and 25-fold increases in the incorporation of FdC, respectively. No detectable incorporation of 5-fluoro-2'-deoxyuridine (FdU) into HEp-2 DNA resulted (detection limit, approximately 5 fmol). In contrast, treatment of HEp-2 cells with 0.1 μM FdU resulted in the incorporation of 1.83 x 10-9 mol of FdU (74.7 fmol detected)/mol of DNA phosphate. A linear incorporation of FdC into the DNA of HEp-2 cells was found with increasing concentrations of FdC and 1.0 mM dH4U. 0.1 μM FdC resulted in the incorporation of 2.39 x 10-6 of FUMP/mol of cytoplasmic RNA phosphate and 2.23 x 10-5 mol of FUMP/mol of nuclear RNA phosphate. Similarly, HEp-2 cells treated with 0.1 μM FdU resulted in the incorporation of 1.10 x 10-5 of FUMP/mol of nuclear RNA phosphate and 9.44 x 10-7 mol of FUMP/mol of cytoplasmic RNA phosphate. In contrast, no detectable FUMP incorporation into either nuclear or cytoplasmic RNAs of HEp-2 cells resulted when H4U or dH4U was coadministered with 0.1 μM FdC. Pool size analyses of log phase HEp-2 cells following a 30-min exposure to FdU or FdC with and without H4U or dH4U were also performed; 0.1 μM FdC treatment resulted in the formation of 169 fmol of FUMP/1.0 x 106 viable HEp-2 cells. Treatment with 0.1 μM FdU produced 253 fmol of FUMP/1.0 x 106 viable HEp-2 cells. In contrast, no detectable FUMP pools were formed when H4U or dH4U was coadministered with 0.1 μM FdC (detection limit, approximately 5 fmol). Pool levels of FdUMP, the inhibitor of thymidylate synthetase, were also assayed; 36.9 fmol of FdUMP/1.0 x 106 viable HEp-2 cells were detected upon administration of 0.1 μM FdC. Coadministration of 1.0 mM H4U with 0.1 μM FdC increased FdUMP pools 2.3-fold, while 1.0 mM dH4U resulted in a 3.2-fold decrease; 0.1 μM FdU resulted in the formation of 55.2 fmol of FdUMP/1.0 x 106 viable HEp-2 cells, 34.3% less than that formed when 1.0 mM H4U was coadministered with 0.1 μM FdC. These studies demonstrate that the coadministration of H4U effectively directs the metabolism of FdC in neoplastic cells through the deoxycytidine kinase-deoxycytidylate deaminase pathway to the formation of FdUMP without the incorporation of FUMP into RNA or the formation of RNA-level antimetabolite pools (i.e., FUra, FUrd, or FUMP); this metabolic restriction was not found to be due to the inhibition of thymidine phosphorylase by H4U.

AB - The metabolism of 5-fluoro-2'-deoxycytidine (FdC) with and without tetrahydrouridine (H4U) or 2'-deoxytetrahydrouridine (dH4U) was examined in log phase HEp-2 cells using HPLC and TLC methods which quantified: (a) the incorporation of FdC-related antimetabolites into RNA and DNA and (b) pool size levels of FdC-related antimetabolites. [3H]-FdC administered to log phase HEp-2 cells at a concentration of 0.01 μM for 24 hr resulted in the incorporation of 5.22 x 10-8 mol of FdC/mol of DNA phosphate, a 0.021% substitution of FdC for dC. Coadministration of 1.0 mM H4U or dH4U resulted in 2- and 25-fold increases in the incorporation of FdC, respectively. No detectable incorporation of 5-fluoro-2'-deoxyuridine (FdU) into HEp-2 DNA resulted (detection limit, approximately 5 fmol). In contrast, treatment of HEp-2 cells with 0.1 μM FdU resulted in the incorporation of 1.83 x 10-9 mol of FdU (74.7 fmol detected)/mol of DNA phosphate. A linear incorporation of FdC into the DNA of HEp-2 cells was found with increasing concentrations of FdC and 1.0 mM dH4U. 0.1 μM FdC resulted in the incorporation of 2.39 x 10-6 of FUMP/mol of cytoplasmic RNA phosphate and 2.23 x 10-5 mol of FUMP/mol of nuclear RNA phosphate. Similarly, HEp-2 cells treated with 0.1 μM FdU resulted in the incorporation of 1.10 x 10-5 of FUMP/mol of nuclear RNA phosphate and 9.44 x 10-7 mol of FUMP/mol of cytoplasmic RNA phosphate. In contrast, no detectable FUMP incorporation into either nuclear or cytoplasmic RNAs of HEp-2 cells resulted when H4U or dH4U was coadministered with 0.1 μM FdC. Pool size analyses of log phase HEp-2 cells following a 30-min exposure to FdU or FdC with and without H4U or dH4U were also performed; 0.1 μM FdC treatment resulted in the formation of 169 fmol of FUMP/1.0 x 106 viable HEp-2 cells. Treatment with 0.1 μM FdU produced 253 fmol of FUMP/1.0 x 106 viable HEp-2 cells. In contrast, no detectable FUMP pools were formed when H4U or dH4U was coadministered with 0.1 μM FdC (detection limit, approximately 5 fmol). Pool levels of FdUMP, the inhibitor of thymidylate synthetase, were also assayed; 36.9 fmol of FdUMP/1.0 x 106 viable HEp-2 cells were detected upon administration of 0.1 μM FdC. Coadministration of 1.0 mM H4U with 0.1 μM FdC increased FdUMP pools 2.3-fold, while 1.0 mM dH4U resulted in a 3.2-fold decrease; 0.1 μM FdU resulted in the formation of 55.2 fmol of FdUMP/1.0 x 106 viable HEp-2 cells, 34.3% less than that formed when 1.0 mM H4U was coadministered with 0.1 μM FdC. These studies demonstrate that the coadministration of H4U effectively directs the metabolism of FdC in neoplastic cells through the deoxycytidine kinase-deoxycytidylate deaminase pathway to the formation of FdUMP without the incorporation of FUMP into RNA or the formation of RNA-level antimetabolite pools (i.e., FUra, FUrd, or FUMP); this metabolic restriction was not found to be due to the inhibition of thymidine phosphorylase by H4U.

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