Metabolic channeling of 5-fluoro-2'-deoxycytidine utilizing inhibitors of its deamination in cell culture.
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: the incorporation of FdC-related antimetabolites into RNA and DNA and pool size levels of FdC-related antimetabolites. [3H]-FdC administered to log phase HEp-2 cells at a concentration of 0.01 microM 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 microM 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 microM FdC resulted in the incorporation of 2.39 X 10(-6) mol 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 microM FdU resulted in the incorporation of 1.10 X 10(-5) mol 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 microM 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 microM FdC treatment resulted in the formation of 169 fmol of FUMP/1.0 X 10(6) viable HEp-2 cells. Treatment with 0.1 microM FdU produced 253 fmol of FUMP/1.0 X 10(6) viable HEp-2 cells. In contrast, no detectable FUMP pools were formed when H4U or dH4U was coadministered with 0.1 microM 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 10(6) viable HEp-2 cells were detected upon administration of 0.1 microM FdC.(ABSTRACT TRUNCATED AT 400 WORDS)
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D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003564:Cytidine Deaminase / Q000037:antagonists & inhibitors*; D004247:DNA / Q000378:metabolism; D003641:Deamination; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D009700:Nucleoside Deaminases / Q000037:antagonists & inhibitors*; D012313:RNA / Q000378:metabolism; D013767:Tetrahydrouridine / Q000494:pharmacology*; D014529:Uridine / Q000031:analogs & derivatives*
D003841:Deoxycytidine; D013767:Tetrahydrouridine; D012313:RNA; D004247:DNA; D009700:Nucleoside Deaminases; D003564:Cytidine Deaminase; C011530:deoxycytidine deaminase; C007746:5-fluoro-2'-deoxycytidine; D014529:Uridine