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: (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.