The effect of derivatives of folic acid on the fluorodeoxyuridylate-thymidylate synthetase covalent complex in human colon xenografts

This study was designed to examine the endogenous concentrations of 5,10-methylenetetrahydrofolate (CH₂FH₄) in human colorectal adenocarcinoma xenografts, and to determine the ability of other folate derivatives to increase the formation of the ternary covalent complex between CH₂FH₄, [6-³H]-5-fluorodeoxyuridylate (FdUMP) and thymidylate synthetase (TS, EC 2.1.1.45). Levels of CH₂FH₄ were determined by measuring the release of [³H]₂O from [5-³H]-dUMP using TS from Lactobacillus casei. The reaction was linear from 1.9 × 10^-13 to 2.4 × 10^-11 mol of CH₂FH₄ assayed. Concentrations of CH₂FH₄ were low, ranging from 66 to 233 nM in cell water. Tetrahydrofolate (FH₄) and dihydrofolate (FH₂) increased complex formation, while 5-formyltetrahydrofolate (5-CHOFH₄) and 5-methyltetrahydrofolate (5-CH₃FH₄) decreased the covalent binding of [6-³H]-FdUMP in vitro. Administration of FH₄ or FH₂ to tumor-bearing mice reduced subsequent formation of the covalent complex in vitro. Since 5-CH₃FH₄ is a major derivative of folate in mammalian tissues, its effect on the covalent binding of [6-³H]-FdUMP was examined further; even in the presence of homocysteine and cyanocobalamin (B₁₂), the formation of the covalent complex was not increased. The fate of [5-¹⁴CH₃]-FH₄ was subsequently examined in vivo. In tumors at 1 hr after injection, 72% of the radiolabel remained as [5-¹⁴CH₃]-FH₄, while 17% had been converted to [¹⁴C]-methionine or incorporated into protein. By contrast, however, the incorporation of radiolabel into the protein fraction of liver was almost 30-fold greater at this time. At 4 hr, radioactivity in tumors (dpm/g) and in the fraction associated with [5-¹⁴CH₃]-FH₄ was decreased by over 60%, while metabolism was increased by only 13%. No polyglutamate forms of [5-¹⁴CH₃]-FH₄ were detected in tumors at 4 hr after treatment.