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

Abstract

This study was designed to examine the endogenous concentrations of 5,10-methylenetetrahydrofolate ( CH 2 FH 4) in human colorectal adenocarcinoma xenografts, and to determine the ability of other folate derivatives to increase the formation of the ternary covalent complex between CH 2 FH 4, [6- 3 H]-5-fluorodeoxyuridylate (FdUMP) and thymidylate synthetase (TS, EC 2.1.1.45). Levels of CH 2 FH 4 were determined by measuring the release of [ 3H] 2O from [5- 3 H]-dUMP using TS from Lactobacillus casei. The reaction was linear from 1.9 × 10 −13 to 2.4 × 10 −11 mol of CH 2 FH 4 assayed. Concentrations of CH 2 FH 4 were low, ranging from 66 to 233 nM in cell water. Tetrahydrofolate ( FH 4) and dihydrofolate ( FH 2) increased complex formation, while 5-formyltetrahydrofolate (5- CHOFH 4) and 5-methyltetrahydrofolate (5- CH 3 FH 4) decreased the covalent binding of [6- 3 H]-FdUMP in vitro. Administration of FH 4 or FH 2 to tumor-bearing mice reduced subsequent formation of the covalent complex in vitro. Since 5- CH 3 FH 4 is a major derivative of folate in mammalian tissues, its effect on the covalent binding of [6- 3 H]-FdUMP was examined further; even in the presence of homocysteine and cyanocobalamin (B 12), the formation of the covalent complex was not increased. The fate of [5- 14 CH 3]- FH 4 was subsequently examined in vivo. In tumors at 1 hr after injection, 72% of the radiolabel remained as [5- 14 CH 3]- FH 4, while 17% had been converted to [ 14 C]-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- 14 CH 3]- FH 4 was decreased by over 60%, while metabolism was increased by only 13%. No polyglutamate forms of [5- 14 CH 3]- FH 4 were detected in tumors at 4 hr after treatment.