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 X 10(-13) to 2.4 X 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.
D000230:Adenocarcinoma / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D003110:Colonic Neoplasms / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D005260:Female; D005468:Fluorodeoxyuridylate / Q000378:metabolism*; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D006710:Homocysteine / Q000494:pharmacology; D006801:Humans; D008780:Methyltransferases / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D009368:Neoplasm Transplantation; D012004:Rectal Neoplasms / Q000378:metabolism*; D013763:Tetrahydrofolates / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000378:metabolism*; D014183:Transplantation, Heterologous; D014805:Vitamin B 12 / Q000494:pharmacology
D003856:Deoxyuracil Nucleotides; D013763:Tetrahydrofolates; D006710:Homocysteine; C013123:5,10-methylenetetrahydrofolic acid; D005468:Fluorodeoxyuridylate; D005492:Folic Acid; D008780:Methyltransferases; D013940:Thymidylate Synthase; D014805:Vitamin B 12