Biochemical determinants of tumor sensitivity to 5-fluorouracil: Ultrasensitive methods for the determination of 5-fluoro-2′-deoxyuridylate, 2′-deoxyuridylate, and thymidylate synthetase

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Abstract
Techniques were developed to measure FdUMP [5-fluro-2''-deoxyuridylate], the active metabolite of 5-FUra [5-flurouracil]; thymidylate synthetase (TMP synthase; 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45), the target enzyme for this antimetabolite; and dUMP [2''-deoxyuridylate], the substrate the competes with FdUMP for binding to TMP synthetase. As little as 0.02 pmol of FdUMP can be quantitated with a competitive ligand binding assay by using homogeneous Lactobacillus casei/MTX [methotrexate resistant] TMP synthetase as a binding protein. A new binding assay for TMP synthetase allows detection of 0.005 pmol of enzyme. The quantitative enzymatic conversion of dUMP to [methyl-14C]-TMP using 5,10-methylene[14C]tetrahydrofolate by pure L. casei TMP synthetase is used as an assay for dUMP with a sensitivity of 10 pmol. Cultured CCRF-CEM human lymphoblastic leukemia cells formed high levels of FdUMP (2.6 nmol/109 cells) within 11 h after exposure to 30 .mu.M 5-FUra. Tumor cell TMP synthetase levels dropped, and then free FdUMP appeared. The intracellular dUMP pool was low (2-5 nmol per 109 cells) in logarithmically growing cultures of several tumor cell lines but expanded rapidly in CCRF-CEM cells on exposure to 5-FUra after enzyme levels decreased. The levels of dUMP found after exposure to 5-FUra are sufficient to severely retard inhibition of TMP synthetase by FdUMP. The methods described are sufficiently sensitive to allow these biochemical parameters of 5-FUra action to be measured in cell culture or in needle biopsy samples of human tumors.