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. 1979 Mar;76(3):1456–1460. doi: 10.1073/pnas.76.3.1456

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

Richard G Moran 1,2,3,*, C Paul Spears 1,2,3, Charles Heidelberger 1,2,3,†
PMCID: PMC383271  PMID: 108681

Abstract

Techniques have been developed to measure FdUMP, the active metabolite of 5-FUra; thymidylate synthetase (TMP synthase; 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45), the target enzyme for this antimetabolite; and dUMP, the substrate that 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 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 per 109 cells) within 11 hr after exposure to 30 μM 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.

Keywords: competitive ligand binding assay, cancer chemotherapy, fluorinated pyrimidines, stoichiometric binding

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Selected References

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