VARIATION IN HUMAN THYMIDYLATE SYNTHASE IS ASSOCIATED WITH RESISTANCETO 5-FLUORO-2'-DEOXYURIDINE

Two human colorectal tumor cell lines are differentially sensitive to growth inhibition by 5-fluorodeoxyuridine (FdUrd); cell line RCA is le ss sensitive to FdUrd than is cell line C. Thymidylate synthase (TS), a target of FdUrd, has been purified to homogeneity from both cell lin es. Because of differences in the avidity for a folate ligand affinity matrix, TS forms from the cells were purified by two different proced ures. Relative to the enzyme from C cells, the enzyme from RCA cells d emonstrated higher K(m) values for the substrates deoxyuridylate and 5 ,10-methylenetetrahydrofolate, a lower rate of association of the inhi bitor 5-fluorodeoxyuridylate (FdUMP), a similar rate of FdUMP dissocia tion, and lower enhancement of covalent FdUMP binding by folate deriva tives. The activities of the enzymes in situ and the catalytic efficie ncies of the purified enzymes were similar. Thus, a cell line that is naturally resistant to FdUrd has been identified that expresses a TS w ith reduced affinity for FdUMP and 5,10-methylenetetrahydrofolate, rel ative to the enzyme expressed in a FdUrd-sensitive cell line.