A naturally occurring variation in thymidylate synthase structure is associated with a reduced response to 5-fluoro-2'-deoxyuridine in a human colon tumor cell line

Inhibition of thymidylate synthase (TS) is an important mechanism of action of fluoropyrimidine antimetabolites. Thus, TS structure and expression are expected to be determinants of response to these agents. The role of TS in fluoropyrimidine response has been analyzed in a panel of human colonic tumor cell lines. Previous work has demonstrated that there is little correlation between TS concentration and sensitivity to 5-fluoro-2'-deoxyuridine (FdUrd) among these cell lines, suggesting that parameters other than the TS levels are responsible for the variations in drug response. One such parameter has been identified in cell line HCT 116. This line, which is relatively resistant to FdUrd, produces two structural forms of TS, as determined by mobility of the enzyme in isoelectric focusing polyacrylamide gels. One form is common to all the cell lines, whereas a variant form, which is more basic and is encoded by a separate structural gene, is unique to HCT 116. Cells expressing one or the other TS form have been isolated and used to demonstrate that the variant form is associated with FdUrd resistance. Kinetic experiments indicate that the variant TS has reduced affinities for 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate, which are ligands involved in formation of a stable inhibitory complex with the enzyme. Thus, the innate resistance of cell line HCT 116 to FdUrd is derived, at least in part, from production of an altered structural form of TS having reduced affinity for ligands.