Mechanisms of acquired resistance to thymidylate synthase inhibitors: The role of enzyme stability

Inhibitors of the enzyme thymidylate synthase (TS), such as the fluoropyrim idines 5-fluorouracil and 5'-fluoro-2'-deoxyuridine (FdUrd) or the antifola tes AG337, ZD1694, and BW1843U89, are widely used in the chemotherapy of ca ncer, particularly cancer of the colon and rectum. Numerous studies have sh own that TS gene amplification, leading to mRNA and enzyme overproduction, is a major mechanism of resistance to these inhibitors. In the present work , we have isolated and characterized FdUrd-resistant derivatives of several human colon tumor cell lines. Although gene amplification was commonly obs erved, the increases in mRNA and enzyme were strikingly discordant. In one drug-resistant line, a deficiency of enzyme relative to mRNA was shown to b e caused by expression of a metabolically unstable TS molecule. The reduced half-life of TS in this line was caused by a Pro-to-Leu substitution at re sidue 303 of the TS polypeptide. The mutant enzyme conferred resistance to FdUrd as well as antifolates in transfected cells. In another FdUrd-resista nt line, which had an excess of enzyme relative to mRNA, the TS molecule wa s more stable than in the parent line. However, no amino acid substitutions were detected in the TS polypeptide from this line, which suggests that th e stabilization must be caused by changes in one or more cellular factors t hat regulate TS degradation. The results indicate that changes in the stabi lity of the TS polypeptide accompany, and even contribute to, acquired resi stance to TS inhibitors in colon tumor cells.