Mechanisms of Acquired Resistance to Thymidylate Synthase Inhibitors: The Role of Enzyme Stability

• Published in: Molecular pharmacology 1999-11, Vol.56 (5), p.1063-1070
• Main Authors: Kitchens, M E, Forsthoefel, A M, Barbour, K W, Spencer, H T, Berger, F G
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Antineoplastic Agents - pharmacology; Cell Line; Colonic Neoplasms - drug therapy; Colonic Neoplasms - enzymology; Drug Resistance, Neoplasm - genetics; Enzyme Inhibitors - pharmacology; Enzyme Stability; Fluorodeoxyuridylate - pharmacology; Fluorouracil - pharmacology; Folic Acid Antagonists - pharmacology; Half-Life; Humans; Peptides - isolation & purification; RNA, Messenger - biosynthesis; Thymidylate Synthase - antagonists & inhibitors; Thymidylate Synthase - genetics; Thymidylate Synthase - metabolism; Tumor Cells, Cultured
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.56.5.1063

Inhibitors of the enzyme thymidylate synthase (TS), such as the fluoropyrimidines 5-fluorouracil and 5′-fluoro-2′-deoxyuridine (FdUrd) or the antifolates AG337, ZD1694, and BW1843U89, are widely used in the chemotherapy of cancer, particularly cancer of the colon and rectum. Numerous studies have shown 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 observed, the increases in mRNA and enzyme were strikingly discordant. In one drug-resistant line, a deficiency of enzyme relative to mRNA was shown to be 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 residue 303 of the TS polypeptide. The mutant enzyme conferred resistance to FdUrd as well as antifolates in transfected cells. In another FdUrd-resistant line, which had an excess of enzyme relative to mRNA, the TS molecule was more stable than in the parent line. However, no amino acid substitutions were detected in the TS polypeptide from this line, which suggests that the stabilization must be caused by changes in one or more cellular factors that regulate TS degradation. The results indicate that changes in the stability of the TS polypeptide accompany, and even contribute to, acquired resistance to TS inhibitors in colon tumor cells.