title: A proposed clinical test for monitoring fluoropyrimidine therapy: detection and stability of thymidylate synthase ternary complexes.
abstract: 5-fluorouracil forms classic (covalent, ternary) complexes consisting of thymidylate synthase, fluoro-deoxyuridine monophosphate, and 5,10-methylene tetrahydrofolate. Despite a high pharmacologic interest in the classic complexes formed in cells treated with fluorouracil anticancer agents, the in vivo stability of the complexes and the possible interference in complex formation by other coadministered compounds have not been adequately described. We visualized classic complexes unaccompanied by unbound thymidylate synthase, inferring complete enzymatic inhibition, in 5-fluorouracil-treated S. cerevisiae and cancer cells in vitro and in murine tumors in vivo treated with 5-fluorouracil. Classic complexes persisted 13 days in cancer cells after a pulse of 5-fluorouracil. Classic complexes were reduced to absent in cancer cells in which the older antifolates methotrexate and aminopterin, or the modern antifolates pemetrexed and tomudex, were coadministered with 5-fluorouracil. Classic complexes were, however, detected when an alternate drug, 5-fluorodeoxyuridine, was administered with methotrexate. We visualized classic complexes at fifteen minutes to seven days after an acute single dose of 5-fluorouracil in mouse tumor models, in tumors and normal tissues. Using the same assay, we detected unbound thymidylate synthase in untreated human tissues, supporting the future use of this assay in evaluating the most appropriate dose of fluoropyrimidine and coadministered agents in clinical settings.
authors.affiliation: Department of Oncology, The Sol Goldman Pancreatic Cancer Research Center and The Sidney Kimmel Comprehensive Cancer Center at The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Brody
authors.forename: Eike
authors.initials: E
authors.lastname: Gallmeier
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yoshimura
authors.forename: Tomas
authors.initials: T
authors.lastname: Hucl
authors.forename: Peter
authors.initials: P
authors.lastname: Kulesza
authors.forename: Marcia I
authors.initials: MI
authors.lastname: Canto
authors.forename: Ralph H
authors.initials: RH
authors.lastname: Hruban
authors.forename: Richard D
authors.initials: RD
authors.lastname: Schulick
authors.forename: Scott E
authors.initials: SE
authors.lastname: Kern
mesh_terms: D000818:Animals; D005260:Female; D005472:Fluorouracil / Q000031:analogs & derivatives / Q000627:therapeutic use*; D005493:Folic Acid Antagonists / Q000627:therapeutic use; D006801:Humans; D008727:Methotrexate / Q000627:therapeutic use; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000254:growth & development; D013763:Tetrahydrofolates / Q000378:metabolism; D013940:Thymidylate Synthase / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays / Q000379:methods*
chemical_list: D005493:Folic Acid Antagonists; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D013940:Thymidylate Synthase; D005472:Fluorouracil; D008727:Methotrexate