5-Fluorouracil-methotrexate synergy: Enhancement of 5-fluorodeoxyuridylate binding to thymidylate synthase by dihydropteroylpolyglutamates

D. J. Fernandes, J. R. Bertino

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Ternary complex formation of thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC, 5-fluorodeoxyuridylate (FdUMP), and poly(γ-glutamyl) conjugates of pteroate and methotrexate (MTX) has been examined as a basis for the sequence-dependent synergism of the 5-fluorouracil-MTX combination in inhibiting viability of L1210 murine tumor cells. A 1.4-log (25-fold) increase in the inhibition of soft agar colony formation was observed when MTX preceded 5-fluorouracil, as compared to the reverse sequence. L1210 cells converted 39% of the total intracellular MTX into MTX poly(γ-glutamate)s within 4 hr of exposure to 1 μM MTX. MTX and MTX(γ-glutamate) formed reversible ternary complexes with FdUMP on one site of thymidylate synthase, whereas with 7,8-dihydropteroylpentaglutamate and l-5,10-methylenetetrahydropteroylpentaglutamate stoichiometric binding of FdUMP to two sites on thymidylate synthase was observed. The dissociation constants for FdUMP in the ternary complexes formed in the presence of MTX, MTX(γ-glutamate) 7,8-dihydropteroylpentaglutamate, and l-5,10-methylenetetrahydropteroylpentaglutamate were estimated to be 370, 27, <10, and <10 nM, respectively, by equilibrium dialysis. We propose that the sequence-dependent effect of MTX plus 5-fluorouracil on L1210 cell viability results from MTX and MTX polyglutamate inhibition of dihyrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC and consequently a trapping of intracellular folates as dihydropteroylpolyglutamates, which increase the extent of FdUMP binding to thymidylate synthase.

Original languageEnglish (US)
Pages (from-to)5663-5667
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number10 II
StatePublished - Dec 1 1980
Externally publishedYes

All Science Journal Classification (ASJC) codes

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