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. 1980 Oct;77(10):5663–5667. doi: 10.1073/pnas.77.10.5663

5-fluorouracil-methotrexate synergy: enhancement of 5-fluorodeoxyridylate binding to thymidylate synthase by dihydropteroylpolyglutamates.

D J Fernandes, J R Bertino
PMCID: PMC350129  PMID: 6160578

Abstract

Ternary complex formation of thymidylate synthase (5,10-methylenetetrahydrofolated:dUMP C-methyltransferase, EC 2.1.1.45), 5-fluorodeoxyuridylate (FdUMP), and poly(gamma-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(gamma-glutamate)s within 4 hr of exposure to 1 microM MTX. MTX and MTX(gamma-glutamate) formed reversible ternary complexes with FdUMP on one site of thymidylate synthase, whereas with 7,8-dihydropteroylpentaglutamate and I-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(gamma-glutamate), 7,8-dihydropteroylpentaglutamate, and I-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 dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) and consequently a trapping of intracellular folates as dihydropteroylpolyglutamates, which increase the extent of FdUMP binding to thymidylate synthase.

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Selected References

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  1. BLAKLEY R. L. The interconversion of serine and glycine; preparation and properties of catalytic derivatives of pteroylglutamic acid. Biochem J. 1957 Feb;65(2):331–342. doi: 10.1042/bj0650331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baugh C. M., Krumdieck C. L., Nair M. G. Polygammaglutamyl metabolites of methotrexate. Biochem Biophys Res Commun. 1973 May 1;52(1):27–34. doi: 10.1016/0006-291x(73)90949-2. [DOI] [PubMed] [Google Scholar]
  3. Bertino J. R., Sawicki W. L., Lindquist C. A., Gupta V. S. Schedule-dependent antitumor effects of methotrexate and 5-fluorouracil. Cancer Res. 1977 Jan;37(1):327–328. [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Brown I., Ward H. W. Therapeutic consequences of antitumour drug interactions: methotrexate and 5-fluorouracil in the chemotherapy of C3H mice with transplanted mammary adenocarcinoma. Cancer Lett. 1978 Nov;5(5):291–297. doi: 10.1016/s0304-3835(78)80027-5. [DOI] [PubMed] [Google Scholar]
  6. Cadman E., Heimer R., Davis L. Enhanced 5-fluorouracil nucleotide formation after methotrexate administration: explanation for drug synergism. Science. 1979 Sep 14;205(4411):1135–1137. doi: 10.1126/science.472732. [DOI] [PubMed] [Google Scholar]
  7. Cashmore A. R., Dreyer R. N., Horváth C., Knipe J. O., Coward J. K., Bertino J. R. Separation of pteroyl-oligo-gamma-L-glutamates by high-performance liquid-chromatography. Methods Enzymol. 1980;66:459–468. doi: 10.1016/0076-6879(80)66489-1. [DOI] [PubMed] [Google Scholar]
  8. Chu M. Y., Fischer G. A. The incorporation of 3H-cytosine arabinoside and its effect on murine leukemic cells (L5178Y). Biochem Pharmacol. 1968 May;17(5):753–767. doi: 10.1016/0006-2952(68)90012-9. [DOI] [PubMed] [Google Scholar]
  9. Coward J. K., Parameswaran K. N., Cashmore A. R., Bertino J. R. 7,8-Dihydropteroyl oligo-gamma-L-glutamates: synthesis and kinetic studies with purified dihydrofolate reductase from mammalian sources. Biochemistry. 1974 Sep 10;13(19):3899–3903. doi: 10.1021/bi00716a013. [DOI] [PubMed] [Google Scholar]
  10. Curthoys N. P., Rabinowitz J. C. Formyltetrahydrofolate synthetase. Binding of adenosine triphosphate and related ligands determined by partition equilibrium. J Biol Chem. 1971 Nov 25;246(22):6942–6952. [PubMed] [Google Scholar]
  11. Danenberg P. V., Langenbach R. J., Heidelberger C. Structures of reversible and irreversible complexes of thymidylate synthetase and fluorinated pyrimidine nucleotides. Biochemistry. 1974 Feb 26;13(5):926–933. doi: 10.1021/bi00702a016. [DOI] [PubMed] [Google Scholar]
  12. Dolnick B. J., Cheng Y. C. Human thymidylate synthetase. II. Derivatives of pteroylmono- and -polyglutamates as substrates and inhibitors. J Biol Chem. 1978 May 25;253(10):3563–3567. [PubMed] [Google Scholar]
  13. Dolnick B. J., Cheng Y. c. Human thymidylate synthetase derived from blast cells of patients with acture myelocytic leukemia. Purification and chracterization. J Biol Chem. 1977 Nov 10;252(21):7697–7703. [PubMed] [Google Scholar]
  14. Friedkin M., Plante L. T., Crawford E. J., Crumm M. Inhibition of thymidylate synthetase and dihydrofolate reductase by naturally occurring oligoglutamate derivatives of folic acid. J Biol Chem. 1975 Jul 25;250(14):5614–5621. [PubMed] [Google Scholar]
  15. Galivan J. H., Maley F., Baugh C. M. Demonstration of separate binding sites for the folate coenzymes and deoxynucleotides with inactivated Lactobacillus casei thymidylate synthetase. Biochem Biophys Res Commun. 1976 Jul 26;71(2):527–534. doi: 10.1016/0006-291x(76)90819-6. [DOI] [PubMed] [Google Scholar]
  16. Galivan J. H., Maley G. F., Maley F. Factors affecting substrate binding in Lactobacillus casei thymidylate synthetase as studied by equilibrium dialysis. Biochemistry. 1976 Jan 27;15(2):356–362. doi: 10.1021/bi00647a018. [DOI] [PubMed] [Google Scholar]
  17. Galivan J., Maley G. F., Maley F. Purification and properties of T2 bacteriophage-induced thymidylate synthetase. Biochemistry. 1974 May 21;13(11):2282–2289. doi: 10.1021/bi00708a007. [DOI] [PubMed] [Google Scholar]
  18. Gewirtz D. A., White J. C., Randolph J. K., Goldman I. D. Formation of methotrexate polyglutamates in rat hepatocytes. Cancer Res. 1979 Aug;39(8):2914–2918. [PubMed] [Google Scholar]
  19. Houlihan C. M., Scott J. M. The identification of pteroylpentaglutamate as the major folate derivative in rat liver and the demonstration of its biosynthesis from exogenous ( 3 H) pteroylglutamate. Biochem Biophys Res Commun. 1972 Sep 26;48(6):1675–1681. doi: 10.1016/0006-291x(72)90908-4. [DOI] [PubMed] [Google Scholar]
  20. Jackson R. C., Harrap K. R. Studies with a mathematical model of folate metabolism. Arch Biochem Biophys. 1973 Oct;158(2):827–841. doi: 10.1016/0003-9861(73)90579-1. [DOI] [PubMed] [Google Scholar]
  21. Jacobs S. A., Derr C. J., Johns D. G. Accumulation of methotrexate diglutamate in human liver during methotrexate therapy. Biochem Pharmacol. 1977 Dec 1;26(23):2310–2313. doi: 10.1016/0006-2952(77)90296-9. [DOI] [PubMed] [Google Scholar]
  22. Kamen B. A., Cashmore A. R., Dreyer R. N., Moroson B. A., Hsieh P., Bertino J. R. Effect of [3H]methotrexate impurities on apparent transport of methotrexate by a sensitive and resistant L1210 cell line. J Biol Chem. 1980 Apr 25;255(8):3254–3257. [PubMed] [Google Scholar]
  23. Kisliuk R. L., Gaumont Y., Baugh C. M. Polyglutamyl derivatives of folate as substrates and inhibitors of thymidylate synthetase. J Biol Chem. 1974 Jul 10;249(13):4100–4103. [PubMed] [Google Scholar]
  24. Klubes P., Connelly K., Cerna I., Mandel H. G. Effects of 5-fluorouracil on 5-fluorodeoxyuridine 5'-monophosphate and 2-deoxyuridine 5'-monophosphate pools, and DNA synthesis in solid mouse L1210 and rat Walker 256 tumors. Cancer Res. 1978 Aug;38(8):2325–2331. [PubMed] [Google Scholar]
  25. Lomax M. I., Greenberg G. R. A new assay of thymidylate synthetase activity based on the release of tritium from deoxyuridylate-5-3-H. J Biol Chem. 1967 Jan 10;242(1):109–113. [PubMed] [Google Scholar]
  26. Matthews R. G., Haywood B. J. Inhibition of pig liver methylenetetrahydrofolate reductase by dihydrofolate: some mechanistic and regulatory implications. Biochemistry. 1979 Oct 30;18(22):4845–4851. doi: 10.1021/bi00589a012. [DOI] [PubMed] [Google Scholar]
  27. Moran R. G., Mulkins M., Heidelberger C. Role of thymidylate synthetase activity in development of methotrexate cytotoxicity. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5924–5928. doi: 10.1073/pnas.76.11.5924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Moran R. G., Werkheiser W. C., Zakrzewski S. F. Folate metabolism in mammalian cells in culture. I Partial characterization of the folate derivatives present in L1210 mouse leukemia cells. J Biol Chem. 1976 Jun 25;251(12):3569–3575. [PubMed] [Google Scholar]
  29. Myers C. E., Young R. C., Chabner B. A. Biochemical determinants of 5-fluorouracil response in vivo. The role of deoxyuridylate pool expansion. J Clin Invest. 1975 Nov;56(5):1231–1238. doi: 10.1172/JCI108199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nixon P. F., Slutsky G., Nahas A., Bertino J. R. The turnover of folate coenzymes in murine lymphoma cells. J Biol Chem. 1973 Sep 10;248(17):5932–5936. [PubMed] [Google Scholar]
  31. Rode W., Scanlon K. J., Hynes J., Bertino J. R. Purification of mammalian tumor (L1210) thymidylate synthetase by affinity chromatography on stable biospecific adsorbent. Stabilization of the enzyme with neutral detergents. J Biol Chem. 1979 Nov 25;254(22):11538–11543. [PubMed] [Google Scholar]
  32. Rosenblatt D. S., Whitehead V. M., Dupont M. M., Vuchich M. J., Vera N. Synthesis of methotrexate polyglutamates in cultured human cells. Mol Pharmacol. 1978 Jan;14(1):210–214. [PubMed] [Google Scholar]
  33. Santi D. V., McHenry C. S., Sommer H. Mechanism of interaction of thymidylate synthetase with 5-fluorodeoxyuridylate. Biochemistry. 1974 Jan 29;13(3):471–481. doi: 10.1021/bi00700a012. [DOI] [PubMed] [Google Scholar]
  34. Shin Y. S., Williams M. A., Stokstad E. L. Identification of folic acid compounds in rat liver. Biochem Biophys Res Commun. 1972 Apr 14;47(1):35–43. doi: 10.1016/s0006-291x(72)80006-8. [DOI] [PubMed] [Google Scholar]
  35. Ullman B., Lee M., Martin D. W., Jr, Santi D. V. Cytotoxicity of 5-fluoro-2'-deoxyuridine: requirement for reduced folate cofactors and antagonism by methotrexate. Proc Natl Acad Sci U S A. 1978 Feb;75(2):980–983. doi: 10.1073/pnas.75.2.980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Washtien W. L., Santi D. V. Assay of intracellular free and macromolecular-bound metabolites of 5-fluorodeoxyuridine and 5-fluorouracil. Cancer Res. 1979 Sep;39(9):3397–3404. [PubMed] [Google Scholar]
  37. White J. C., Goldman I. D. Mechanism of action of methotrexate. IV. Free intracellular methotrexate required to suppress dihydrofolate reduction to tetrahydrofolate by Ehrlich ascites tumor cells in vitro. Mol Pharmacol. 1976 Sep;12(5):711–719. [PubMed] [Google Scholar]

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