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. 2000 Nov 22;83(11):1510–1515. doi: 10.1054/bjoc.2000.1456

Prolonged and enhanced suppression of thymidylate synthase by weekly 24-h infusion of high-dose 5-fluorouracil

K-H Yeh 1,3, S-H Yeh 3, C-H Hsu 1,3, T-M Wang 3, I-F Ma 3, A-L Cheng 1,2,3
PMCID: PMC2363429  PMID: 11076661

Abstract

We have recently demonstrated that HDFL (high-dose 5-FU 2600 mg m–2 week–1 and leucovorin 500 mg m–2 week–1, weekly 24-h infusion) is highly active in the treatment of gastric cancer. To further clarify the possible mechanism underlying the improved activity of HDFL compared with conventional 5-FU regimens, we conducted in vitro studies examining the effect of these regimens on the differential regulation of thymidylate synthase (TS) in NCI-N87, a human gastric cancer cell line. The expected serum concentrations of 5-FU are 100–200 mM (lasting for less than 30 min) and 5–10 mM (lasting for 24 h) for the conventional 5-FU regimens (bolus injection or short intravenous infusion of 5-FU 370–500 mg m–2) and the HDFL regimens, respectively. Western blot analysis revealed that 24-h exposure of NCI-N87 to 2.5–10.0 mM of 5-FU resulted in a dose-dependent depletion of free TS, lasting for more than 24 h. In contrast, 30-min exposure of NCI-N87 to 200 mM of 5-FU resulted in a less than 12-h depletion of free TS. Moreover, 24-h exposure to 5-FU resulted in a higher S-phase blockade and enhanced cytotoxicity. In both modes of 5-FU treatment, the initial rapid depletion of free TS was accompanied by a rapid increment of a higher-molecular-weight TS molecule, suggesting that rapid formation of the ternary complex was the key mechanism of 5-FU action during this period. Northern blot analysis showed that the steady-state mRNA of TS was not affected by either of the schedules. We conclude that 24-h exposure of gastric cancer cells to low concentration of 5-FU resulted in better suppression of free TS, a higher degree of S-phase blockade, and enhanced cytotoxicity compared to 30-min exposure to high concentration of 5-FU. These in vitro results may help explain the improved clinical efficacy of HDFL regimens compared to conventional 5-FU regimens. © 2000 Cancer Research Campaign http://www.bjcancer.com

Keywords: thymidylate synthase suppression, high-dose 5-fluorouracil

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Selected References

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  1. Ardalan B., Chua L., Tian E. M., Reddy R., Sridhar K., Benedetto P., Richman S., Legaspi A., Waldman S., Morrell L. A phase II study of weekly 24-hour infusion with high-dose fluorouracil with leucovorin in colorectal carcinoma. J Clin Oncol. 1991 Apr;9(4):625–630. doi: 10.1200/JCO.1991.9.4.625. [DOI] [PubMed] [Google Scholar]
  2. Banerjee D., Schnieders B., Fu J. Z., Adhikari D., Zhao S. C., Bertino J. R. Role of E2F-1 in chemosensitivity. Cancer Res. 1998 Oct 1;58(19):4292–4296. [PubMed] [Google Scholar]
  3. Beerblock K., Rinaldi Y., André T., Louvet C., Raymond E., Tournigand C., Carola E., Favre R., de Gramont A., Krulik M. Bimonthly high dose leucovorin and 5-fluorouracil 48-hour continuous infusion in patients with advanced colorectal carcinoma. Groupe d'Etude et de Recherche sur les Cancers de l'Ovaire et Digestifs (GERCOD). Cancer. 1997 Mar 15;79(6):1100–1105. [PubMed] [Google Scholar]
  4. Cheng A. L., Yeh K. H., Lin J. T., Hsu C., Liu M. Y. Cisplatin, etoposide, and weekly high-dose 5-fluorouracil and leucovorin infusion (PE-HDFL)--a very effective regimen with good patients' compliance for advanced gastric cancer. Anticancer Res. 1998 Mar-Apr;18(2B):1267–1272. [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Chu E., Allegra C. J. The role of thymidylate synthase in cellular regulation. Adv Enzyme Regul. 1996;36:143–163. doi: 10.1016/0065-2571(95)00004-6. [DOI] [PubMed] [Google Scholar]
  7. Chu E., Drake J. C., Koeller D. M., Zinn S., Jamis-Dow C. A., Yeh G. C., Allegra C. J. Induction of thymidylate synthase associated with multidrug resistance in human breast and colon cancer cell lines. Mol Pharmacol. 1991 Feb;39(2):136–143. [PubMed] [Google Scholar]
  8. Chu E., Koeller D. M., Casey J. L., Drake J. C., Chabner B. A., Elwood P. C., Zinn S., Allegra C. J. Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8977–8981. doi: 10.1073/pnas.88.20.8977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dolnick B. J., Black A. R., Winkler P. M., Schindler K., Hsueh C. T. rTS gene expression is associated with altered cell sensitivity to thymidylate synthase inhibitors. Adv Enzyme Regul. 1996;36:165–180. doi: 10.1016/0065-2571(95)00009-7. [DOI] [PubMed] [Google Scholar]
  10. Fraile R. J., Baker L. H., Buroker T. R., Horwitz J., Vaitkevicius V. K. Pharmacokinetics of 5-fluorouracil administered orally, by rapid intravenous and by slow infusion. Cancer Res. 1980 Jul;40(7):2223–2228. [PubMed] [Google Scholar]
  11. Grem J. L., McAtee N., Murphy R. F., Balis F. M., Steinberg S. M., Hamilton J. M., Sorensen J. M., Sartor O., Kramer B. S., Goldstein L. J. A pilot study of interferon alfa-2a in combination with fluorouracil plus high-dose leucovorin in metastatic gastrointestinal carcinoma. J Clin Oncol. 1991 Oct;9(10):1811–1820. doi: 10.1200/JCO.1991.9.10.1811. [DOI] [PubMed] [Google Scholar]
  12. Hsu C. H., Yeh K. H., Chen L. T., Liu J. M., Jan C. M., Lin J. T., Chen Y. C., Cheng A. L. Weekly 24-hour infusion of high-dose 5-fluorouracil and leucovorin in the treatment of advanced gastric cancers. An effective and low-toxic regimen for patients with poor general condition. Oncology. 1997 Jul-Aug;54(4):275–280. doi: 10.1159/000227702. [DOI] [PubMed] [Google Scholar]
  13. Johnston P. G., Drake J. C., Trepel J., Allegra C. J. Immunological quantitation of thymidylate synthase using the monoclonal antibody TS 106 in 5-fluorouracil-sensitive and -resistant human cancer cell lines. Cancer Res. 1992 Aug 15;52(16):4306–4312. [PubMed] [Google Scholar]
  14. Johnston P. G., Fisher E. R., Rockette H. E., Fisher B., Wolmark N., Drake J. C., Chabner B. A., Allegra C. J. The role of thymidylate synthase expression in prognosis and outcome of adjuvant chemotherapy in patients with rectal cancer. J Clin Oncol. 1994 Dec;12(12):2640–2647. doi: 10.1200/JCO.1994.12.12.2640. [DOI] [PubMed] [Google Scholar]
  15. Johnston P. G., Lenz H. J., Leichman C. G., Danenberg K. D., Allegra C. J., Danenberg P. V., Leichman L. Thymidylate synthase gene and protein expression correlate and are associated with response to 5-fluorouracil in human colorectal and gastric tumors. Cancer Res. 1995 Apr 1;55(7):1407–1412. [PubMed] [Google Scholar]
  16. Johnston P. G., Liang C. M., Henry S., Chabner B. A., Allegra C. J. Production and characterization of monoclonal antibodies that localize human thymidylate synthase in the cytoplasm of human cells and tissue. Cancer Res. 1991 Dec 15;51(24):6668–6676. [PubMed] [Google Scholar]
  17. Köhne C. H., Schöffski P., Wilke H., Käufer C., Andreesen R., Ohl U., Klaasen U., Westerhausen M., Hiddemann W., Schott G. Effective biomodulation by leucovorin of high-dose infusion fluorouracil given as a weekly 24-hour infusion: results of a randomized trial in patients with advanced colorectal cancer. J Clin Oncol. 1998 Feb;16(2):418–426. doi: 10.1200/JCO.1998.16.2.418. [DOI] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Lenz H. J., Leichman C. G., Danenberg K. D., Danenberg P. V., Groshen S., Cohen H., Laine L., Crookes P., Silberman H., Baranda J. Thymidylate synthase mRNA level in adenocarcinoma of the stomach: a predictor for primary tumor response and overall survival. J Clin Oncol. 1996 Jan;14(1):176–182. doi: 10.1200/JCO.1996.14.1.176. [DOI] [PubMed] [Google Scholar]
  20. Li W., Fan J., Hochhauser D., Banerjee D., Zielinski Z., Almasan A., Yin Y., Kelly R., Wahl G. M., Bertino J. R. Lack of functional retinoblastoma protein mediates increased resistance to antimetabolites in human sarcoma cell lines. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10436–10440. doi: 10.1073/pnas.92.22.10436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Navalgund L. G., Rossana C., Muench A. J., Johnson L. F. Cell cycle regulation of thymidylate synthetase gene expression in cultured mouse fibroblasts. J Biol Chem. 1980 Aug 10;255(15):7386–7390. [PubMed] [Google Scholar]
  22. Peters G. J., van der Wilt C. L., van Groeningen C. J., Smid K., Meijer S., Pinedo H. M. Thymidylate synthase inhibition after administration of fluorouracil with or without leucovorin in colon cancer patients: implications for treatment with fluorouracil. J Clin Oncol. 1994 Oct;12(10):2035–2042. doi: 10.1200/JCO.1994.12.10.2035. [DOI] [PubMed] [Google Scholar]
  23. Tsujinaka T., Kido Y., Shiozaki H., Iijima S., Homma T., Sakaue M., Mori T. Schedule-dependent inhibition of thymidylate synthase by 5-fluorouracil in gastric cancer. Cancer. 1992 Dec 15;70(12):2761–2765. doi: 10.1002/1097-0142(19921215)70:12<2761::aid-cncr2820701206>3.0.co;2-f. [DOI] [PubMed] [Google Scholar]
  24. Vanhoefer U., Wilke H., Weh H. J., Clemens M., Harstrick A., Stahl M., Hossfeld D. K., Seeber S. Weekly high-dose 5-fluorouracil and folinic acid as salvage treatment in advanced gastric cancer. Ann Oncol. 1994 Nov;5(9):850–851. doi: 10.1093/oxfordjournals.annonc.a059017. [DOI] [PubMed] [Google Scholar]
  25. Weh H. J., Wilke H. J., Dierlamm J., Klaassen U., Siegmund R., Illiger H. J., Schalhorn A., Kreuser E. D., Hilgenfeld U., Steinke B. Weekly therapy with folinic acid (FA) and high-dose 5-fluorouracil (5-FU) 24-hour infusion in pretreated patients with metastatic colorectal carcinoma. A multicenter study by the Association of Medical Oncology of the German Cancer Society (AIO). Ann Oncol. 1994 Mar;5(3):233–237. doi: 10.1093/oxfordjournals.annonc.a058799. [DOI] [PubMed] [Google Scholar]
  26. Wilke H., Klaassen U., Achterrath W., Losch M., Vanhoefer U., Hayungs J., Harstrick A., Stahl M., Eberhardt W., Becher R. Phase I/II study with a weekly 24-hour infusion of 5-fluorouracil plus high-dose folinic acid (HD-FU/FA) in intensively pretreated patients with metastatic breast cancer. Ann Oncol. 1996 Jan;7(1):55–58. doi: 10.1093/oxfordjournals.annonc.a010478. [DOI] [PubMed] [Google Scholar]
  27. Yeh K. H., Chen Y. C., Yeh S. H., Chen C. P., Lin J. T., Cheng A. L. Detection of circulating cancer cells by nested reverse transcription-polymerase chain reaction of cytokeratin-19 (K19)--possible clinical significance in advanced gastric cancer. Anticancer Res. 1998 Mar-Apr;18(2B):1283–1286. [PubMed] [Google Scholar]
  28. Yeh K. H., Cheng A. L. An alternative method to overcome central venous portable external infusion pump blockage in patients receiving weekly 24-hour high-dose fluorouracil and leucovorin. J Clin Oncol. 1994 Apr;12(4):875–876. doi: 10.1200/JCO.1994.12.4.875. [DOI] [PubMed] [Google Scholar]
  29. Yeh K. H., Cheng A. L. Gastric cancer associated with acute disseminated intravascular coagulation: successful initial treatment with weekly 24-hour infusion of high-dose 5-fluorouracil and leucovorin. Br J Haematol. 1998 Mar;100(4):769–772. doi: 10.1046/j.1365-2141.1998.00613.x. [DOI] [PubMed] [Google Scholar]
  30. Yeh K. H., Cheng A. L., Lin M. T., Hong R. L., Hsu C. H., Lin J. F., Chang K. J., Lee P. H., Chen Y. C. A phase II study of weekly 24-hour infusion of high-dose 5-fluorouracil and leucovorin (HDFL) in the treatment of recurrent or metastatic colorectal cancers. Anticancer Res. 1997 Sep-Oct;17(5B):3867–3871. [PubMed] [Google Scholar]
  31. Yeh K. H., Shun C. T., Chen C. L., Lin J. T., Lee W. J., Lee P. H., Chen Y. C., Cheng A. L. High expression of thymidylate synthase is associated with the drug resistance of gastric carcinoma to high dose 5-fluorouracil-based systemic chemotherapy. Cancer. 1998 May 1;82(9):1626–1631. doi: 10.1002/(sici)1097-0142(19980501)82:9<1626::aid-cncr5>3.0.co;2-8. [DOI] [PubMed] [Google Scholar]
  32. de Gramont A., Bosset J. F., Milan C., Rougier P., Bouché O., Etienne P. L., Morvan F., Louvet C., Guillot T., François E. Randomized trial comparing monthly low-dose leucovorin and fluorouracil bolus with bimonthly high-dose leucovorin and fluorouracil bolus plus continuous infusion for advanced colorectal cancer: a French intergroup study. J Clin Oncol. 1997 Feb;15(2):808–815. doi: 10.1200/JCO.1997.15.2.808. [DOI] [PubMed] [Google Scholar]
  33. de Gramont A., Louvet C., André T., Tournigand C., Krulik M. A review of GERCOD trials of bimonthly leucovorin plus 5-fluorouracil 48-h continuous infusion in advanced colorectal cancer: evolution of a regimen. Groupe d'Etude et de Recherche sur les Cancers de l'Ovaire et Digestifs (GERCOD). Eur J Cancer. 1998 Apr;34(5):619–626. doi: 10.1016/s0959-8049(97)00364-x. [DOI] [PubMed] [Google Scholar]

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