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. 1994 Dec;70(6):1075–1084. doi: 10.1038/bjc.1994.451

Determinants of trimetrexate lethality in human colon cancer cells.

J L Grem 1, D M Voeller 1, F Geoffroy 1, E Horak 1, P G Johnston 1, C J Allegra 1
PMCID: PMC2033700  PMID: 7981057

Abstract

We examined the cytotoxicity and biochemical effects of the lipophilic antifol trimetrexate (TMQ) in two human colon carcinoma cell lines, SNU-C4 and NCI-H630, with different inherent sensitivity to TMQ. While a 24 h exposure to 0.1 microM TMQ inhibited cell growth by 50-60% in both cell lines, it did not reduce clonogenic survival. A 24 h exposure to 1 and 10 microM TMQ produced 42% and 50% lethality in C4 cells, but did not affect H630 cells. Dihydrofolate reductase (DHFR) and thymidylate synthase were quantitatively and qualitatively similar in both lines. During drug exposure, DHFR catalytic activity was inhibited by > or = 85% in both cell lines; in addition, the reduction in apparent free DHFR binding capacity (< or = 20% of control), depletion of dTTP, ATP and GTP pools and inhibition of [6-3H]deoxyuridine incorporation into DNA were similar in C4 and H630 cells. TMQ produced a more striking alteration of the pH step alkaline elution profile of newly synthesised DNA in C4 cells compared with 630 cells, however, indicating greater interference with DNA chain elongation or more extensive DNA damage. When TMQ was removed after a 24 h exposure to 0.1 microM, recovery of DHFR catalytic activity and apparent free DHFR binding sites was evident over the next 24-48 h in both cell lines. With 1 and 10 microM, however, persistent inhibition of DHFR was evident in C4 cells, whereas DHFR recovered in H630 cells. These data suggest that, although DHFR inhibition during TMQ exposure produced growth inhibition, DHFR catalytic activity 48 h after drug removal was a more accurate predictor of lethality in these two cell lines. Several factors appeared to influence the duration of DHFR inhibition after drug removal, including initial TMQ concentration, declining cytosolic TMQ levels after drug removal, the ability to acutely increase total DHFR content and the extent of TMQ-mediated DNA damage. The greater sensitivity of C4 cells to TMQ-associated lethality may be attributed to the greater extent of TMQ-mediated DNA damage and more prolonged duration of DHFR inhibition after drug exposure.

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Selected References

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  1. Allegra C. J., Chabner B. A., Drake J. C., Lutz R., Rodbard D., Jolivet J. Enhanced inhibition of thymidylate synthase by methotrexate polyglutamates. J Biol Chem. 1985 Aug 15;260(17):9720–9726. [PubMed] [Google Scholar]
  2. Allegra C. J., Fine R. L., Drake J. C., Chabner B. A. The effect of methotrexate on intracellular folate pools in human MCF-7 breast cancer cells. Evidence for direct inhibition of purine synthesis. J Biol Chem. 1986 May 15;261(14):6478–6485. [PubMed] [Google Scholar]
  3. Assaraf Y. G., Molina A., Schimke R. T. Sequential amplification of dihydrofolate reductase and multidrug resistance genes in Chinese hamster ovary cells selected for stepwise resistance to the lipid-soluble antifolate trimetrexate. J Biol Chem. 1989 Nov 5;264(31):18326–18334. [PubMed] [Google Scholar]
  4. Assaraf Y. G., Schimke R. T. Identification of methotrexate transport deficiency in mammalian cells using fluoresceinated methotrexate and flow cytometry. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7154–7158. doi: 10.1073/pnas.84.20.7154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Assaraf Y. G., Slotky J. I. Characterization of a lipophilic antifolate resistance provoked by treatment of mammalian cells with the antiparasitic agent pyrimethamine. J Biol Chem. 1993 Feb 25;268(6):4556–4566. [PubMed] [Google Scholar]
  6. BERTINO J. R., DONOHUE D. R., GABRIO B. W., SILBER R., ALENTY A., MEYER M., HUENNEKENS F. M. Increased level of dihydrofolic reductase in leucocytes of patients treated with amethopterin. Nature. 1962 Jan 13;193:140–142. doi: 10.1038/193140a0. [DOI] [PubMed] [Google Scholar]
  7. Baram J., Allegra C. J., Fine R. L., Chabner B. A. Effect of methotrexate on intracellular folate pools in purified myeloid precursor cells from normal human bone marrow. J Clin Invest. 1987 Mar;79(3):692–697. doi: 10.1172/JCI112872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bastow K. F., Prabhu R., Cheng Y. C. The intracellular content of dihydrofolate reductase: possibilities for control and implications for chemotherapy. Adv Enzyme Regul. 1984;22:15–26. doi: 10.1016/0065-2571(84)90006-2. [DOI] [PubMed] [Google Scholar]
  9. Bertino J. R., Donohue D. M., Simmons B., Gabrio B. W., Silber R., Huennekens F. M. THE "INDUCTION" OF DIHYDROFOLIC REDUCTASE ACTIVITY IN LEUKOCYTES AND ERYTHROCYTES OF PATIENTS TREATED WITH AMETHOPTERIN. J Clin Invest. 1963 Apr;42(4):466–475. doi: 10.1172/JCI104735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Chu E., Takimoto C. H., Voeller D., Grem J. L., Allegra C. J. Specific binding of human dihydrofolate reductase protein to dihydrofolate reductase messenger RNA in vitro. Biochemistry. 1993 May 11;32(18):4756–4760. doi: 10.1021/bi00069a009. [DOI] [PubMed] [Google Scholar]
  12. Chu E., Zinn S., Boarman D., Allegra C. J. Interaction of gamma interferon and 5-fluorouracil in the H630 human colon carcinoma cell line. Cancer Res. 1990 Sep 15;50(18):5834–5840. [PubMed] [Google Scholar]
  13. Cowan K. H., Goldsmith M. E., Levine R. M., Aitken S. C., Douglass E., Clendeninn N., Nienhuis A. W., Lippman M. E. Dihydrofolate reductase gene amplification and possible rearrangement in estrogen-responsive methotrexate-resistant human breast cancer cells. J Biol Chem. 1982 Dec 25;257(24):15079–15086. [PubMed] [Google Scholar]
  14. Cowan K. H., Goldsmith M. E., Ricciardone M. D., Levine R., Rubalcaba E., Jolivet J. Regulation of dihydrofolate reductase in human breast cancer cells and in mutant hamster cells transfected with a human dihydrofolate reductase minigene. Mol Pharmacol. 1986 Jul;30(1):69–76. [PubMed] [Google Scholar]
  15. Cowan K. H., Jolivet J. A methotrexate-resistant human breast cancer cell line with multiple defects, including diminished formation of methotrexate polyglutamates. J Biol Chem. 1984 Sep 10;259(17):10793–10800. [PubMed] [Google Scholar]
  16. Curt G. A., Jolivet J., Carney D. N., Bailey B. D., Drake J. C., Clendeninn N. J., Chabner B. A. Determinants of the sensitivity of human small-cell lung cancer cell lines to methotrexate. J Clin Invest. 1985 Oct;76(4):1323–1329. doi: 10.1172/JCI112106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dedhar S., Freisheim J. H., Hynes J. B., Goldie J. H. Further studies on substituted quinazolines and triazines as inhibitors of a methotrexate-insensitive murine dihydrofolate reductase. Biochem Pharmacol. 1986 Apr 1;35(7):1143–1147. doi: 10.1016/0006-2952(86)90151-6. [DOI] [PubMed] [Google Scholar]
  18. Diddens H., Niethammer D., Jackson R. C. Patterns of cross-resistance to the antifolate drugs trimetrexate, metoprine, homofolate, and CB3717 in human lymphoma and osteosarcoma cells resistant to methotrexate. Cancer Res. 1983 Nov;43(11):5286–5292. [PubMed] [Google Scholar]
  19. Domin B. A., Grill S. P., Bastow K. F., Cheng Y. C. Effect of methotrexate on dihydrofolate reductase activity in methotrexate-resistant human KB cells. Mol Pharmacol. 1982 Mar;21(2):478–482. [PubMed] [Google Scholar]
  20. Domin B. A., Grill S. P., Cheng Y. Establishment of dihydrofolate reductase-increased human cell lines and relationship between dihydrofolate reductase levels and gene copy. Cancer Res. 1983 May;43(5):2155–2158. [PubMed] [Google Scholar]
  21. Drake J. C., Allegra C. J., Curt G. A., Chabner B. A. Competitive protein-binding assay for trimetrexate. Cancer Treat Rep. 1985 Jun;69(6):641–644. [PubMed] [Google Scholar]
  22. Erickson L. C., Ross W. E., Kohn K. W. Isolation and purification of large quantities of DNA replication intermediates by pH step alkaline elution. Chromosoma. 1979;74(2):125–139. doi: 10.1007/BF00292268. [DOI] [PubMed] [Google Scholar]
  23. Fry D. W., Besserer J. A. Characterization of trimetrexate transport in human lymphoblastoid cells and development of impaired influx as a mechanism of resistance to lipophilic antifolates. Cancer Res. 1988 Dec 15;48(24 Pt 1):6986–6991. [PubMed] [Google Scholar]
  24. Grem J. L., Allegra C. J. Enhancement of the toxicity and DNA incorporation of arabinosyl-5-azacytosine and 1-beta-D-arabinofuranosylcytosine by cyclopentenyl cytosine. Cancer Res. 1990 Nov 15;50(22):7279–7284. [PubMed] [Google Scholar]
  25. Grem J. L., Fischer P. H. Augmentation of 5-fluorouracil cytotoxicity in human colon cancer cells by dipyridamole. Cancer Res. 1985 Jul;45(7):2967–2972. [PubMed] [Google Scholar]
  26. Grem J. L., Mulcahy R. T., Miller E. M., Allegra C. J., Fischer P. H. Interaction of deoxyuridine with fluorouracil and dipyridamole in a human colon cancer cell line. Biochem Pharmacol. 1989 Jan 1;38(1):51–59. doi: 10.1016/0006-2952(89)90148-2. [DOI] [PubMed] [Google Scholar]
  27. Hayman R., McGready R., Van der Weyden M. B. A rapid radiometric assay for dihydrofolate reductase. Anal Biochem. 1978 Jul 1;87(2):460–465. doi: 10.1016/0003-2697(78)90696-6. [DOI] [PubMed] [Google Scholar]
  28. Jackson R. C., Fry D. W., Boritzki T. J., Besserer J. A., Leopold W. R., Sloan B. J., Elslager E. F. Biochemical pharmacology of the lipophilic antifolate, trimetrexate. Adv Enzyme Regul. 1984;22:187–206. doi: 10.1016/0065-2571(84)90014-1. [DOI] [PubMed] [Google Scholar]
  29. Jackson R. C., Hart L. I., Harrap K. R. Intrinsic resistance to methotrexate of cultured mammalian cells in relation to the inhibition kinetics of their dihydrololate reductases. Cancer Res. 1976 Jun;36(6):1991–1997. [PubMed] [Google Scholar]
  30. Jackson R. C., Niethammer D., Hart L. I. Reactivation of dihydrofolate reductase inhibted by methotrexate or aminopterin. Arch Biochem Biophys. 1977 Aug;182(2):646–656. doi: 10.1016/0003-9861(77)90545-8. [DOI] [PubMed] [Google Scholar]
  31. Kamen B. A., Eibl B., Cashmore A., Bertino J. Uptake and efficacy of trimetrexate (TMQ, 2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl] quinazoline), a non-classical antifolate in methotrexate-resistant leukemia cells in vitro. Biochem Pharmacol. 1984 May 15;33(10):1697–1699. doi: 10.1016/0006-2952(84)90298-3. [DOI] [PubMed] [Google Scholar]
  32. Kaufman R. J., Schimke R. T. Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line. Mol Cell Biol. 1981 Dec;1(12):1069–1076. doi: 10.1128/mcb.1.12.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Klohs W. D., Steinkampf R. W., Besserer J. A., Fry D. W. Cross resistance of pleiotropically drug resistant P338 leukemia cells to the lipophilic antifolates trimetrexate and BW 301U. Cancer Lett. 1986 Jun;31(3):253–260. doi: 10.1016/0304-3835(86)90145-x. [DOI] [PubMed] [Google Scholar]
  34. Krishan A. Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol. 1975 Jul;66(1):188–193. doi: 10.1083/jcb.66.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Lin J. T., Bertino J. R. Trimetrexate: a second generation folate antagonist in clinical trial. J Clin Oncol. 1987 Dec;5(12):2032–2040. doi: 10.1200/JCO.1987.5.12.2032. [DOI] [PubMed] [Google Scholar]
  37. McMartin K. E., Virayotha V., Tephly T. R. High-pressure liquid chromatography separation and determination of rat liver folates. Arch Biochem Biophys. 1981 Jun;209(1):127–136. doi: 10.1016/0003-9861(81)90264-2. [DOI] [PubMed] [Google Scholar]
  38. Mini E., Moroson B. A., Franco C. T., Bertino J. R. Cytotoxic effects of folate antagonists against methotrexate-resistant human leukemic lymphoblast CCRF-CEM cell lines. Cancer Res. 1985 Jan;45(1):325–330. [PubMed] [Google Scholar]
  39. 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]
  40. Myers C. E., Lippman M. E., Elliot H. M., Chabner B. A. Competitive protein binding assay for methotrexate. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3683–3686. doi: 10.1073/pnas.72.9.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Park J. G., Oie H. K., Sugarbaker P. H., Henslee J. G., Chen T. R., Johnson B. E., Gazdar A. Characteristics of cell lines established from human colorectal carcinoma. Cancer Res. 1987 Dec 15;47(24 Pt 1):6710–6718. [PubMed] [Google Scholar]
  42. Pizzorno G., Mini E., Coronnello M., McGuire J. J., Moroson B. A., Cashmore A. R., Dreyer R. N., Lin J. T., Mazzei T., Periti P. Impaired polyglutamylation of methotrexate as a cause of resistance in CCRF-CEM cells after short-term, high-dose treatment with this drug. Cancer Res. 1988 Apr 15;48(8):2149–2155. [PubMed] [Google Scholar]
  43. Roberts D. An isotopic assay for thymidylate synthetase. Biochemistry. 1966 Nov;5(11):3546–3548. doi: 10.1021/bi00875a022. [DOI] [PubMed] [Google Scholar]
  44. Ross D. D., Chen S. R., Cuddy D. P. Effects of 1-beta-D-arabinofuranosylcytosine on DNA replication intermediates monitored by pH-step alkaline elution. Cancer Res. 1990 May 1;50(9):2658–2666. [PubMed] [Google Scholar]
  45. Schuetz J. D., Matherly L. H., Westin E. H., Goldman I. D. Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line. J Biol Chem. 1988 Jul 15;263(20):9840–9847. [PubMed] [Google Scholar]
  46. Sharma R. C., Assaraf Y. G., Schimke R. T. A phenotype conferring selective resistance to lipophilic antifolates in Chinese hamster ovary cells. Cancer Res. 1991 Jun 1;51(11):2949–2959. [PubMed] [Google Scholar]
  47. Swain S. M., Lippman M. E., Egan E. F., Drake J. C., Steinberg S. M., Allegra C. J. Fluorouracil and high-dose leucovorin in previously treated patients with metastatic breast cancer. J Clin Oncol. 1989 Jul;7(7):890–899. doi: 10.1200/JCO.1989.7.7.890. [DOI] [PubMed] [Google Scholar]
  48. Van Mouwerik T. J., Pangallo C. A., Willson J. K., Fischer P. H. Augmentation of methotrexate cytotoxicity in human colon cancer cells achieved through inhibition of thymidine salvage by dipyridamole. Biochem Pharmacol. 1987 Mar 15;36(6):809–814. doi: 10.1016/0006-2952(87)90168-7. [DOI] [PubMed] [Google Scholar]
  49. White J. C., Goldman I. D. Methotrexate resistance in al L1210 cell line resulting from increased dihydrofolate reductase, decreased thymidylate synthetase activity, and normal membrane transport. Computer simulations based on network thermodynamics. J Biol Chem. 1981 Jun 10;256(11):5722–5727. [PubMed] [Google Scholar]
  50. Yalowich J. C., Kalman T. I. Rapid determination of thymidylate synthase activity and its inhibition in intact L1210 leukemia cells in vitro. Biochem Pharmacol. 1985 Jul 1;34(13):2319–2324. doi: 10.1016/0006-2952(85)90788-9. [DOI] [PubMed] [Google Scholar]
  51. van der Veer L. J., Westerhof G. R., Rijksen G., Schornagel J. H., Jansen G. Cytotoxicity of methotrexate and trimetrexate and its reversal by folinic acid in human leukemic CCRF-CEM cells with carrier-mediated and receptor-mediated folate uptake. Leuk Res. 1989;13(11):981–987. doi: 10.1016/0145-2126(89)90005-2. [DOI] [PubMed] [Google Scholar]

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