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. 1991 Nov;117(6):519–525. doi: 10.1007/BF01613282

A human leukemia cell line made resistant to two folate analogues, trimetrexate andN 10-propargyl-5,8-dideazafolic acid (CB3717)

Yuzuru Takemura 1,✉, Takao Ohnuma 2, Hayato Miyachi 3, Susumu Sekiguchi 1
PMCID: PMC12201284  PMID: 1683871

Abstract

We established a novel human acute lympho-blastic leukemia cell line made resistant to two folate analogues, trimetrexate (TMQ) andN 10-propargyl-5,8-dideazafolic acid (CB3717), by sequential exposure of the 200-fold TMQ-resistant cells (MOLT-3/TMQ200) to CB3717. A 30-fold-resistant subline to CB3717 was selected from the TMQ-resistant cells and designated as MOLT-3/TMQ200-CB371730. This double-folate-resistant cell line was 15-fold more resistant to methotrexate (MTX) than MOLT-3/TMQ200; however, TMQ resistance was decreased to 10-fold as compared to MOLT-3/TMQ200. The doubly resistant cells also showed 2-fold cross-resistance to 5-fluorouracil (5-FU). Equimolar concentrations of leucovorin almost completely reversed the inhibitory effect of MTX on the doubly resistant cells and partially that of CB3717 and TMQ; on the other hand, leucovorin enhanced the inhibitory effect of 5-FU. Thymidylate synthase activities demonstrated little or no difference among these three cell lines, being consistent with no overexpression of mRNA for this enzyme in the doubly resistant cells. MOLT-3/TMQ200 cells displayed classical multidrug resistance; sequential development of CB3717 resistance in the TMQ-resistant cells resulted in an enhancement of the multidrug-resistance phenotype and a concomitant increase ofMDR1 mRNA. The development of a complex resistance pattern seen in this double-folate-resistant subline indicates intricacy in the study of drug resistance after multidrug chemotherapy.

Key words: Trimetrexate (TMQ); N10; Propargyl-5,8-dideazafolic acid (CB3717); Multidrug chemotherapy; Drug resistance

Abbreviations

TMQ

trimetrexate 2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline

MTX

methotrexate, 4-amino-N 10-methylpteroylglutamic acid

CB3717

N 10-propargyl-5,8-dideazafolic acid

MDR1

the gene for multidrug resistance

ID50

50% inhibiting drug concentration for cell growth on day 3

Footnotes

This work was supported in part by the Japan Research Foundation for Clinical Pharmacology and by USPHS research grant CA-15936 from the National Cancer Institute, Bethesda, Md., USA. Yuzuru Takemura was an Iwama Memorial Sony International Fellow

References

  1. Arkin H, Ohnuma T, Kamen BA, Holland JF, Vallabhajosula S (1989) Multidrug resistance in a human leukemic cell line selected for resistance to trimetrexate. Cancer Res 49:6556–6561 [PubMed] [Google Scholar]
  2. Assaraf YG, Molina A, Schimke RT (1989) Amplification of mdr1 and/or DHFR genes in hamster cells resistant to the lipophilic antifolates trimetrexate and piritrexim. Proc Am Assoc Cancer Res 30:477 [Google Scholar]
  3. Ayusawa D, Shimizu K, Koyama H, Kaneda S, Takeishi K, Seno T (1986) Cell-cycle-directed regulation of thymidylate synthase messenger RNA in human diploid fibroblasts stimulated to proliferate. J Mol Biol 190:559–567 [DOI] [PubMed] [Google Scholar]
  4. Bertino JR, Sawicki WL, Morosin BA, Cashmore AR, Elslager EF (1979) 2,4-Diamino-5-methyl-6-(3,4,5-trimethoxyanilino)-methyl quinazoline (TMQ), a potent non-classical folate inhibitor. 1. Effect of dihydrofolate reductase and rodent tumors in vitro and in viv Biochem Pharmacol 28:1983–1987 [DOI] [PubMed] [Google Scholar]
  5. Bird OD, Vaitkus JW, Clarke J (1970) 2-Amino-4-hydroxy quinazolines as inhibitors of thymidylate synthetase. Mol Pharmacol 6:573–575 [PubMed] [Google Scholar]
  6. Calvert AH, Jones TR, Jackman AL, Brown SJ, Harrap KR (1980) An approach to the design of antimetabolites active against cells resistant to conventional agents illustrated by quinazoline antifolates with N-10 substitutions. In: Davis W, Harrap KR, Stathopoulos G (eds) Advances in tumour prevention, detection and characterization, vol 5. Human cancer, its characterization and treatment. Excerpta Medica, Amsterdam, pp 272–283 [Google Scholar]
  7. Carlin SC, Rosenberg RN, VandeVenter L, Friedkin M (1974) Quinazoline antifolate as inhibitors of growth, dihydrofolate reductase and thymidylate synthetase of mouse neuroblastoma cells in culture. Mol Pharmacol 10:194–203 [PubMed] [Google Scholar]
  8. Chirgwin JM, Przybyla AE, Macdonald RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299 [DOI] [PubMed] [Google Scholar]
  9. Cowan KH, Jolivet J (1984) A methotrexate-resistant human breast cancer cell line with multiple defects, including diminished formation of methotrexate polyglutamates. J Biol Chem 259:10793–10800 [PubMed] [Google Scholar]
  10. Frei E III, Rosowsky A, Wright JE, Cucchi CA, Lippke JA, Ervin TJ, Jolivet J, Haseltine WA (1984) Development of methotrexate resistance in a human squamous cell carcinoma of the head and neck in culture. Proc Natl Acad Sci USA 81: 2873–2877 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fry DW, Besserer JA (1988) Characterization of trimetrexate transport in human lymphoblastoid cells and development of impaired influx as a mechanism of resistance to lipophilic antifolates. Cancer Res 48:6986–6991 [PubMed] [Google Scholar]
  12. Galivan J, Nimec Z, Rhee M (1987) Synergistic growth inhibition of rat hepatoma cells exposed in vitro toN10-propargyl-5,8-dideazafolate with methotrexate or the lipophilic antifolates trimetrexate or metoprine. Cancer Res 47:5256–5260 [PubMed] [Google Scholar]
  13. Jackman AL, Grezelakowska-Sztabert B, Newell DR, Calvert AH, Harrap KR (1985) Transport studies with [3H]-CB3717. Br J Cancer 52:431 [Google Scholar]
  14. Jackman AL, Alison DL, Calvert AH, Harrap KR (1986) Increased thymidylate synthase in L1210 cells possessing acquired resistance toN10-propargyl-5,8-dideazafolic acid (CB3717): develpment, characterization, and cross-resistance studies. Cancer Res 46:2810–2815 [PubMed] [Google Scholar]
  15. Jansen G, Westerhof GR, van Emmerik N, Kathmann I, Rijksen G, Jackman AL, Schornagel JH (1990) Effects of antifolates on L1210 cells exhibiting carrier- and receptor-mediated folate transport systems. Proc Am Assoc Cancer Res 31:341 [PubMed] [Google Scholar]
  16. Jones TR, Calvert AH, Jackman AL, Brown SJ, Jones M, Harrap KR (1981) A potent antitumour quinazoline inhibitor of thymidylate synthetase: Synthesis, biological properties and therapeutic results in mice. Eur J Cancer 17:11–19 [DOI] [PubMed] [Google Scholar]
  17. Kamen BA, Eibl B, Cashmore A, Bertino J (1984) Uptake and efficacy of trimetrexate (TMQ, 2,4-diamino-5-methyl-6-[(2,4,5-trimethoxyanilino)methyl]quinazoline), a nonclassical antifolate in methotrexate-resistant leukemia cells in vitro. Biochem Pharmacol 33:1697–1699 [DOI] [PubMed] [Google Scholar]
  18. Kano Y, Ohnuma T, Holland JF (1986) Folate requirements of methotrexate-resistant human acute lymphoblastic leukemia cell lines. Blood 68:586–591 [PubMed] [Google Scholar]
  19. Kobayashi H, Takemura Y, Miyachi H, Ohnuma T (1991) The alterations of dihydrofolate reductase (DHFR) and MDR1 genes in human leukemia sublines resistant to various folate analogues. Proc Am Assoc Cancer Res 32:326 [Google Scholar]
  20. Minowada J, Ohnuma T, Moore GE (1972) Rosette-forming human lymphoid cell lines: I. Establishment and evidence for origin of thymus-derived lymphocytes. J Natl Cancer Inst 49:891–895 [PubMed] [Google Scholar]
  21. Morandi C, Masters JN, Mottes M, Attardi G (1982) Multiple forms of human dihydrofolate reductase messenger RNA. Cloning and expression inEscherichia coli of their DNA coding sequence. J Mol Biol 156:583–607 [DOI] [PubMed] [Google Scholar]
  22. Ohnoshi T, Ohnuma T, Takahashi I, Scanlon KJ, Kamen BA, Holland JF (1982) Establishment of methotrexate-resistant human acute lymphoblastic leukemia cells in culture and effects of folate antagonists. Cancer Res 42:1655–1660 [PubMed] [Google Scholar]
  23. Ohnuma T, Lo RJ, Scanlon KJ, Kamen BA, Ohnoshi T, Wolman SR, Holland JF (1985) Evolution of methotrexate resistance of human acute lymphoblastic leukemia cells in vitro. Cancer Res 45:1815–1822 [PubMed] [Google Scholar]
  24. Pawelczak K, Jones TR, Kempny M, Jackman AL, Newell DR, Krzyzanowski L, Rzeszotarska B (1989) Quinazoline antifolates inhibiting thymidylate synthase: Synthesis of four oligo(l-γ-glutamyl) conjugates ofN10-propargyl-5,8-dideazafolic acid and their enzyme inhibition. J Med Chem 32:160–165 [DOI] [PubMed] [Google Scholar]
  25. Pizzorno G, Mini E, Coronnello M, McGuire JJ, Moroson BA, Cashmore AR, Dreyer RN, Lin JT, Mazzei T, Periti P, Bertino JR (1988) Impaired polyglutamylation of methotrexate as a cause of resistance in CCRF-CEM cells after short-term, high-dose treatment with this drug. Cancer Res 48:2149–2155 [PubMed] [Google Scholar]
  26. Roberts D (1966) An isotopic assay for thymidylate synthetase. Biochemistry 5:3546–3548 [DOI] [PubMed] [Google Scholar]
  27. Rode W, Scanlon KJ, Moroson B, Bertino JR (1980) Regulation of thymidylate synthetase in mouse leukemia cells (L1210). J Biol Chem 255:1305–1311 [PubMed] [Google Scholar]
  28. Sikora E, Jackman AL, Newell DR, Calvert AH (1988) Formation and retention and biological activity ofN10-propargyl-5,8-dideazafolic acid (CB3717) polyglutamates in L1210 cells in vitro. Biochem Pharmacol 37:4047–4054 [DOI] [PubMed] [Google Scholar]
  29. Singer-Sam J, Simmer RL, Keith DH, Shively L, Teplitz M, Itakura K, Gartler SM, Riggs AD (1983) Isolation of a cDNA clone for human X-linked 3-phosphoglycerate kinase by use of a mixture of synthetic oligodeoxyribonucleotides as a detection probe. Proc Natl Acad Sci USA 80:802–806 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sirotnak FM, Moccio DM, Goutas LJ, Kelleher LE, Montgomery JA (1982) Biochemical correlates of responsiveness and collateral sensitivity of some methotrexate-resistant murine tumors to the lipophilic antifolate, metoprine. Cancer Res 42:924–928 [PubMed] [Google Scholar]
  31. Takemura Y, Ohnuma T, Arkin H, Holland JF, Calvert AH (1985) Biological effects of N-(4-(N-((2-amino-4-hydroxy-6-quinazolinyl)methyl)prop-2-ynylamino)benzoyl)-l-glutamic acid (CB3717) on human acute lymphocytic leukemia cell lines resistant to methotrexate (MTX) or trimetrexate (TMQ). Proc Am Assoc Cancer Res 26:230 [Google Scholar]
  32. Ueda K, Clark DP, Chen CJ, Roninson IB, Gottesman MM, Pastan I (1987) The human multidrug resistance (mdr-1) gene. cDNA cloning and transcription initiation. J Biol Chem 262:505–508 [PubMed] [Google Scholar]

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