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Amplification of the thymidylate synthase gene in anN 10-propargyl-5,8-dideazafolic-acid-resistant human leukemia, MOLT-3 cell line developed in pteroylglutamic acid, but not in leucovorin

  • Original Paper
  • Experimental Oncology
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Abstract

The types of folates used during the development of resistance to methotrexate have been suggested to play an important role in the mechanisms of established resistance. In this study, effects of reduced and oxidized folates on the development of resistance to a thymidylate synthase (TS) inhibitor,N 10-propargyl-5,8-dideazafolic acid (CB3717), were exāmined in the human leukemia cell line MOLT-3. MOLT-3 cells were made resistant to CB3717 by soft-agar cloning in RPMI-1640 medium with either pteroylglutamic acid (PGA) or a more physiological folate (10 nM leucovorin). A 40-fold CB3717-resistant subline developed in PGA (MOLT-3/CB371740-PGA) showed amplification of the TS gene with a concomitant increased level in the gene expression. A 200-fold CB3717-resistant subline (MOLT-3/CB3717200-PGA), which was derived from MOLT-3/CB371740-PGA, showed further enhancement of amplification of the TS gene. In contrast, even a 200-fold CB3717-resistant subline developed in leucovorin (MOLT-3/CB3717200-LV) showed neither amplification nor overexpression of the TS gene. Both MOLT-3/CB3717200-PGA and MOLT-3/CB3717200-LV cells showed decreased membrane transport of PGA as well as methotrexate. These results suggest that the types of folates used during the development of CB3717 resistance may play a role in resistance, and that impaired transport of PGA, in CB3717-resistant MOLT-3 cells developed in PGA, might have accelerated amplification of the TS gene.

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Abbreviations

CB3717:

N 10-propargyl-5,8-dideazafolic acid

DHFR :

dihydrofolate reductase

FCS :

fetal calf serum

IC 50 :

50% inhibitory concentration for cell growth by MTT assay

LV :

leucovorin, 5-formyltet-rahydrofolate

MTT :

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

PGA :

pteroylglutamic acid

TS :

thymidylate synthase

References

  • 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

    PubMed  Google Scholar 

  • Bisset G, Pawelczak K, Jackman AL, Dix Perkin EE, Calvert AH, Hughes LR (1989) The synthesis and thymidylate synthase inhibitory activity of the poly-γ-glutamyl derivatives of some analogues ofN 10-propargyl-5,8-dideazafolate (CB3717). Br J Cancer 60:501

    Google Scholar 

  • Erlichman C (1991) Novel chemotherapeutic agents in clinical development. Curr Opin Oncol 3:1037–1042

    PubMed  Google Scholar 

  • Harrap KR, Jackman AL, Newell DR, Taylor GA, Hughes LR, Calvert AH (1989) Thymidylate synthase, a target for anticancer drug design. Adv Enzyme Regul 29:161–179

    PubMed  Google Scholar 

  • Herbert V, Larabee AR, Buckanan JM (1962) Studies on the identification of a folate compound of human serūm. J Clin Invest 41:1134–1138

    PubMed  Google Scholar 

  • Hills BT, Bailey BD, White JC, Goldman ID (1979) Characteristics of transport of 4-amino antifolates and folate compounds by two lines of L5178Y lymphoblasts, one with impaired transport of methotrexate. Cancer Res 39:2440–2446

    PubMed  Google Scholar 

  • Hoffbrand AV, Newcombe BF (1967) Leucocyte folate in vitamin B12 folate deficiency and in leukaemia. Br J Haematol 13:954–966

    PubMed  Google Scholar 

  • Jackman AL, Alison DL, Calvert AH, Harrap KR (1986) Increased thymidylate synthase in L1210 cells possessing acquired resistance toN 10-propargyl-5,8-dideazafolic acid (CB3717): development, characterization, and cross-resistance studies. Cancer Res 46:2810–2815

    PubMed  Google Scholar 

  • Jackman AL, Taylor GA, O'Connor BM, Bishop JA, Moran RG, Calvert AH (1990) Activity of thymidylate synthase inhibitor 2-desamino-N 10-propargyl-5,8-dideazafolic acid and related compounds in murine (L1210) and human (W1L2) systems in vitro and in L1210 in vivo. Cancer Res 50:5212–5218

    PubMed  Google Scholar 

  • Jackman AL, Taylor GA, Gibson W, Kimbell R, Brown M, Calvert AH, Judson IR, Hughes LR (1991) ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that is a potent inhibitor of L1210 tumor cell growth in vitro and in vivo: a new agent for clinical study. Cancer Res 51:5579–5586

    PubMed  Google Scholar 

  • Jackson RC, Jackman AL, Calvert AH (1983) Biochemical effects of a quinazoline inhibitor of thymidylate synthetase, N-{4-(N-[(2-amino-4-hydroxy-6-quinazolinyl)methyl] prop-2-ynylamino) benzoyl}-l-glutamic acid (CB3717), on human lymphoblastoid cells. Biochem Pharmacol 32:3783–3790

    PubMed  Google Scholar 

  • Jansen G, Schornagel JH, Westerhof GR, Rijksen G, Newell DR, Jackman AL (1990) Multiple membrane transport systems for the uptake of folate-based thymidylate synthase inhibitors. Cancer Res 50:7544–7548

    PubMed  Google Scholar 

  • Jolivet J, Cowan KH, Curt GA, Clendeninn NJ, Chabner BA (1983) The pharmacology and clinical use of methotrexate. N Engl J Med 309:1094–1104

    PubMed  Google Scholar 

  • Jones TR, Calvert AH, Jackman AL, Brown SJ, Jones M, Harrap KR (1981) A potent antitumor quinazoline inhibitor of thymidylate synthetase: synthesis, biological properties and therapeutic results in mice. Eur J Cancer 17:11–19

    PubMed  Google Scholar 

  • Krishan A (1975) Rapid flow cytoflurometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 66:188–193

    PubMed  Google Scholar 

  • Masters JN, Yang JK, Cellini A, Attardi G (1983) A human dihydrofolate reductase pseudogene and its relationship to the multiple forms of specific messenger RNA. J Mol Biol 167:23–26

    PubMed  Google Scholar 

  • McGuire JJ, Bertino JR (1981) Enzymatic synthesis and function of folylpolyglutamates. Mol Cell Biochem 38:19–48

    PubMed  Google Scholar 

  • Mehta BM, Gisolfi AL, Hutchinson DJ, Nierenberg A, Kellick M, Rosen G (1978) Serum distribution of citrovorum factor and 5-methyltetrahydrofolate following oral and i.m. administration of calcium leucovorin in normal adults. Cancer Treat Rep 62:345–350

    PubMed  Google Scholar 

  • 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 

  • Miyachi H, Takemura Y, Ando Y, Scanlon KJ (1992a) The role of folates in the development of methotrexate resistance in human leukemia cell line K562. J Cancer Res Clin Oncol 119:101–105

    PubMed  Google Scholar 

  • Miyachi H, Jiao L, Sowers LC, Scanlon KJ (1992b) Collateral sensitivity to azidothymidine in methotrexate resistant human leukemia cells. In Vivo 6:7–22

    Google Scholar 

  • Moran RG, Werkheiser WC, Zakrzewski SF (1976) Folate metabolism in mammalian cells in culture. J Biol Chem 251:3569–3575

    PubMed  Google Scholar 

  • Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J, Immunol Methods 65:55–63

    Google Scholar 

  • O'Connor BM, Jackman AL, Crossley PH, Freemantle SE, Lunec J, Calvert AH (1992) Human lymphoblastoid cells with acquired resistance to C2-desamino-C2-methyl-N 10-propargyl-5,8-dideazafolic acid: a novel folate-based thymidylate synthase inhibitor. Cancer Res 52:1137–1143

    PubMed  Google Scholar 

  • 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 

  • 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 treatment with this drug. Cancer Res 48:2149–2155

    PubMed  Google Scholar 

  • Rader JI, Niethammer D, Huennekens FM (1974) Effects of sulfhydryl inhibitors upon transport of folate compounds into L1210 cells. Biochem Pharmacol 23:2057–2059

    PubMed  Google Scholar 

  • Scanlon KJ, Cashmore AR, Kashani-Sabet M, Pallai M, Dreyer RN, Moroson BA, Saketos M (1987) Inhibition of methionine uptake by methotrexate in mouse leukemia L1210 cells. Cancer Chemother Pharmacol 19:21–24

    PubMed  Google Scholar 

  • Schimke RT (1988) Gene amplification in cultured cells. J Biol Chem 263:5989–5992

    PubMed  Google Scholar 

  • Sharma RC, Schimke RT (1989) Enhancement of the frequency of methotrexate resistance by γ-radiation in chinese hamster ovary and mouse 3T6 cells. Cancer Res 49:3861–3866

    PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Clinical Pathology, Tokai University School of Medicine, Bohseidai, 259-11, Isehara, Kanagawa, Japan

    Hayato Miyachi, Yuzuru Takemura, Hiroyuki Kobayashi & Yasuhiko Ando

  2. Department of Laboratory Medicine, National Defense Medical College, 3-2 Namiki, 359, Tokorozawa, Saitama, Japan

    Yuzuru Takemura & Hiroyuki Kobayashi

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  1. Hayato Miyachi
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  2. Yuzuru Takemura
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  3. Hiroyuki Kobayashi
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  4. Yasuhiko Ando
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Miyachi, H., Takemura, Y., Kobayashi, H. et al. Amplification of the thymidylate synthase gene in anN 10-propargyl-5,8-dideazafolic-acid-resistant human leukemia, MOLT-3 cell line developed in pteroylglutamic acid, but not in leucovorin. J Cancer Res Clin Oncol 122, 659–664 (1996). https://doi.org/10.1007/BF01209028

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  • DOI: https://doi.org/10.1007/BF01209028

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