Skip to main content
NCBI home page
Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 1992 Feb;119(2):101–105. doi: 10.1007/BF01209664

The role of folates in the development of methotrexate resistance in human leukemia cell line K562

Hayato Miyachi 1,✉, Yuzuru Takemura 2, Yasuhiko Ando 1, Kevin J Scanlon 3
PMCID: PMC12200529  PMID: 1429825

Abstract

The effect of reduced and oxidized folates on the development of methotrexate (MTX) resistance has been examined in human leukemia cell line K562 (K562/S). K562/S cells were made resistant to MTX by soft-agar cloning either in RPMI-1640 medium (K562/MTX-PGA) or in folic-acidfree RPMI-1640 medium containing 10 nM leucovorin (K562/MTX-LV). The optimal concentrations of leucovorin for the growth of K562/S, K562/MTX-PGA and K562/MTX-LV cells were 1 nM, 5 nM and 10 nM respectively. K562/MTX-PGA cells were 24-fold resistant to MTX as noted by impaired MTX transport. In contrast, K562/MTX-LV cells were 26-fold resistant to MTX as noted by gene amplification of dihydrofolate reductase. Furthermore cross-resistance to cytosine arabinoside was only demonstrated in K562/MTX-PGA, while the K562/MTX-LV cells showed no significant cross-resistance to cytosine arabinoside. These results suggest that the type and level of folates used during the development of MTX resistance may play a role in the mechanism for MTX resistance. Leukemia cells that are grown in leucovorin might serve as a model for acqured MTX resistance in vivo.

Key words: Methotrexate, Gene amplification, Folate

Abbreviations

MTX

methotrexate

PGA

pteroylglutamic acid

PDDF

N 10-propargyl-5,8-dideazafolate

MTT

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

IC50

50% inhibitory concentration for cell growth by MTT assay

FCS

fetal calf serum

References

  1. 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]
  2. Brown PC, Tlsty TD, Schimke RT (1983) Enhancement of methotrexate resistance and dihydrofolate reductase gene amplification by treatment of mouse 3T6 cells with hydroxyurea. Mol Cell Biol 3:1097–1107 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carman MD, Schornagel JH, Rivest RS, Srimatkandada S, Portlock CS, Duffy T, Bertino JR (1984) Resistance to methotrexate due to gene amplification in a patient with acute leukemia. J Clin Oncol 2:16–20 [DOI] [PubMed] [Google Scholar]
  4. Diddens H, Niethammer D, Jackson RC (1983) Patterns of cross-resistance to the antifolate drugs trimetrexate, metoprine, homofolate, and CB3717 in human lymphoma and osteosarcoma cells resistant to methotrexate. Cancer Res 43:5286–5292 [PubMed] [Google Scholar]
  5. Galivan J (1979) Transport and metabolism of methotrexate in normal and resistant cultured rat hepatoma cells. Cancer Res 39:735–743 [PubMed] [Google Scholar]
  6. Henderson GB, Tsuji JM, Kumar HP (1987) Transport of folate compounds by leukemic cells. Evidence for a single influx carrier for methotrexate, 5-methyltetrahydrofolate, and folate in CCRF-CEM human lymphoblasts. Biochem Pharmacol 36:3007–3014 [DOI] [PubMed] [Google Scholar]
  7. Herbert V, Larabee AR, Buckanan JM (1962) Studies on the identification of a folate compound of human serum. J Clin Invest 41:1134–1138 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Hoffbrand AV, Newcombe BF (1967) Leucocyte folate in vitamin B12 folate deficiency and in leukaemia. Br J Haematol 13:954–966 [DOI] [PubMed] [Google Scholar]
  10. Hoffbrand AV, Tripp E, Catovsky D, Das KC (1973) Transport of methotrexate into normal haematopoietic cells and into leukaemic cells and its effects on DNA synthesis. Br J Haematol 25:497–511 [DOI] [PubMed] [Google Scholar]
  11. Horns RC, Dower WJ, Schimke RT (1984) Gene amplification in a leukemic patient treated with methotrexate. J Clin Oncol 2:2–7 [DOI] [PubMed] [Google Scholar]
  12. Huennekens FM, Vitols KS, Henderson GB (1978) Transport of folate compounds in bacteria and mammalian cells. Adv Enzymol 47:313–346 [DOI] [PubMed] [Google Scholar]
  13. Jackson RC, Niethammer D, Huennekens FM (1975) Enzymic and transport mechanisms of amethopterin resistance in L1210 mouse leukaemia cells. Cancer Biochem Biophys. 1:151–155 [Google Scholar]
  14. 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 [DOI] [PubMed] [Google Scholar]
  15. Johnston RN, Feder J, Hill AB, Sherwood SW, Schimke RT (1986) Transient inhibition of DNA synthesis results in increased dihydrofolate reductase synthesis and subsequent increased DNA content per cell. Mol Cell Biol 6:3373–3381 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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 [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Krishan A (1975) Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 66:188–193 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lozzio CB, Lozzio BB (1960) Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood 45:321–334 [PubMed] [Google Scholar]
  20. Lu Y, Han J, Scanlon KJ (1988) Biochemical and molecular properties of cisplatin-resistant A2780 cells grown in folinic acid. J Biol Chem 263:4891–4894 [PubMed] [Google Scholar]
  21. 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 [DOI] [PubMed] [Google Scholar]
  22. Mehta BM, Gisolfi AL, Hutchinson DJ, Nierenberg A, Kellick M, Rosen G (1978) Serum distribution of citrovorum factor and 5-methyltetrahydrofolate following oral and im administration of calcium leucovorin in normal adults. Cancer Treat Rep 62:345–350 [PubMed] [Google Scholar]
  23. Mini E, Moroson BA, Franco CT, Bertino JR (1985) Cytotoxic effects of folate antagonists against methotrexate-resistant human leukemic lymphoblast CCRF-CEM cell lines. Cancer Res 45:325–330 [PubMed] [Google Scholar]
  24. Miyachi H, Scanlon KJ (1989) A human lymphoblastic leukemia CCRF-CEM cell line (CEM-S) resistant to methotrexate (CEM-MTX) has increased sensitivity to fluoropyrimidines when grown in leucovorin (LV). Pro Am Assoc Cancer Res 30:480 [Google Scholar]
  25. Miyachi H, Han H, Scanlon KJ (1991) Dihydrofolate reductase gene expression characterized by the PCR assay in human leukemia cells. In Vivo 5:7–12 [PubMed] [Google Scholar]
  26. Miyachi H, Jiao L, Sowers LC, Scanlon KJ (1992) Collateral sensitivity to azidothymidine in methotrexate resistant human leukemia cells. In Vivo 6:17–22 [PubMed] [Google Scholar]
  27. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63 [DOI] [PubMed] [Google Scholar]
  28. Ohnoshi T, Ohnuma T, Takahashi I, Scanlon K, 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]
  29. 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]
  30. Rader JI, Niethammer D, Huennekens FM (1974) Effects of sulfhydryl inhibitors upon transport of folate compounds into L 1210 cells. Biochem Pharmacol 23:2057–2059 [DOI] [PubMed] [Google Scholar]
  31. 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 [DOI] [PubMed] [Google Scholar]
  32. Schimke RT (1988) Gene amplification in cultured cells. J Biol Chem 263:5989–5992 [PubMed] [Google Scholar]
  33. Sharma RC, Schimke RT (1989) Enhancement of the frequency of methotrexate resistance by γ-radiation in chinese hamester ovary and mouse 3T6 cells. Cancer Res 49:3861–3866 [PubMed] [Google Scholar]
  34. Sirotnak FM, Noccio DM, Kelleher LE, Goutas LJ (1981) Relative frequency and kinetic propeties of transport-defective phenotypes among methotrexate-resistant L1210 clonal cell lines derived in vivo. Cancer Res 41:4447–4452 [PubMed] [Google Scholar]
  35. Trent JM, Buick RN, Olson S, Horns JrRC, Schimke RT (1984) Cytologic evidence for gene amplification in methotrexate-resistant cells obtained from a patient with ovarian adenocarcinoma. J Clin Oncol 2:8–15 [DOI] [PubMed] [Google Scholar]

Articles from Journal of Cancer Research and Clinical Oncology are provided here courtesy of Springer

RESOURCES