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Japanese Journal of Cancer Research : Gann logoLink to Japanese Journal of Cancer Research : Gann
. 1988 Nov;79(11):1230–1237. doi: 10.1111/j.1349-7006.1988.tb01549.x

Impairment of Methotrexate (MTX)‐Polyglutamate Formation of MTX‐resistant K562 Cell Lines

Shoichi Koizumi 1
PMCID: PMC5917644  PMID: 2465288

Abstract

We examined the mechanism of methotrexate (MTX) resistance in five K562 cell subclones resistant to MTX. Based on a clonogenic assay, the IC50s of these MTX‐resistant clones were 10 to 40μMMTX, indicating 2,000 to 5,000‐fold resistance as compared to that of the parent cell line. The doubling times of these MTX‐resistant K562 cell lines are longer (27–60 hr) than that of the parent K562 cell line (24 hr). One‐hour MTX accumulation in the resistant cells was 70–80% of that in parent cells. To investigate the formation of MTX‐polyglutamates (MTX‐PGs), resistant cells were incubated with 3H‐MTX (1 or 1OμM) for 24 hr in the presence of thymidine and deoxyinosine to prevent cytotoxicity. MTX (‐Glu1) and the polyglutamate metabolites (MTX‐Glu2, ‐Glu3, ‐Glu4 and ‐Glu5) were analyzed by a high‐pressure liquid chromatography (HPLC) technique. After a 24‐hr incubation with 10μM MTX, the total concentration of intracellular MTX reached 39 to 89 nmol/g protein, only 20 to 40% of the MTX level of the parent K562 cells. The HPLC analysis revealed that less than 2% of intracellular MTX was in the form of high‐molecular MTX‐PGs (MTX‐Glu3, ‐Glu4 and ‐Glu5) in the five MTX‐resistant K562 cell lines, while in the parent cells MTX‐Glu3–5 comprised 46% of the total intracellular MTX. These data indicate the possibility that impairment of MTX‐PG formation, with transport alteration, may be a special mechanism for the high level of resistance to this agent in human leukemic cells.

Keywords: Methotrexate, Polyglutamation, Drug resistance, K562 cells

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References

  • 1. ) Jolivet , J. , Cowan , K. H. , Curt , G. A. , Clendeninn , N. J. and Chabner , B. A.The pharmacology and clinical use of methotrexate . N. Engl. J. Med. , 309 , 1094 – 1104 ( 1983. ). [DOI] [PubMed] [Google Scholar]
  • 2. ) Chabner , B. A.Methotrexate . In “ Pharmacologic Principles of Cancer Treatment ,” ed. Chabner B. A. , pp. 229 – 255 ( 1982. ). W. B. Saunders Co. , Philadelphia . [Google Scholar]
  • 3. ) Curt , G. A. , Clendeninn , N. J. and Chabner , B. A.Drug resistance in cancer . Cancer Treat. Rep. , 68 , 87 – 99 ( 1984. ). [PubMed] [Google Scholar]
  • 4. ) Hill , B. T. , Bailey , B. D. , White , J. C. and Goldman , I. D.Characteristics of transport of 4‐amino anti‐folates and folate compounds by two lines of L5178Y lymphoblasts, one with impaired transport of methotrexate . Cancer Res. , 39 , 2440 – 2446 ( 1979. ). [PubMed] [Google Scholar]
  • 5. ) Ohnoshi , T. , Ohnuma , T. , Takehashi , I. , Scanlon , K. , Kamen , B. A. and Holland , J. F.Establishment of methotrexate‐resistant human acute lymphoblastic leukemia cells in culture and effects of folate antagonists . Cancer Res. , 42 , 1655 – 1660 ( 1982. ). [PubMed] [Google Scholar]
  • 6. ) Sirotnak , F. M. , Maccio , D. M. , Kelleher , L. E. and Goutas , L. J.Relative frequency and kinetic properties of transport‐defective phenotypes among methotrexate‐resistant L‐1210 clonal cell lines derived in vivo . Cancer Res. , 41 , 4447 – 4452 ( 1981. ). [PubMed] [Google Scholar]
  • 7. ) Jackson , R. C. and Niethammer , D.Acquired methotrexate resistance in lymphoblasts resulting from altered kinetic properties of dihydrofolate reductase . Eur. J. Cancer , 13 , 567 – 575 ( 1977. ). [DOI] [PubMed] [Google Scholar]
  • 8. ) Flintoff , W. F. and Essani , K.Methotrexate‐resistant Chinese hamster ovary cells contain a dihydrofolate reductase with an altered affinity for methotrexate . Biochemistry , 19 , 4321 – 4327 ( 1980. ). [DOI] [PubMed] [Google Scholar]
  • 9. ) Goldie , J. H. , Krystal , G. , Hartley , D. , Gudauskas , G. and Dedhar , S.A methotrexate insensitive variant of folate reductase present in two lines of methotrexate‐resistant L5178Y cells . Eur. J. Cancer , 16 , 1539 – 1546 ( 1980. ). [DOI] [PubMed] [Google Scholar]
  • 10. ) White , J. C. and Goldman , I. D.Methotrexate resistance in an L1210 cell line resulting from increased dihydrofolate reductase, decreased thymidylate synthetase activity, and normal membrane transport . J. Biol. Chem. , 256 , 5722 – 5727 ( 1981. ). [PubMed] [Google Scholar]
  • 11. ) Alt , F. W. , Kellems , R. E. , Bertino , J. R. and Schimke , R. T.Selective multiplication of dihydrofolate reductase genes in methotrexate‐resistant variants of cultured murine cells . J. Biol. Chem. , 253 , 1357 – 1370 ( 1978. ). [PubMed] [Google Scholar]
  • 12. ) Moran , R. G. , Mulkins , M. and Heidelberger , C.Role of thymidylate synthetase activity in development of methotrexate cytotoxicity . Proc. Natl. Acad. Sci. USA , 76 , 5924 – 5928 ( 1979. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. ) Ayusawa , D. , Koyama , H. and Seno , T.Resistance to methotrexate in thymidylate synthetase‐deficient mutants of cultured mouse mammary tumor FM3A cells . Cancer Res. , 41 , 1497 – 1501 ( 1981. ). [PubMed] [Google Scholar]
  • 14. ) Washtien , W. L.Thymidylate synthetase levels as a factor in 5‐fluorodeoxyuridine and methotrexate cytotoxicity in gastrointestinal tumor cells . Mol. Pharmacol. , 21 , 723 – 728 ( 1982. ). [PubMed] [Google Scholar]
  • 15. ) Chabner , B. A. , Allegra , C. J. , Curt , G. A. , Clendeninn , N. J. , Baram , J. , Koizumi , S. , Drake , J. C. and Jolivet , J.Polyglutamation of methotrexate. Is methotrexate a prodrug ? J. Clin. Invest. 76 , 907 – 912 ( 1985. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. ) Allegra , C. J. , Drake , J. C. , Jolivet , J. and Chabner , B. A.Inhibition of phosphoribosylaminoimidazolecarboxamide transformylase by methotrexate and dihydrofolic acid polyglutamates . Proc. Natl. Acad. Sci. USA. , 82 , 4881 – 4885 ( 1985. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. ) Allegra , C. J. , Chabner , B. A. , Drake , J. C. , Lutz , R. , Rodbard , D. and Jolivet , J.Enhanced inhibition of thymidylate synthase by methotrexate polyglutamates . J. Biol. Chem. , 260 , 9720 – 9726 ( 1985. ). [PubMed] [Google Scholar]
  • 18. ) Baugh , C. M. , Krumdieck , C. L. and Nair , M. G.Polygammaglutamyl metabolites of methotrexate . Biochem. Biophys. Res. Commun. , 52 , 27 – 34 ( 1973. ). [DOI] [PubMed] [Google Scholar]
  • 19. ) Jacobs , S. A. , Derr , C. J. and Johns , D. G.Accumulation of methotrexate diglutamate in human liver during methotrexate therapy . Biochem. Pharmacol. , 26 , 2310 – 2313 ( 1977. ). [DOI] [PubMed] [Google Scholar]
  • 20. ) Koizumi , S. , Curt , G. A. , Fine , R. L. , Griffin , J. D. and Chabner , B. A.Formation of methotrexate polyglutamates in purified myeloid precursor cells from normal human bone marrow . J. Clin. Invest. , 75 , 1008 – 1014 ( 1985. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. ) Rosenblatt , D. S. , Whitehead , V. M. , Vera , N. , Pettier , A. , Dupont , M. and Vuchich , M. J.Prolonged inhibition of DNA synthesis associated with the accumulation of methotrexate polyglutamates by cultured human cells . Mol. Pharmacol. , 14 , 1143 – 1147 ( 1978. ). [PubMed] [Google Scholar]
  • 22. ) Whitehead , V. M. , Perrault , M. M. and Stelener , S.Tissue‐specific synthesis of methotrexate polyglutamates in the rat . Cancer Res. , 35 , 2985 – 2990 ( 1975. ). [PubMed] [Google Scholar]
  • 23. ) Whitehead , V. M.Synthesis of methotrexate polyglutamates in L1210 murine leukemia cells . Cancer Res. , 37 , 408 – 412 ( 1977. ). [PubMed] [Google Scholar]
  • 24. ) Galivan , J.Evidence for the cytotoxic activity of polyglutamate derivatives of methotrexate . Mol. Pharmacol. , 17 , 105 – 110 ( 1980. ). [PubMed] [Google Scholar]
  • 25. ) Schilsky , R. L. , Bailey , B. D. and Chabner , B. A.Methotrexate polyglutamate synthesis by cultured human breast cancer cells . Proc. Natl. Acad. Sci. USA , 77 , 2919 – 2922 ( 1980. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. ) Frei , E. , III , Rosowsky , A. , Wright , J. E. , Cucchi , C. A. , Lippke , J. A. , Ervin , T. J. , Jolivet , J. and Haseltine , W. A.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 ( 1984. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. ) Poser , R. G. , Sirotnak , F. M. and Chello , P. L.Differential synthesis of methotrexate polyglutamates in normal proliferative and neoplastic mouse tissues in vivo . Cancer Res. , 41 , 4441 – 4446 ( 1981. ). [PubMed] [Google Scholar]
  • 28. ) Fry , D. W. , Anderson , L. A. , Borst , M. and Goldman , I. D.Analysis of the role of membrane transport and polyglutamation of methotrexate in gut and the Ehrlich tumor in vivo as factors in drug sensitivity and selectivity . Cancer Res. , 43 , 1087 – 1092 ( 1983. ). [PubMed] [Google Scholar]
  • 29. ) Curt , G. A. , Jolivet , J. , Bailey , B. D. , Carney , D. N. and Chabner , B. A.Synthesis and retention of methotrexate polyglutamates by human small cell lung cancer . Biochem. Pharmacol. , 33 , 1682 – 1685 ( 1984. ). [DOI] [PubMed] [Google Scholar]
  • 30. ) Jolivet , J. , Schilsky , R. L. , Bailey , B. D. , Drake , J. C. and Chabner , B. A.Synthesis, retention, and biological activity of methotrexate polyglutamates in cultured human breast cancer cells . J. Clin. Invest. , 70 , 351 – 360 ( 1982. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. ) Curt , G. A. , Jolivet , J. , Carney , D. N. , Bailey , B. D. , Drake , J. C. , Clendeninn , N. J. and Chabner , B. A.Determinants of the sensitivity of human small‐cell lung cancer cell lines to methotrexate . J. Clin. Invest. , 76 , 1323 – 1329 ( 1985. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. ) Cowan , H. K. and Jolivet , J.A methotrexate‐resistant human breast cancer cell line with multiple defects, including diminished formation of methotrexate polyglutamates . J. Biol. Chem. , 259 , 10793 – 10800 ( 1984. ). [PubMed] [Google Scholar]
  • 33. ) Lowry , O. H. , Rosebrough , N. J. , Farr , A. L. and Randall , R. J.Protein measurement with the Folin phenol reagent . J. Biol. Chem. , 193 , 265 – 275 ( 1951. ). [PubMed] [Google Scholar]
  • 34. ) Bielder , J. L. and Spengler , B. A.Metaphase chromosome anomaly: association with drug resistance and cell‐specific products . Science , 191 , 185 – 187 ( 1976. ). [DOI] [PubMed] [Google Scholar]
  • 35. ) Allegra , C. J. , Fine , R. L. , Drake , J. C. and 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. , 261 , 6478 – 6485 ( 1986. ). [PubMed] [Google Scholar]

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