Skip to main content
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1985 Mar;75(3):1008–1014. doi: 10.1172/JCI111761

Formation of methotrexate polyglutamates in purified myeloid precursor cells from normal human bone marrow.

S Koizumi, G A Curt, R L Fine, J D Griffin, B A Chabner
PMCID: PMC423648  PMID: 2579975

Abstract

Immature myeloid precursor cells were preferentially selected from normal human bone marrow by using immune rosette techniques that employed monoclonal antibodies against mature granulocytes, monocytes, T and B lymphocytes, and erythroid precursors (Mo5, M3, OKT3, B1, and EP1, respectively). We examined the formation, retention, and cytotoxic effects of methotrexate (MTX) polyglutamates (MTX-PGs) in these purified myeloid precursor cells. After 1- and 24-h exposures to MTX, with thymidine and deoxyinosine as rescue, the intracellular MTX-PG profile was examined by high-pressure liquid chromatography. Efflux patterns of MTX-PGs were also studied after additional 1- and 24-h incubations in drug-free media. Cytotoxic effects of retained MTX-PGs on bone marrow myeloid precursors were examined by colony formation in drug-free semisolid agar. Normal myeloid precursor cells converted MTX to MTX-PGs in a concentration- and time-dependent manner, preferentially retaining MTX-PGs with three to five glutamyl moieties. At low concentrations of MTX (1 microM), MTX-PG formation was insufficient to maintain saturation of the target enzyme dihydrofolate reductase after removal of drug from the incubation medium, and there was no decrease in myeloid colony formation. At higher concentrations of MTX (10 microM), formation of higher molecular weight polyglutamates was sufficient to allow for 24-h saturation of intracellular binding capacity after removal of extracellular drug and resulted in a 35% reduction in the formation of colony-forming units in culture. Comparison of MTX metabolism in normal bone marrow cells and the MTX-sensitive HL-60 human leukemia cell line showed twofold greater PG formation by these tumor cells after 24-h exposure to 1 or 10 microM MTX, and a marked (greater than 30-fold) increase in cytotoxicity for the HL-60 cells as compared with normal myeloid precursors, suggesting that the MTX polyglutamation may be important to its selective antitumor action.

Full text

PDF
1008

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Balinska M., Nimec Z., Galivan J. Characteristics of methotrexate polyglutamate formation in cultured hepatic cells. Arch Biochem Biophys. 1982 Jul;216(2):466–476. doi: 10.1016/0003-9861(82)90235-1. [DOI] [PubMed] [Google Scholar]
  2. Baugh C. M., Krumdieck C. L., Nair M. G. Polygammaglutamyl metabolites of methotrexate. Biochem Biophys Res Commun. 1973 May 1;52(1):27–34. doi: 10.1016/0006-291x(73)90949-2. [DOI] [PubMed] [Google Scholar]
  3. Breitman T. R., Collins S. J., Keene B. R. Replacement of serum by insulin and transferrin supports growth and differentiation of the human promyelocytic cell line, HL-60. Exp Cell Res. 1980 Apr;126(2):494–498. doi: 10.1016/0014-4827(80)90296-7. [DOI] [PubMed] [Google Scholar]
  4. Chabner B. A., Young R. C. Threshold methotrexate concentration for in vivo inhibition of DNA synthesis in normal and tumorous target tissues. J Clin Invest. 1973 Aug;52(8):1804–1811. doi: 10.1172/JCI107362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Covey J. M. Polyglutamate derivatives of folic acid coenzymes and methotrexate. Life Sci. 1980 Mar 3;26(9):665–678. doi: 10.1016/0024-3205(80)90256-8. [DOI] [PubMed] [Google Scholar]
  6. Curt G. A., Jolivet J., Bailey B. D., Carney D. N., Chabner B. A. Synthesis and retention of methotrexate polyglutamates by human small cell lung cancer. Biochem Pharmacol. 1984 May 15;33(10):1682–1685. doi: 10.1016/0006-2952(84)90292-2. [DOI] [PubMed] [Google Scholar]
  7. Fabre I., Fabre G., Goldman I. D. Polyglutamylation, an important element in methotrexate cytotoxicity and selectivity in tumor versus murine granulocytic progenitor cells in vitro. Cancer Res. 1984 Aug;44(8):3190–3195. [PubMed] [Google Scholar]
  8. Fry D. W., Anderson L. A., Borst M., 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. 1983 Mar;43(3):1087–1092. [PubMed] [Google Scholar]
  9. Fry D. W., Anderson L. A., Borst M., 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. 1983 Mar;43(3):1087–1092. [PubMed] [Google Scholar]
  10. Fry D. W., Yalowich J. C., Goldman I. D. Rapid formation of poly-gamma-glutamyl derivatives of methotrexate and their association with dihydrofolate reductase as assessed by high pressure liquid chromatography in the Ehrlich ascites tumor cell in vitro. J Biol Chem. 1982 Feb 25;257(4):1890–1896. [PubMed] [Google Scholar]
  11. Galivan J. Evidence for the cytotoxic activity of polyglutamate derivatives of methotrexate. Mol Pharmacol. 1980 Jan;17(1):105–110. [PubMed] [Google Scholar]
  12. Gewirtz D. A., White J. C., Randolph J. K., Goldman I. D. Transport, binding, and polyglutamation of methotrexate in freshly isolated rat hepatocytes. Cancer Res. 1980 Mar;40(3):573–578. [PubMed] [Google Scholar]
  13. Goldman I. D., Lichtenstein N. S., Oliverio V. T. Carrier-mediated transport of the folic acid analogue, methotrexate, in the L1210 leukemia cell. J Biol Chem. 1968 Oct 10;243(19):5007–5017. [PubMed] [Google Scholar]
  14. Griffin J. D., Beveridge R. P., Schlossman S. F. Isolation of myeloid progenitor cells from peripheral blood of chronic myelogenous leukemia patients. Blood. 1982 Jul;60(1):30–37. [PubMed] [Google Scholar]
  15. Jacobs S. A., Derr C. J., Johns D. G. Accumulation of methotrexate diglutamate in human liver during methotrexate therapy. Biochem Pharmacol. 1977 Dec 1;26(23):2310–2313. doi: 10.1016/0006-2952(77)90296-9. [DOI] [PubMed] [Google Scholar]
  16. Jolivet J., Chabner B. A. Intracellular pharmacokinetics of methotrexate polyglutamates in human breast cancer cells. Selective retention and less dissociable binding of 4-NH2-10-CH3-pteroylglutamate4 and 4-NH2-10-CH3-pteroylglutamate5 to dihydrofolate reductase. J Clin Invest. 1983 Sep;72(3):773–778. doi: 10.1172/JCI111048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jolivet J., Cowan K. H., Curt G. A., Clendeninn N. J., Chabner B. A. The pharmacology and clinical use of methotrexate. N Engl J Med. 1983 Nov 3;309(18):1094–1104. doi: 10.1056/NEJM198311033091805. [DOI] [PubMed] [Google Scholar]
  18. Jolivet J., Schilsky R. L., Bailey B. D., Drake J. C., Chabner B. A. Synthesis, retention, and biological activity of methotrexate polyglutamates in cultured human breast cancer cells. J Clin Invest. 1982 Aug;70(2):351–360. doi: 10.1172/JCI110624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jolivet J., Schilsky R. L. High-pressure liquid chromatography analysis of methotrexate polyglutamates in cultured human breast cancer cells. Biochem Pharmacol. 1981 Jun 1;30(11):1387–1390. doi: 10.1016/0006-2952(81)90330-0. [DOI] [PubMed] [Google Scholar]
  20. Koizumi S., Yamagami M., Miura M., Horita S., Sano M., Ikuta N., Taniguchi N. Expression of Ia-like antigens defined by monoclonal OKIal antibody of hemopoietic progenitor cells in cord blood: a comparison with human bone marrow. Blood. 1982 Oct;60(4):1046–1049. [PubMed] [Google Scholar]
  21. Koizumi S., Yamagami M., Ueno Y., Miura M., Taniguchi N. Resistance of human bone marrow CFUC to high-dose methotrexate cytotoxicity. Exp Hematol. 1980 May;8(5):635–640. [PubMed] [Google Scholar]
  22. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  23. Pike B. L., Robinson W. A. Human bone marrow colony growth in agar-gel. J Cell Physiol. 1970 Aug;76(1):77–84. doi: 10.1002/jcp.1040760111. [DOI] [PubMed] [Google Scholar]
  24. Poser R. G., Sirotnak F. M., Chello P. L. Differential synthesis of methotrexate polyglutamates in normal proliferative and neoplastic mouse tissues in vivo. Cancer Res. 1981 Nov;41(11 Pt 1):4441–4446. [PubMed] [Google Scholar]
  25. Rosenblatt D. S., Whitehead V. M., Vera N., Pottier A., Dupont M., Vuchich M. J. Prolonged inhibition of DNA synthesis associated with the accumulation of methotrexate polyglutamates by cultured human cells. Mol Pharmacol. 1978 Nov;14(6):1143–1147. [PubMed] [Google Scholar]
  26. Whitehead V. M., Perrault M. M., Stelcner S. Tissue-specific synthesis of methotrexate polyglutamates in the rat. Cancer Res. 1975 Nov;35(11 Pt 1):2985–2990. [PubMed] [Google Scholar]
  27. Whitehead V. M., Rosenblatt D. S. Methotrexate metabolism by bone marrow cells from patients with leukemia. Adv Exp Med Biol. 1983;163:287–303. doi: 10.1007/978-1-4757-5241-0_22. [DOI] [PubMed] [Google Scholar]
  28. Whitehead V. M. Synthesis of methotrexate polyglutamates in L1210 murine leukemia cells. Cancer Res. 1977 Feb;37(2):408–412. [PubMed] [Google Scholar]
  29. Yokochi T., Brice M., Rabinovitch P. S., Papayannopoulou T., Stamatoyannopoulos G. Monoclonal antibodies detecting antigenic determinants with restricted expression on erythroid cells: from the erythroid committed progenitor level to the mature erythroblast. Blood. 1984 Jun;63(6):1376–1384. [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES