Synthesis, Binding and Intracellular Retention of Methotrexate Polyglutamates by Cultured Human Breast Cancer Cells

  • Richard L. Schilsky
  • Jacques Jolivet
  • Brenda D. Bailey
  • Bruce A. Chabner
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 163)


Synthesis, binding, and intracellular retention of methotrexate polyglutamates by cultured human breast cancer cells were investigated by gel filtration and high-pressure liquid chromatography to separate methotrexate from its metabolites. MCF-7, ZR-75–1, and MDA-231 human breast cancer cells were found to readily convert methotrexate to higher polyglutamates during a 24-hr incubation period, although at differing rates. Examination of that portion of intracellular methotrexate specifically bound to dihydrofolate reductase revealed that, with prolonged incubation, methotrexate polyglutamates become the predominant drug form bound to the enzyme. Similarly, methotrexate polyglutamates accumulated free in the cyto-sol and when cells were suspended in drug-free medium, were retained intracellularly both bound to dihydrofolate reductase and in the unbound fraction, indicating their slow passage through the cell membrane. Studies of methotrexate polyglutamate binding to purified bacterial dihydrofolate reductase revealed high affinity binding for compounds with up to 6 additional glutamyl residues. These studies demonstrate that methotrexate polyglutamates are readily formed in human breast cancer cells, bind intracellularly to dihydrofolate reductase, and are selectively retained both bound to the enzyme and free in the cell cytosol.


Human Breast Cancer Cell Dihydrofolate Reductase Cell Cytosol Intracellular Retention Competitive Protein Binding Assay 
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Copyright information

© Springer Science+Business Media New York 1983

Authors and Affiliations

  • Richard L. Schilsky
    • 1
  • Jacques Jolivet
    • 1
  • Brenda D. Bailey
    • 1
  • Bruce A. Chabner
    • 1
  1. 1.Clinical Pharmacology Branch, Clinical Oncology Program, Division of Cancer TreatmentNational Cancer InstituteBethesdaUSA

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