Formation of 7-hydroxymethotrexate polyglutamyl derivatives and their cytotoxicity in human chronic myelogenous leukemia cells, in vitro.
The rapid synthesis of poly-gamma-glutamyl derivatives of 7-hydroxymethotrexate (7-OH-MTX) and their selective intracellular retention are reported in human chronic myelogenous leukemia cells, K-562. After a 30-min exposure to 5 microM [3H]7-OH-MTX, three different polyglutamyl derivatives were detected by high-performance liquid chromatography. When extracellular 7-OH-MTX was removed, the 7-OH-MTX diglutamate level declined slowly in comparison to the monoglutamate, but the higher polyglutamyl derivative levels increased. Within 10 min after exposure of cells to 7-OH-MTX, the level of these polyglutamyl derivatives far exceeds the dihydrofolate reductase binding capacity. Gel filtration or charcoal binding analysis followed by high-performance liquid chromatography analysis of the bound component showed intracellular binding of virtually all 7-OH-MTX tetraglutamate at a level 4-fold higher than that of the dihydrofolate reductase binding capacity. No bound 7-OH-MTX diglutamate or triglutamate could be detected. Treatment of the 7-OH-MTX tetraglutamate: protein complex with 100 microM unlabeled methotrexate (MTX) for 15 min resulted in only a partial dissociation of this complex to an extent compatible with the dihydrofolate reductase level. The residual 7-OH-MTX tetraglutamate remained bound to a site with a molecular weight of approximately 25,000 to 35,000 as assessed by Bio-Gel P-60 analysis and could not be displaced by folic acid, 5-formyltetrahydrofolate, 7-OH-MTX, or the tetraglutamate of MTX. 7-OH-MTX and MTX cytotoxicities were compared by clonogenic assay in agar and by their effects on cell growth. After a 2-hr exposure, the 50% inhibitory concentrations for 7-OH-MTX and MTX in cells growing in agar were 10(-5) and 10(-6) M, respectively. A 10-fold difference in cytotoxicity was also observed in cells growing in suspension. Continuous exposure to glycine: adenosine: thymidine completely protects cells from a sustained exposure to 7-OH-MTX over the entire period of clonal growth. However, even a brief exposure to 7-OH-MTX also requires continuous exposure to glycine: adenosine: thymidine for protection. This suggests that, as observed for MTX, the 7-OH-MTX polyglutamyl derivatives that are retained within the cells have a sustained cytotoxic effect after the monoglutamate is removed.
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D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D005493:Folic Acid Antagonists / Q000633:toxicity*; D006801:Humans; D007700:Kinetics; D007951:Leukemia, Myeloid / Q000473:pathology*; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity; D010455:Peptides / Q000378:metabolism*; D011099:Polyglutamic Acid / Q000031:analogs & derivatives / Q000378:metabolism* / Q000633:toxicity; D014410:Tumor Stem Cell Assay
D005493:Folic Acid Antagonists; D010455:Peptides; D011099:Polyglutamic Acid; C011864:7-hydroxymethotrexate; D008727:Methotrexate