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Formation of 7-Hydroxymethotrexate Polyglutamyl Derivatives and Their Cytotoxicity in Human Chronic Myelogenous Leukemia Cells, in Vitro¹

Departments of Medicine and Pharmacology, Medical College of Virginia, Richmond, Virginia 23298

The rapid synthesis of poly--glutamyl derivatives of 7-hydroxy-methotrexate (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 µM [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 µM 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.

¹ This investigation was supported by USPHS Grant CA-16906 awarded by the National Cancer Institute and NIH.

² Exchange Scientist under the Clinical Cancer Research Program Area of the United States—France (NCHNSERM) Cancer Program (NO. G5-0111). Present address: INSERM SC 16, Laboratoire de Pharmacocinetique et Toxicocinetique, Faculte de Pharmacie, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France.

³ To whom requests for reprints should be addressed.

Received 3/ 5/84. Accepted 9/25/84.

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