Synthesis of Methotrexate Polyglutamates in Cultured Human Cells

We describe the biosynthesis of po1y-γ-glutamyl metabolites of methotrexate in cultured human fibroblasts. The accumulation of polyglutamates by these cells is dependent on the concentration of methotrexate in the culture medium, duration of incubation, and stage of the culture cycle. When [ 3 H]methotrexate is replaced with equimolar unlabeled methotrexate, label is found in successively longer polyglutamates. Synthesis of the polyglutamates is dependent on the continued presence of methotrexate in the culture medium.