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In vivo folylpolyglutamate pools of the wild-type (Px4) and methotrexate-resistant (MTX161) Datura innoxia cell lines were detected by incorporation of [14C]p-aminobenzoate into folates. The folylpolyglutamate derivatives were cleaved to p-aminobenzoylpolyglutamates and separated according to glutamyl chain length by high-performance liquid chromatography. Hexaglutamates were the predominant form in both Datura cell lines. The proportions of individual folylpolyglutamates were unaffected by culturing the cells in medium containing products of one-carbon metabolism such as glycine, adenine, thymidine, or methionine. Radiolabeling of the hexaglutamates was greatly reduced in the presence of 10-8 M methotrexate (MTX) in the Px4 cells but not in the MTX161 cells. Tetrahydrofolate, 5, 10-methylenetetrahydrofolate, and folinic acid were effective substrates for the folylpolyglutamate synthetase from Datura cells in vitro, whereas MTX and folate were poor substrates. In vivo, MTX can be slowly converted into its polyglutamate derivatives up to MTXGlu4 or MTXGlu5 in Datura cells in the longer term. Significantly lower levels of MTX polyglutamates in MTX161 cells were found compared with those of Px4 cells during prolonged (10 d) exposure to MTX. Although in vivo and in vitro folylpolyglutamate synthesis was found to be similar in both cell lines, about a 4-fold increase in specific activity of [gamma]-glutamyl hydrolase (GGH) was detected in the MTX161 cell line. The increase in GGH in the resistant cells suggested that breakdown of polyglutamylated forms of MTX may play a role in acquired MTX resistance.

Folate Metabolism in Datura innoxia (In Vivo and in Vitro Folylpolyglutamate Synthesis in Wild-Type and Methotrexate-Resistant Cells)