FOLATE METABOLISM IN DATURA-INNOXIA - IN-VIVO AND IN-VITRO FOLYLPOLYGLUTAMATE SYNTHESIS IN WILD-TYPE AND METHOTREXATE-RESISTANT CELLS

In vivo folylpolyglutamate pools of the wild-type (Px4) and methotrexa te-resistant (MTX161) Datura innoxia cell lines were detected by incor poration of [C-14]p-aminobenzoate into folates. The folylpolyglutamate derivatives were cleaved to p-aminobenzoylpolyglutamates and separate d according to glutamyl chain length by high-performance liquid chroma tography. Hexaglutamates were the predominant form in both Datura cell lines. The proportions of individual folylpolyglutamates were unaffec ted by culturing the cells in medium containing products of one-carbon metabolism such as glycine, adenine, thymidine, or methionine. Radiol abeling of the hexaglutamates was greatly reduced in the presence of 1 0(-8) M methotrexate (MTX) in the Px4 cells but not in the MTX161 cell s. Tetrahydrofolate, 5,10-methylenetetrahydrofolate, and folinic acid were effective substrates for the folylpolyglutamate synthetase from D atura cells in vitro, whereas MTX and folate were poor substrates. In vivo, MTX can be slowly converted into its polyglutamate derivatives u p to MTXGlu(4) or MTXGlu(5) in Datura cells in the longer term. Signif icantly lower levels of MTX polyglutamates in MTX161 cells were found compared with those of Px4 cells during prolonged (10 d) exposure to M TX. Although in vivo and in vitro folylpolyglutamate synthesis was fou nd to be similar in both cell lines, about a 4-fold increase in specif ic activity of gamma-glutamyl hydrolase (GGH) was detected in the MTX1 61 cell line. The increase in CGH in the resistant cells suggested tha t breakdown of polyglutamylated forms of MTX may play a role in acquir ed MTX resistance.