FOLYLPOLYGLUTAMATE SYNTHETASE EXPRESSION IN ANTIFOLATE-SENSITIVE AND ANTIFOLATE-RESISTANT HUMAN CELL-LINES

Synthesis of poly(gamma-glutamyl) metabolites of many antifolates, suc h as methotrexate (MTX), by folylpolyglutamate synthetase (FPGS) is of ten essential to their cytotoxic activity. FPGS expression in the MTX- sensitive human T-lymphoblastic leukemia Cell line CCRF-CEM and a numb er of MTX-resistant sublines was previously investigated at the DNA, R NA, and activity levels. Using an FPGS peptide deduced from its cDNA s equence, a rabbit polyclonal antibody to FPGS has now been elicited, i mmunoaffinity purified, and used to quantitate FPGS protein expression by chemiluminescent Western immunoblot analysis. The antibody was use d to determine the half-life of human FPGS protein (3.7 +/- 1.1 h) in parental CCRF-CEM cells. A subline resistant to MTX as a result of amp lified dihydrofolate reductase expression shows no change in FPGS prot ein or activity relative to CCRF-CEM. An MTX transport-defective line, however, displays both higher FPGS protein and activity levels. For s everal sublines in which the only apparent mechanism of MTX resistance is decreased FPGS activity, the FPGS protein level is decreased propo rtionally. However, we previously showed that these sublines have the same gene copy number, restriction map, and mRNA size and levels as th e parent. Evidently, in these MTX-resistant sublines the mRNA is poorl y translated and/or the protein turns over more rapidly.