POSTTRANSCRIPTIONALLY MEDIATED DECREASES IN FOLYLPOLYGLUTAMATE SYNTHETASE GENE-EXPRESSION IN SOME FOLATE ANALOG-RESISTANT VARIANTS OF THE L1210 CELL - EVIDENCE FOR AN ALTERED COGNATE MESSENGER-RNA IN THE VARIANTS AFFECTING THE RATE OF DE-NOVO SYNTHESIS OF THE ENZYME

L1210 cell variants resistant to edatrexate (EDX) were isolated by sel ection in vivo during therapy with this folate analogue. Among the var iants selected, seven (L1210/EDX-4 to -7 and L1210/EDX-12 to -14) were found to exhibit 2-23-fold lower levels of folylpolyglutamate synthet ase (FPGS) activity compared with parental L1210 cells. Lower levels o f FPGS activity in cell-free extract from these variants using EDX as substrate were characterized by the same relative decrease in value fo r V-max with no change in apparent K-m. The results of an analysis of FPGS activity in mixtures of variant and parental cell extract suggest ed that no endogenous inhibitors in the variant cells or stimulatory f actors in parental cells accounted for the differences observed. Also, FPGS hom variant and parental cells showed no difference in thermosta bility. Decreases in a 60-61-kDa protein as shown by immunoblotting wi th anti-FPGS peptide antibody were found to occur commensurately with the decrease in FPGS activity in cell extract from the variants compar ed with parental cells. However, no evidence was obtained for a differ ence in turnover of FPGS protein during measurement of the decay of FP GS activity in cycloheximide-treated variant and parental cells. In ad dition, Northern blotting of poly(A)(+) RNA did not reveal any differe nce in the size or level of FPGS mRNA among these various cell types, Studies of in vitro translation of hybridization-selected FPGS mRNA fr om L1210 cells showed that both mitochondrial and cytosolic forms of F PGS were generated during the reaction. Moreover, FPGS mRNA from the v ariant cells was significantly less effective in mediating formation o f the FPGS peptide product in a manner correlating with FPGS activity and protein found in the cytosol of the various cell types, These resu lts suggest that FPGS gene expression in these variants is posttranscr iptionally altered at the level of the cognate mRNA itself and that th is alteration constitutively down-regulates the steady-state level of FPGS in these variants.