Molecular analysis of murine leukemia cell lines resistant to 5,10-dideazatetrahydrofolate identifies several amino acids critical to the function offolylpolyglutamate synthetase

Four L1210 murine leukemia cell lines resistant to 5,10-dideazatetrahydrofo late (DDATHF) and other folate analogs, but sensitive to continuous exposur e to methotrexate, were developed by chemical mutagenesis followed by DDATH F selective pressure. Endogenous folate pools were modestly reduced but pol yglutamate derivatives of DDATHF and ALIMTA (LY231514, MTA) were markedly d ecreased in these mutant cell lines. Membrane transport was not a factor in drug resistance; rather, folypolyglutamate synthetase (FPGS) activity was decreased by >98%. In each cell line, FPGS mRNA expression was unchanged bu t both alleles of the FPGS gene bore a point mutation in highly conserved d omains of the coding region. Four mutations were in the predicted ATP-, fol ate-, and/or glutamate-binding sites of FPGS, and two others were clustered in a peptide predicted to be beta sheet 5, based on the crystal structure of the Lactobacillus casei enzyme. Transfection of cDNAs for three mutant e nzymes into FPGS-null Chinese hamster ovary cells restored a reduced level of clonal growth, whereas a T339I mutant supported growth at a level compar able to that of the wild-type enzyme. The two mutations predicted to be in beta sheet 5, and one in the loop between NH2- and COOH-terminal domains di d not support cell growth. When sets of mutated cDNAs were co-transfected i nto FPGS-null cells to mimic the genotype of drug-selected resistant cells, clonal growth was restored. These results demonstrate for the first time t hat single amino acid substitutions in several critical regions of FPGS can cause marked resistance to tetrahydrofolate antimetabolites, while still a llowing cell survival.