Human Lymphoblastoid Cells with Acquired Resistance to C2-Desamino-C2- methyl-A^-propargyl-S^-dideazafolic Acid: A Novel Folate-based Thymidylate Synthase Inhibitor1

We describe the characterization of human lymphoblastoid cell lines with acquired resistance (>20,000-fold) to a novel folate-based thymidylate synthase (TS) (EC 2.1.1.45) inhibitor, C2-desamino-C2-methylW°-propargyl-5,8-dideazafolic acid (ICI198S83). This acquired resist ance was associated with a 64-fold amplification of the TS gene, a similar elevation in the corresponding mKNA, and an --200-fold increase in both TS activity and TS protein. This amplification was maintained when the cells were grown in the absence of the selective agent, ICI198583, for 340 generations. TS isolated from one of the resistant cell lines, WlL2:C1, displayed inhibition kinetic parameters similar to those of TS isolated from the parent W1-L2 cell line. It thus appears unlikely that resistance is due to an altered TS enzyme having a lower affinity for ICI198583. The resistant cell line, W1-L2:C1, was cross-resistant to other folate-based TS inhibitors but was as sensitive as the parent cell line, W1-L2, to 5-fluorodeoxyuridine. The W1-L2:C1 cell line was col laterally sensitive to the classical dihydrofolate reducÃ-ase(EC 1.5.1.3) inhibitor methotrexate as well as to the lipophilic dihydrofolate reducÃ-ase inhibitors metoprine and 2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline glucuronic acid salt (also called trimetrexate). When the W1-L2 and W1-L2:C1 cell lines were exposed to 1 JIM ICI 198583 for 24 h they accumulated the same concentration of total cellular ICI198583 polyglutamates despite the fact that the latter cell line accumulated a 300-fold greater concentration of ICI 198583 mono glutamate. As polyglutamates, the tetraand pentaglutamate forms pre dominated in the W1-L2 cell line, whereas the diglutamate form predom inated in the W1-L2:C1 cell line, with few higher polyglutamates being detected. The lack of triand higher polyglutamates of ICI 198583 (i.e., the more active species) in the W1-L2:C1 cell line may also contribute to the observed resistance. These findings may have important implica tions in light of the rapid onset of resistance to antifolates in the clinic.