ESTABLISHMENT OF METHOTREXATE-RESISTANT HUMAN ACUTE LYMPHOBLASTIC LEUKEMIA-CELLS IN CULTURE AND EFFECTS OF FOLATE ANTAGONISTS

Abstract

A human acute lymphoblastic T-cell line, MOLT-3, was fed with RPMI 1640-10% fetal bovine serum-antibiotics, containing increasing concentrations of methotrexate (MTX). After 10 mo. of feeding, the cells became resistant to 10-7 M MTX; resistance was not reversed when the cells were placed in the original MTX-free medium. At 10-7 M MTX, the concentration which produced complete inhibition of the parent MOLT-3 cell growth, the resistant cells were not inhibited at all. On a 50% inhibitory concentration basis, the resistant cells were .apprx. 30-fold more resistant to MTX. The parent MOLT-3 and the resistant line had the same doubling time of .apprx. 36 h. There were no differences in light microscopic morphology. MOLT-3 produced loose colonies on 0.5% agar enriched with fetal bovine serum; the colonies of the resistant line were tightly packed. The development of resistance was accompanied by a 4- to 5-fold decrease in [3H]MTX transport (MOLT-3/MTXt). Kinetic analysis of MTX uptake showed that the resistant subline did not have an altered Km for MTX (6.6 .mu.M), but had a decreased Vmax of about 20% of the parent cell line. Apparently, either the number of folate transport sites or the turnover rate of these sites was reduced in the MTX-resistant cell line. Dihydrofolate reductase was only minimally elevated in the resistant cell line. The MTX-resistant cell line was cross-resistant to dichloromethotrexate. The sensitivity of the resistant line to the substituted 2,4-diaminoquinazoline and pyrimidine compounds, 2,4-diamino-5-methyl-6-[(3'',4'',5''-trimethoxyanilino) methyl] quinazoline (JB-11) and 2,4-diamino-5-(3'',4''-dichlorophenyl)-6-methylpyrimidine, increased > 3-fold. While leucovorin equally reversed the MTX effects on the parent and resistant cells, leucovorin reversal of 2,4-diamino-5-methyl-6-[(3'',4'',5''-trimethoxyanilino)methyl] quinazoline and 2,4-diamino-5-(3'',4''-dichlorophenyl)-6-methylpyrimidine effects was limited only to the parent cell line. 2,4-Diamino-5-methyl-6-[(3'',4'',5''-trimethoxyanilino)methyl]quinazoline or 2,4-diamino-5-(3'',4''-dichlorophenyl)-6-methylpyrimidine plus leucovorin might prove to be unique in treating patients with acute lymphoblastic leukemia when the leukemic cells develop transport resistance to MTX.