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Establishment of Methotrexate-resistant Human Acute Lymphoblastic Leukemia Cells in Culture and Effects of Folate Antagonists¹

Departments of Neoplastic Disease [T. Oi., T. Oa., I. T., J. F. H.] and Medicine (Oncology) [K. S., J. F. H.], Mt. Sinai School of Medicine, New York, N. Y. 10029, and Departments of Pediatrics and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510. [B. A. K.]

A human acute lymphoblastic T-cell line, MOLT-3, was fed with Roswell Park Memorial Institute Medium 1640-10% fetal bovines serum-antibiotics, containing increasing concentrations of methotrexate (MTX). After 10 months 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 approximately 30-fold more resistant to MTX. The parent MOLT-3 and the resistant line had the same doubling time of approximately 36 hr. There were no differences in light microscopic morphology. MOLT-3 produced loose colonies on 0.5% agar enriched with fetal bovine serum, whereas 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/MTX_t). Kinetic analysis of MTX uptake showed that the resistant subline did not have an altered K_m for MTX (6.6 µM) but had a decreased V_max of about 20% of the parent cell line. These data suggest that either the number of folate transport sites or the turnover rate of these sites has been 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 more than 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] quiazoline 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.

¹ Supported by USPHS research Grants CA-25865 from the National Cancer Institute and ROI-AM-16690-06 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, NIH, Bethesda Md.; by the Chemotherapy Foundation, Inc., New York, N. Y.; by the United Leukemia Fund, Inc., New York, N. Y.; and by the T. J. Martell Memorial Foundation, New York, N. Y.

² To whom requests for reprints should be addressed, at the Department of Neoplastic Diseases, Mt. Sinal School of Medicine, One Gustave L. Levy Place, New York, N. Y. 10029.

³ Leukemia Society Fellow.

Received 4/20/81. Accepted 1/20/81.