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Impaired Polyglutamylation of Methotrexate as a Cause of Resistance in CCRF-CEM Cells after Short-Term, High-Dose Treatment with This Drug¹

Departments of Pharmacology and Medicine, Yale University School of Medicine, New Haven, Connecticut 06510 [G. P., J. J. M., B. A. M., A. R. C., R. N. D., J. T. L., J. R. B.], and Dipartimento di Farmacologia Preclinica e Clinica, Cattedra di Chemioterapia, Universita' degli Studi, 50100 Firenze, Italy [E. M., M. C., T. M., P. P.]

Two methotrexate-resistant sublines, CCRF-CEM R3/7 and CCRF-CEM R30/6, were selected from the human leukemia T-lymphoblast cell line, CCRF-CEM, after repeated exposures (7 and 6 times, respectively) for 24 h to constant concentrations (3 and 30 µM) of the drug. Analysis of the mechanism of resistance revealed no differences in levels of dihydrofolate reductase activity, its binding affinity for methotrexate, or in methotrexate transport between the CCRF-CEM parent and methotrexate-resistant cell lines. The development of resistance to methotrexate was associated with a marked decrease in the intracellular level of methotrexate polyglutamates. Although the resistant sublines were able to form substantial amounts of folate polyglutamates when measured with [³H]folic acid, the level of polyglutamates formed was decreased to about 50% of that formed by the parent cell line. No qualitative differences in folate polyglutamates formed were noted between the parental and resistant sublines.

This is the first example of a cell line which displays resistance which is solely attributable to defective methotrexate polyglutamate synthesis.

¹ Supported by USPHS Grant CA 08010, Italian Consiglio Nazionale delle Ricerche Grant 86.00479.44, and an AIRC grant.

² Fellow of the Associazione Italiana per la Ricerca sul Cancro (AIRC), Milano, Italy.

³ Scholar of the Leukemia Society of America. Present address: Grace Cancer Drug Center, Roswell Park Memorial Institute, 666 Elm St., Buffalo, NY 14263.

⁴ American Cancer Society Professor. Present address: Memorial Sloan-Kettering Cancer Center, New York, NY 10021. To whom requests for reprints should be addressed.

Received 10/ 5/87. Revised 12/29/87. Accepted 1/15/88.

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