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Multidrug Resistance in a Human Leukemic Cell Line Selected for Resistance to Trimetrexate¹

Departments of Neoplastic Diseases [H. A., T. O., J. F. H.] and Radiology [S. V.] and the Derald H. Ruttenberg Cancer Center [T. O., J. F. H., S. V.], Mount Sinai School of Medicine, New York, New York 10029, and Department of Pediatrics, University of Texas, Southwestern Medical Center, Dallas, Texas 75235 [B. A. K.]

Trimetrexate (TMQ) is a lipophilic antifolate shown to have antitumor activity in humans. TMQ-resistant sublines of the MOLT-3 human acute lymphoblastic leukemia cell line were developed and were designated as MOLT-3/TMQ₂₀₀, MOLT-3/TMQ₈₀₀, and MOLT-3/TMQ₂₅₀₀ based on degrees of resistance to TMQ. The TMQ resistance was accompanied by 5- to 7-fold increases in dihydrofolate reductase activity and markedly reduced cellular TMQ accumulation. Methotrexate accumulation was not impaired in TMQ-resistant cells. TMQ retention (efflux) was unchanged in these TMQ-resistant cells. Verapamil enhanced the TMQ accumulation in the resistant cells to the level seen in the parent cells but had no effects on the TMQ retention.

These sublines were cross-resistant not only to methotrexate but also to vincristine, doxorubicin, daunorubicin, and mitoxantrone. There was no cross-resistance to bleomycin or cisplatin. Resistance to vincristine, doxorubicin, daunorubicin, and mitoxantrone was reversed by verapamil. TMQ resistance was only minimally reversed by verapamil and methotrexate resistance not affected at all. Both cellular accumulation and retention of vincristine and daunorubicin in the TMQ-resistant cells were markedly decreased. Verapamil enhanced both accumulation and retention of the drug.

Plasma membrane fractions of the TMQ-resistant cells analyzed by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue revealed the presence of a distinct band with a molecular weight of 170,000. Immunoblot analysis with ¹²⁵I-labeled monoclonal antibody raised against P-glycoprotein of multidrug-resistant Chinese hamster ovary cells (C219) cross-reacted with the M_r 170,000 protein of the TMQ-resistant cells.

These results show that the TMQ-resistant cells displayed not only decreased TMQ uptake and increased dihydrofolate reductase but also characteristics associated with a classical multidrug-resistant phenotype. Multidrug resistance includes lipophilic antifolate.

¹ This work was supported in part by the United States Public Health Service Grant CA-15936 from the National Cancer Institute, Bethesda, MD; by the United Leukemia Fund, Rego Park, NY; by the Chemotherapy Foundation, Inc., New York, NY; and by the T. J. Martell Foundation for Leukemia and Cancer Research, New York, NY.

² To whom requests for reprints should be addressed, at Department of Neoplastic Diseases, Mail Box 1126, Mount Sinai Medical Center, One Gustave L. Levy Place, New York, NY 10029.

Received 3/16/89. Revised 8/21/89. Accepted 8/28/89.

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