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Characterization of Multidrug Resistance by Fluorescent Dyes¹

Departments of Pharmacology [D. K., V. S.] and Pathology [D. Ku.], Wayne State University, Detroit, Michigan 48201, and Department of Pharmacology [W. B.], St. Jude Children's Hospital, Memphis, Tennessee 38101

Fluorimetric techniques were used to examine accumulation of fluorescent probes by the P388 murine leukemia and an anthracycline-resistant subline, P388/Adriamycin(ADR), which expresses the multidrug-resistant phenotype. P388 could be differentiated from P388/ADR on the basis of fluorescence intensity measurements using 3 classes of cationic dyes that are sensitive to membrane potential differences: rhodamine esters, cyanines, and styrylpyridinium dyes. But fluorescence intensity differences were also observed with potential-insensitive dyes: zwitterionic rhodamines and an acridine orange derivative. In all cases, fluorescence intensity differences were caused by impaired dye accumulation, and could be eliminated by treatment of P388/ADR cells with verapamil. Moreover, fluorescence signals from 2 anionic potential-sensitive dyes, merocyanine 540 and a bis-oxonol, were identical in P388 and P388/ADR. None of these dyes could be used to delineate CCRF-CEM, a lymphoblastic leukemia of human origin from the CEM/VM-1 subline that exhibits a markedly atypical drug resistance pattern not based on an enhanced outward transport. But accumulation of both neutral and cationic dyes was impaired in CEM/VLB₁₀₀, a subline of CCRF-CEM expressing mdr. These studies show that many cationic and neutral fluorescent probes are substrates for the enhanced outward drug transport system associated with P388/ADR cells, and cannot be used to probe membrane-potential differences in cells expressing the mdr phenotype. With several dyes, differences in fluorescence intensity were sufficient so that flow cytometry could be used to delineate P388 from P388/ADR and CCRF-CEM from CEM-VLB₁₀₀. The latter technique may be useful for identifying malignant cell populations expressing multidrug resistance in patients with neoplastic disease.

¹ This work was supported in part by Grants CA 23378, CA 40570, and CA 21765 from the National Cancer Institute, NIH, Department of Health and Human Services, and by a grant from the American Lebanese Associated Charities.

² To whom requests for reprints should be addressed.

Received 2/22/91. Accepted 6/12/91.

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