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Subcellular Distribution of Daunorubicin in P-Glycoprotein-positive and -negative Drug-resistant Cell Lines Using Laser-assisted Confocal Microscopy¹

Division of Medical Oncology, Department of Medicine, Columbia University, New York, New York 10032 [J. E. G., R. N. T., S. F., S. K., M. R.]; Winthrop University Hospital, Division of Oncology/Hematology, Mineola, New York 11501 [A. A. H.]; and Toronto General Hospital, Toronto, Ontario, Canada M5G 1L7 [M. A. B.]

Four well defined multidrug-resistant cell lines and their drug-sensitive counterparts were examined for intracellular distribution of daunorubicin (DNR) by laser-assisted confocal fluorescence microscopy: P-glycoprotein-negative HL-60/AR cells, and P-glycoprotein-positive P388/ADR, KBV-1, and MCF-7/ADR cells.

Both drug sensitive cell lines (HL-60/S, P388/S, KB3-1, and MCF-7/S) and drug-resistant cell lines (HL-60/AR, P388/ADR, KBV-1, and MCF-7/ADR) exposed to DNR showed a similar rapid distribution of drug from the plasma membrane to the perinuclear region within the first 2 min. From 2–10 min, the drug sensitive HL-60/S, P388/S, and MCF-7/S cells redistributed drug to the nucleus and to the cytoplasm in a diffuse pattern. In contrast, drug-resistant HL-60/AR, P388/ADR, and MCF-7/ADR redistributed DNR from the perinuclear region into vesicles distinct from nuclear structures, thereby assuming a "punctate" pattern. This latter redistribution could be inhibited by glucose deprivation (indicating energy dependence), or by lowering the temperature of the medium below 18°C. The differences in distribution between sensitive and resistant cells did not appear to be a function of intracellular DNR content, nor the result of drug cytotoxicity.

Drug-sensitive KB3-1 and -resistant KBV-1 cells did not fully follow this pattern in that they demonstrated an intracellular DNR distribution intermediate between HL-60/S and HL-60/AR cells with both "punctate" and nuclear/cytoplasmic uptake sometimes in the same cell. These data indicate that the intracellular distribution of DNR is an important determinant of drug resistance regardless of the overexpression of P-glycoprotein. The intracellular movement of drug requires the presence of glucose and a temperature above 18°C, implicating energy-dependent processes and vesicle fusion in the distribution process. This intracellular transport of DNR away from the nucleus in multidrug-resistant cells may protect putative cell targets such as DNA against drug toxicity.

¹ Supported by Grants ACS CH-357, CA-31761, CA-42450, and CA-40188, and the William J. Matheson Foundation, the Medical Research Council of Canada, and the National Cancer Institute of Canada.

² To whom requests for reprints should be addressed, at Columbia University Cancer Center, 630 168th Street, VC 12-213, New York, NY 10032.

Received 9/21/90. Accepted 7/ 9/91.

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