Subcellular Distribution of the Anticancer Drug Mitoxantrone in Human and Drug-resistant Murine Cells Analyzed by Flow Cytometry and Confocal Microscopy and Its Relationship to the Induction of DNA Damage1

Flow cytometry and laser scanning confricai imaging have been used to analyze the uptake of the anticancer topoisomerase II poison mitoxantrone by intact mammalian cells and the results correlated with the induction of DNA damage. Unlike Adriamycin, mitoxantrone displays only minimal levels of red fluorescence when excited at 514 nm wave length. However, using these excitation and emission conditions, flow cytometry could detect low levels of fluorescence in human transformed fibroblasts exposed to high concentrations (5-20 /IM) of mitoxantrone for 1 h. Over this dose range whole cell fluorescence was a function of cell size and increased with drug concentration while drug-induced DNA-protein cross-linking showed saturation. Confocal microscopy re vealed the timeand dose-dependent appearance of fluorescence, inter preted here as reflecting the disposition of drug molecules, preferentially within the cytoplasm, nuclear membrane, and nucleoli. This pattern contrasted with the intense intranuclear fluorescence observed in Adriamycin-treated human cells. Loss of the nuclear membrane during mitosis resulted in an apparent increase in chromatin-associated fluo rescence. Photon counting procedures revealed a predominantly cytoplasmic, possibly lysosontal, location for fluorescence from human cells exposed for l h to a low but cytotoxic concentration (0.1 MM,yielding approximately 90% cell kill) of mitoxantrone. At this low concentration, human cells displayed minimal levels of DNA strand cleavage or DNAprotein cross-linking. Murine cells, displaying mitoxantrone resistance as part of the P-glycoprotein-mediated multidrug resistance phenotype, showed specific extinction of mitoxantrone-associated fluorescence from inside nuclei but not from within extranuclear compartments. The study demonstrates the feasibility of high resolution studies on the intracellular distribution of mitoxantrone in intact living cells. We suggest a mechanism by which cytoplasmic sequestration of mitoxantrone may be important in determining the responses of normal and multidrug-resistant cells as they attempt to progress through mitosis.