title: 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 damage.
abstract: Flow cytometry and laser scanning confocal 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 wavelength. 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 microM) 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 revealed the time- and dose-dependent appearance of fluorescence, interpreted 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 fluorescence. Photon counting procedures revealed a predominantly cytoplasmic, possibly lysosomal, location for fluorescence from human cells exposed for 1 h to a low but cytotoxic concentration (0.1 microM, yielding approximately 90% cell kill) of mitoxantrone. At this low concentration, human cells displayed minimal levels of DNA strand cleavage or DNA-protein 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 response of normal and multidrug-resistant cells as they attempt to progress through mitosis.
authors.affiliation: MRC Clinical Oncology and Radiotherapeutics Unit, MRC Centre, Cambridge, United Kingdom.
authors.forename: P J
authors.initials: PJ
authors.lastname: Smith
authors.forename: H R
authors.initials: HR
authors.lastname: Sykes
authors.forename: M E
authors.initials: ME
authors.lastname: Fox
authors.forename: I J
authors.initials: IJ
authors.lastname: Furlong
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D002470:Cell Survival; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage; D004351:Drug Resistance; D005240:Feasibility Studies; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D005453:Fluorescence; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008942:Mitoxantrone / Q000493:pharmacokinetics*
chemical_list: D004247:DNA; D008942:Mitoxantrone