LOCALIZATION AND MOLECULAR-INTERACTIONS OF MITOXANTRONE WITHIN LIVINGK562 CELLS AS PROBED BY CONFOCAL SPECTRAL IMAGING ANALYSIS

Studying mechanisms of drug antitumor action is complicated by the lac k of noninvasive methods enabling direct monitoring of the state and i nteractions of the drugs within intact viable cells. Here we present a confocal spectral imaging (CSI) technique as a method of overcoming t his problem. We applied this method to the examination of localization and interactions of mitoxantrone (1, 4-dihydroxy-5, droxyethyl)-amino ]ethyl)amino-9,10-anthracenedione dihydrochloride), a potent antitumor drug, in living K562 cells. A two-dimensional set of fluorescence spe ctra of mitoxantrone (MITOX) recorded with micron resolution within a drug-treated cell was analyzed to reveal formation of drug-target comp lexes and to create the maps of their intracellular distribution. The analysis was based on detailed in vitro modeling of drug-target (DNA, RNA, DNA topoisomerase II) interactions and environmental effects affe cting drug fluorescence. MITOX exposed to aqueous intracellular enviro nment, MITOX bound to hydrophobic cellular structures, complexes of MI TOX with nucleic acids, as well as the naphtoquinoxaline metabolite of MITOX were simultaneously detected and mapped in K562 cells. These st ates and complexes are known to be immediately related to the antitumo r action of the drug. The results obtained present a basis for the sub sequent quantitative analysis of concentration and time-dependent accu mulation of free and bound MITOX within different compartments of livi ng cancer cells.