Selective determination of the doxorubicin content of individual acidic organelles in impure subcellular fractions.
Since organelle preparations often contain more than one organelle type (e.g., acidic organelles and mitochondria), techniques that measure the properties of a given organelle type while avoiding biases caused by ancillary subcellular compartments are highly desirable. We report here the use of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) dual-channel detection to identify acidic organelles containing doxorubicin (DOX) in crude subcellular fractions from CCRF-CEM and CEM/C2 cell lines. As confirmed by confocal microscopy, acidic organelles are identified by their accumulation of fluorescently labeled nanospheres. Using CE-LIF analysis, individually detected organelles are classified into three kinds: acidic organelles containing only nanospheres, acidic organelles containing nanospheres and DOX, and other organelles containing DOX (e.g., mitochondria) with no detectable nanospheres. Electrophoretic mobility, DOX fluorescence intensity, and nanosphere fluorescence intensity distributions of individual acidic organelles and other organelles containing DOX are determined in the same CE-LIF run. The acidic organelle mobilities range from (-0.7 to -2.0) x 10(-4) cm(2) V(-1) s(-1) while those of the other organelles spread from (-0.6 to -3.5) x 10(-4) cm(2) V(-1) s(-1). In addition, by calibrating the detector response, DOX content in individual acidic organelles and other organelles can be estimated. The average amounts of DOX per acidic organelle in CEM/C2 and CCRF-CEM cells are 11.1 +/- 0.5 and 10.6 +/- 0.4 zmol, respectively. This first report of an analysis of the accumulation of DOX in individual acidic organelles presents a procedure for analyzing the accumulation of fluorescent compounds in acidic organelles that could be useful for investigating acidic organelle maturation and the role of these organelles in drug resistance.
Chen
Yun
Y
Department of Chemistry, University of Minnesota, 207 pleasant Street S.E., Minneapolis, Minnesota 55455, USA.
Walsh
Richard J
RJ
Arriaga
Edgar A
EA
D000903:Antibiotics, Antineoplastic / Q000032:analysis; D004317:Doxorubicin / Q000032:analysis* / Q000378:metabolism; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D007091:Image Processing, Computer-Assisted; D007834:Lasers; D036103:Nanotechnology / Q000379:methods; D015388:Organelles / Q000737:chemistry* / Q000145:classification; D013347:Subcellular Fractions; D014407:Tumor Cells, Cultured
D000903:Antibiotics, Antineoplastic; D004317:Doxorubicin