Determination of intracellular organelles implicated in daunorubicin cytoplasmic sequestration in multidrug-resistant MCF-7 cells using fluorescence microscopy image analysis

Background: Anthracycline resistance is known to be mediated by P-glycoprot ein (P-gp) or multidrug-resistance related protein (MRP) as well as intrace llular sequestration of drugs. Methods: The resistance phenotype of doxorubicin-selected MCF-7(DXR) human breast adenocarcinoma cell line was characterized by cellular and nuclear d aunorubicin efflux, P-ey and MRP expression and apoptosis induction. Daunor ubicin sequestration was investigated through organelle markers (lysosomes, endoplasmic reticulum and Golgi apparatus) and daunorubicin co-localizatio n by dual-color image analysis fluorescence microscopy using high numerical aperture objective lenses to achieve the smallest field depth and the best lateral resolution. Signal to noise and specificity ratios were optimized for daunorubicin and organelle fluorescent probes labeling. Results: tin original image analysis procedure was developed to investigate daunorubicin and organelles co-localization. The reliability of the image analysis was controlled through chromatic shift and intensity linearity mea surement using calibrated microbeads. The main contribution (65%) of Golgi vesicles in daunorubicin sequestration was demonstrated Although no rationa l relationship could be established between daunorubicin sequestration and apoptosis induction, no apoptosis was observed in MCF-7(DXR) cells. Conclusions: In addition to P-glycoprotein mediated drug efflux and without MRP overexpression, MCF-7(DXR) daunorubicin resistance phenotype involves drug sequestration within intracellular resides identified as Golgi vesicle s and resistance to apoptosis induction. (C) 2000 Wiley-Liss, Inc.