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-glycoprotein (P-gp) or multidrug-resistance related protein (MRP) as well as intracellular sequestration of drugs.


METHODS
The resistance phenotype of doxorubicin-selected MCF-7(DXR) human breast adenocarcinoma cell line was characterized by cellular and nuclear daunorubicin efflux, P-gp and MRP expression and apoptosis induction. Daunorubicin sequestration was investigated through organelle markers (lysosomes, endoplasmic reticulum and Golgi apparatus) and daunorubicin co-localization 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
An 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 measurement using calibrated microbeads. The main contribution (65%) of Golgi vesicles in daunorubicin sequestration was demonstrated. Although no rational 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 vesicles identified as Golgi vesicles and resistance to apoptosis induction.
Bour-Dill
C
C
Laboratoire de Recherche en Oncologie, Centre Alexis Vautrin, Vandoeuvre-les-Nancy cedex, France.
Gramain
M P
MP
Merlin
J L
JL
Marchal
S
S
Guillemin
F
F
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis; D000903:Antibiotics, Antineoplastic / Q000378:metabolism*; D017209:Apoptosis; D003593:Cytoplasm / Q000378:metabolism*; D003630:Daunorubicin / Q000378:metabolism*; D018432:Drug Resistance, Multiple; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015388:Organelles / Q000378:metabolism*; D011786:Quality Control; D015203:Reproducibility of Results; D013347:Subcellular Fractions; D014407:Tumor Cells, Cultured; D020394:Vault Ribonucleoprotein Particles / Q000096:biosynthesis; D000099187:Major Vault Protein
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D003630:Daunorubicin; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein