title: Determination of intracellular organelles implicated in daunorubicin cytoplasmic sequestration in multidrug-resistant MCF-7 cells using fluorescence microscopy image analysis.
abstract: 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.
authors.affiliation: Laboratoire de Recherche en Oncologie, Centre Alexis Vautrin, Vandoeuvre-les-Nancy cedex, France.
authors.forename: C
authors.initials: C
authors.lastname: Bour-Dill
authors.forename: M P
authors.initials: MP
authors.lastname: Gramain
authors.forename: J L
authors.initials: JL
authors.lastname: Merlin
authors.forename: S
authors.initials: S
authors.lastname: Marchal
authors.forename: F
authors.initials: F
authors.lastname: Guillemin
mesh_terms: 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
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D003630:Daunorubicin; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein