title: Detection and characterisation of multi-drug resistance protein 1 (MRP-1) in human mitochondria.
abstract: BACKGROUND
  Overexpression of plasma membrane multi-drug resistance protein 1 (MRP-1) can lead to multidrug resistance. In this study, we describe for the first time the expression of mitochondrial MRP-1 in untreated human normal and cancer cells and tissues.
  
  
  METHODS
  MRP-1 expression and subcellular localisation in normal and cancer cells and tissues was examined by differential centrifugation and western blotting, and immunofluorescence microscopy. Viable mitochondria were isolated and MRP-1 efflux activity measured using the calcein-AM functional assay. MRP-1 expression was increased using retroviral infection and specific overexpression confirmed by RNA array. Cell viability was determined by trypan blue exclusion and annexin V-propidium iodide labelling of cells.
  
  
  RESULTS
  MRP-1 was detected in the mitochondria of cancer and normal cells and tissues. The efflux activity of mitochondrial MRP-1 was more efficient (55-64%) than that of plasma membrane MRP-1 (11-22%; P<0.001). Induced MRP-1 expression resulted in a preferential increase in mitochondrial MRP-1, suggesting selective targeting to this organelle. Treatment with a non-lethal concentration of doxorubicin (0.85 nM, 8 h) increased mitochondrial and plasma membrane MRP-1, increasing resistance to MRP-1 substrates. For the first time, we have identified MRP-1 with efflux activity in human mitochondria.
  
  
  CONCLUSIONS
  Mitochondrial MRP-1 may be an exciting new therapeutic target where historically MRP-1 inhibitor strategies have limited clinical success.
authors.affiliation: Children's Cancer Research Group, Leeds Institute of Molecular Medicine, St. James's University Hospital, Beckett Street, Leeds LS9 7TF, UK. e.a.roundhill@leeds.ac.uk
authors.forename: E A
authors.initials: EA
authors.lastname: Roundhill
authors.forename: S A
authors.initials: SA
authors.lastname: Burchill
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D001693:Biological Transport, Active; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D005047:Etoposide / Q000494:pharmacology; D017313:Fenretinide / Q000494:pharmacology; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D021381:Protein Transport; D012512:Sarcoma, Ewing / Q000378:metabolism / Q000473:pathology; D014750:Vincristine / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; C018551:glucose-regulated proteins; D017313:Fenretinide; D003609:Dactinomycin; D014750:Vincristine; D005047:Etoposide; D004317:Doxorubicin; C007740:fluorexon