title: Induction of lysosomal dilatation, arrested autophagy, and cell death by chloroquine in cultured ARPE-19 cells.
abstract: OBJECTIVE
  To characterize and investigate the mechanism of chloroquine (CQ) retinotoxicity in human retinal pigment epithelium-derived ARPE-19 cells.
  
  
  METHODS
  Cultured ARPE-19 cells were exposed to 10 to 250 μM CQ, and cell death was quantified using a lactate dehydrogenase release assay. Autophagy was studied in ARPE-19 cells transfected with GFP-LC3. Lysosomes in living cells were stained and observed by live-cell confocal microscopy.
  
  
  RESULTS
  After exposure to CQ, ARPE-19 cells developed cytosolic vacuoles within 1 hour and underwent cell lysis within 24 hours. The levels of LC3-II, beclin-1 and, p62, as well as the number GFP-LC3- and RPF-LC3-positive autophagic vacuoles (AVs), increased after CQ treatment, indicating that autophagy was activated. However, lysosomal staining revealed that almost all AVs were separate from lysosomes; thus, fusion between AVs and lysosomes was completely blocked. In addition, the levels of ubiquitinated proteins and GFP-mHttp aggregates in ARPE-19 cells were increased by CQ, providing further evidence that autophagic degradation was inhibited.
  
  
  CONCLUSIONS
  CQ induces vacuole formation and cell death in ARPE-19 cells. Initially, vacuoles developed from enlarged lysosomes, followed by the activation of upstream steps in the autophagy pathway and the formation of LC3-positive AVs. Because CQ blocked the fusion of AVs with lysosomes, autophagic protein degradation was inhibited, indicating that CQ-induced retinotoxicity may be caused by the accumulation of potentially toxic ubiquitinated proteins.
authors.affiliation: Department of Ophthalmology, University of Ulsan, College of Medicine, Seoul, Korea.
authors.forename: Young Hee
authors.initials: YH
authors.lastname: Yoon
authors.forename: Kyung Sook
authors.initials: KS
authors.lastname: Cho
authors.forename: Jung Jin
authors.initials: JJ
authors.lastname: Hwang
authors.forename: Sook-Jeong
authors.initials: SJ
authors.lastname: Lee
authors.forename: Jeong A
authors.initials: JA
authors.lastname: Choi
authors.forename: Jae-Young
authors.initials: JY
authors.lastname: Koh
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000962:Antimalarials / Q000633:toxicity*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D000071186:Beclin-1; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death; D002478:Cells, Cultured; D002738:Chloroquine / Q000633:toxicity*; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects* / Q000473:pathology; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D017382:Reactive Oxygen Species / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D000071456:Sequestosome-1 Protein; D014162:Transfection; D014617:Vacuoles / Q000187:drug effects / Q000473:pathology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000962:Antimalarials; D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D017382:Reactive Oxygen Species; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D049452:Green Fluorescent Proteins; D002738:Chloroquine; D007770:L-Lactate Dehydrogenase; D053148:Caspase 3