Induction of lysosomal dilatation, arrested autophagy, and cell death by chloroquine in cultured ARPE-19 cells.
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.
Yoon
Young Hee
YH
Department of Ophthalmology, University of Ulsan, College of Medicine, Seoul, Korea.
Cho
Kyung Sook
KS
Hwang
Jung Jin
JJ
Lee
Sook-Jeong
SJ
Choi
Jeong A
JA
Koh
Jae-Young
JY
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
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