Portal de Periódicos da CAPES

Induction of Lysosomal Dilatation, Arrested Autophagy, and Cell Death by Chloroquine in Cultured ARPE-19 Cells

2010; Cadmus Press; Volume: 51; Issue: 11 Linguagem: Inglês

10.1167/iovs.10-5278

1552-5783

Young Hee Yoon, Kyung Sook Cho, Jung Jin Hwang, Sook-Jeong Lee, Jeong A Choi, Jae‐Young Koh,

Ocular Diseases and Behçet’s Syndrome

Purpose.: 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.