J R Sparrow1, B Cai. 1. Department of Ophthalmology, Columbia University, 630 W. 168th Street, New York, NY 10032, USA. jrs88@columbia.edu
Abstract
PURPOSE: The lipofuscin fluorophore A2E has been shown to mediate blue light-induced damage to retinal pigment epithelial (RPE) cells. The purpose of this study was to evaluate caspase-3 and Bcl-2 as executor and modulator, respectively, of the cell death program that is initiated in A2E-containing cells in response to blue light. METHODS: Human RPE cells (ARPE-19) that had accumulated A2E were exposed to blue light. Caspase-3 activity was assayed by observing cleavage of a fluorogenic peptide substrate, and the effect of a peptide inhibitor of caspase-3 (Z-DEVD-fmk) on the quantity of apoptotic nuclei was determined. ARPE-19 cells were transfected with either a neomycin-selectable expression vector containing Bcl-2 cDNA or a control neomycin-selectable expression vector without Bcl-2 cDNA. Expression of Bcl-2 transcripts by independently derived clones was established by in situ hybridization, and Bcl-2 protein expression was confirmed by Western blot analysis. Cell viability was assayed by TdT-dUTP terminal nick-end labeling (TUNEL) in conjunction with 4'6'-diamidino-2-phenylindole (DAPI) staining and by fluorescence staining of the nuclei of membrane-compromised cells. RESULTS: In RPE cells that had previously accumulated A2E, caspase-3 activity was detected within 5 hours of blue light exposure. The incidence of apoptotic nuclei was attenuated when A2E-containing RPE cells were exposed to blue light in the presence of caspase-3 inhibitor and in A2E-loaded RPE cells that had been stably transfected with Bcl-2. CONCLUSIONS: Blue light illumination of RPE in the setting of intracellular A2E initiates a cell death program that is executed by a proteolytic caspase cascade and that is regulated by Bcl-2.
PURPOSE: The lipofuscin fluorophore A2E has been shown to mediate blue light-induced damage to retinal pigment epithelial (RPE) cells. The purpose of this study was to evaluate caspase-3 and Bcl-2 as executor and modulator, respectively, of the cell death program that is initiated in A2E-containing cells in response to blue light. METHODS:Human RPE cells (ARPE-19) that had accumulated A2E were exposed to blue light. Caspase-3 activity was assayed by observing cleavage of a fluorogenic peptide substrate, and the effect of a peptide inhibitor of caspase-3 (Z-DEVD-fmk) on the quantity of apoptotic nuclei was determined. ARPE-19 cells were transfected with either a neomycin-selectable expression vector containing Bcl-2 cDNA or a control neomycin-selectable expression vector without Bcl-2 cDNA. Expression of Bcl-2 transcripts by independently derived clones was established by in situ hybridization, and Bcl-2 protein expression was confirmed by Western blot analysis. Cell viability was assayed by TdT-dUTP terminal nick-end labeling (TUNEL) in conjunction with 4'6'-diamidino-2-phenylindole (DAPI) staining and by fluorescence staining of the nuclei of membrane-compromised cells. RESULTS: In RPE cells that had previously accumulated A2E, caspase-3 activity was detected within 5 hours of blue light exposure. The incidence of apoptotic nuclei was attenuated when A2E-containing RPE cells were exposed to blue light in the presence of caspase-3 inhibitor and in A2E-loaded RPE cells that had been stably transfected with Bcl-2. CONCLUSIONS: Blue light illumination of RPE in the setting of intracellular A2E initiates a cell death program that is executed by a proteolytic caspase cascade and that is regulated by Bcl-2.
Authors: Nicole M Haralampus-Grynaviski; Laura E Lamb; Christine M R Clancy; Christine Skumatz; Janice M Burke; Tadeusz Sarna; John D Simon Journal: Proc Natl Acad Sci U S A Date: 2003-02-28 Impact factor: 11.205
Authors: So Ra Kim; Nathan Fishkin; Jian Kong; Koji Nakanishi; Rando Allikmets; Janet R Sparrow Journal: Proc Natl Acad Sci U S A Date: 2004-07-26 Impact factor: 11.205