Blue light-induced apoptosis of A2E-containing RPE: involvement of caspase-3 and protection by Bcl-2.
OBJECTIVE
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.
Sparrow
J R
JR
Department of Ophthalmology, Columbia University, 630 W. 168th Street, New York, NY 10032, USA. jrs88@columbia.edu
Cai
B
B
D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects*; D015153:Blotting, Western; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002470:Cell Survival / Q000502:physiology; D019254:Genes, bcl-2 / Q000235:genetics; D006801:Humans; D017403:In Situ Hybridization; D020287:In Situ Nick-End Labeling; D008027:Light; D008062:Lipofuscin / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection
D061945:Caspase Inhibitors; D008062:Lipofuscin; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases