Blue light-induced apoptosis of A2E-containing RPE: Involvement of caspase-3 and protection by bcl-2

PURPOSE. The lipofuscin fluorophore A2E has been shown to mediate blue ligh t-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, r espectively, 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 fluo rogenic 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 w ithout Bcl-2 cDNA. Expression of Bcl-2 transcripts by independently derived clones was established by in situ hybridization, and Bcl-2 protein express ion was confirmed by Western blot analysis. Cell viability was assayed by T dT-dUTP terminal nick-end labeling (TUNEL) in conjunction with 4'6'-diamidi no-2-phenylindole (DAPI) staining and by fluorescence staining of the nucle i of membrane-compromised cells. RESULTS. In RPE cells that had previously accumulated A2E, caspase-3 activi ty was detected within 5 hours of blue light exposure. The incidence of apo ptotic 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 cel ls 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 caspa se cascade and that is regulated by Bcl-2.