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Janet R. Sparrow, Bolin Cai; Blue Light–Induced Apoptosis of A2E-Containing RPE: Involvement of Caspase-3 and Protection by Bcl-2. Invest. Ophthalmol. Vis. Sci. 2001;42(6):1356-1362. doi: https://doi.org/.
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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
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
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