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

Invest Ophthalmol Vis Sci. 2001 May;42(6):1356-62.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Apoptosis / drug effects
  • Apoptosis / radiation effects*
  • Blotting, Western
  • Caspase 3
  • Caspase Inhibitors
  • Caspases / metabolism*
  • Cell Survival / physiology
  • Genes, bcl-2 / genetics
  • Humans
  • In Situ Hybridization
  • In Situ Nick-End Labeling
  • Light
  • Lipofuscin / metabolism*
  • Oligopeptides / pharmacology
  • Pigment Epithelium of Eye / drug effects
  • Pigment Epithelium of Eye / metabolism*
  • Pigment Epithelium of Eye / pathology
  • Proto-Oncogene Proteins c-bcl-2 / metabolism*
  • RNA, Messenger / metabolism
  • Transfection

Substances

  • Caspase Inhibitors
  • Lipofuscin
  • Oligopeptides
  • Proto-Oncogene Proteins c-bcl-2
  • RNA, Messenger
  • benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone
  • CASP3 protein, human
  • Caspase 3
  • Caspases