Abstract
Abstract: :
Purpose: Macular degeneration (MD), a leading cause of vision loss in humans, leads to apoptotic death of cones. However, little is known regarding the execution of cell death in cones. Therefore, our aim is to generate an in vitro model of cone cells that will undergo death upon light exposure. Methods: About 24,000 661W cells (or their bcl–2 transfected counterparts) were plated in DMEM media in each well of 96–well plates and allowed to attach overnight. Next day, 10 µM of 9–cis–retinal were added and cells were allowed to incubate for 4 hours in the dark in presence of the chromophore. Plates were then placed on a light box and exposed to 30,000 Lux for varying times. Then an MTT viability test was performed. Oxidative stress in the cells was analyzed by Western analysis with 4–hydroxynonenal (HNE) antibody and looking for the presence of HNE–adducts. NF–ΚB protein expression was analyzed by performing Western analysis. Results: Light exposure for 4 hours, induced death of ∼80% of 661W cells in the presence of the chromophore while 80% of Bcl–2–expressing 661W cells survived. NF–ΚB, an anti–apoptotic protein is greatly reduced upon light stress and seems to be specifically cleaved into a smaller fragment upon light exposure in the presence of chromophore. Formation of HNE–adducts (hydroxy–nonenal–protein adducts), a marker of ROS (reactive oxygen species), was observed and the presence of two proteins that were HNE–adducts shows that the oxidative modification of proteins is specific. Conclusions: We have successfully created an assay that causes light induced apoptosis in 661W that is chromophore dependant. Bcl2 expression protects the cells from undergoing cell death. Light stress results in oxidative stress in the cells. We are now in the process of identifying the proteins oxidized as a result of oxidative stress.
Keywords: apoptosis/cell death • retinal culture • photoreceptors