Bcl-2, Bax, and c-Fos expression correlates to RPE cell apoptosis induced by UV-light and daunorubicin

Purpose. The aim of this study was to determine the role of Bcl-2, Bcl-X-L, Bax, and c-Fos in regulation of apoptosis. induced by ultraviolet-light A (UV-A) and daunorubicin (DNR), in retinal pigment epithelium (RPE) cells gr own on bovine extracellular matrix (ECM)-coated or uncoated plastic dishes. Methods. Apoptosis in confluent RPE cells cultured on ECM-coated or uncoate d dishes was induced by UV-A or DNR. Apoptosis was detected by 7-amino-acti nomycin D labeling followed by flow cytometry and by terminal deoxy-transfe rase mediated X-dUTP nick end labeling (TUNEL). Cellular expression of Bcl- 2, Bcl-X-L, Bax and c-Fos was determined by the use of antibodies and flow cytometry, Western blot analysis, and immunocytochemical staining. Results. Both UV-A and DNR induce apoptosis in human RPE cells in vitro. Hu man fetal RPE cells grown on ECM-coated dishes were significantly more resi stant to UV-A or DNR induced apoptosis than cells grown on uncoated dishes. RPE cells grown on ECM-coated dishes expressed higher Bcl-2 levels and low er Bax levels compared to cells grown on uncoated dishes. However, Bcl-X-L and c-Fos levels were comparable in the two cultures. After UV-A or DNR tre atment, Bcl-2, Bcl-X-L, Bax, and c-Fos levels were differently regulated in cells grown on ECM-coated dishes compared to cells grown on uncoated dishe s. Conclusion. A significant protection against apoptosis of RPE cells grown o n ECM compared to cells grown on uncoated plastic dishes was found after ex posure to W-A or DNR. This protection was found to be proportionally correlated to the anti-apopt otic protein Bcl-2 and inversely correlated to the expression of Bax. Furth ermore a sustained induction and expression of c-Fos was found to correlate to a higher percentage of apoptotic cells of RPE cells grown on plastic. T hese findings demonstrate that ECM is of great importance for RPE cell surv ival during noxious stimuli and points out the essential role for a healthy Bruch's Membrane (BM) for RPE survival.