Density-dependent resistance to apoptosis in retinal cells

Purpose. To study effects of cell density on retinal cell survival. Methods. Apoptotic cell death was induced in cultured retinal cells seeded at higher or lower density by various stimuli including simulated ischemia, excitotoxicity and antibody against heat shock protein 27 (hsp27). Quantit ative analysis of apoptotic cells was performed using terminal deoxynucleot idyl transferase-mediated dUTP nick end labeling technique and flow cytomet ry. Cytoskeleton was examined using immunocytochemistry and specific staini ng of actin by phalloidin and DNase I. In addition, alterations in the cyto skeletal proteins, bcl-2 family of proteins and hsp27 were studied using we stern blotting. Results. Incubation of the cells under apoptotic stimuli caused higher rate s of apoptosis in lower density cultures as determined by TUNEL technique a nd flow cytometric analysis. Both morphologic examination of cytoskeleton a nd western blotting revealed that after incubation with various stressors, degradation of actin and tubulin was more prominent in lower density cultur es compared to higher density cultures. The expression of bcl-2 and bcl-xL was higher and the expression of bar was lower in lower density cultures co mpared to higher density cultures at basal condition. After incubation with stressors, bcl-2 and bcl-xL expressions decreased and bar expression incre ased in both lower and higher density cultures. However, we observed that t he expression of hsp27 was higher in higher density cultures than in lower density cultures in the presence or absence of apoptotic stimuli. Conclusions. These findings demonstrate that retinal cells are more resista nt to apoptosis in higher density cultures, independent of the inducer. Thi s might be partly due to protective activity of endogenous hsp27 in the cel ls at higher density, which contributes to cytoskeletal integrity in respon se to apoptotic stimuli.