β-Galactosidase histochemistry and telomere loss in senescent retinal pigment epithelial cells

Purpose. To investigate the relation of senescence-related β- galactosidase activity and telomere shortening to replicative senescence in cultured human retinal pigment epithelial (RPE) cells. Methods. A human RPE cell line was serially passaged until 80% of cells were nondividing in a 72- hour 5-bromo-2'-deoxyuridine (BrdU) labeling study. Early- and late-passage cells were double-stained for BrdU and senescence-related β-galactosidase activity (pH 6). The average chromosomal telomere length at several population doublings was estimated by Southern blot analysis after double digestion of DNA with RsaI and HinfI and using a telomere-specific probe. Results. BrdU-β-galactosidase double-staining revealed an inverse correlation between the number of BrdU-labeled nuclei and β-galactosidase- labeled cells as a function of population doubling level (PDL). At PDL 58, only 20% of all cells labeled for BrdU, whereas 57% stained for β- galactosidase. The mean terminal restriction fragment length (TRF) was reduced from 10 kb in early (PDL 12) cultures to 4 kb in late (PDL 57) cultures. Conclusions. Senescence-related β-galactosidase activity and mean TRF length may prove useful in studying the senescence of RPE cells in vitro. These techniques may be valuable in determining senescence of the retinal pigment epithelium in vivo, where senescent RPE cells could be involved in the development of age-related maculopathy and age-related macular degeneration.