Senescence-associated beta-galactosidase reflects an increase in lysosomalmass during replicative ageing of human endothelial cells

Senescence-associated beta -galactosidase is widely used as a biomarker of replicative senescence. However, it remains unknown whether this is a disti nct enzyme active at pH 6, and differentially expressed in senescence, or a manifestation of an increase in the classic acid lysosomal beta -galactosi dase, Here we have investigated the origin of senescence-associated-beta -g alactosidase activity by modifying the intracellular and lysosomal pH of yo ung and senescent human umbilical vein endothelial cells and examining the effect of these manipulations on the levels of activity, using a flow cytom etric assay, Lysosomal alkalinisation with chloroquine or bafilomycin Al, a s well as equilibration of the intracellular milieu to pH 6 with nigericin, caused a profound (92-99%) inhibition of the total intracellular beta -gal actosidase activity. However, independent of pH alterations, senescent cell s showed levels of beta -galactosidase activity three- to sixfold higher th an young cells, This increase in activity occurred in parallel to an increa se in beta -galactosidase protein levels, Acridine Orange staining revealed an increase in lysosomal content with replicative age, which correlated wi th the increase in beta -galactosidase. These findings demonstrate that sen escence-associated beta -galactosidase is a manifestation of residual lysos omal activity at a suboptimal pH, which becomes detectable due to the incre ased lysosomal content in senescent cells.