The limitations and validities of senescence associated-β-galactosidase activity as an aging marker for human foreskin fibroblast Hs68 cells

The senescence associated-β-galactosidase (SA-βG) assay has become one of the most commonly used markers of cell-aging. However, the reliability of the assay is questionable because the enzyme is a non-specific marker for cell-aging. In this study, we found that the SA-βG activity increased with cell age as well as in confluent quiescent cells or cells under serum starvation, and in cells treated with H2O2. Importantly, we found that SA-βG activity was irreversibly increased in the senescent cells or H2O2-teated cells, but was reversible in quiescent cells induced by serum starvation or confluence. Using fluorescein di-β-d-galactopyranoside (FDG) method for SA-βG detection, we showed that senescent human foreskin fibroblast Hs68 cells did not express a specific enzyme that has a maximal activity at pH 6.0. In the pH profile of the cellular βG activity in senescent Hs68 cells, only a single peak was found (with maximum at pH 4.6), and no addition peak was found at or around pH 6.0 that could be attributed to the SA-βG activity. These results support the contention that SA-βG is the lysosomal βG that is detectable at suboptimal pH (i.e. pH 6.0) and demonstrate that cell-aging is not the only factor that can increase SA-βG activity, rendering SA-βG activity unspecific for cell-aging. Thus, the assay for cell-aging is only reliable when these confounding factors are controlled or excluded.