Elsevier

Experimental Gerontology

Volume 40, Issue 10, October 2005, Pages 813-819
Experimental Gerontology

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

https://doi.org/10.1016/j.exger.2005.07.011Get rights and content

Abstract

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.

Introduction

Eukaryotic β-galactosidase (βG) is a hydrolase localized in the lysosome that cleaves β-d-galactose residues in β-d-galactosides. The activity of the enzyme is maximal at acid pH 3–5 and is species-, organ-, substrate- and buffer-dependent (Krishna et al., 1999). In the year 1995, a report suggested that the senescent cells express a specific type of the enzyme β-galactosidase referred to senescence associated-β-galactosidase (SA-βG) that has an optimal pH at 6.0 (Dimri et al., 1995). To date, SA-βG assay has become one of the most commonly used markers of cell-aging in vitro. For examples, the assay has been used to investigate the accelerating effect on cell-aging of homocysteine (Xu et al., 2000), ceramide (Mouton and Venable, 2000), sublethal oxidative stress (Dumont et al., 2000) and cardiolipin (Arivazhagan et al., 2004), as well as the effect for anti-cell-aging of carnosine (Mcfarland and Holliday, 1994), nicotinamide (Matuoka et al., 2001), and HDTIC-1 and HDTIC-2 (two compounds extracted from Astragali radix, a traditional Chinese anti-aging medicine) (Wang et al., 2003). Despite its wide application, the reliability of the assay is questionable because of a lack of specificity. For instance, SA-βG activity can increase in the immortalized cells following serum starvation (Yegorov et al., 1998), oxidative treatment with H2O2 (Severino et al., 2000), and in confluent non-transformed fibroblast cultures (Severino et al., 2000). To date, it has not been established whether a distinct enzyme expresses specifically at cell-aging. SA-βG has been reported as a manifestation of residual lysosomal βG activity at a suboptimal pH, and the enzyme activity becomes detectable owing to increased lysosomal βG in senescent cells (Kurz et al., 2000). This seems reasonable because lysosomal contents including several enzymes have been proposed to increase with cell-aging (Kurz et al., 2000). However, more evidence is needed to support this contention.

To address the specificity and reliability issues of the SA-βG assay as a cell-aging marker, we set out to examine the relationship between SA-βG activity and cell-aging in human foreskin fibroblast Hs68 cells. We also investigated possible interfering factors of the SA-βG activity, such as serum starvation, cell confluence, and oxidative damage by H2O2, and we provided solutions to these interferences. Here, we used a modified method for SA-βG detection by using di-β-d-galactopyranoside (FDG) as substrate (Yang and Hu, 2004) to examine our hypothesis that, if senescent cells express SA-βG, there will be two different optimal pH values for the cellular βG activity, i.e. one at or around pH 4.0 and the other at or around pH 6.0, because the lysosomal βG and the SA-βG have maximal activities at around pH 4.0 and 6.0, respectively (Kurz et al., 2000).

Section snippets

Chemical

All chemicals used were of analytical grade. NaCl, KCl, Na2HPO4·2H2O, KH2 PO4, dimethylsulfoxide (DMSO), formaldehyde were purchased from Merck (Germany). 5-Bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-Gal), NN-dimethyl formamide, citric acid, potassium ferricyanide, potassium ferrocyanide, fluorescein, glutaraldehyde were obtained from Sigma (St Louis, MO, USA). Commercial βG from Aspergillus oryzae (G5610) and Escherichia coli (G5635) were also purchased from Sigma (St Louis, MO, USA).

Effect of confluence and cell age on SA-βG

Hs68 cells at p20 with 4×105 and 1.5×105 were plated onto a 75-cm2 flasks and cultured until confluence, and the cellular SA-βG activity was detected by X-Gal staining method. The results showed that confluence with a high cell density affected the SA-βG activity profoundly. The SA-βG activity of Hs68 cells at p20 was lower before confluence than at confluence, as shown by fewer cells stained positive of the former (Fig. 1a and d, blue pigment). Cells that had reached confluence showed a 100%

Discussion

The data presented here demonstrate that senescent Hs68 cells do not have a distinct enzyme SA-βG and that this so-called SA-βG in Hs68 cells is probably the same as the lysosomal βG. First, we found that, in addition to cell senescence and H2O2 treatment, serum starvation and confluent culture increased the enzyme activity, indicating that the enzyme is not specific for cell-aging. In fact, serum starvation, confluent culture, and cell-aging have been reported to increase lysosomal βG activity

Conclusion

We have demonstrated that several factors, including serum starvation, confluent culture, H2O2 treatment, and cell ages, induce SA-βG activity in Hs68 cells and that the SA-βG is likely the lysosomal βG that is detected at pH 6.0. We also demonstrate that the increase in SA-βG activity induced by serum starvation and cell confluence is reversible, whereas, the activity induced by cell senescence or by H2O2-treatment is not. Therefore, the SA-βG assay can only be a reliable marker for cell-aging

Acknowledgements

This research was supported by grants from the National Science Council (NSC-92-2320-B-005-004).

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