Status of mTOR activity may phenotypically differentiate senescence and quiescence

Abstract

SA β-Gal activity is a key marker of cellular senescence. The origin of this activity is the lysosomal β-galactosidase, whose activity has increased high enough to be detected at suboptimal pH. SA β-Gal is also expressed in the cells in quiescence driven by serum-starvation or a high confluency, and it has been hypothesized that SA β-Gal positivity is rather a surrogate marker of high lysosome content or activity. In this study, it was determined how SA β-Gal activity is expressed in quiescence and how lysosome content and activities are differently maintained in senescence and quiescence using DNA damage-induced senescence and serum starvation-induced quiescence as study models. Lysosome content increased to facilitate SA β-Gal expression in both the conditions but with a big difference in the levels of the change. Lipofuscins whose accumulation leads to an increase in residual bodies also increased but with a smaller difference between the two conditions. Meanwhile, lysosome biogenesis was actively ongoing only in senescence progression, indicating that the difference in the lysosome contents may largely be due to lysosome biogenesis. Further, the cells undergoing senescence progression but not the ones in quiescence maintained high mTOR and low autophagy activities. Overall, the results indicate that, although SA β-Gal is expressed due to the elevated lysosome content in both cellular senescence and quiescence, senescence differs from quiescence with high lysosome biogenesis and low autophagy activity, and mTOR activity might be involved in these differences.

Fig. 1.

Changes in SA β-Gal activity in the cells undergoing senescence progression and serum starvation. H460 cells pulsed with adriamycin to induce senescence (A, C) or cultured without FBS (B, D) were incubated for the indicated time points and either stained for SA β-Gal activity in situ (A, B) or collected and lysed to measure β-galactosidease activity. An equal number of cells were lysed and assayed either at pH 4.5 (•) or pH 6.0 (○). The activities measured at the two pH conditions changed almost indetically. A mean of three biological repeats were plotted.

Fig. 2.

Changes in lysosome content and the levels of lysosomal proteins. (A) Cells in both conditions were stained with LTR for 30 min and then applied to flow cytometry. Mean values from three biological repeats were normalized against that of the 0 day control and plotted. (-○- senescence; -•- serum starvation). (B, C) Cells undergoing senescence progression (B) or serum starvation (C) were collected at indicated time points, lysed, and applied to Western blotting assay for Lamp1, Lamp2, LIMP2, Rab7, and cathepsin D, and β-acin.

Fig. 3.

Changes in lysosomal enzyme activities. (A) After staining with DQ™ Red BSA for 30 min at 37°C, cells were applied to flow cytometry. Mean values from three biological repeats were normalized against that of the 0 day control and plotted. (-○- senescence; -•- serum starvation). (B) To detect the intracellular cathepsin B activity in situ, cells were stained with Magic Red™ Cathepsin B (MR-(RR)2) for 1 h at 37°C and observed by confocal microscopy. The brightness of red spots shows the degree of cathepsin B activity within in lysosomes. [(-), day 0 control] [(-), untreated control; top panels, senescence; bottom panels, serum starvation].

Fig. 4.

Changes in autofluorescence and mRNA levels of lysosomal proteins. (A) Cells undergoing senescence progression (-○-) or serum-starved (-•-) were applied to flow cytometry to determine autofluorescene. Mean values from three biological repeats were normalized against that of the 0 day control and plotted. (B, C) mRNA isolated from cells cultured for indicated time points of senescence progression (B) or serum starvation (C) were converted to cDNA and applied for quantitative real-time PCR analysis for the indicated genes of lysosomal proteins (n = 3, *p < 0.05). ASAH1 N-acylsphingosine amidohydrolase; GLB1, β-galactosidase 1; CTSB, cathepsin B; TPP1, tripeptidyl peptidase I; PSAP, prosaponin; SIAE sialic acid acetylesterase.

Fig. 5.

Changes in TFEB activity. (A, B) Cells were seeded on a cover-slip in 24 wells, and cultured for either senescence progression (A) or serum starvation (B), stained with primary TFEB antibody or DAPI at indicated time point, and observed by confocal microscopy. (C, D) Proteins from either senescence progression (C) or serum starvation (D) were applied to Western blotting for TFEB. TFEB protein appeared in multiple bands due to different phosphorylation status. It has been suggested that one of the fast migrating bands is the active one (Pena-Llopis et al., 2011).

Fig. 6.

Cells undergoing either senescence progression (A) or serum starvation (B) were collected at indicated time points and lyzed. Equal amount of proteins were applied to Western blotting for phosphorylated S6K, S6K, phosphorylated S6, 4E-BP1, phosphorylated mTOR, LC3, p62, and beta-actin protein.

Fig. 7.

Changes in LC3 in punctae formation and colocalization with lysosome. H460 cells seeded on a coverslip in 24 wells were treated for senescence (A) or for serum starvation (B), and processed for immunofluorescence staining either LC3 (green) or Lamp1 (red) proteins at indicated time points. Typical field in confocal microscopic fields were photographed. The image at day 5 of the serum starvation was enlarged and presented at the bottom box (× 200).