Increased expression of SIRT2 is a novel marker of cellular senescence and is dependent on wild type p53 status

Abstract

Sirtuins (SIRT) belonging to the NAD+ dependent histone deacetylase III class of enzymes have emerged as master regulators of metabolism and longevity. However, their role in prevention of organismal aging and cellular senescence still remains controversial. In the present study, we now report upregulation of SIRT2 as a specific feature associated with stress induced premature senescence but not with either quiescence or cell death. Additionally, increase in SIRT2 expression was noted in different types of senescent conditions such as replicative and oncogene induced senescence using multiple cell lines. Induction of SIRT2 expression during senescence was dependent on p53 status as depletion of p53 by shRNA prevented its accumulation. Chromatin immunoprecipitation revealed the presence of p53 binding sites on the SIRT2 promoter suggesting its regulation by p53, which was also corroborated by the SEAP reporter assay. Overexpression or knockdown of SIRT2 had no effect on stress induced premature senescence, thereby indicating that SIRT2 increase is not a cause of senescence; rather it is an effect linked to senescence-associated changes. Overall, our results suggest SIRT2 as a promising marker of cellular senescence at least in cells with wild type p53 status.

Keywords: SIRT2; Sirtuins; doxorubicin; p53; senescence.

Figure 1.

Doxorubicin induced premature senescence and expression of Sirtuin isoforms. (A) U2OS cells were treated with doxorubicin (Dox, 1 µM, 2 h), grown in fresh culture medium for 120 h and assayed for SA-βgal (blue). Untreated cells served as control. (B) Bar diagram showing percentage SA-βgal positive cells in control and doxorubicin treated U2OS cells at 120 h (*P < 0.05). (C) Immunoblots showing expression of senescence-associated markers viz., p53, p21, PAI-1 and Lamin B1 in control and senescent cells at 120 h. (D) Time kinetics showing cell cycle distribution of control and doxorubicin treated cells. (E) Bar diagram showing relative fold change in expression levels of various Sirtuin transcripts in control and senescent U2OS cells at 120 h. The transcript levels of SIRT1-SIRT7 were quantified by real-time PCR and normalized to GAPDH mRNA (*P < 0.05). (F) Immunoblots showing expression of various Sirtuin isoforms in control vs. senescent cells at 120 h. (G) Bar diagram showing the fold increase in expression levels of Sirtuin proteins in senescent cells in comparison to control cells. Note the presence of 2 distinct isoforms for both SIRT2 (39 and 43 kDa) and SIRT7 (45 and 47.5 kDa) on the immunoblot. To calculate the total fold change, the corresponding increase in both the isoforms were normalized first to GAPDH and the values obtained were averaged together to represent the total pool of either SIRT2 or SIRT7 in the bar graph (*P < 0.05).

Figure 2.

Increased expression of SIRT2 is specific only to stress-induced premature senescence but not to conditions of quiescence, cell death, or G2/M phase cell cycle arrest. (A) Representative immunoblots showing expression levels of SIRT2, SIRT4 and p21 in control, doxorubicin (Dox) induced senescent cells (1 µM, 120 h) and serum starved growth arrested quiescent U2OS cells (0.1% serum, 96 h). (B, C) Bar diagram showing fold change in expression levels of total SIRT2 and SIRT4 in control, senescent and quiescent cells (*P < 0.05). (D) Flow cytometry analysis showing cell cycle phase distribution of U2OS cells treated with doxorubicin (Dox) and nocodazole (Noc), a specific mitotic inhibitor. Following nocodazole treatment (300 ng/ml, 16 h), the loosely attached cells were collected by shake off (Noc-F) method and adherent cells (Noc-A) by trypsinization. (E) Immunoblots showing expression of SIRT2 in doxorubicin induced senescent cells (72 h) and nocodazole (Noc-F and Noc-A) treated G2/M arrested cells (16 h). Note a mobility shift of SIRT2 isoforms (indicated by arrow) in nocodazole arrested G2/M cells. (F) SA-βgal staining of control and doxorubicin treated (0.1 µM, 1 µM and 10 µM) U2OS cells at 72 h. (G) Immunoblot showing expression of SIRT2, p21 and PARP cleavage in senescent inducing doses (0.1 µM, 1 µM) and cell death inducing dose (10 µM) of doxorubicin. Paclitaxel was used as positive control to induce programmed cell death. (H) Bar diagram showing fold change in expression level of total SIRT2 in control, doxorubicin and paclitaxel treated U2OS cells at 72 h (*P < 0.05).

Figure 3.

SIRT2 increase in doxorubicin treated cells does not coincide with early DNA damage but associated with induction of senescence and correlates with expression of Lamin B1. (A) Immunoblots showing expression of SIRT2 and γH2AX at early time (4 h) point following treatment of U2OS cells with varying doses of doxorubicin (Dox). (B-D) Quantitative analysis of the time course of SIRT2, Lamin B1 and p21 expression levels during the induction of senescence following doxorubicin treatment. The bar graph represents the results of 3 independent experiments (*P < 0.05). (E) Increase in SIRT2 expression in doxorubicin induced senescent cells is accompanied with decrease in acetylation (Ac) status of its well-known targets, tubulin, p65 and histone H4, at their respective lysine residues. GAPDH and total histone H3 served as loading controls. (F-H) Bar graph showing fold change in acetylation status of tubulin, p65 and histone H4 following doxorubicin treatment at varying time intervals (*P < 0.05).

Figure 4.

SIRT2 expression in various modes of senescent conditions viz., stress-, replicative- and oncogene- induced. (A) SA-βgal staining of U2OS cells at 120 h following treatment with DNA damaging agents such as doxorubicin (Dox, 1 µM, 2 h), camptothecin (Campto, 1 µM, 2 h), etoposide (Etopo, 1 µM, 72 h) or oxidative stress agent, H₂O₂ (200 µM, 2 h). (B) Immunoblots showing expression of SIRT2 and p21 in U2OS control and senescent cells (120 h) induced by various agents. (C) Bar diagram showing fold change in expression level of SIRT2 in stress-induced senescence by different agents (*P < 0.05). (D) SA-βgal activity in human primary adult dermal fibroblasts undergoing replicative senescence. (E) Immunoblots showing expression of SIRT2 and Lamin B1 in young (passage 4, 17) and senescent fibroblasts (passage 57). The values below the blots indicate the relative fold change in total SIRT2 and Lamin B1 levels. (F) SA-βgal staining in untransfected U2OS cells and transfected with either vector alone or mutant RAS (HRAS-V12). (G) Immunoblots showing expression of RAS, SIRT2 and Lamin B1 in the untransfected cells (U2OS) and those transfected with either vector or HRAS-V12. (H) Bar graph showing relative change in total SIRT2 levels in oncogene-induced senescent cells compared to vector control cells (*P < 0.05).

Figure 5.

Treatment with antioxidant NAC alleviated stress induced premature senescence by doxorubicin and hydrogen peroxide. (A) SA-βgal staining (blue) of cells treated with doxorubicin (0.2 µM) and H₂O₂ (200 µM) alone or in combination with NAC (50mM). Note fewer senescent cells in NAC treated cells. (B) Bar diagram showing percentage SA-βgal positive cells in various treatment conditions: control, doxorubicin (0.2 µM), H₂O₂ (200 µM) and in combination with NAC (50 mM). (C) Representative immunoblots showing expression of SIRT2 and p21 in doxorubicin (0.2 µM) and H₂O₂ (200 µM) treated U2OS cells alone or in combination with NAC (50 mM). Tubulin served as a loading control. Values below the blot indicate fold change in levels of p21 normalized to tubulin. (D) Bar diagram showing quantification of levels of SIRT2 in various treatment conditions. (*P <0.05).

Figure 6.

p53 regulates expression of SIRT2. (A) SA-βgal staining in p53 null cells (Saos-2) treated with doxorubicin (0.5 µM, 1 h or 50 nM, 48 h) at 120 h. (B) Immunoblots showing expression of total SIRT2 in control and senescent Saos-2 cells at 120 h. Values below the blot indicate fold change in total level of SIRT2 normalized to GAPDH. (C) U2OS cells transfected with either p53 shRNA or non-target shRNA (NT shRNA) were treated with doxorubicin (1 µM, 2 h) and SA-βgal staining was performed at 120 h. (D) Immunoblots showing expression of p53, SIRT2 and p21 in U2OS cells with or without p53 depletion (p53 shRNA) treated with varying doses of doxorubicin. (E) Quantification of fold change in expression of SIRT2 in NT shRNA and p53 depleted cells (*P < 0.05). (F) SA-βgal staining of U2OS cells treated with doxorubicin, and nutlin either alone or in combination. (G) Immunoblot analysis of p53, SIRT2 and p21 in U2OS cells treated with doxorubicin (0.2 µM, 2 h) and nutlin (5 µM) either alone or in combination. (H) Quantification of fold change of total SIRT2 in doxorubicin and nutlin treated U2OS cells either alone or together.

Figure 7.

SIRT2 promoter has a p53 binding site. (A) Schematic representation of chromosome 19 showing location of SIRT2 gene (39370265-39390629 bp) and p53 binding site at its promoter region [+348 bp to +368 bp from transcription start site (TSS)]. Positions of primers used in ChIP assay are indicated by the arrows (+239 bp to +487 bp from TSS). (B, C) ChIP assay was performed in control and senescent U2OS cells using a p53 specific antibody or mouse IgG as control, followed by quantitative PCR with primers specific for SIRT2 and p21 promoter regions. Bar diagram indicates the fold enrichment of p53 binding at SIRT2/p21 promoter in control and doxorubicin induced senescent cells (*P < 0.05). (D) U2OS control and doxorubicin induced senescent cells were transfected with either SEAP plasmid alone (Vector) or with SEAP plasmid containg SIRT2 promoter with intact p53 binding site (SIRT2) or with deletion of p53 binding region (SIRT2_p53 del). Later at 24 h post transfection, SEAP assay was performed using the culture-conditioned medium from both control and senescent cells. Bar diagram indicates the SEAP activity (plotted as fold change over control) in control and senescent U2OS cells (* P < 0.05).

Figure 8.

Effect of SIRT2 overexpression or knockdown in regulation of stress induced premature senescence in U2OS cells. (A) Immunoblot showing expression of endogenous and exogenously overexpressed SIRT2 in U2OS cells transfected with either GFP or SIRT-GFP vector. Note a specific deacetylation of tubulin in SIRT2-GFP cells. (B) Bar graph represents the SA-βgal positive cells treated with varying doses doxorubicin (0.2 µM – 1 µM) in U2OS cells expressing either GFP or SIRT2-GFP, at 120 h. (C) Immunoblot showing expression of LaminB1 and p21 in SIRT2-GFP and GFP overexpressing cells treated with different doses of doxorubicin (Dox) at 120 h. (D) Immunoblot showing knockdown of SIRT2 following transfection of U2OS cells with specific shRNA for SIRT2 which was accompanied by increase in levels of acetylated (Ac) tubulin. Non target shRNA (NT shRNA) was used as a control. Values below the blot indicate fold change in levels of SIRT2 and acetylated tubulin normalized to GAPDH. (E) Percentage SA-βgal positive cells after doxorubicin treatment in non-target shRNA and SIRT2 shRNA U2OS cells at 120 h. (F) Immunoblots showing expression of Lamin B1 and p21 in SIRT2 depleted cells (SIRT2 shRNA) and control non target shRNA transfected cells (NT shRNA) treated with different doses of doxorubicin at 120 h. (G) Immunoblot showing SIRT2 expression at 120 h following doxorubicin treatment in control (NT shRNA) and SIRT2 depleted cells (SIRT2 shRNA). (H) Quantification of SIRT2 levels after doxorubicin treatment (0.1 µM and 1 µM) in control (NT shRNA) and SIRT2 depleted cells (SIRT2 shRNA) at 120 h (*P < 0.05).