Rapamycin decelerates cellular senescence

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Feb 22, 2009 - peroxide did not alter S6 phosphorylation (Fig. 1A). As a result, ... In agreement with previous reports,5,6 IPTG caused G1 and G2 arrest in HT-p21 cells. ... Cells were treated with IPTG for 3 days plus rapamycin either for the entire period of .... 6, “9” versus “9 + R9” would require a log-scale to be apparent).
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Rapamycin decelerates cellular senescence Zoya N. Demidenko,1 Svetlana G. Zubova,2 Elena I. Bukreeva,2 Valery A. Pospelov,2 Tatiana V. Pospelova2 and Mikhail V. Blagosklonny1,3,* 1Oncotarget;

Albany, New York USA; 2Institute of Cytology; Russian Academy of Sciences; St-Petersburg, Russia; 3Ordway Research Institute; Albany, New York USA

Key words: cellular senescence, cell cycle arrest, aging, rapamycin, mTOR, TOR

When the cell cycle is arrested but cellular growth is not, then cells senesce, permanently losing proliferative potential. Here we demonstrated that the duration of cell cycle arrest determines a progressive loss of proliferative capacity. In human and rodent cell lines, rapamycin (an inhibitor of mTOR) dramatically decelerated loss of proliferative potential caused by ectopic p21, p16 and sodium butyrate-induced p21. Thus, when the cell cycle was arrested by these factors in the presence of rapamycin, cells retained the capacity to resume proliferation, once p21, p16 or sodium butyrate were removed. While rapamycin prevented the permanent loss of proliferative potential in arrested cells, it did not force the arrested cells into proliferation. During cell cycle arrest, rapamycin transformed the irreversible arrest into a reversible condition. Our data demonstrate that senescence can be pharmacologically suppressed.

Introduction Cellular senescence is characterized by irreversible loss of proliferative potential and a distinct morphology (SA-βGal staining, enlarged and flattened cell morphology). Numerous cellular stresses induce CDK inhibitors such as p21WAF1/CIP1 (abbreviated as p21) and p16, causing cellular senescence.1-3 Still, the same stresses can cause reversible arrest of the cell cycle. We have recently demonstrated that serum-stimulation is required for acquiring the senescent phenotype upon cell cycle arrest caused by DNA damage and p21 overexpression.4 Serum stimulates the mTOR pathway, which is involved in cellular (mass) growth. Inhibition of mTOR by rapamycin partially decreased SA-βGal staining and cellular hypertrophy but did not affect the flat cell morphology.4 The significance of morphologic markers of senescence remains unclear. For example, it is not clear whether rapamycin merely exerts superficial “cosmetic” effects without affecting the essential features of cellular senescence. Therefore, we investigated whether rapamycin can prevent irreversible loss of the proliferative potential.

*Correspondence to: Mikhail V. Blagosklonny; Roswell Park Cancer Institute; Buffalo, New York USA; Email: [email protected] Submitted: 02/22/09; Accepted: 04/01/09 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/8606

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Results Rapamycin decreased cell senescence in RPE cells. First, we investigated cellular senescence in ARPE-19, a spontaneously immortalized cell line of human retinal pigment epithelium (RPE). ARPE-19 cells undergo reversible quiescence upon serum withdrawal or irreversible senescence upon DNA damage.4 Stimulation of mTOR by serum was required for cellular senescence caused by DNA damage.4 Here, we investigated whether rapamycin can prevent cellular senescence caused by doxorubicin (DOX) and hydrogen peroxide (peroxide). DOX and peroxide blocked cell proliferation by different mechanisms: DOX induced p53, while peroxide did not (Fig. 1A). In proliferating ARPE-19 cells, S6 was highly phosphorylated, a marker of mTOR activation (Fig. 1A). While inhibiting cell proliferation (Fig. 1B), doxorubicin and peroxide did not alter S6 phosphorylation (Fig. 1A). As a result, cells acquired large-cell morphology with an intense SA-β-Gal staining (data not shown). Rapamycin completely inhibited S6 phosphorylation in the presence of DOX and peroxide. Rapamycin did not force cells to proliferate in the presence of DOX and peroxide (Fig. 1B). However, rapamycin partially decreased SA-β-Gal staining caused by DOX and peroxide (Fig. 1C). Although rapamycin partially decreased SA-β-Gal staining, it did not affect the flat-cell morphology. Therefore, we next addressed the question as to whether rapamycin could prevent irreversible lose of proliferative potential, a well-established marker of cellular senescence. Rapamycin did not force cells to proliferate in the presence of DOX and peroxide, which block the cell cycle. In order to investigate the effect of rapamycin on the proliferative potential, DOX and peroxide should be removed. Doxorubicin is a DNA intercalating agent and cannot be easily washed out. Therefore, we choose to examine the effects of peroxide in these experiments. ARPE-19 cells were treated with peroxide for 1 h and then the medium was replaced. In the absence of rapamycin, cells stressed by peroxide become permanently arrested (Fig. 2). In contrast, rapamycin allowed cells to resume proliferation after a 4 day-lag period (Fig. 2). Thus, rapamycin partially prevented loss of proliferative capacity. This effect was incomplete. Hydrogen peroxide can cause damage, which may persist even after peroxide removal. Therefore, next we employed cells with IPTG-inducible p21WAF1/CIP1 (p21). This model allowed us to switch p21 on and off without causing DNA damage.

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Figure 2. Effects of rapamycin on the loss of proliferative potential in ARPE19 cells caused by oxidative-stress. ARPE-19 cells were plated in 100-mm plates. On day 0, cells were pulse-treated with hydrogen peroxide for 1 hr in the presence (closed squares) or absence (closed circles) of rapamycin. As a negative control, cells were treated with rapamycin alone (open squares) or left untreated (open circles). Cells were washed and then cultured with fresh medium. Cells were counted daily.

Figure 1. Effects of rapamycin on stress-induced senescence in RPE cells. ARPE-19 cells were treated with either 100 ng/ml DOX or 150 μM hydrogen peroxide (H2O2). After 30 min, hydrogen peroxide was removed and cells were cultivated in the fresh medium. (A) Next day, cells were lysed and immunoblotted for p53, pS6, S6 and tubulin was performed as described in Methods. Note: p53 induction is a positive control for DOX effects. (B) After 3 days, cells were counted and results are shown as a percent of control. (C) After 3 days, cells were fixed, stained for beta-Gal and beta-Gal-positive cells were counted.

Effects of rapamycin on p21-induced senescence. HT1080-p21 cell line (abbreviated as HT-p21 cells) is a derivative of p16-deficient HT1080 human fibrosarcoma cells, where the p21 expression can be turned on or off using a physiologically neutral agent isopropylβ-thio-galactosidase (IPTG).5,6 Treatment with IPTG induced p21 followed by G1 and G2 arrest as well as cell senescence characterized by SA-β-Gal staining, large and flat cell morphology.5,6 Rapamycin did not change levels of IPTG-induced p21.4 Rapamycin slightly slowed progression through G1 phase (Fig. 3). This was transient because the cells were able to proliferate and form colonies in the presence of rapamycin. In agreement with previous reports,5,6 IPTG caused G1 and G2 arrest in HT-p21 cells. Rapamycin did not affect cell cycle arrest caused by IPTGinduced p21. So, rapamycin did not force cell cycle progression in the presence of IPTG. Treatment with IPTG for 3 days caused irreversible arrest. The term irreversible arrest means that HT-p21 cells could not resume proliferation and, thus, could not form colonies even following IPTG removal. Here we investigated whether rapamycin prevents loss of clonogenicity. As shown in Figure 4A, HT-p21 cells had clonogenicity of approximately 100%. Thus each cell forms a www.landesbioscience.com

visible colony after 7–8 days. Treatment with rapamycin only marginally affected the clonogenicity (Fig. 4B). As expected, IPTG completely blocked colony formation (Fig. 4C). Treatment with IPTG alone caused a profound loss of clonogenicity: there were a few colonies even when IPTG was washed out (Fig. 4E). When the cells were cultured with a combination of IPTG and rapamycin, they retained the proliferative potential (Fig. 4F). When IPTG was washed out, the released cells proliferated and formed colonies (Fig. 4F). Thus, a 3-day treatment with IPTG caused senescence, whereas rapamycin can prevent senescence. The anti-aging effect of rapamycin during senescence induction. Next we investigated whether rapamycin could prevent senescence, if added with a delay during senescence-induction. Cells were treated with IPTG for 3 days plus rapamycin either for the entire period of 3 days, or for the last 2 days or for the last day. Also, cells were treated with IPTG without rapamycin (0). As controls, cells were treated with rapamycin alone for the entire period of 3 days, or for the last 2 days or for only the last day. First, we evaluated S6 phosphorylation, a marker of mTOR activity. S6 was highly phosphorylated in proliferating cells (Fig. 5A, no IPTG). Rapamycin completely blocked S6 phosphorylation and this effect lasted at least 3 days after addition of rapamycin. S6 phosphorylation remained high 3 days after addition of IPTG (Fig. 5A, +IPTG). Noteworthy, such cells with active mTOR were senescent as evidenced by SA-βGal staining (Fig. 5B, 0). Rapamycin completely blocked S6 phosphorylation in the presence of IPTG too. Rapamycin did not affect Erk phosphorylation in senescent cells (Fig. 5A). Rapamycin decreased SA-β-Gal staining in IPTG-treated cells (Fig. 5B). This effect was observed even when rapamycin was added for the last 30 hrs (1 day) of a 3-day period of senescenceinduction by IPTG. However, the effect on SA-βGal staining was not complete even when rapamycin was added from the beginning. The effect was diminished, if rapamycin was added with delay

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Figure 3. Effects of rapamycin on p21-induced cell cycle arrest. HT-p21 cells were incubated overnight with: (A) no treatment (control), (B) IPTG, (C) Rapamycin + IPTG, (D) Rapamycin + IPTG (R + I). Flow cytometry was performed as described in Methods.

(Fig. 5B, 1). There was no pronounced effect of rapamycin on large-cell morphology. We next evaluated colony formation. The ability of rapamycin to prevent loss of clonogenicity was evident, even when rapamycin was added for the last 30 hrs of a 3-day period (Fig. 5C). So rapamycin partially prevented senescence during its induction. Effects of rapamycin on senescent HT-p21 cells. When added to already senescent cells, rapamycin neither reverted senescent morphology nor decreased SA-β-Gal staining (data not shown). However, it decreased the further progression of the senescent phenotype. As previously suggested, the longer the arrest, the deeper the senescence. In comparison with cells arrested for 3 days, cells arrested for 9 days showed an increased intensity of SA-β-Gal staining. While rapamycin decreased SA-βGal staining during 3-day-arrest, its effect on SA-β-Gal staining was minimal during 9-day-arrest (not shown). There was no change in flat cell morphology (not shown). Also, during the 9-day-arrest, the effects of rapamycin on proliferative potential and clonogenicity were also weakened. As shown in Figure 6, the ability to resume proliferation was decreased after prolonged treatment with IPTG (compare 3, 6 and 9 days with IPTG on Fig. 6). This is consistent with the model that prolonged hypertrophic type of cell cycle arrest leads to cellular senescence. Rapamycin dramatically prevented the loss of clonogenic capability and proliferative potential during a 3-day induction of senescence. Rapamycin also partially preserved the proliferative potential when cells were treated with IPTG for 6 days and 9 days. The presence of rapamycin for the entire period of senescence-induction was more effective than its delayed addition for the last 3 days of senescence-induction. Still even when added at the last 3 days of a 9-day induction, rapamycin decreased loss of clonogenicity 2–3 folds (Fig. 6, “9” versus “9 + R9” would require a log-scale to be apparent). So the later rapamycin was added, the less effective it was. This is consistent with the scenario that rapamycin 1890

decelerates senescence rather then reverses it. Thus, a 3-day senescence induction (IPTG treatment) in the absence of rapamycin and a 9-day senescence induction in the presence of rapamycin caused comparable changes in clonogenic and proliferative potentials (Fig. 6). In other words, rapamycin partially prevented (decelerated) senescence or “slowed down time” approximately 3 fold. Rapamycin prevents loss of clonogenicity caused by p16. Next, we investigated whether rapamycin can preserve proliferative potential during p16-induced senescence. IPTG-induced p16 causes G1 arrest and cellular senescence in this cell line.5 IPTG blocked colony formation in this cell line. Loss of proliferative potential was irreversible. Most cells could neither proliferate nor form colonies, even when IPTG was washed out (Fig. 7). Rapamycin significantly preserved the proliferative capacity of p16-arrested cells (Fig. 7). Rapamycin prevented NaB-induced senescence in HT1080. Next we investigated whether rapamycin would prevent cellular senescence caused by sodium butyrate (NaB) in parental HT1080 cells. NaB caused G1 and G2 arrest (Fig. 8A) and senescent morphology (Fig. 8B). Consistently, NaB completely blocked proliferation of HT1080 cells (Fig. 8C). Senescent morphology included large-cell morphology (cellular hypertrophy) and intense SA-β-Gal staining (Fig. 9B). This was accompanied by permanent loss of proliferative potential (Fig. 8D). When NaB was washed out, cells did not resume proliferation (Fig. 8D). Rapamycin alone slightly inhibited G1 progression, resulting in a modest increase of G1 fraction (Fig. 8A) and an insignificant decrease in cell proliferation (Fig. 8C). When added together with NaB, rapamycin did not abrogate NaB-induced arrest (Fig. 8A) and did not force cells to proliferate in the presence of NaB (Fig. 8C). Rapamycin significantly decreased SA-β-Gal staining and hypertrophy caused by NaB (Fig. 8B). Most importantly, rapamycin prevented the loss of proliferative potential. Thus, when cells were arrested with NaB alone, they lost the ability to proliferate upon NaB removal (Fig. 8D). In contrast, when cells were arrested by NaB in the presence of rapamycin, they resumed proliferation upon NaB removal (Fig. 8D). Rapamycin prevented cellular senescence in rodent cells. Next we extended the study to include rodent fibroblasts, using the model of NaB-induced cellular senescence described previously.7 Sodium butyrate (NaB) caused irreversible cell cycle arrest that was p21Waf1-dependent.7 Rapamycin alone did not affect cell cycle in this cell line (Fig. 9). NaB rapidly caused both G1- and G2 arrest. Rapamycin did not affect NaB-induced arrest and did not force cells to proliferate in the presence of NaB (Fig. 9). Despite inhibition of cell proliferation, mTOR activity remained unchanged in NaB-arrested cells (Fig. 10A). In agreement with the role of mTOR in cell mass growth, cells become hypertrophic. Thus, NaB caused large-cell morphology and increased the amount of protein per cell. This was accompanied by permanent loss of proliferative potential and intensive SA-β-Gal staining by day 5 (Fig. 10B). Co-treatment with rapamycin only marginally decreased SA-β-Gal staining (Fig. 10B). There was a modest effect of rapamycin on cellular morphology, manifested mainly as a slight decrease of cell size.

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Figure 4. Effects of rapamycin on p21-induced loss of clonogenicity. Macroscopic colonies. 2 x 103 HT-p21 cells were plated in 100 mm plates. The next day, 50 μM IPTG was added, if indicated (IPTG). Rapamycin was added if indicated (RAPA). After 3 days, the plates were washed to remove IPTG and RAPA, if indicated “Wash”. Then cells were incubated until colonies become visible: 5 days for control and RAPA alone; and 8 days for all other plates.

Figure 5. Effects of rapamycin during senescence-induction period. HT-p21 cells were plated in 6 well plates (A and B) or in 100 mm dishes (C). The next day, cells were treated: (A) with IPTG, if indicated, for 3 days. Rapamycin (RAPA) was added either simultaneously with IPTG for all 3 days (3), for the last 2 days (2), or for the last 30 h day (1), as indicated. Then cells were lysed and immunoblot was performed. (B) corresponds to “+IPTG 3 d column” in (A). All plates were treated with IPTG for 3 days. Rapamycin (RAPA) was added either simultaneously with IPTG for all 3 days (3), for the last 2 days (2), or for the last 1 day (1), as indicated. Then cells were fixed and beta-Gal-staining was performed. (C) corresponds to (B). All 100-mm dishes were treated with IPTG for 3 days. Rapamycin (RAPA) was added either simultaneously with IPTG for all 3 days (3), for the last 2 days (2), or for the last 1 day (1), as indicated. Then plates were washed and incubated in the fresh medium until colonies become visible (7 days). Plates were fixed and stained with fixed and stained with. [Note, a few colonies would appear in “0” at later time points, as shown in other figures].

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Figure 7. Effects of rapamycin on p16-induced loss of clonogenicity. 2 x 103 HT-p16 cells were plated in 100-mm plates. Next day, 50 μM IPTG was added, if indicated (IPTG). Rapamycin was added, if indicated (RAPA). After 3 days, the plates were washed to remove IPTG and RAPA, if indicated Wash. Then cells were incubated until colonies become visible: 5 days for control and RAPA alone; and 8 days for IPTG-treated cells. Figure 6. Effects of the duration of treatment with IPTG and rapamycin on cellular senescence. Two thousand HT-p21 cells were plated in 100-mm plates. Next day, cells were treated with IPTG for 3 days, 6 days or 9 days, as shown in bold “3”, “6” or “9”. If indicated, “R”, rapamycin was added for the last 3, 6 or 9 days (+3R, +6R, +9R). Specifically: “3”—IPTG for 3 days; “3 + 3R”—IPTG plus rapamycin for 3 days; “6”—IPTG for 6 days; “6 + 3R”—IPTG for 6 days plus rapamycin for the last 3 days; “6 + 6R”—IPTG plus rapamycin for 6 days; “9”—IPTG for 9 days; “9 + 3R”—IPTG for 9 days plus rapamycin for the last 3 days; “9 + 6R”—IPTG for 9 days plus rapamycin for the last 6 days; “9 + 9R”—IPTG plus rapamycin for 9 days. Then plates were washed and incubated without drugs for 7 days. (A) Colonies. Plates were stained and colonies were counted. (B) Cell numbers. Plates were trypsinized and cells were counted.

Yet, rapamycin prevented the loss of proliferative potential. Thus, when cells were arrested (for at least 5 days) with NaB alone, they completely lost the ability to proliferate upon NaB removal (Fig. 10C). In contrast, when cells were arrested with NaB in the presence of rapamycin, they resumed proliferation upon NaB removal (Fig. 10C). It is important to emphasize that the longer arrest, the longer a lag-period before cells can resume ­proliferation (Fig. 10C). Rapamycin decelerated the loss of proliferative potential rather than prevented it completely.

Discussion Loss of proliferative potential is the most accepted marker of cellular senescence in cell culture. Using immortalized human RPE cells, human fibrosarcoma HT-1080 cells and transformed rodent fibroblasts, we demonstrated that rapamycin partially prevented loss of proliferative potential caused by oxidative stress, ectopic p21 or p16 expression, or sodium butyrate, respectively. Specifically, in HT-1080 cells with IPTG-inducible p21 (HT-p21 cells), p21 could be switched on and off by addition and removal of IPTG. Induction of p21 for 3 days ensured irreversible loss of proliferative capacity in most cells. In senescent cells, TOR remained active, consistent with cellular growth in size. When added during senescence-induction, rapamycin partially reduced SA-β-Gal staining. The longer arrest, the deeper cell senescence. This is consistent with the notion that duration of cell cycle arrest determines cell senescence.8,9 Prolonged arrest is “converted” into senescence. 1892

Here we demonstrated that after a prolonged arrest, fewer cells were capable of resuming proliferation upon removal of the initial inhibitors. There was a gradual loss of proliferative potential over time. Rapamycin decelerated this loss. Even when added at the last 3 days of a 9-day senescence-induction, rapamycin partially preserved clonogenic and proliferative potential. HT-p21 cells arrested either for 3 days without rapamycin or for 9 days with rapamycin lost the proliferative potential to the same extent (Fig. 6). Figuratively speaking, rapamycin slowed or decelerated aging approximately 3-fold. In summary, we can formulate three provisional rules. First, the longer arrest (9 days instead of 3–5 days), the more profound loss of clonogenicity. Second, rapamycin is less effective during prolonged arrest (9 days instead of 3–5 days). Third, its anti-aging effect was diminished, when rapamycin was added after a delay. Similar results were obtained in parental HT-1080 cells treated with sodium butyrate (NaB). In theory, rapamycin may restore GF-responsiveness and preserve proliferative potential in stem and wound-healing cells.10 As we demonstrated here, rapamycin did not force a cell to proliferate if cell cycle was arrested by p21. Cells remained arrested in the presence of rapamycin unless IPTG and NaB were removed. We conclude that rapamycin prevents irreversible loss of proliferative potential, a marker of senescence. But in order to proliferate, the initial cell cycle inhibitors must be removed. This is principally different from all previous models of senescence reversal, known as escape, by-pass or avoidance of senescence. Previously, reversal of cellular senescence was based on genetic abrogation of cell cycle arrest (by loss of tumor suppressors, for instance). Rapamycin does not reverse cell cycle arrest. It suppresses inappropriate activation of growth-promoting mTOR pathway. As we discussed, cellular senescence requires not only cell cycle arrest but also inappropriate growth-stimulation via mTOR. And agents that suppress senescence without escaping from cell cycle arrest can be named aging-suppressants. It was reported that rapamycin inhibited aging in yeast.11,12 Amazingly, senescence of mammalian cells and the yeast turns out to be remarkably similar. Importantly, rapamycin is a clinically approved drug, which is considered for therapy of many

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Figure 8. Effects of rapamycin on NaB-induced senescence in HT1080 cells. HT1080 cells were incubated with 2 mM sodium butyrate (NaB), rapamycin (Rap), or Rapamycin + NaB (NaB + Rap), or left untreated: (A) Flow cytometry was performed after 1 day. Cells were washed with PBS and permeabilized for 30 min with 0.01% saponin. Then the cells (105) were incubated with 40 μg/ml of propidium iodide, 0.1 mg/ml RNase A for 15 min at 37°C prior to analysis using ATC300 (Brucker) and Coulter Epics XL cytometers. (B) After 5 days, SA-βGal staining was performed, as described previously.7 (C) Cell proliferation. ERas cells were cultured in the presence of either NaB, Rap, NaB + Rap or no drug (control) and cells were counted and shown as Cell numbers x Cell numbers x 10-4. (D) Proliferative potential. HT1080 cells were cultured in the presence of either NaB or NaB + Rap for 5 days. Then the drugs were washed out and cells were cultivated in the fresh medium. Cells were counted at 3 after removal of the drugs. Cell numbers x 10-4 are shown.

age-related diseases.13-16 Perhaps, one drug is indicated for so many age-related diseases because it decelerates cellular senescence.

Materials and Methods Cell lines and reagents. ARPE-19 (spontaneously immortalized cell line of retinal pigmental epithelium) cells were provided by Sally Temple and originally obtained from ATCC. ARPE-19 cells were cultured in MEM plus 10% FC2 serum. HT-p21 cells are a derivative of p16-deficient HT1080 human fibrosarcoma cells, where p21 expression can be turned on or off using a physiologically neutral agent isopropyl-β-thio-galactosidase (IPTG).5 HT-p21 cells were cultured in DMEM medium supplemented with FC2 serum. HT-p16, a derivative of p16-deficient HT1080 human fibrosarcoma cells, where p16 expression can be turned on or off using a physiologically neutral agent isopropyl-β-thiogalactosidase (IPTG).5 E1A + Ras (Eras) cells were established by calcium phosphate transfection of rat and mouse embryonic fibroblasts by E1A ad5 and activated c-Ha-Ras oncogenes as overgrowth foci on fibroblast monolayers.7 The transformed cell lines were www.landesbioscience.com

Figure 9. Effects of rapamycin on NaB-induced cell cycle arrest in rodent cells. ERas cells were incubated with: (A) no treatment (control), (B) Rapamycin, (C) NaB, (D) Rapamycin + NaB (R + NaB). Flow cytometry was performed after 1 day. Cells were washed with PBS and permeabilized for 30 min with 0.01% saponin. Then the cells (105) were incubated with 40 μg/ml of propidium iodide, 0.1 mg/ml RNase A for 15 min at 37°C prior to analysis using ATC300 (Brucker) and Coulter Epics XL cytometers.

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Figure 10. Effects of rapamycin on NaB-induced senescence in rodent cells. (A) Immunoblot. ERas cells were treated with NaB (1 and 5 days), irradiated (radiation) or treated with rapamyin for 5 days (Rap 5d). Immunoblots for p-S6 and S6 were performed. (B) SA-βGal staining. ERas cells were treated with NaB and NaB + Rap for 5 days or left untreated. (C) Proliferative potential. ERas cells were cultured in the presence of either NaB or NaB + Rap for 5 days and 9 days. Then the drugs were washed out and cells were cultivated in the fresh medium. Cells were counted at days 0, 3 and 5 after removal of the drugs.

maintained in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum and gentamycin. Rapamycin was obtained from LC Laboratories and dissolved in DMSO as 2 mM solution and was used at final concentration of 500 nM, unless otherwise indicated. Doxorubicin (DOX), IPTG, sodium butyrate, hydrogen peroxide, and FC2 were obtained from Sigma-Aldrich (St. Louis, MO). DOX was dissolved in DMSO, as a 2 mg/ml stock solution and used at final concentration 100 ng/ ml. IPTG was dissolved in water as 50 mg/ml stock solution and used in cell culture at final concentration of 50 μg/ml. Western blot analysis. Cells were lysed and soluble proteins were harvested as previously described.17 Immunoblot analysis was performed using mouse monoclonal anti p21, p53, mouse monoclonal anti p-Erk: phospho-p44/42 MAPK (Thr202/Tyr204) (Cell Signaling, MA, USA), mouse monoclonal anti phospho-S6 Ser240/244 (Cell Signaling, MA, USA), rabbit polyclonal anti S6 (Cell Signaling, MA, USA), mouse monoclonal anti-tubulin antibodies as previously described.4,7,17 Flow cytometry. Cells were harvested by trypsinization, washed with PBS and resuspended in 75% ethanol at 4°C for at least 30 minutes. Thereafter, cells were washed in PBS and incubated for 30 min in a PBS solution containing 0.05 mg/ml propidium iodide (Sigma), 1 mM EDTA, 0.1% Triton-X-100 and 1 mg/ml 1894

RNAse A. The suspension was passed through a nylon mesh filter and analyzed on a flow cytometer (FACscan: Becton Dickinson Immunocytometry Systems, San Jose, CA) as previously described.4,7 Determination of cell viability. 20,000 cells were plated in 24-well plates in 1 ml of medium. The next day, cells were treated with the drugs. After 3–4 days, cells were harvested and counted in triplicates on a Coulter Z1 cell counter (Hialeah, FL). In addition, cells were incubated with trypan blue and a number of blue (dead) cells and transparent (live) cells were counted by microscopy. Colony formation assay. Two thousand HT-p21 cells were plated per 100 mm dishes. On the next day, cells were treated with 50 μg/ml IPTG and/or 500 nM rapamycin, as indicated. After 3 days, the medium was removed; cells were washed and cultivated in the fresh medium. When colonies become visible, plates were fixed and stained with 0.1% crystal violet (Sigma). Plates were photographed and the number of colonies were determined as previously described.4 SA-β-Gal staining. Cells were fixed for 5 min in β-galactosidase fixative (2% formaldehyde; 0.2% glutaraldehyde in PBS), and washed in PBS and stained in β-galactosidase solution (1 mg/ ml 5-bromo-4-chloro-3-indolyl-beta-gal (X-gal) in 5 mM potassium ferricyamide, 5 mM potassium ferrocyamide, 2 mM MgCl2

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in PBS) at 37°C until beta-Gal staining become visible in either experiment or control plates. Thereafter, cells were washed in PBS, and the number of β-galactosidase activity-positive cells (blue staining) were counted under bright field illumination. Live cell multimode time-lapse imaging. Phase contrast timelapse sequences were collected on a Leica ASMDW imaging system equipped with Roeper HQ cooled camera and xenon monochromator using 40x/0.55 NA long-working distance objective. Cells were contained CO2-independent medium in a temperaturecontrolled box stabilized at 37°C. Images were collected every 3 minutes for 72 hours using Leica ASMDW software. Acknowledgements

This work was supported in part by NIH 1 R41 EY018520-01 to Oncotarget and grants of the Russian Foundation for Basic Research (RFBR) (07-04-01537), CGP grant from CRDF-RFBR (RUB1-2868-ST-07/07-04-91154) and grant of the Russian Academy of Sciences (MCB RAS). We thank Deputy Editors of the journal for editing and handling MS submission. References 1. Itahana K, Dimri G, Campisi J. Regulation of cellular senescence by p53. Eur J Biochem 2001; 268:2784-91. 2. Serrano M, Blasco MA. Putting the stress on senescence. Curr Opin Cell Biol 2001; 13:748-53. 3. Campisi J, D’Adda di Fagagna F. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol 2007; 8:729-40. 4. Demidenko ZN, Blagosklonny MV. Growth stimulation leads to cellular senescence when the cell cycle is blocked. Cell Cycle 2008; 7:3355-61. 5. Chang BD, Broude EV, Dokmanovic M, Zhu H, Ruth A, Xuan Y, et al. A senescencelike phenotype distinguishes tumor cells that undergo terminal proliferation arrest after exposure to anticancer agents. Cancer Res 1999; 59:3761-7. 6. Chang BD, Broude EV, Fang J, Kalinichenko TV, Abdryashitov R, Poole JC, Roninson IB. p21Waf1/Cip1/Sdi1-induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells. Oncogene 2000; 19:2165-70. 7. Abramova MV, Pospelova TV, Nikulenkov FP, Hollander CM, Fornace AJJ, Pospelov VA. G1/S arrest induced by histone deacetylase inhibitor sodium butyrate in E1A + Rastransformed cells is mediated through downregulation of E2F activity and stabilization of beta-catenin. J Biol Chem 2006; 281:21040-51. 8. Blagosklonny MV. Cell senescence and hypermitogenic arrest. EMBO Rep 2003; 4:358-62; 209:592-7. 9. Blagosklonny MV. Cell senescence: hypertrophic arrest beyond restriction point. J Cell Physiol 2006. 10. Blagosklonny MV. Aging, stem cells and mammalian target of rapamycin: a prospect of pharmacologic rejuvenation of aging stem cells. Rejuvenation Res 2008; 11:801-8. 11. Powers RW, Kaeberlein M, Caldwell SD, Kennedy BK, Fields S. Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev 2006; 20:174-84. 12. Kaeberlein M, Powers RW, Steffen KK, Westman EA, Hu D, Dang N, et al. Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients. Science 2005; 310:1193-6. 13. Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell 2006; 124:471-84. 14. Blagosklonny MV. Aging and immortality: quasi-programmed senescence and its pharmacologic inhibition. Cell Cycle 2006; 5:2087-102. 15. Tsang CK, Qi H, Liu LF, Zheng XFS. Targeting mammalian target of rapamycin (mTOR) for health and diseases. Drug Disc Today 2007; 12:112-24. 16. Blagosklonny MV. An anti-aging drug today: from senescence-promoting genes to antiaging pill. Drug Disc Today 2007; 12:218-24. 17. Giannakakou P, Robey R, Fojo T, Blagosklonny MV. Low concentrations of paclitaxel induce cell type-dependent p53, p21 and G1/G2 cell cycle arrest instead of mitotic arrest: molecular determinants of paclitaxel-induced cytotoxicity. Oncogene 2001; 20:3806-13.

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