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, 2 (12), 924-35

DNA Damaging Agents and p53 Do Not Cause Senescence in Quiescent Cells, While Consecutive Re-Activation of mTOR Is Associated With Conversion to Senescence

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DNA Damaging Agents and p53 Do Not Cause Senescence in Quiescent Cells, While Consecutive Re-Activation of mTOR Is Associated With Conversion to Senescence

Olga V Leontieva et al. Aging (Albany NY).

Abstract

When the cell cycle is arrested, growth-promoting pathways such as mTOR (Target of Rapamycin) drive cellular senescence, characterized by cellular hyper-activation, hypertrophy and permanent loss of the proliferative potential. While arresting cell cycle, p53 (under certain conditions) can inhibit the mTOR pathway. Senescence occurs when p53 fails to inhibit mTOR. Low concentrations of DNA-damaging drugs induce p53 at levels that do not inhibit mTOR, thus causing senescence. In quiescence caused by serum starvation, mTOR is deactivated. This predicts that induction of p53 will not cause senescence in such quiescent cells. Here we tested this prediction. In proliferating normal cells, etoposide caused senescence (cells could not resume proliferation after removal of etoposide). Serum starvation prevented induction of senescence, but not of p53, by etoposide. When etoposide was removed, such cells resumed proliferation upon addition of serum. Also, doxorubicin did not cause senescent morphology in the absence of serum. Re-addition of serum caused mTOR-dependent senescence in the presence of etoposide or doxorubicin. Also, serum-starvation prevented senescent morphology caused by nutlin-3a in MCF-7 and Mel-10 cells. We conclude that induction of p53 does not activate the senescence program in quiescent cells. In cells with induced p53, re-activation of mTOR by serum stimulation causes senescence, as an equivalent of cellular growth.

Figures

Figure 1.
Figure 1.. Rapamycin pretreatment prevents loss of proliferative potential during etoposide treatment.
A-C. WI-38t cells were plated at 10000 cells/well in 24-well plates, and the next day were either left untreated (A) or pretreated with 10 nM Rapamycin (B). The next day, cells were treated with either 2.5 μM nutlin-3a or 1 μg/ml etoposide or left untreated. After 4 days, cells were trypsinized and 10% of cells were plated in fresh drug-free medium (blue bars). 6 days later cells were counted (red bars). In C the results for etoposide treatment (Et) with or without rapamycin (R) pretreatment are shown in the same scale. (D) Cells were lysed after 24 hr treatment with etoposide (E), rapamycin (R), or both (R+E) and immunoblot was performed.
Figure 2.
Figure 2.. Experimental schema: transient induction of p53 in proliferating versus quiescent cells
Cells are treated (or left untreated) under different condi-tions [control (10% serum), 0% serum or rapamycin] with etoposide for 4 days. Cells are counted twice: 1) at the time of etoposide removal to measure inhibition of proliferation and 2) 6-11 days after wash to measure proliferative potential (PP). PP should not be confused with proliferation. Thus, rapamycin and 0% serum inhibit proliferation but preserve (increase) proliferative potential in etoposide-treated cells.
Figure 3.
Figure 3.. Effects of rapamycin and serum starvation on etoposide-induced senescence in WI-38t cells.
A-C. WI-38t cells were plated at 5000/well in 12 well plates, and the next day either treated with 10 nM rapamycin in complete medium (R), or placed in serum-free medium (no serum or 0), or left in complete medium (control). The next day, 1 μg/ml etoposide (Et) was added, as indicated. A. After 4 days, cells were stained for beta-Gal and microphotographed (bar - 50 micron). B. After 4 days, cells were counted: control (C), rapamycin (R), no serum (0). C. Proliferative potential. In replicate plates, cells were washed and incubated in complete, drug-free medium for 6 days and then counted (black bars). Note: red bars correspond to red bars in panel B. Fold (f) increase in a cell number after drug removal. D. Immunoblot. Cells were plated in 6 well plates. The next day, cells were treated with 1 μg/ml etoposide (Et) for 24 hrs: control -C, rapamycin - R, no serum −0.
Figure 4.
Figure 4.. Effects of rapamycin and serum starvation on etoposide-induced senescence in RPE cells.
A-C. RPE cells were plated at 5000/well in 12 well plates, and the next day either treated with 10 nM rapamycin in complete medium (R), or placed in serum-free medium (no serum or 0), or left in complete medium (control). The next day, 0.5 μg/ml etoposide (Et) was added, as indicated. B. After 4 days, cells were stained for beta-Gal and micro-photographed (bar - 50 micron) C. Proliferative potential. In replicate plates, cells were washed and incubated in complete, drug-free medium for 6 days and then counted (black bars). Note: red bars corre-spond to red bars in panel B. Fold (f) increase in a cell number after drug removal. D. Immunoblot. Cells were plated in 6 well plates. The next day, cells were treated with 0.5 μg/ml etoposide (Et) for 24 hrs: control -C, rapamycin -R, no serum −0.
Figure 5.
Figure 5.. Effects of rapamycin and serum starvation on senescence caused by a higher concentration of etoposide.
A. Immunoblot: WI-38t and RPE cells were treated with 0.5 μg/ml and 10 μg/ml etoposide (Et) or left untreated (-). The next day, cells were lysed and immunoblot was performed. B-C: WI-38t and RPE cells were plated at 25000/well in 12 well plates, the next day cells were either pretreated with 10 nM rapamycin (Rapa), placed in serum free medium (no serum) or left in complete medium with 10% serum (control). The next day, 0.5 μg/ml and 10 μg/ml etoposide (Et) was added: in complete medium (control) or with 10 nM Rapamycin (Rapa) or in serum free medium (no serum). After 5 days, cells were washed and cultured in fresh, drug free medium for 11 days and then trypsinized and counted. (in panel C): Before trypsinization, cells treated with 10 μg/ml etoposide (under three conditions: control, Rapa and no serum) were microphotographed.
Figure 6.
Figure 6.. Schema. Serum stimulation of quiescent cells locked by p53.
Figure 7.
Figure 7.. Serum stimulation of etoposide-locked WI-38t cells results in mTOR-dependent sensecence.
A-B. WI38t cells were treated with 1μg/ml etoposide in the absence of serum as shown in Figure 6. Then, 10% serum was added either with 10 nM rapamycin (+R) or alone. No serum indicates that cells were continuously incubated with etoposide in serum free medium. 24 h after serum stimulation, cells were lysed and subjected to immuno-blotting as indicated (A). 4 days after serum stimulation cells were microphotographed (B).
Figure 8.
Figure 8.. Serum stimulation converts Dox-locked quiescence into senescence in WI-38t cells.
WI38t cells were treated with 100 ng/ml doxorubicin (low-Dox) or left untreated in serum-free medium (no serum) for 3 days, and then 10% serum was added. After 3 days of serum stimulation cells were stained for beta-gal and microphotographed. Bar - 50 micron.
Figure 9.
Figure 9.. Serum stimulation of etoposide-locked RPE cells results in mTOR-dependent senescence.
A-B. RPE cells were treated with 0.5 μg/ml etoposide in the absence of serum as shown in Figure 6. Then, 10% serum was added either with 10 nM rapamycin (+R) or alone. No serum indicates that cells were continuously incubated with etoposide in serum free medium. 24 h after serum stimulation, cells were lysed and subjected to immunoblotting as indicated (A). 4 days after serum stimulation cells were microphotographed (B).
Figure 10.
Figure 10.. Serum stimulation converts Dox-locked quiescence into senescence in RPE cells.
RPE cells were treated with 50 ng/ml doxorubicin (low-Dox) or left untreated in serum-free medium (no serum) for 3 days, then 10% serum was added. After 3 days of serum stimulation cells were stained for beta-Gal and microphotographed. Bar - 50 micron.
Figure 11.
Figure 11.. Serum stimulation is required for senescent morphology in nutlin-treated MEL-10 cells.
A. MEL-10 cells treated with 2.5 μM nutlin-3a in the presence (Nutlin) or absence (No serum+Nutlin) of serum for 3 days were stained for beta-Gal and microphotographed (left panels). In parallel, 10% serum was added to a replicate well. After 3 days of serum stimulation cells were stained for beta-Gal and microphotographed (right lower panel). Bars - 50 micron. B. MEL-10 cells treated with 2.5 μM Nutlin-3a (N) in the absence or presence of 10% serum for 24 hr were lysed and subjected to immunoblotting, as indicated. Treatment with 10 nM rapamycin (R) is used as a control for mTOR inhibition.
Figure 12.
Figure 12.. Rapamycin and serum starvation prevents nutlin-induced senescence in MCF-7 cells.
A-B. MCF7 cells were plated at 5000 or 10000/well in 12 well plates, allowed to attach and then were either pretreated with 500 nM rapamycin (R), placed in serum free medium or left untreated in complete medium (control). The next day, 5μM nutlin-3a was added. After 5 days, cells were stained for beta-Gal and microphotographed (bars −50 micron) (A). (B) In replicate plate, cells were counted. C. MCF7 cells were treated as indicated for 24 hr, and immunoblot was performed.

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