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. 2014 Dec 30;5(24):12715-27.
doi: 10.18632/oncotarget.3011.

Tumor promoter-induced cellular senescence: cell cycle arrest followed by geroconversion

Olga V Leontieva  1 , Mikhail V Blagosklonny  1
Affiliations

Tumor promoter-induced cellular senescence: cell cycle arrest followed by geroconversion

Olga V Leontieva et al. Oncotarget. .

Abstract

Phorbol ester (PMA or TPA), a tumor promoter, can cause either proliferation or cell cycle arrest, depending on cellular context. For example, in SKBr3 breast cancer cells, PMA hyper-activates the MEK/MAPK pathway, thus inducing p21 and cell cycle arrest. Here we showed that PMA-induced arrest was followed by conversion to cellular senescence (geroconversion). Geroconversion was associated with active mTOR and S6 kinase (S6K). Rapamycin suppressed geroconversion, maintaining quiescence instead. In this model, PMA induced arrest (step one of a senescence program), whereas constitutively active mTOR drove geroconversion (step two). Without affecting Akt phosphorylation, PMA increased phosphorylation of S6K (T389) and S6 (S240/244), and that was completely prevented by rapamycin. Yet, T421/S424 and S235/236 (p-S6K and p-S6, respectively) phosphorylation became rapamycin-insensitive in the presence of PMA. Either MEK or mTOR was sufficient to phosphorylate these PMA-induced rapamycin-resistant sites because co-treatment with U0126 and rapamycin was required to abrogate them. We next tested whether activation of rapamycin-insensitive pathways would shift quiescence towards senescence. In HT-p21 cells, cell cycle arrest was caused by IPTG-inducible p21 and was spontaneously followed by mTOR-dependent geroconversion. Rapamycin suppressed geroconversion, whereas PMA partially counteracted the effect of rapamycin, revealing the involvement of rapamycin-insensitive gerogenic pathways. In normal RPE cells arrested by serum withdrawal, the mTOR/pS6 pathway was inhibited and cells remained quiescent. PMA transiently activated mTOR, enabling partial geroconversion. We conclude that PMA can initiate a senescent program by either inducing arrest or fostering geroconversion or both. Rapamycin can decrease gero-conversion by PMA, without preventing PMA-induced arrest. The tumor promoter PMA is a gero-promoter, which may be useful to study aging in mammals.

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Conflict of interest statement

Conflicts of Interests

No conflict to declare.

Figures

Figure 1
Figure 1. PMA-induced senescence in SKBr3 cells
A. Beta-gal staining. SKBR3 cells were treated with 100 nM PMA either in serum-free or in complete (10% FBS) medium. After 4 days drug was washed out and cells were cultured in drug-free medium and stained for beta-gal. Bar – 100 μm. B. Immunoblot analysis. SKBR3 cells were treated with 100 nM PMA in either serum-free or complete medium for times indicated and lysed. Results shown were obtained from 2 separate gels. C-D. RP (reversibility potential) of SKBR3 treated with PMA. C – Schema of experiment; D – RP: SKBR3 cells were plated at low density and treated with 100 nM PMA either in serum-free medium or in complete medium (10% FBS). After 4 days drug was washed out and cells were incubated in drug-free complete medium (10% FBS) for 6 days and counted. Fold increase in cell number was calculated relative to initially plated numbers. Data presented as mean ±SD from triplicates.
Figure 2
Figure 2. Suppression of PMA-induced senescence by rapamycin in SkBR3 cells
A. Beta-gal staining. SkBR3 cells were treated with PMA +/− rapamycin (20 nM) for 5 days, then drugs were washed out and cells were cultured for another 3 days and stained for beta-gal. Bar – 100 μm. B. RP (reversibility potential). SkBR3 cells were plated at low density and treated with 100 nM PMA −/+ rapamycin (20 nM). After 4 days cells were washed and colonies were allowed to regrow in drug-free medium and stained with Crystal Violet after 13 days in culture. C. Schema: PMA-induced senescence and its suppression by rapamycin (Rapa).
Figure 3
Figure 3. PMA-induced activation of the mTOR pathway in SkBr3 cells
A. Immunoblot analysis. SkBR3 cells were treated with 100 nM PMA for the times indicated and lysed. One set was pre-treated (and co-treated) with 100 nM rapamycin for 16 h before adding PMA, as indicated at the bottom (+ Rapa). B. Immunoblot analysis. SkBR3 cells were pre-treated and co-treated with either 10 μMU0126 (U), rapamycin 100 nM (R) or their combination, or with Torin 1 (100 nM) for 24 h. Then 100 nM PMA was added for 1 h and cells were lysed.
Figure 4
Figure 4. PMA-activated pathways
Figure 5
Figure 5. Effects of rapamycin plus U0126 on senescent morphology
Beta-gal staining. SkBR3 cells were pre-treated with rapamycin (100 nM) or its combination with U126 (10 μM) for 24 h before adding 100 nM PMA. After 3-day treatment with PMA drugs were washed out and cells were cultured for another 3 days in drug-free medium and stained for beta-gal. Bar – 100 μm.
Figure 6
Figure 6. Effects of PMA on IPTG-induced senescence in HT-p21 cells
A. Schema of experiment. Rapamycin – R. B-C. HT-p21 cells were plated at low density and treated with IPTG, rapamycin (R) (500 nM) and PMA (100 nM) as indicated in Schema (A). After 3 days drugs were washed out, cells were incubated in drug-free medium for another 3 days and stained for beta-gal (B) (bar – 100 μm) and colonies were stained 7 days after wash (C). As indicated in the Schema, rapamycin was added 1 h before PMA.
Figure 7
Figure 7. PMA induced rapamycin-insensitive p-S6 in HT-p21 cells A
Immunoblot analysis. HT-p21 cells were pre-treated with IPTG in the presence of either rapamycin (500 nM), U126 (10 μM) or their combination or torin 1 (100 nM) for 24 h, then 100 nM PMA was added for 1 h and cells were lysed. All treatments were performed in the presence of IPTG to match conditions shown in fig. 6 and panel 7B. B. RP: HT-p21 cells were pre-treated with IPTG in the presence of different drugs as in panel A for 24 h, then 100 nM PMA was added. After 3 day-treatment with PMA (4 days with IPTG and other drugs), drugs were washed out and colonies were allowed to grow and stained with Crystal violet after 9 days in culture and counted in triplicates. Data are presented as mean ± SD. C – cells treated with IPTG alone; R – treated with IPTG in the presence of rapamycin; U – treated with IPTG in the presence of U126; U+R – treated with IPTG in the presence of combination of rapamycin and U126.
Figure 8
Figure 8. mTOR-dependent geroconversion in RPE cells by PMA
A. Immunoblot analysis. RPE cells were incubated in serum-free MEM overnight and then treated with 100 nM PMA for the times indicated. B. Beta-gal staining. RPE cells were pre-incubated in serum free medium before being treated with 100 nM PMA. After 2 day-treatment PMA was washed out, cells were incubated in drug-free medium for another 2 days and stained for beta-gal. Bar – 100 μm. C. Mechanism.
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