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Mitotic Stress Is an Integral Part of the Oncogene-Induced Senescence Program that Promotes Multinucleation and Cell Cycle Arrest.

Dikovskaya D, Cole JJ, Mason SM, Nixon C, Karim SA, McGarry L, Clark W, Hewitt RN, Sammons MA, Zhu J, Athineos D, Leach JD, Marchesi F, van Tuyn J, Tait SW, Brock C, Morton JP, Wu H, Berger SL, Blyth K, Adams PD - Cell Rep (2015)

Bottom Line: ERK-dependent transcriptional upregulation of Mcl1 was, at least in part, responsible for enhanced survival and slippage of cells with mitotic defects.Importantly, mitotic slippage and oncogene signaling cooperatively induced senescence and key senescence effectors p21 and p16.In summary, activated Ras coordinately triggers mitotic disruption and enhanced cell survival to promote formation of multinucleate senescent cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer Sciences, CR-UK Beatson Laboratories, University of Glasgow, Glasgow G61 1BD, UK. Electronic address: ddikovskaya@gmail.com.

No MeSH data available.


Related in: MedlinePlus

Mitotic Slippage and Low-Level Oncogene Signaling Cooperate to Upregulate p21 and p16 and Induce Senescence-Associated Cell Cycle Arrest(A) Titration of ER-H-RasV12 level in ERRAS cells by differential 4OHT concentration. Lysates from ERRAS cells induced for 7 days with indicated 4OHT concentrations (0–100 nM) were blotted with anti-Ras antibody that recognizes both endogenous Ras (asterisk) and ER-RasV12 fusion. Actin is a loading control.(B) Combination of mitotic slippage and low-level (subthreshold for OIS onset) H-RasV12 expression induces effective cell-cycle arrest in ERRAS cells. The experiment was conducted as in Figure 6A but using several concentrations of 4OHT. Percentage of replicating cells measured by EdU incorporation in control ERRAS cells (0, Cntr) or ERRAS cells treated with indicated concentrations of 4OHT are shown at 9 days after slippage (14 days of H-RasV12 induction). Data indicate means ± SD from seven replicate experiments. d, days.(C and D) Immunoblots of the lysates from samples shown in (B) collected at 2 (C) or 9 (D) days after slippage, corresponding to 7 (C) or 14 (D) days of Ras induction, to detect changes in protein amounts of p53, p21, and p16. Actin is a loading control.(E) Activation of p16-promoter-driven luciferase expression in p16Luc/wt transgenic mice, treated with a combination of ectopic DMBA-TPA protocol and Eg5 inhibitor SB-743921. Mice received DMBA (week 0, not shown) followed by TPA during weeks 1–3. Three i.p. injections of SB-743921 or vehicle were given during the first week of TPA (shown as asterisks). Luciferase activity was measured weekly using the IVIS in vivo imaging system. Average increases in luciferase activity (as compared to week 0) ± SEM in vehicle-treated cohort (n = 8, blue) or in SB-743921-treated cohort (n = 9, red) are shown. Difference between cohorts is significant at week 2, with p < 0.05. Changes in luciferase expression in two individual vehicle-treated (gray) and two individual SB-743921-treated (green) mice without DMBA-TPA application are shown as controls.(F) Example of p16-promoter-driven luciferase expression activated by the DMBA-TPA protocol (third week) with (left) or without (right) SB-743921 injections in p16Luc/wt mice, as measured by luminescence in the presence of D-licuferin substrate. The color key to radiance of luminescence is on the right. The measurement regions (cycles) corresponding to the areas subjected to DMBA/TPA applications are shown.See also Figures S4 and S5.
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fig7: Mitotic Slippage and Low-Level Oncogene Signaling Cooperate to Upregulate p21 and p16 and Induce Senescence-Associated Cell Cycle Arrest(A) Titration of ER-H-RasV12 level in ERRAS cells by differential 4OHT concentration. Lysates from ERRAS cells induced for 7 days with indicated 4OHT concentrations (0–100 nM) were blotted with anti-Ras antibody that recognizes both endogenous Ras (asterisk) and ER-RasV12 fusion. Actin is a loading control.(B) Combination of mitotic slippage and low-level (subthreshold for OIS onset) H-RasV12 expression induces effective cell-cycle arrest in ERRAS cells. The experiment was conducted as in Figure 6A but using several concentrations of 4OHT. Percentage of replicating cells measured by EdU incorporation in control ERRAS cells (0, Cntr) or ERRAS cells treated with indicated concentrations of 4OHT are shown at 9 days after slippage (14 days of H-RasV12 induction). Data indicate means ± SD from seven replicate experiments. d, days.(C and D) Immunoblots of the lysates from samples shown in (B) collected at 2 (C) or 9 (D) days after slippage, corresponding to 7 (C) or 14 (D) days of Ras induction, to detect changes in protein amounts of p53, p21, and p16. Actin is a loading control.(E) Activation of p16-promoter-driven luciferase expression in p16Luc/wt transgenic mice, treated with a combination of ectopic DMBA-TPA protocol and Eg5 inhibitor SB-743921. Mice received DMBA (week 0, not shown) followed by TPA during weeks 1–3. Three i.p. injections of SB-743921 or vehicle were given during the first week of TPA (shown as asterisks). Luciferase activity was measured weekly using the IVIS in vivo imaging system. Average increases in luciferase activity (as compared to week 0) ± SEM in vehicle-treated cohort (n = 8, blue) or in SB-743921-treated cohort (n = 9, red) are shown. Difference between cohorts is significant at week 2, with p < 0.05. Changes in luciferase expression in two individual vehicle-treated (gray) and two individual SB-743921-treated (green) mice without DMBA-TPA application are shown as controls.(F) Example of p16-promoter-driven luciferase expression activated by the DMBA-TPA protocol (third week) with (left) or without (right) SB-743921 injections in p16Luc/wt mice, as measured by luminescence in the presence of D-licuferin substrate. The color key to radiance of luminescence is on the right. The measurement regions (cycles) corresponding to the areas subjected to DMBA/TPA applications are shown.See also Figures S4 and S5.

Mentions: Next, we asked if mitotic slippage can promote establishment of OIS. To visualize any contribution of mitotic slippage to OIS-associated cell-cycle arrest, we decreased expression of oncogenic H-RasV12 in induced ERRAS cells by titrating down tamoxifen (Figure 7A). Resulting low levels of H-RasV12 were unable to induce cell-cycle arrest (Figure 7B, 12.5 nM and 6.25 nM tamoxifen, no slippage). Likewise, slippage alone (protocol as in Figure 6A, −4OHT), failed to induce stable proliferation arrest characteristic of senescence, and instead caused only transient cell-cycle arrest in cells that survived DME treatment (Figures 7B, S4A, and S4B). Enlarged nuclear volume and lobulated nuclei or micronucleation in the proliferating EdU-positive cells confirmed that these cells had likely slipped out of DME-induced mitotic arrest (Figure S4C). In addition, these slipped cells displayed only weak SA-β-gal staining (Figure S4D, left panel) and little p16 (Figure S4E, left panels), underscoring that they are not senescent. However, combined low-level H-RasV12 expression and DME-enforced slippage effectively induced long-term cell-cycle arrest (Figure 7B, 12.5 nM and 6.25 nM 4OHT + slippage; Figure S4A). Thus, mitotic slippage synergizes with low-level oncogenic Ras in establishing senescence-associated long-term cell-cycle arrest.


Mitotic Stress Is an Integral Part of the Oncogene-Induced Senescence Program that Promotes Multinucleation and Cell Cycle Arrest.

Dikovskaya D, Cole JJ, Mason SM, Nixon C, Karim SA, McGarry L, Clark W, Hewitt RN, Sammons MA, Zhu J, Athineos D, Leach JD, Marchesi F, van Tuyn J, Tait SW, Brock C, Morton JP, Wu H, Berger SL, Blyth K, Adams PD - Cell Rep (2015)

Mitotic Slippage and Low-Level Oncogene Signaling Cooperate to Upregulate p21 and p16 and Induce Senescence-Associated Cell Cycle Arrest(A) Titration of ER-H-RasV12 level in ERRAS cells by differential 4OHT concentration. Lysates from ERRAS cells induced for 7 days with indicated 4OHT concentrations (0–100 nM) were blotted with anti-Ras antibody that recognizes both endogenous Ras (asterisk) and ER-RasV12 fusion. Actin is a loading control.(B) Combination of mitotic slippage and low-level (subthreshold for OIS onset) H-RasV12 expression induces effective cell-cycle arrest in ERRAS cells. The experiment was conducted as in Figure 6A but using several concentrations of 4OHT. Percentage of replicating cells measured by EdU incorporation in control ERRAS cells (0, Cntr) or ERRAS cells treated with indicated concentrations of 4OHT are shown at 9 days after slippage (14 days of H-RasV12 induction). Data indicate means ± SD from seven replicate experiments. d, days.(C and D) Immunoblots of the lysates from samples shown in (B) collected at 2 (C) or 9 (D) days after slippage, corresponding to 7 (C) or 14 (D) days of Ras induction, to detect changes in protein amounts of p53, p21, and p16. Actin is a loading control.(E) Activation of p16-promoter-driven luciferase expression in p16Luc/wt transgenic mice, treated with a combination of ectopic DMBA-TPA protocol and Eg5 inhibitor SB-743921. Mice received DMBA (week 0, not shown) followed by TPA during weeks 1–3. Three i.p. injections of SB-743921 or vehicle were given during the first week of TPA (shown as asterisks). Luciferase activity was measured weekly using the IVIS in vivo imaging system. Average increases in luciferase activity (as compared to week 0) ± SEM in vehicle-treated cohort (n = 8, blue) or in SB-743921-treated cohort (n = 9, red) are shown. Difference between cohorts is significant at week 2, with p < 0.05. Changes in luciferase expression in two individual vehicle-treated (gray) and two individual SB-743921-treated (green) mice without DMBA-TPA application are shown as controls.(F) Example of p16-promoter-driven luciferase expression activated by the DMBA-TPA protocol (third week) with (left) or without (right) SB-743921 injections in p16Luc/wt mice, as measured by luminescence in the presence of D-licuferin substrate. The color key to radiance of luminescence is on the right. The measurement regions (cycles) corresponding to the areas subjected to DMBA/TPA applications are shown.See also Figures S4 and S5.
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fig7: Mitotic Slippage and Low-Level Oncogene Signaling Cooperate to Upregulate p21 and p16 and Induce Senescence-Associated Cell Cycle Arrest(A) Titration of ER-H-RasV12 level in ERRAS cells by differential 4OHT concentration. Lysates from ERRAS cells induced for 7 days with indicated 4OHT concentrations (0–100 nM) were blotted with anti-Ras antibody that recognizes both endogenous Ras (asterisk) and ER-RasV12 fusion. Actin is a loading control.(B) Combination of mitotic slippage and low-level (subthreshold for OIS onset) H-RasV12 expression induces effective cell-cycle arrest in ERRAS cells. The experiment was conducted as in Figure 6A but using several concentrations of 4OHT. Percentage of replicating cells measured by EdU incorporation in control ERRAS cells (0, Cntr) or ERRAS cells treated with indicated concentrations of 4OHT are shown at 9 days after slippage (14 days of H-RasV12 induction). Data indicate means ± SD from seven replicate experiments. d, days.(C and D) Immunoblots of the lysates from samples shown in (B) collected at 2 (C) or 9 (D) days after slippage, corresponding to 7 (C) or 14 (D) days of Ras induction, to detect changes in protein amounts of p53, p21, and p16. Actin is a loading control.(E) Activation of p16-promoter-driven luciferase expression in p16Luc/wt transgenic mice, treated with a combination of ectopic DMBA-TPA protocol and Eg5 inhibitor SB-743921. Mice received DMBA (week 0, not shown) followed by TPA during weeks 1–3. Three i.p. injections of SB-743921 or vehicle were given during the first week of TPA (shown as asterisks). Luciferase activity was measured weekly using the IVIS in vivo imaging system. Average increases in luciferase activity (as compared to week 0) ± SEM in vehicle-treated cohort (n = 8, blue) or in SB-743921-treated cohort (n = 9, red) are shown. Difference between cohorts is significant at week 2, with p < 0.05. Changes in luciferase expression in two individual vehicle-treated (gray) and two individual SB-743921-treated (green) mice without DMBA-TPA application are shown as controls.(F) Example of p16-promoter-driven luciferase expression activated by the DMBA-TPA protocol (third week) with (left) or without (right) SB-743921 injections in p16Luc/wt mice, as measured by luminescence in the presence of D-licuferin substrate. The color key to radiance of luminescence is on the right. The measurement regions (cycles) corresponding to the areas subjected to DMBA/TPA applications are shown.See also Figures S4 and S5.
Mentions: Next, we asked if mitotic slippage can promote establishment of OIS. To visualize any contribution of mitotic slippage to OIS-associated cell-cycle arrest, we decreased expression of oncogenic H-RasV12 in induced ERRAS cells by titrating down tamoxifen (Figure 7A). Resulting low levels of H-RasV12 were unable to induce cell-cycle arrest (Figure 7B, 12.5 nM and 6.25 nM tamoxifen, no slippage). Likewise, slippage alone (protocol as in Figure 6A, −4OHT), failed to induce stable proliferation arrest characteristic of senescence, and instead caused only transient cell-cycle arrest in cells that survived DME treatment (Figures 7B, S4A, and S4B). Enlarged nuclear volume and lobulated nuclei or micronucleation in the proliferating EdU-positive cells confirmed that these cells had likely slipped out of DME-induced mitotic arrest (Figure S4C). In addition, these slipped cells displayed only weak SA-β-gal staining (Figure S4D, left panel) and little p16 (Figure S4E, left panels), underscoring that they are not senescent. However, combined low-level H-RasV12 expression and DME-enforced slippage effectively induced long-term cell-cycle arrest (Figure 7B, 12.5 nM and 6.25 nM 4OHT + slippage; Figure S4A). Thus, mitotic slippage synergizes with low-level oncogenic Ras in establishing senescence-associated long-term cell-cycle arrest.

Bottom Line: ERK-dependent transcriptional upregulation of Mcl1 was, at least in part, responsible for enhanced survival and slippage of cells with mitotic defects.Importantly, mitotic slippage and oncogene signaling cooperatively induced senescence and key senescence effectors p21 and p16.In summary, activated Ras coordinately triggers mitotic disruption and enhanced cell survival to promote formation of multinucleate senescent cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer Sciences, CR-UK Beatson Laboratories, University of Glasgow, Glasgow G61 1BD, UK. Electronic address: ddikovskaya@gmail.com.

No MeSH data available.


Related in: MedlinePlus