Pseudo-DNA damage response in senescent cells - PubMed (original) (raw)

Pseudo-DNA damage response in senescent cells

Tatyana V Pospelova et al. Cell Cycle. 2009.

Abstract

Cellular senescence is currently viewed as a response to DNA damage. In this report, we showed that non-damaging agents such as sodium butyrate-induced p21 and ectopic expression of either p21 or p16 cause cellular senescence without detectable DNA breaks. Nevertheless, senescent cells displayed components of DNA damage response (DDR) such as gammaH2AX foci and uniform nuclear staining for p-ATM. Importantly, there was no accumulation of 53BP1 in gammaH2AX foci of senescent cells. Consistently, comet assay failed to detect DNA damage. Rapamycin, an inhibitor of mTO R, which was shown to suppress cellular senescence, decreased gammaH2AX foci formation. Thus, cellular senescence leads to activation of atypical DDR without detectable DNA damage. Pseudo-DDR may be a marker of general over-activation of senescent cells.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Immunofluorescence for γH2AX and p-ATM in NaB-treated E1A + Ras cells. Representative images of E1A + Ras cells stained for γH2AX (Ser139) and p-ATM (Ser 1981) at various time points. Cells were treated with NaB and then fixed at 24, 72, 120 hr. Immunofluorescence of γH2AX (red) and p-ATM (green), nuclei were stained with DAPI (blue), scale 10 m. Bottom panel: Higher magnification (scale 5 m). γH2AX foci accumulation at 5 days of NaB treatment.

Figure 2

Figure 2

53BP1 and γH2AX foci in NaB-treated and irradiated E1A + Ras cells. Representative images of E1A + Ha-ras cells stained for γH2AX (Ser139) and 53BP1 following NaB treatment (5 d) or irradiation (positive control). Immunofluorescence of γH2AX (red) and 53BP1 (green), nuclei were stained with DAPI (blue), scale 10 m. Bottom panel: Irradiation.

Figure 3

Figure 3

53BP1 foci area in NaB treated and irradiated E1A + Ras cells. Cells were treated with NaB 15 min (15’), 1 day and 5 days or irradiated (6 Gy) and incubated for 15 min or 30 min before fixation. IPLab software was used for quantification of the focus area. Approximately 100 cells were analyzed for each time point. Bars indicate Standard Error (SE). Results are shown as percent of control (no treatment).

Figure 4

Figure 4

Effect of rapamycin on γH2AX foci accumulation in NaB treated E1A + Ras cells. Cells were treated with NaB for 5 days or irradiated (1 Gy) for 15 min and 30 min (X-rays: 15 and 30) before fixation (positive control). IPLab software was used for quantification of the focus area. Approximately 100 cells were analyzed for each time point. Bars indicate Standard Error (SE). Results are shown as percent of control (no treatment).

Figure 5

Figure 5

Analysis of DNA breaks in E1A + Ras cells. E1A + Ras cells were subjected to single-cell gel electrophoresis under denaturing conditions (comet assay). Cells were gamma-irradiated for 10 min and 20 min or treated with NaB, Rapamycin (Rapa) or a combination of NaB and Rapa at indicated time points. Images of ethidium bromide-stained DNA comets and nuclei were obtained at magnification x200.

Figure 6

Figure 6

p21- and p16-induced γH2AX phosphorylation in senescent HT-p21a and HT-p16 cells. HT-p21a cells (upper) and HT-p16 cells (low) were treated with IPTG for 3 days and 5 days or left untreated (control). Results are present as DNA content (X-axis) vs. γH2AX levels (Y-axis).

Figure 7

Figure 7

Analysis of DNA breaks in senescent HT-p21a cells by comet assay. HT-p21a cells were treated with IPTG for 4 days or left untreated (control). As a positive control cells were treated with DNA-damaging drugs (DDD): doxorubicin (Dox), which causes double strand breaks, or topotecan (Top), which causes mostly single strand breaks. Then cells were subjected to single-cell gel DNA electrophoresis (at neutral and alkaline conditions). Bottom panel: deleted

Figure 8

Figure 8

Effect of rapamycin on γH2AX phosphorylation in senescent cells. HT-p21a cells (deleted) and HT-p16 cells (low) were treated with IPTG, rapamycin (RAPA) or IPTG + Rapa for 4 days. (A) Left column: DNA content (X-axis) vs. H2AX levels (Y-axis). (B) Right column: γH2AX levels (X-axis) vs. cell count (Y-axis).

Figure 9

Figure 9

Effects of rapamycin on accumulation of γH2AX foci in HT-p21a senescent cells. HT-p21a cells were treated with IPTG, rapamycin (RAPA) or IPTG + Rapa for 3 days. Slides were fixed and stained for γH2AX. Foci per nucleus were counted. Results are shown as percent of control (no treatment).

Comment in

References

    1. Celeste A, Petersen S, Romanienko PJ, Fernandez-Capetillo O, Chen HT, Sedelnikova OA, et al. Genomic instability in mice lacking histone H2AX. Science. 2002;296:922–7. - PMC - PubMed
    1. Bassing CH, Alt FW. H2AX May Function as an Anchor to Hold Broken Chromosomal DNA Ends in Close Proximity. Cell Cycle. 2008;3:149–53. - PubMed
    1. Fernandez-Capetillo O, Celeste A, Nussenzweig A. Focusing on foci: H2AX and the recruitment of DNA-damage response factors. Cell Cycle. 2003;2:426–7. - PubMed
    1. Demidenko ZN, Blagosklonny MV. Growth stimulation leads to cellular senescence when the cell cycle is blocked. Cell Cycle. 2008;7:3355–61. - PubMed
    1. Demidenko ZN, Zubova SG, Bukreeva EI, Pospelov VA, Pospelova TV, Blagosklonny MV. Rapamycin decelerates cellular senescence. Cell Cycle. 2009;8:1888–95. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources