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. 2007 Jan 1;21(1):43-8.
doi: 10.1101/gad.1487307.

The DNA damage signaling pathway is a critical mediator of oncogene-induced senescence

Frédérick A Mallette  1 , Marie-France Gaumont-Leclerc, Gerardo Ferbeyre
Affiliations

The DNA damage signaling pathway is a critical mediator of oncogene-induced senescence

Frédérick A Mallette et al. Genes Dev. .

Abstract

Here we report that RNA interference against ATM inhibited p53 accumulation in cells expressing oncogenic STAT5 and cooperated with Rb inactivation to suppress STAT5A-induced senescence. Knocking down ATM was also effective to bypass E2F1-induced senescence and in combination with Rb inactivation, inhibited RasV12-induced senescence. Cells that senesced in response to ca-STAT5A or RasV12 accumulated DNA damage foci and activated ATM, ATR, Chk1, and Chk2, indicating that aberrant oncogene activation induces a DNA damage signaling response. Intriguingly, bypassing oncogene-induced senescence by inactivation of p53 and Rb did not eliminate the accumulation of oncogene-induced DNA damage foci (ODDI), suggesting a mechanism that may limit transformation in immortalized cells.

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Figures

Figure 1.
Figure 1.
ATM is critical for oncogene-induced senescence. (A) SA-β-gal staining of human diploid fibroblasts IMR90 infected with control vector or ca-STAT5A. The percent of SA-β-gal-positive cells is indicated at the bottom of each panel. The percent of cells that incorporate BrdU is shown below the panels. (B) Indirect immunofluorescence showing p53 induction by ca-STAT5A in cells fixed 8 d after infection. (C) Quantitation of ATM protein levels in IMR90 infected with a control hairpin or the anti-ATM hairpin vector. (D) Indirect p53 immunofluorescence in IMR90 cells expressing the indicated vectors. The staining intensity is translated into a color code using Metamorph software. (E) Rescue of ca-STAT5A-induced senescence by shRNA against ATM measured by SA-β-gal staining. IMR90 cells bearing the empty vector LXSN or its derivatives expressing E6 or E7 were infected with an empty vector or its derivative expressing an shRNA against ATM together with STAT5A1*6. The percent and standard deviation of SA-β-gal-positive cells are indicated at the bottom right of each panel. Data represent three independent experiments done with cells collected 6 d post-selection. (F) Rescue of RasV12-induced senescence by shRNA against ATM measured by SA-β-gal staining. Fibroblasts bearing the empty vector LXSN or its derivative expressing E7 were infected with an empty vector or its derivative expressing an shRNA against ATM together with RasV12. The percent and standard deviation of SA-β-gal-positive cells are indicated at the bottom right of each panel. Data represent three independent measurements done with cells fixed 14 d post-selection.
Figure 2.
Figure 2.
DNA damage foci in oncogene-induced senescence. (A) Indirect immunofluorescence for γH2AX foci in cells expressing control vector, RasV12, or STAT5A1*6. As a positive control, cells were treated with hydrogen peroxide. (B) Quantitation of the number of γH2AX foci. Histogram indicates the number of cells containing one, two to five, six to 10, and >10 foci. The data represent the average and standard deviation of three independent counts of 100 cells each.
Figure 3.
Figure 3.
Characterization of the DNA damage signaling pathway in STAT5A-induced senescence. (A) Colocalization 53BP1 foci with phospho-ATMS1981 in STAT5A1*6-expressing cells by confocal immunofluorescence. (B) Colocalization γH2AX foci with phospho-S/TQ epitopes in STAT5A1*6-expressing cells by confocal immunofluorescence. (C) Quantitation of DNA damage foci; asterisk represents a p < 0.001 using the Student’s t-test. (D) Immunoblots for Chk1 and Chk2 phosphorylation. (V) Vector; (R) RasV12; (5A) STAT5A. (E) ATM is required for phosphorylation of Chk2 at Thr 68 in ca-STAT5A-induced senescence.
Figure 4.
Figure 4.
ODDI persist after inactivation of the senescence program. Confocal immunofluorescence of cells stained with anti-53BP1 and anti-γH2AX. Two nuclei, representative of the cell population, are shown.
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Comment in

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