Oncogenic ras induces multiple regulators of mitochondrial biogenesis. Results are shown for real-time PCR measuring mRNA levels of the indicated genes at days 2 and 7 after selection with a retroviral vector control or its derivative expressing oncogenic ras.

Mitochondrial dysfunction in RasV12-expressing cells: JC-1 fluorescence. Red fluorescence indicates a normal membrane potential and green fluorescence indicates mitochondrial membrane depolarization. A decrease in the ratio of red/green fluorescence measured by FACS is a measure of the extent of mitochondrial membrane depolarization. PC, phase-contrast images.

p53 and Rb pathways can independently connect oncogenic ras signals to the mitochondria. Fluorescence intensity of cells taken 8 days after selection for the indicated vectors and analyzed by flow cytometry after staining with MitoFluor Green (A), DCFDA (B), and DHE (C). (D) Immunoblots for p53, Ras, and tubulin for the cells used for panels A to C. V, empty vector.

Mitochondrial dysfunction as an effector mechanism of OIS and replicative senescence (short telomeres). The oncogenic activity of the Ras/mitogen-activated protein kinase pathway induces a replication stress (12) and mitochondrial biogenesis leading to DNA damage and the activation of the p53 and Rb pathways. The activity of these tumor suppressors converge in the mitochondria to induce a senescence effector pathway characterized here as a metabolic checkpoint and oxidative stress. This is not a linear pathway, since the effects of mitochondrial dysfunction can activate the p53 and Rb pathways, generating positive feedback mechanisms that we propose contribute to keeping cells senescent.

Mitochondrial dysfunction after knocking down mitochondrial RISP. (A) Fluorescence intensity of cells taken 6 days after infection with shRNA control of shRNA against RISP and analyzed by flow cytometry after being stained with the indicated fluorescent probes. (B) mtDNA levels in cells described for panel A. (C) Relative ATP levels of cells expressing shRNA control of shRNA against RISP and treated with oligomycin (olig.; 7 μg/ml for 24 h) or vehicle. (D and E) Activation of AMPK as measured using an antibody against phospho-AMPK (phAMPK) (D) or phospho-ACC (phACC) (E).

Senescence after knocking down mitochondrial RISP. (A) Growth curves. Cells were plated in triplicate 2 days after infection with a retroviral vector expressing shRNA control or shRNA against RISP. Cell counts were estimated every 2 days using a crystal violet staining assay. (B) SA-β-Gal assay of cells described for panel A and plated 8 days after infection with the indicated vectors. (C) BrdU/PI profiles of cells described for panel A and plated 8 days after infection. (D and E) Immunofluorescence for γH2AX (D) or PML bodies (E) of cells described for panel A plated 6 days after infection. (F) Immunoblots for RISP and members of the p53 and Rb pathways in cells described for panel A 8 days after infection.

Mitochondrial changes in Ras-expressing cells. (A) MitoSox fluorescence images of Ras-expressing cells and cells with a control vector obtained from cells 2 and 8 days after selection. (B) DCFDA fluorescence images of cells as described for panel A. Nuclei were counterstained with Hoechst 33342. PC means phase-contrast images, and the arrows point to vacuoles that characterize the cytoplasm of Ras-senescent cells. (C) Magnified immunofluorescence image to visualize the vacuoles in the cytoplasm of Ras-senescent cells stained with DFCDA. (D to F) Fluorescence intensity of cells analyzed by flow cytometry and stained with DHE, DCFDA, or Mitofluor Green as indicated. (G) Real-time PCR measuring the relative levels of mtDNA versus nuDNA in cells with control vector or its derivative expressing oncogenic ras.

Rotenone and oligomycin induce a cell cycle arrest with some characteristics of senescence. (A) For growth curves, 5 × 10⁴ cells were plated in 12-well plates. Twenty-four hours later, rotenone (1.5 μM) or oligomycin (7 μg/ml) was added (day 0). Drugs were added with fresh medium every 2 days, and cells were counted at the indicated time points. (B) SA-β-Gal was added 5 days after treatment. (C) PML bodies were detected by immunofluorescence. We quantified the number of cells having 5 or more PML bodies, and the numbers are indicated at the bottom right of each panel. Rot. 5d, rotenone administration for 5 days; Olig. 5d, oligomycin administration for 5 days. (D) Fluorescence intensity of cells analyzed by flow cytometry after being stained with the indicated fluorescent probes. (E) Relative ATP levels of cells treated with rotenone or oligomycin.

Oxidative DNA damage during Ras-induced senescence. (A) 8-OHdG detection in Ras-expressing cells. Cells were fixed 8 days after selection with a control vector or its derivative expressing RasV12 and were left untreated or were treated with HCl. 8-OHdG was visualized by indirect immunofluorescence using an antibody against 8-OHdG. Representative images of cells in the population were obtained by immunofluorescence microscopy. Nuclei were visualized by DAPI. (B) Real-time qPCR indicating relative levels of SOD2 mRNA at the designated times. (C) Immunofluorescence for catalase in cells fixed at day 8 post selection. (D) ATP content of control growing cells with an empty vector (V) (set to 100%) or cells expressing oncogenic ras (R). Cells also were treated with oligomycin (olig) at 3.5 μg/ml for 24 h or with vehicle. Data are means ± standard deviations. (E) Activation of AMPK as measured using an antibody against phospho-AMPK (pAMPK). (F) Total levels of the α1 and α2 subunits of AMPK (tAMPK) as measured by immunoblots with a specific antibody. (G) Glucose consumption in cells expressing oncogenic ras or a control vector. Cells were taken at day 6 after selection. The value for control cells was taken as a reference. Data represent the means and standard deviations from four measurements.