Oncogenic ras and p53 cooperate to induce cellular senescence - PubMed (original) (raw)

Oncogenic ras and p53 cooperate to induce cellular senescence

Gerardo Ferbeyre et al. Mol Cell Biol. 2002 May.

Abstract

Oncogenic activation of the mitogen-activated protein (MAP) kinase cascade in murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the ARF/p53 tumor suppressor pathway. To investigate whether p53 is sufficient to induce senescence, we introduced a conditional murine p53 allele (p53(val135)) into p53-null mouse embryonic fibroblasts and examined cell proliferation and senescence in cells expressing p53, oncogenic Ras, or both gene products. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras or activated mek1 with p53 enhanced both p53 levels and activity relative to that observed for p53 alone and produced an irreversible cell cycle arrest that displayed features of cellular senescence. p19(ARF) was required for this effect, since p53(-/-) ARF(-/-) double-null cells were unable to undergo senescence following coexpression of oncogenic Ras and p53. Although the levels of exogenous p53 achieved in ARF-null cells were relatively low, the stabilizing effects of p19(ARF) on p53 could not explain the cooperation between oncogenic Ras and p53 in promoting senescence. Hence, enforced p53 expression without oncogenic ras in p53(-/-) mdm2(-/-) double-null cells produced extremely high p53 levels but did not induce senescence. Taken together, our results indicate that oncogenic activation of the MAP kinase pathway in murine fibroblasts converts p53 into a senescence inducer through both quantitative and qualitative mechanisms.

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Figures

FIG. 1.

Constitutive activation of the MAP kinase cascade engages the ARF/p53 pathway to promote senescence of primary MEFs. (A) Growth curves of MEFs obtained from wild-type, _ARF_−/−, and _p53_−/− knockout mice transduced with an empty retroviral vector (V) or its derivatives expressing oncogenic ras (R) or oncogenic mek1 (M). Each experiment was performed three times with similar results. The data represent the mean (± the standard deviation) of triplicate time points of a representative experiment. (B) p19ARF, p53, and Mdm2 immunoblots of wild-type MEFs expressing an empty vector or oncogenic ras and treated with the MEK inhibitor PD98059 (PD) diluted in DMSO. Untreated cells and cells treated with DMSO alone were used as controls. (C) p53 and Mdm2 immunoblots of cellular lysates from wild-type (wt) and _ARF_-null (_ARF_−/−) MEFs containing the empty retroviral vector pBabe (V) or its H-RasV12-expressing derivative (R). (D) Northern blot of total RNA purified from wild-type (wt), _ARF_-null (_ARF_−/−) and _p53_-null (_p53_−/−) MEFs expressing either the empty retroviral vector pBabe (V) or its derivative expressing oncogenic ras (R). α-Tub., anti-tuberculin antibody.

FIG. 2.

Senescence markers. (A) Wild-type MEFs transduced with pBabe (V) or its derivative expressing oncogenic ras (R) were fixed 8 days after infection and stained for SA β-Gal. Fifty percent of the cells expressing oncogenic Ras were SA β-Gal positive versus 5% of control cells. (B) Cells prepared as described above were stained with an anti-PML antibody and an FITC-conjugated secondary antibody. Images were obtained by immunofluorescence microscopy. Expression of oncogenic ras increased the number of PML bodies from 7 ± 3 in control cells to 32 ± 5.

FIG. 3.

Oncogenic activation of the MAP kinase pathway modifies the outcome of a p53-dependent cell cycle arrest. (A) Temperature shift assay evaluating the effect of the genetic background on a p53-mediated cell cycle arrest. MEFs expressing p53val135 can be grown at 39°C and arrested at 32°C. The arrested populations are then shifted to 39°C to inactivate p53val135 and investigate their recovery from a cell cycle arrest. (B) [3H]thymidine incorporation assay of TSP-vector (V), TSP-Ras (R), and TSP-Mek cells (M). All values are compared to the value scored by TSP-vector cells (V) growing at 39°C, which is taken as 100%. Shown is the amount of incorporation of cells growing at 39°C (day 0) or arrested at 32°C for 1 day and then the incorporation of the cells shifted to 39°C after being arrested at 32°C for 1, 2, 4, and 8 days (recovery). (C) In situ BrdU incorporation of TSP cells containing the empty vector (V) or a derivative with oncogenic ras (R). The cells were pulsed with 10 μM BrdU for 3 h 24 h after plating. Results are presented as the percentage of BrdU-positive nuclei.

FIG. 4.

Colony formation assay. (A) Cells growing at 39°C (day 0) or arrested at 32°C for 1, 2, 4, or 8 days were plated at a density of 500 cells per 6-cm-diameter plate and grown at 39°C for 10 days. Colonies were visualized by staining with crystal violet. (B) Same data as above summarizing three independent measures and the standard deviation. The data were normalized to the number of colonies obtained with cells growing at 39°C (day 0), which was taken as 100%.

FIG. 5.

Oncogenic Ras or Mek1 potentiates the activity of p53val135. Immunoblots of cellular lysates (20 μg of total protein) from TSP-vector (V), TSP-Ras (R), and TSP-Mek (M) cells. Cells were collected after incubation first for 4 days at 32°C and then for 4 days at 39°C.

FIG. 6.

Oncogenic Ras and p53 cooperate to promote senescence. (A) SA β-Gal staining. Cells were grown at 39°C, arrested for 8 days at 32°C, and then fixed for staining. (B) PML bodies. Cells were grown at 39°C and arrested for 4 days at 32°C. They were then fixed on coverslips and incubated with anti-PML and anti-p53 primary antibodies. After staining with FITC- or Texas Red-conjugated secondary antibodies, the cells were imaged by confocal immunofluorescence microscopy.

FIG. 7.

Oncogenic ras cooperates with wild-type (wt) p53 to induce senescence. p53−/− MEFs were coinfected with retroviruses expressing wild-type human p53 (pBabep53) and either a vector control, MSCVGFP (p53 + V), or its derivative expressing oncogenic ras (p53 + R) and analyzed 6 days postinfection. (A) Representative immunofluorescence staining with an anti-PML antibody. Samples were photographed at identical exposures. (B) Western blot analysis showing expression of the indicated proteins. pS15 indicates an anti-human phospho-serine-15 p53 antibody. (C) SA β-Gal staining of representative fields. (D) Quantitation of SA β-Gal activity. See Material and Methods for experimental details. α-tub, anti-tubulin antibody.

FIG. 8.

Coexpression of p53val135 and oncogenic Ras in _p53_−/− _ARF_−/− and _p53_−/− _mdm2_−/− MEFs. See Fig. 1 for experimental details. (A and D) Assay of [3H]thymidine incorporation by TSPA (A) and TSPM (D) cells containing the empty pBabe vector (V) or its derivative expressing Ras-V12 (R). (B and E) Summary of the number of colonies formed by TSPA (B) and TSPM (E) cells containing the empty pBabe vector (V) or its derivative expressing Ras-V12 (R) after their arrest at 32°C for the numbers of days indicated. (C and F) Immunoblots for p53 and p21 of TSPA (C) and TSPM (F) cells containing either the empty vector (V) or Ras-expressing retroviruses (R).

FIG. 9.

Analysis of senescence markers in _ARF_-null and _mdm2_-null MEFs. (A) Summary of SA β-Gal-positive cells among TSP, TSPA, and TSPM cells expressing the empty vector (V) or its derivative expressing oncogenic ras (R). (B) PML bodies in TSP, TSPA, and TSPM cells. Cells were fixed after 4 days of arrest at 32°C and stained for PML as described in Materials and Methods.

FIG. 10.

p53 levels do not correlate with the senescence phenotype. (A) p53 immunoblots after a long exposure (LE) and a short exposure (SE), p21 and p19ARF immunoblots of cellular lysates (20 μg of total protein) from wild-type (WT) MEFs, TSP cells, TSPA cells, and TSPM cells bearing the empty vector (V) or its derivative with oncogenic ras (R). Cells were collected after 4 days at 32°C. Tub, tubulin. (B) p53 levels in different cell lines expressing p53val135 are not dependent on mRNA levels. Northern blot of p53val135 expressed in TSP cells (_p53_−/−), three clones of TSPA cells (_p53_−/− _ARF_−/−), and two clones of TSPM cells (_p53_−/− _mdm2_−/−). ts, temperature sensitive.

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Oncogenic ras and p53 cooperate to induce cellular senescence - PubMed (original) (raw)