Chk2 activation in fibroblasts expressing E6/E7 and/or hTERT. (A) Immunoblots for abundance of phospho-Thr68-Chk2, Chk2 and p21 of fibroblasts expressing HPV-E6/E7 grown beyond senescence up to near crisis. Young HFF and those expressing HPV-E6/E7 (+E6/E7) were untreated (−) or treated (+) with bleomycin (bleo). Serially passaged HFF expressing E6/E7 were analysed at PD85 up to PD122. Reduction of the endogenous level of p21, a direct transcriptional target of p53, served as a marker for the activity of E6 in E6/E7 cells. Loading control (LC) is the scan of the amido-black-stained membrane. (B) Chk2 phosphorylation of fibroblasts expressing HPV-E6/E7 grown beyond senescence up to near crisis. Phosphorylation at Thr68, Ser26/Thr28 and Thr387 was examined with phospho-specific antibodies on immunoblots of Chk2 immunoprecipitates from lysates of young fibroblasts expressing E6/E7 at PD25 untreated (−) or treated (+) with bleomycin, and of E6/E7 cells serially passaged and analysed at PD85 up to PD122. Phospho-specific immunoblots are from independent blot of the same lysates. Recovery of immunoprecipitated Chk2 was monitored by immunoblotting with total Chk2 (lower panel). IP, immunoprecipitation; WB, Western blot. (C) Similar analysis as in (A) for young and IMR90 fibroblasts expressing E6/E7 at the indicated PD. (D) hTERT expression prevents Chk2 activation in fibroblasts expressing E6/E7. Immunoblots for Chk2 phosphorylated at Thr68 and total levels of Chk2, p21 in E6/E7 cells, young (Y) and at crisis (Cri) compared to immortalised E6/E7 cells through ectopic expression of hTERT (hT) untreated (−) or treated (+) with bleomycin (bleo). (E) hTERT expression prevents Chk2 activation in fibroblasts. Immunoblots for Rb, Chk2 and p21 in young (Y) and senescent (S) cells compared to immortalised fibroblasts through ectopic expression of hTERT (hT) untreated (−) or treated (+) with bleomycin (bleo). Loading control (LC) is the scan of the amido-black-stained membrane.

Association of γ-H2AX, but not Chk2, with telomeric DNA in post-senescent cells as detected by ChIP. (A) Dot blot analysis of DNA co-immunoprecipitated using the indicated antibodies or beads only (control) of crosslinked chromatin from cells expressing E6/E7, young (at PD25) and post-senescent (at PD96 and PD117). We used two antibodies to H2AX, a monoclonal antibody (m) and a polyclonal antibody (p). Duplicate dot blots were hybridised with a telomeric or Alu probe and membranes were exposed to PhosphorImager. (B) Quantification of the ChIP experiments shown in (A). The histograms give a quantitation of the signal recovered in each immunoprecipitate and detected by PhosphorImager as a percentage of the total amount of telomeric DNA used in each immunoprecipitation.

Abrogation of senescence-associated replicative arrest by Chk2 siRNA. (A) Senescent fibroblasts at PD80 were repeatedly transfected with Chk2 siRNA (Chk2) or with control siRNA (control). After 7 days, cells were collected and immunoblotted with anti-Chk2, anti-Chk1 and anti-β-tubulin antibodies used as an internal control (LC). (B) Immunoblots for pRb, cyclin A and p21 of senescent fibroblasts transfected with control siRNA or Chk2 siRNA at day 7. (C) BrdU incorporation and SA-βgal activity of senescent fibroblasts transfected with control siRNA or Chk2 siRNA at day 7. The percentage of cells incorporating BrdU (24 h labelling) and staining positive for SA-βgal is indicated in the bottom left of each photograph. Procedure as in Figure 1A. Representative photographs of stained field are shown.

Chk2 activation at replicative senescence. (A) Abundance and status of pRb, Chk2 and p21 of fibroblasts at different PDs up to senescence. Western blot analysis of young fibroblasts at PD35 untreated (−) or treated (+) with bleomycin (bleo) (used here as positive control) and ageing fibroblasts at PD74, PD82 and PD87 (cell population fully senescent). Loading control (LC) is the scan of amido-black-stained membrane, which showed equal amounts of protein transferred. The phosphorylation status of Rb is used to monitor cell cycle regulation. The percentages of cells positive for BrdU (24 h labelling) and SA-βgal are shown below. The standard deviations were 1–3%. Positive cells were scored by microscopic observation after indirect immunostaining of BrdU incorporation and histochemical assessment of β-galactosidase activity at pH 6. (B) Similar analysis for young and aged IMR90 fibroblasts at PD60 and PD68 (cell population fully senescent). (C) Chk2 phosphorylation at senescence. Phosphorylation at Thr68, Ser26/Thr28 and Thr387 was examined with phospho-specific antibodies on immunoblots of Chk2 immunoprecipitates from lysates of young fibroblasts untreated (−) or treated (+) with bleomycin (bleo), and of aged and young contact-inhibited (CI) fibroblasts. Recovery of immunoprecipitated Chk2 was monitored by immunoblotting with total Chk2. IP, immunoprecipitation; WB, Western blot.

Accumulation of phosphorylated H2AX (γ-H2AX) foci in senescent and E6/E7-expressing fibroblasts grown beyond senescence. (A, C) Immunofluorescence images of fibroblasts (HFF) and fibroblasts expressing E6/E7 (+E6/E7) immunostained with anti-γ-H2AX antibodies. (A) Young at PD39 and senescent at PD82 fibroblasts. (C) Young E6/E7 cells at PD43, post-senescent E6/E7 cells at PD89 and PD115 and immortalised through ectopic expression of hTERT (hTERT). Treatment of young cells with bleomycin is indicated (young+bleo). Right panels show quantification of γ-H2AX foci. The number of γ-H2AX foci per nucleus was counted for each sample and nuclei were categorised as indicated. Note that quantification shown for post-senescent E6/E7 cells refers to cells at PD115. Panels are representative of four independent experiments. (B) Age-dependent increase in the incidence of γ-H2AX foci in HFF cultures. The percentage of cells with γ-H2AX foci (closed symbols) versus the percentage of cells incorporating BrdU (open symbols) is shown at the indicated PD. Bars: standard deviation.

Senescence-associated replicative arrest impaired by dominant-negative Chk2 expression. (A) Validation of the retroviral vector encoding dominant-negative Chk2 in young HFF. Young HFF at PD28 were infected by a retroviral vector encoding Chk2DN (Chk2DN) or neo-only (control) and pooled drug-resistant cell populations were untreated (−) or treated (+) with bleomycin (bleo). Immunoblot analysis of Chk2 activation indicated by appearance of a slower migrating species using an antibody to total Chk2 (upper panel) and by band intensity using a phospho-specific antibody to Thr387-Chk2 (lower panel). Two exposures for Chk2 detection are shown in order to better visualise the absence of mobility shift in cells expressing Chk2DN after DNA damage (LE: long exposure; SE: short exposure). The line numbered 2 depicts the slower shifted Thr387 band. Note that Thr387 antibody revealed a faster migrating form of Chk2 in cells expressing Chk2DN, which probably reflects basal phosphorylation at Thr387 (line numbered 1). (B) Cell morphology of early colonies of fibroblasts stably expressing Chk2DN compared with HFF expressing p53DN and neo-only. Near-senescent HFF were infected with virus encoding Chk2DN, p53DN and neo-only (Cont), drug selected and photographed on day 20 post-infection. (C) Stable expression of mutant Chk2 in early colonies as revealed by increased nuclear Chk2 protein content. Indirect immunoperoxidase immunostaining of Chk2 protein. Only background signal was observed in senescent controls. (D) Growth curves of near-senescent HFF cultures infected with Chk2DN or p53DN. Near-senescent HFF were infected with retrovirus expressing the indicated proteins, drug-selected, serially passaged over the indicated period of time and cell number determined at each passage, as described in Materials and methods. (E) Immunoblot analysis of post-senescent HFF expressing Chk2DN. Cell lysates were prepared from culture of HFF at the time of infection (PD73) and of HFF expressing Chk2DN or p53DN that had entered into an extended lifespan phase (at day 20 post-infection) and were terminally growth arrested. Comparison was carried out with young (y) and age-matched control culture of HFF. Expression of pRb, p53, Chk2, cyclin A and p21 as indicated was analysed by Western blotting. Loading control (LC) is the scan of the amido-black-stained membrane. The percentages of BrdU-positive cells within these same populations are shown at the bottom. Procedure as in Figure 1A. The asterisk indicates a nonspecific band.

A model connecting telomere damage signalling and senescence-associated replicative arrest. As a consequence of cumulative cell divisions, a DNA DSB signal is generated either directly via exposure of critically shortened telomere or indirectly via chromosomal breaks, which may occur as a consequence of chromosome fusion and breakage following critical telomere shortening. These events elicit an ATM-dependent and -independent phosphorylation of targets including H2AX and Chk2. Activated Chk2 could further propagate the signal via a downstream substrate such as p53, which would in turn activate the transcription of the cell cycle inhibitor p21, leading to replicative arrest. Other intermediates and co-factors in the signalling pathway from the senescence-associated DNA damage to p53 activation are undoubtedly necessary.