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      Chronic senolytic treatment alleviates established vasomotor dysfunction in aged or atherosclerotic mice


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          While reports suggest a single dose of senolytics may improve vasomotor function, the structural and functional impact of long‐term senolytic treatment is unknown. To determine whether long‐term senolytic treatment improves vasomotor function, vascular stiffness, and intimal plaque size and composition in aged or hypercholesterolemic mice with established disease. Senolytic treatment (intermittent treatment with Dasatinib + Quercetin via oral gavage) resulted in significant reductions in senescent cell markers ( TAF + cells) in the medial layer of aorta from aged and hypercholesterolemic mice, but not in intimal atherosclerotic plaques. While senolytic treatment significantly improved vasomotor function (isolated organ chamber baths) in both groups of mice, this was due to increases in nitric oxide bioavailability in aged mice and increases in sensitivity to NO donors in hypercholesterolemic mice. Genetic clearance of senescent cells in aged normocholesterolemic INKATTAC mice phenocopied changes elicited by D+Q. Senolytics tended to reduce aortic calcification (alizarin red) and osteogenic signaling ( qRTPCR, immunohistochemistry) in aged mice, but both were significantly reduced by senolytic treatment in hypercholesterolemic mice. Intimal plaque fibrosis (picrosirius red) was not changed appreciably by chronic senolytic treatment. This is the first study to demonstrate that chronic clearance of senescent cells improves established vascular phenotypes associated with aging and chronic hypercholesterolemia, and may be a viable therapeutic intervention to reduce morbidity and mortality from cardiovascular diseases.

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          The Achilles’ heel of senescent cells: from transcriptome to senolytic drugs

          The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age-related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro-survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL-xL, or plasminogen-activated inhibitor-2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM-MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation-exposed, and progeroid Ercc1 −/Δ mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1 −/Δ mice, delaying age-related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.
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            Telomeres are favoured targets of a persistent DNA damage response in ageing and stress-induced senescence

            Cellular senescence is defined as the irreversible loss of division potential in somatic cells and is marked by a number of distinct phenotypic changes1. It is believed that senescence has an important role in vivo, protecting organisms on one hand against a build-up of cells containing DNA damage and on the other hand contributing to age-dependent tissue dysfunction1. Evidence is mounting that cells bearing senescent markers accumulate in several tissues with age2 3, as well as in several age-related diseases4 5. DNA damage, especially double-strand breaks (DSBs), induces a DNA damage response (DDR)6 that leads to repair, apoptosis or permanent growth arrest (senescence). The senescent phenotype develops slowly over several days after the initial DNA damage7 8 and is maintained by an ongoing DDR7 9. This response is characterized by activation of sensor kinases (ATM/ATR, DNA-PK), formation of DNA damage foci containing phosphorylated histone H2A.X (γH2A.X) and ultimately induction of checkpoint proteins, such as p53 (TP53) and the CDK inhibitor p21 (CDKN1A), which contribute to cell-cycle arrest. The described signalling pathway continues to actively contribute to the stability of the cell-cycle arrest long after induction of senescence9. Similar to replicative senescence, persistent DNA damage foci have also been observed as a result of stress-induced senescence induced by irradiation7 8, and while they are thought to be important in the maintenance of the phenotype, the nature of this irreparable DNA damage is still unclear. Telomeres are specialized structures at the ends of linear chromosomes, and consist of tandem TTAGGG repeats that bind proteins to form a complex named shelterin10. This complex sequesters the telomeric DNA and prevents it from being recognized as DNA damage11. Telomere-induced foci can be induced by experimental disruption of shelterin components such as TRF2 and POT112 13, telomere shortening during replicative senescence9 or spontaneously in cancer cells14. Uncapped telomeres have been shown to become associated with DDR factors such as 53BP1, MRE11, phosphorylated forms of H2AX and ATM12, and activate a signalling cascade that culminates in cell-cycle arrest9. Telomeres are distinct from the rest of the genome in their DNA repair capacity15 and have been shown to inhibit non-homologous end joining (NHEJ) as a mechanism to prevent end-to-end fusions16. NHEJ is a major pathway for the repair of DSBs, which allows the rejoining of two DNA ends and has been reported to be inhibited in vitro by shelterin components, such as TRF217. For these reasons, we speculated that telomeric repeats might be the preferential locations for unresolved DSBs, following exposure to DNA-damaging agents. The inability to repair DSBs by NHEJ at telomeric regions could be a major contributor to a persistent DDR7. Here, we have investigated the role of genotoxic and oxidative stress in the persistence of a DDR during cellular senescence and its potential role in mammalian ageing. We show, both by immuno-fluorescent in situ hybridization (FISH) and ChIP, that DNA damage foci in senescence induced by exogenous DNA-damaging agents are preferentially located at telomeres, irrespective of telomerase activity. Importantly, using a combination of an AcGFP-53BP1c fusion protein and telomere PNA probes in live cells, we show that all persistent foci (surviving for longer than 3 h) are associated with telomeres. Moreover, in order to test the importance of DNA damage at telomeres in the ageing process, we investigated the presence of telomere-associated foci (TAF) in the gut and liver of mice, which have long telomeres and active telomerase but show increases of DNA damage foci frequencies in both tissues with age. We found an age-dependent increase in the frequencies of TAF in both the gut and liver. These data indicate that telomeres are important targets for genotoxic stress in vitro and in vivo with specific relevance for the induction of stress-induced senescence. Results TAF are persistent in stress-induced senescence We first tested whether persistent foci following X-ray irradiation were located at telomeres. We treated proliferation-competent human MRC5 fibroblasts and mouse embryonic fibroblasts (MEFs) with X-ray radiation (20 Gy and 10 Gy, respectively). As previously shown7, we confirmed that these levels of irradiation abolished cell proliferation and labelling indices for BrdU and Ki67, and caused increased activity of senescence-associated β-galactosidase (Sen-β-Gal) (results not shown). In order to test the sensitivity of telomeres to irradiation-induced DNA damage, we exposed MRC5 fibroblasts to increasing levels of X-ray irradiation (Fig. 1a,b). Frequencies of γH2A.X foci per nucleus increased with irradiation dose as expected (Fig. 1a). However, some DNA damage foci colocalized with telomeres as shown by immuno-FISH (Fig. 1b,c and Supplementary Fig. S1). As the DNA damage foci we observe are the result of random DSB caused by irradiation, we have defined them as TAF, as opposed to telomere-induced foci, which are generally attributed to telomere uncapping. As shown previously in MRC5 fibroblasts, only high doses of irradiation (20 Gy) can induce permanent cell arrest7, suggesting that TAF numbers might be the determining factor in cell-cycle arrest. While frequencies of non-TAF decreased rapidly following irradiation, the mean number of TAF per cell remained stable with time in human fibroblasts (Fig. 1d,e). These changes resulted in a time-dependent increase in the fraction of TAF (Fig. 1d). In young non-irradiated cells, as previously reported18, we found very low frequencies of cells positive for γH2A.X foci and overall low numbers of TAF (Fig. 1e). We also found that TAF were significantly larger than non-TAF at all time points analysed (Supplementary Fig. S2), which is consistent with the reports that larger foci are persistent19. To assess independently the location of DDR proteins at telomeres following irradiation, we performed chromatin immunoprecipitation (ChIP) on cross-linked chromatin from MRC5 fibroblasts non-irradiated and 10 days after irradiation using an antibody against γH2A.X. We first confirmed the specificity of the ChIP by western blotting immunoprecipitated γH2A.X (Supplementary Fig. S3) retrieved from phenol fraction following the ChIP assay. ChIP was followed by quantitative real-time PCR of sub-telomeric regions using a set of primers that had been previously validated for replicative senescent human fibroblasts and showed excellent concordance to ChIP–ChIP20. The results show robust enrichment of γH2A.X at sub-telomeric regions in chromosome 12 (10 days following irradiation when compared with non-irradiated controls) and that this enrichment increases abruptly in the direction of the end of the chromosome at 0.24 Mbp (Fig. 1f), similar to what was shown for replicative senescent fibroblasts. Moreover, in order to demonstrate that γH2A.X was in fact binding telomeric repeats and not just sub-telomeric regions, we performed quantitative real-time PCR for telomere repeats following γH2A.X ChIP. Our results show an average 14-fold enrichment of γH2A.X at telomere repeats 10 days after irradiation when compared with non-irradiated fibroblasts in two independent ChIP experiments (Fig. 1g). This confirms that colocalization observed by immuno-FISH is due to a physical association with telomeres and not mere proximity in the three-dimensional nuclear space. To determine that enrichment of γH2A.X foci at telomeres was not restricted to X-ray irradiation treatment, we have also incubated MRC5 fibroblasts with H2O2 and neocarzinostatin (a protein–chromophore complex known for its anti-tumour activity). Similar to irradiation, we found evidence for both treatments of persistent DNA damage foci located at telomeres by immuno-FISH (Fig. 2a,b) at concentrations that induce cellular senescence (Fig. 2c–f). Overexpression of hTERT, the catalytic subunit of telomerase, has been shown to counteract telomere shortening, extend the replicative potential and prevent replicative senescence in cells21. To test the effects of hTERT overexpression on irradiation-induced telomere damage, we transfected proliferating-competent MRC5 human fibroblasts (PD25-30) with hTERT and subjected them to 20 Gy irradiation. MRC5 fibroblasts overexpressing hTERT showed high levels of telomerase activity, stable telomere length and extended replicative lifespan (results not shown and Ahmed et al.22). We found that the presence of telomerase did not have a significant impact on numbers and percentage of TAF for up to 7 days after irradiation (Fig. 3a–c), cells still arrested irreversibly as shown by increased Sen-β-Gal activity and lack of the proliferation marker Ki67 (Fig. 3d). Accordingly, in MEFs that have long telomeres and telomerase activity, we found similar numbers and percentage of TAF as in human fibroblasts following irradiation (Fig. 4a,b). As in human fibroblasts, we observed that the frequencies of non-TAF decreased rapidly following irradiation and that the mean number of TAF per cell remained stable with time (Fig. 4b,c). TAF are longer-lived than non-TAF In order to establish whether telomeric location was necessary for foci persistence, we used kinetic live-cell microscopy. DNA damage foci lifespan was tracked by using a AcGFP-53BP1c fusion protein, which allowed us to quantitatively report DNA damage foci formation and degradation at the level of the individual focus23. We combined this with microbead-mediated incorporation of a fluorescently labelled PNA probe, which specifically tags telomere repeats. Owing to their stability, PNA probes can form PNA/DNA heteromultiplexes and allow visualization of telomeres in live cells for several hours24. To test whether the observed signals were indeed located at telomeres, after incorporation of a Cy3-labelled telomere probe in live cells, cells were fixed and hybridized with a FITC-labelled telomere probe. Results show that the majority of spots colocalize (Supplementary Fig. S4a). Metaphase spreads generated after microbead-mediated incorporation of a PNA telomere probe also reveal specific labelling of the ends of chromosomes (Supplementary Fig. S4b). For the first time, time-resolved live-cell microscopy allowed the kinetic tracking of telomeric and non-telomeric DNA damage foci independent from each other in the same cell after induction of senescence (Fig. 4d). We ensured proper tracking of individual foci by using a wide z-stack range (4.5 μm) and a high imaging frequency (every 10 min.). Videos were deconvolved using Huygens (SVI), and individual foci lifespan and colocalization with telomeres were tracked manually in MEFs starting at 3 days after irradiation. Live-cell microscopy detected similar numbers of telomeres in MEFs as FISH in fixed cells (Supplementary Fig. S4a). Moreover, no significant difference in average numbers of TAF per nucleus was observed by live-cell microscopy and by immuno-FISH at 3 days after 10 Gy irradiation (4.02±0.18 versus 3.1±0.28, P=0.07 two-tailed t-test). It is possible that incorporation of telomere PNA probes would induce TAF by eliciting dissociation of telomere-binding proteins from telomeres. However, we found in proliferating non-irradiated MEFs transfected with AcGFP-53BP1c fusion protein no evident induction of TAF after incorporation of PNA probes and generally very low number of 53BP1 DNA damage foci (Supplementary Fig. S4b,c). By manual tracking of foci, we constructed Kaplan–Maier survival curves for telomeric and non-telomeric foci in MEFs starting at 72 h after 10 Gy irradiation (Fig. 4e). The data show that all non-telomeric foci were short-lived, with a maximum lifespan of about 3 h. In contrast, only foci that colocalize with telomeric signals were able to survive for more than 3 h, and about 50% of TAF had a lifespan of >6 h (Fig. 4e,f). Previous work by our group had shown by live-cell imaging that foci lifespan increased in both replicative senescence and irradiation-induced senescence of human fibroblasts with half of all DDR foci being short-lived and dependent on oxidative stress7. Our recent data show that most, if not all, long-lived foci in stress-induced senescence colocalize with telomeres. This is in apparent contrast to a recent study19 that reported no colocalization between TRF2 and 53BP1 10 days after irradiation in human fibroblasts. This inconsistency may be due to non-telomeric location of TRF2 as previously shown25. TAF increase with age irrespective of telomere length Our findings of persistent DNA damage foci at telomeres irrespective of telomerase activity led us to speculate whether random telomere damage could lead to accumulation of TAF in mouse tissues with age. So far, an age-dependent increase in the percentage of cells harbouring TAF has only been reported in skin fibroblasts from baboons2. An age-dependent increase in DNA damage foci in mice in several tissues, including enterocytes in intestinal crypts and hepatocytes (which we confirm here, Supplementary Fig. S5a,b), together with increased oxidative stress markers has been reported3. Using high-resolution imaging of tissue sections, in depth Z stacking (a minimum of 40 optical slices with ×100 objective) followed by Huygens (SVI) deconvolution and manual quantification of colocalization by five independent analysts, we show that both liver hepatocytes and intestinal crypt enterocytes of C57BL/6 wild-type mice contain TAF and that the mean number of TAF per cell increases exponentially with age in both tissues (Fig. 5a,c,d,f and g). Moreover, the percentage of colocalizing foci per cell (%TAF) increases linearly with age in both tissues (Fig. 5b,e). Preliminary analysis of longitudinal gut sections revealed that most TAF-positive cells in the intestinal crypts of old animals were enterocytes in the transient amplifying zone of the crypts while enterocytes at the bottom of the crypts (where stem cells are located) generally have a low degree of colocalization (not shown). In both the intestine and liver, frequencies and localization of TAF-positive cells as demonstrated here (Fig. 5) correspond well with frequencies of senescent cells in these tissues3 26. However, we found that the number of non-colocalizing foci per cell (non-TAF) does not increase significantly with age in either tissue (Fig. 5i,j), supporting the idea that TAF are persistent and accumulate with age, while non-TAF do not. It is not clear at present whether TAF as seen in tissues of ageing mice are the result of telomere shortening or random telomeric damage. Mouse gut is a highly proliferative tissue, and an age-dependent decrease in telomere length in C57BL/6 mice intestine has been reported3 27, which can increase the probability of telomere uncapping and detection by DDR proteins. To test whether the presence of TAF could be explained by telomere shortening, we quantified telomere intensity of both colocalizing (TAF) and non-colocalizing (non-TAF) telomeres. In old mice intestinal crypts, we found no statistically significant difference between telomere length distributions in TAF and non-TAF (Mann–Whitney test, P=0.08) and a slight increase in median telomere length in the liver TAF (Mann–Whitney test, P=0.002) (Fig. 5h), suggesting that telomere length is not the sole defining factor in the induction of TAF. Our results show for the first time that TAF increases with age in mouse liver. As very low frequencies of proliferating hepatocytes were observed at any age, we hypothesize that oxidative stress may be responsible for increased telomere damage. In effect, we have previously reported that hepatocytes containing DDR foci were also positive for oxidative stress markers26. Discussion Maintenance of genomic stability requires that ends of chromosomes possess special characteristics, which make them distinct from DSBs. While these characteristics are essential for prevention of telomere end-to-end fusions, our results suggest that they might have detrimental consequences in terms of DNA repair capacity following random DNA damage whether it is caused endogenously by oxidative stress or exogenously by DNA-damaging agents. Previous work has attributed a key role for TRF2 in the distinction between telomeres and DSBs. Displacement of TRF2 from telomeres by overexpression of TRF2ΔBΔM or conditional deletion of TRF2 in mouse cells has been shown to result in ligase-IV-dependent telomere fusions16 28 29. It has also been described in vitro that TRF2 and its binding partner RAP1 are required to prevent NHEJ-dependent telomeric DNA fusions by inhibiting DNA-PK and ligase-IV-mediated end joining17. Telomere-binding proteins may also impact on regions adjacent to telomere repeats. This DSB repair inhibition was demonstrated in Saccharomyces cerevisiae where signalling from DNA breaks was inhibited up to 0.6 kb from telomeres30. Thus, while our Immuno-FISH results point to DDR occurring specifically at telomeric repeats following genotoxic stress, it is possible that regions adjacent to telomeres may have been affected. However, it is methodologically difficult to establish the exact position of the DNA DSB as it has been shown that telomere-induced foci extend well beyond telomere repeats. In fact, ChIP–Chip assays on replicatively senescent cells detected spreading of γH2A.X hundreds of kilobases in several chromosome ends9 20 similar to our results in chromosome 12p following irradiation. While our data show direct binding of γH2A.X to telomeric repeats, we are unable to determine experimentally whether a persistent DNA damage is occurring at the chromosome end or within telomeric repeats. For that reason, several scenarios can be proposed to explain our data: it is possible that genotoxic stress contributes to the loss of telomere loop (t-loop) conformation and leads to a DDR at telomere ends. T-loop has been hypothesized to be maintained by binding of shelterin components10 31 and dependent on telomere length32. Our data point to a persistent DDR occurring irrespectively of telomere length following genotoxic stress and during ageing in mice, which would be inconsistent with the idea of short telomeres inducing loss of protective cap. However, it is still possible that random oxidative damage to telomere repeats will affect binding ability to shelterin components as supported by in vitro assays, which show that binding of TRF1 and TRF2 to telomeres is reduced by oxidative DNA damage33. An additional scenario has been proposed by Cesare et al.14 They demonstrated in cancer cells that a spontaneous DDR at telomeres could occur in the absence of chromosome fusions, suggesting the existence of an intermediary state of telomere capping, which retains sufficient TRF2 or other shelterin components to suppress NHEJ without inhibiting a DDR at telomere ends14. This scenario, the authors have shown, can occur partly independent of telomere length in ALT (alternative lengthening of telomeres)-positive cancer cell lines14. This could explain persistent DDR at telomeres irrespectively of length in the absence of NHEJ. A final scenario is that random DNA damage generated by genotoxic stress occurs within telomeric repeats and does not lead to recognition of chromosome terminal ends by DDR. Shelterin components (such as TRF2) would then act to repress NHEJ in telomeric repeats throughout their length, while allowing a persistent DDR. This would be consistent with in vitro data showing that telomeric repeats are resistant to NHEJ17 and that a DDR can occur in long telomeres concomitantly with inhibition of NHEJ14. Persistence of TAF following X-ray irradiation-induced senescence were observed independently by d'Adda di Fagagna's group and the authors suggest that TRF2 plays a role in inhibition of NHEJ repair (ref. 34) Our data also suggest that telomeric regions, despite occupying a minute fraction of the genome, are favoured targets for random DNA damage. In fact, previous work has demonstrated that telomeres are particularly sensitive to oxidative stress when compared with the bulk of the genome15 and less efficiently repaired when subjected to single-stranded breaks and UV-induced damage35. It has been argued that this sensitivity to single-stranded breaks might be due to the fact that telomeric repeats contain guanine triplets that are remarkably sensitive to oxidative modifications36 37. These factors, coupled with the reported protection of telomeres from repair activities, may contribute to their specific targeting and persistent damage as a consequence of genotoxic stress. However, further work needs to be conducted in order to elucidate the characteristics of telomeres that make them particularly susceptible to damage. We conclude that persistent telomere-associated damage is a frequent outcome of genotoxic stress and a component of stress-induced senescence, which is independent of telomerase activity and telomere length, and may contribute to age-related decline in the tissue function. This highlights the relevance of telomeres as targets of stress-related ageing. Methods Cell culture and treatments Human embryonic lung MRC5 fibroblasts were obtained from ECACC (Salisbury, UK) and grown in DMEM (Sigma, Dorset, UK) supplemented with 10% heat-inactivated foetal calf serum, penicillin/streptomycin and 2 mM glutamine under atmospheric oxygen conditions (air plus 5% CO2). Mouse embryonic fibroblasts (MEFs) were obtained from C57BL/6 wild-type mice and cultured in DMEM (Sigma) plus 10% FSC, penicillin/streptomycin and 2 mM glutamine at 3% ambient oxygen in a 3-gas cell culture incubator (Zapf Instruments, Sarstedt, Germany). MRC5 fibroblasts (PD 25–30) were transfected retrovirally with the catalytic subunit of telomerase hTERT as described22. MEFs at 50% confluency were transiently transfected with a 53BP1 reporter protein using plasmid pG-AcGFP-53BP1c using Fugene6 (Roche, Welwyn, UK) at a ratio of 3 μl fugene: 2 μg DNA following the manufacturer's instructions. Stress-induced senescence in MRC5 fibroblasts was induced by X-ray irradiation with 20 Gy, neocarzinostatin (80 ng ml−1) for 1 h or H2O2 (400 μM) in serum-free media for 1 h. Following treatment, culture medium was refreshed. Senescence was induced in MEFs by X-ray irradiation with 10 Gy. Animals Three male C57BL/6 mice per age group (3, 12, 22, 36 and 42 months) were killed. Tissues were immediately perfusion fixed. Formalin-fixed paraffin-embedded 3-μm sections were prepared from the liver and gut. All work was complied with the guiding principles for the care and use of laboratory animals. The project was approved by the Faculty of Medical Sciences Ethical Review Committee, Newcastle University. Project licence number 60/3864. Immunofluorescence For immunostaining, cells grown on sterile coverslips were fixed in 2% paraformaldehyde and incubated with a primary antibody for 1 h at room temperature, followed by either an Alexa Fluor® 594 or Alexa Fluor® 488 secondary antibody (Invitrogen) 45-min incubation. Antibodies used: rabbit polyclonal anti-Ki67 antibody (ab15580; 4 μg ml−1 Abcam), mouse monoclonal anti-γ-H2A.X antibody (AB10022; 0.25 μg ml–1 Upstate Millipore), rabbit monoclonal anti-γ-H2A.X IgG (S139) (#9718; 0.4 μg ml–1 Cell Signaling), rabbit polyclonal anti-53BP1 antibody (#4937; 0.2 μg ml–1 Cell Signaling) and rabbit polyclonal anti-53BP1 (NB100-904 4 μg ml–1 Novus Biologicals). Immuno-FISH Immuno-FISH was performed as described18. Briefly, cells grown on sterile coverslips were fixed with 2% paraformaldehyde and incubated with anti- γ-H2A.X (Upstate Biotechnology) or anti-53BP1 (Cell Signaling) antibodies for 1 h at room temperature as described above. After application of the secondary antibody, cells were washed with PBS and FISH was performed. 20 μl of Cy-3-labelled telomere specific (C3TA2)3 peptide nucleic acid (PNA) probe (4 ng μl−1) (Panagene) was applied to the cells followed by co-denaturation at 80 °C and hybridization for 2 h at room temperature in the dark. Cells were then washed three times for 10 min with wash buffer made from 70 ml formamide (70%), 30 ml SSC 2% (3 ml stock SSC 20%+27 ml H20) and three times for 5 min with TBS-Tween 0.05%. Cells were incubated with 4,6-diamidino-2-phenylindole (DAPI), mounted and imaged using a Leica DM5500B fluorescence microscope. In depth Z stacking was used (images were captured as stacks separated by 0.247 μm with ×100 objective) followed by Huygens (SVI) deconvolution. For Immuno-FISH in formalin-fixed paraffin-embedded liver and gut mice tissues, sections were hydrated by incubation in 100% Histoclear, 100, 95 and 70% methanol for 5 min (twice) and finally in distilled water for 5 min (twice). For antigen retrieval, the slides were placed in 0.1 M citrate buffer and heated until boiling for 10 min. After cooling down to room temperature, the slides were washed twice with distilled water for 5 min. After blocking in normal goat serum (1:60) in BSA/PBS, primary antibody (rabbit γ-H2A.X 1:250) was applied and incubated at 4 °C overnight. The next day, slides were washed three times in PBS, incubated with secondary antibody for 30 min, washed three times in PBS and incubated with Avidin DCS (1:500) for 20 min. Following incubation, slides were washed three times in PBS and dehydrated with 70, 90 and 100% ethanol for 3 min each. Sections were denatured for 5 min at 80 °C in hybridization buffer (70% formamide (Sigma), 25 mM MgCl2, 1 M Tris pH 7.2, 5% blocking reagent (Roche)) containing 2.5 μg ml−1 Cy-3-labelled telomere specific (CCCTAA) peptide nuclei acid probe (Panagene), followed by hybridization for 2 h at room temperature in the dark. The slides were washed twice, for 15 min each, with 70% formamide in 2×SSC, followed by 2×SSC and PBS washes for 10 min. Sections were incubated with DAPI, mounted and imaged. In depth Z stacking was used (a minimum of 40 optical slices with ×100 objective) followed by Huygens (SVI) deconvolution. Crosslinked ChIP assay Crosslinked ChIP assay was carried out using 100-μg crosslinked chromatin prepared from control or irradiated MRC5 fibroblasts (10 days after 20 Gy) as described previously38. Antibody used for immunoprecipitation raised against γH2A.X was purchased from Upstate Biotechnology. Isotype and species-matched control ChIP grade IgG was obtained from Abcam. The 10 μg of each antibody were used in each ChIP reaction. Real-time PCR Primers used for the detection of relevant sub-telomeric sequences of chromosome 12p were as published in ref. 20. Real-time PCR specific for telomeric repeats was performed as described in ref. 39. Briefly, 4 μl of ChIP eluate was added to a final volume of 13 μl quantitative PCR reaction containing 6.5 μl Jumpstart SYBR master mix (Sigma) and 10 pmol appropriate primer mix. Telomere and sub-telomere PCR reaction conditions were: 10 s at 95 °C followed by 40 cycles of 10 s at 95 °C, 30 s at 55 °C and 30 s at 72 °C followed by ABI7500 Fast Real-Time PCR System machine predetermined melt curve analysis (Applied Biosystems). Each PCR reaction was performed in triplicate from two independent ChIP experiments. All reactions were normalized to the control antibody and fold enrichment above background was calculated using the following equation: (1/(2A)) ×100. The values were then adjusted to the values of total input and expressed as log to the base 2. Extraction of immunoprecipitated γH2A.X from phenol fraction Following reversal of crosslinks after the ChIP assay, eluate was mixed with equal amount of phenol and centrifuged at 13,000 r.p.m. for 5 min. DNA-containing fraction was removed, cleaned up and used in PCR, whereas 500 μl of phenol was mixed with concentrated HCl (final concentration of 0.4 M) and ten volumes of acetone. This mix was allowed to precipitate at −80 °C overnight. Precipitated histones were pelleted at 3,500 r.p.m. for 20 min at 4 °C. The pellets were washed three times in acetone, vacuum dried and resuspended in 10 μl of 8 M urea/10% β-mercaptoethanol. Samples were heated to 65 °C for 10 min, then 10 μl of SDS–polyacrylamide gel electrophoresis loading buffer was added, mixed and heated to 100 °C for 5 min before loading on a 12% SDS–polyacrylamide gel electrophoresis gel. Western blots were carried out using routine protocol from this point onwards. Telomere PNA probes incorporation and live-cell imaging Incorporation of telomere-specific PNA probe by glass beads was performed as described24. Briefly, the Cy-3-labelled telomere-specific (C3TA2)3 peptide nucleic acid (PNA) probe (Panagene) was dissolved in 80 mM KCl, 10 mM K2PO4, 4 mM NaCl, pH 7.2, to a final concentration of 1 μM. MEFs were grown in glass-bottomed dishes and PNA probe was pipetted onto the culture. A 0.12 g of 75 μm alkali-washed glass beads (Sigma) were carefully and evenly sprinkled onto the surface of the dish, and cells were allowed to recover for 2 h in DMEM (10% FSC). To confirm that PNA probe was located at telomeres, following Cy-3-labelled telomere-specific PNA probe incorporation in live MEFs, cells were fixed in 2% paraformaldehyde and FISH was performed, as described above, using a FITC-labelled telomere specific (C3TA2)3 peptide nucleic acid (PNA) probe (Panagene). For live-cell timelapse microscopy of the 53BP1 reporter and PNA probe, a Zeiss LSM510 confocal microscope equipped with a Solent incubator (Solent Scientific) at 37 °C with humidified 5% CO2, using a ×63 (NA=1.4) objective, was used as described23. MEFs were plated in Iwaki glass-bottomed dishes (Iwaki) and imaged using a 63×1.3 NA oil objective. AcGFP-53BP1c foci and telomere PNA probes were tracked manually using ImageJ (http://rsb.info.nih.gov/ij/). Metaphase spreads Metaphase spreads from MEFs were prepared by treatment of subconfluent cells with 10 μg ml−1 colcemid for 2 h at 37 °C, followed by 60 mM KCl for 15 min at room temperature and fixation in ethanol:acetic acid (3:1) as described in ref. 18. Slides were air-dried, stained with DAPI and imaged using a Leica DM5500B fluorescence microscope. Senescence-associated β-galactosidase staining For senescence-associated β-galactosidase staining, cells were fixed with 2% formaldehyde in PBS for 5 min and stained as described in ref. 40. Briefly, following fixation, cells were washed once with PBS and incubated at 37 °C for 16 h in freshly prepared senescence-associated β-galactosidase staining solution containing 2 mM magnesium chloride, 150 mM sodium chloride, 40 mM citric acid, 12 mM sodium phosphate dibasic, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide and 1 mg l−1 5-bromo-4-chloro-3-inolyl-β-d-galactoside (X-Gal) at pH 6.0. Following staining, nuclei were stained with DAPI and cells were imaged. Statistical analyses We conducted two-tailed t-tests, linear and non-linear regression analysis, and Gehan–Breslow test using Sigma Plot vs11.0. Wilcoxon–Mann–Whitney tests were conducted using IBM SPSS Statistics 19. Author contributions G.H. performed and quantified immuno-FISH in fibroblasts and contributed to the writing of the manuscript, D.J. performed and quantified immuno-FISH in mouse tissues, F.D.M.M. imaged and quantified mouse tissue and fibroblast immuno-FISH and live-cell imaging, and contributed to study design, C.C.M. and R.A. performed immunostainings and Sen-βGal in fibroblasts, T.H. and A.G. performed ChIP experiments, M.T. developed imaging method for visualizing TAF in the gut, J.M. supervised and analysed ChIP experiments, J.F.P. designed, supervised the study, wrote the paper and conducted live-cell imaging. Additional information How to cite this article: Hewitt, G. et al. Telomeres are favoured targets of a persistent DNA damage response in ageing and stress-induced senescence. Nat. Commun. 3:708 doi: 10.1038/ncomms1708 (2012). Supplementary Material Supplementary Information Supplementary Figures S1-S5
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              Is Open Access

              Identification of a novel senolytic agent, navitoclax, targeting the Bcl‐2 family of anti‐apoptotic factors

              Summary Clearing senescent cells extends healthspan in mice. Using a hypothesis‐driven bioinformatics‐based approach, we recently identified pro‐survival pathways in human senescent cells that contribute to their resistance to apoptosis. This led to identification of dasatinib (D) and quercetin (Q) as senolytics, agents that target some of these pathways and induce apoptosis preferentially in senescent cells. Among other pro‐survival regulators identified was Bcl‐xl. Here, we tested whether the Bcl‐2 family inhibitors, navitoclax (N) and TW‐37 (T), are senolytic. Like D and Q, N is senolytic in some, but not all types of senescent cells: N reduced viability of senescent human umbilical vein epithelial cells (HUVECs), IMR90 human lung fibroblasts, and murine embryonic fibroblasts (MEFs), but not human primary preadipocytes, consistent with our previous finding that Bcl‐xl siRNA is senolytic in HUVECs, but not preadipocytes. In contrast, T had little senolytic activity. N targets Bcl‐2, Bcl‐xl, and Bcl‐w, while T targets Bcl‐2, Bcl‐xl, and Mcl‐1. The combination of Bcl‐2, Bcl‐xl, and Bcl‐w siRNAs was senolytic in HUVECs and IMR90 cells, while combination of Bcl‐2, Bcl‐xl, and Mcl‐1 siRNAs was not. Susceptibility to N correlated with patterns of Bcl‐2 family member proteins in different types of human senescent cells, as has been found in predicting response of cancers to N. Thus, N is senolytic and acts in a potentially predictable cell type‐restricted manner. The hypothesis‐driven, bioinformatics‐based approach we used to discover that dasatinib (D) and quercetin (Q) are senolytic can be extended to increase the repertoire of senolytic drugs, including additional cell type‐specific senolytic agents.

                Author and article information

                Aging Cell
                Aging Cell
                Aging Cell
                John Wiley and Sons Inc. (Hoboken )
                05 August 2016
                October 2016
                : 15
                : 5 ( doiID: 10.1111/acel.2016.15.issue-5 )
                : 973-977
                [ 1 ] Department of SurgeryMayo Clinic Rochester MNUSA
                [ 2 ] Kogod Center on AgingMayo Clinic Rochester MNUSA
                [ 3 ] Newcastle University Institute for AgingNewcastle University Newcastle Upon TyneUK
                [ 4 ] Department of Physiology & Biomedical EngineeringMayo Clinic Rochester MNUSA
                Author notes
                [*] [* ] Correspondence

                Jordan D. Miller, Department of Surgery, Kogod Center on Aging, Department of Physiology & BME, Mayo Clinic, 200 First St. SW, Rochester, MN 55905, USA. Tel.: 507‐255‐7621; fax: 507‐255‐7378; e‐mail : miller.jordan@ 123456mayo.edu


                James L. Kirkland, Kogod Center on Aging, Department of Internal Medicine, Department of Physiology & BME, Mayo Clinic, 200 First St. SW, Rochester, MN 55905, USA. Tel.: 507‐293‐3466; fax: 507‐255‐7378; e‐mail : kirkland.james@ 123456mayo.edu


                Denotes equal contribution by these authors.

                © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

                This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                Page count
                Pages: 5
                Funded by: Mayo Clinic Center for Regenerative Medicine
                Award ID: R01 HL111121
                Award ID: R01 AG013925
                Award ID: F30 AG046061
                Funded by: Connor Group, Noaber Foundation, and Ted Nash Foundation
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                Custom metadata
                October 2016
                Converter:WILEY_ML3GV2_TO_NLMPMC version:4.9.4 mode:remove_FC converted:06.09.2016

                Cell biology
                aging,atherosclerosis,endothelial function,calcification,fibrosis,senescence
                Cell biology
                aging, atherosclerosis, endothelial function, calcification, fibrosis, senescence


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