Increased p16INK4a Expression Research Articles

Implications of Senescent Cell Burden and NRF2 Pathway in Uremic Calcification: A Translational Study.

Increased senescent cell burden and dysregulation of the nuclear factor erythroid 2-related factor 2 (NRF2) pathway have been associated with numerous age-related pathologies; however, their role in promoting vascular calcification (VC) in chronic kidney disease (CKD) has yet to be determined. We investigated whether senescence and NRF2 pathways may serve as drivers of uremia-induced VC using three complementary approaches: a novel model of induced VC in 5/6-nephrectomized rats supplemented with high phosphate and vitamin D; epigastric arteries from CKD patients with established medial calcification; and vascular smooth muscle cells (VSMCs) incubated with uremic serum. Expression of p16Ink4a and p21Cip1, as well as γ-H2A-positive cells, confirmed increased senescent cell burden at the site of calcium deposits in aortic sections in rats, and was similarly observed in calcified epigastric arteries from CKD patients through increased p16Ink4a expression. However, uremic serum-induced VSMC calcification was not accompanied by senescence. Expression of NRF2 and downstream genes, Nqo1 and Sod1, was associated with calcification in uremic rats, while no difference was observed between calcified and non-calcified EAs. Conversely, in vitro uremic serum-driven VC was associated with depleted NRF2 expression. Together, our data strengthen the importance of senescence and NRF2 pathways as potential therapeutic options to combat VC in CKD.

P16INK4a in the Gut Mucosa Associates with NFKB1 Gene Polymorphisms and Decreased Serum Levels of Inflammation-Associated Proteins in Patients with Gastrointestinal Acute Graft Versus Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Cellular senescence is characterized by irreversible cell cycle arrest and pro-inflammatory secretion, mostly regulated by NF-kB. Expression of p16INK4a - a cell cycle inhibitor, is considered a hallmark of cellular senescence. Based on overlapping features observed in inflammatory signaling of both GVHD and cellular senescence, we have postulated a hypothesis whether genetic variants of the NFKB1 gene and expression of p16INK4a in intestinal mucosa of the recipients have impact on transplant-related inflammatory complications. Here we present 52 consecutive patients with symptoms of lower gastrointestinal GVHD after allogeneic stem cell transplantation. The patients were genotyped for GVHD-prone polymorphisms of the NFKB1 gene, rs3774937 C/T and rs3774959 A/G. All patients underwent diagnostic proctosigmoidoscopy with gut mucosa biopsy. Immunohistochemical detection of p16INK4a expression was evaluated in normal intestinal crypts and stroma. On day +14 after graft infusion serum levels of inflammation - associated proteins were analyzed. Increased p16INK4a expression in intestinal crypts was independently associated with late manifestation of lower gastrointestinal symptoms. Patients with and without p16INK4a expression in intestinal crypts developed lower gastrointestinal symptoms in median of 43 days (range,12-140) and 24 days (range,13-80), respectively. The effect was observed in the whole study cohort (HR 2.3 [95%CI 0.99-5.47];p=0.006) as well as in a subgroup of patients who fulfilled the clinical diagnostic criteria of GVHD only (HR 3.1 [95%CI 0.95-10.01];p=0.002). Increased risk of histological grade 3-4 aGVHD was observed in patients without stromal p16INK4a expression (HR 3.45 [95%CI 1.26-9.42];p=0.01). The impact was confirmed by means of multivariate Cox regression (p=0.009). No clinical variable had effect on p16INK4a expression but NFKB1 genotype. The GVHD-prone genotypes rs3774937 CC and rs3774959 AA were significantly less frequent in patients with increased p16INK4a expression (p=0.04 and p=0.02, respectively). In 13 patients, measurement of serum levels of 92 inflammation-associated proteins from the Proseek Multiplex lnflammation - I kit on day +14 after graft infusion was performed. Patients with increased expression of p16INK4a in intestinal crypts at the time of gut biopsy had significantly lower serum levels of proinflammatory proteins (IL-17A, IL-18, IL-4, CXCL10 and TNFRSF9) on day +14. Using the STRING web engine we performed the enrichment analysis with statistically significant confirmation of interactions among these proteins suggesting various T-cell cytokine production or Type -2 immune response from the gene ontology analysis. Cellular senescence is considered a program of innate immunity. In GVHD, innate immunity becomes seriously decontrolled. Cellular senescence may thus bring novel approaches towards GVHD diagnostics and therapy. Supported by Ministry of Health, Czech Republic - conceptual development of research organization (FNOl, 00098892) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The elevated level of IL-1α in the bone marrow of aged mice leads to MSC senescence partly by down-regulating Bmi-1.

Osteoporosis is becoming increasingly prevalent with individual aging. Recent studies found that bone marrow mesenchymal stem cells (MSCs) undergo senescence along with the progression of age-related osteoporosis, leading to a decreased rate of new bone formation and fracture repair. The underlying mechanism of MSC senescence in the aged bone marrow has not been clarified yet. Here we found that MSCs from aged mice (12-month-old, O-MSCs) exhibited apparent senescent phenotypes compared with those from young controls (2-month-old, Y-MSCs), including lower proliferation rate, impaired self-renewal capacity, increased p16Ink4a expression and shifted differentiation balance to favor adipocytes over osteoblasts. Bmi-1, one of the main factors that regulate stem cell self-renewal, is dramatically decreased in O-MSCs. Knocking-down of Bmi-1 in Y-MSCs lead to cellular senescence, while over-expression of it rejuvenated O-MSCs. We further showed that the level of IL-1α is much higher in the bone marrow fluid of aged mice, which significantly inhibited Bmi-1 expression in MSCs. Our present study indicated that IL-1α, a key component of the senescence-associated secretory phenotype (SASP), is elevated in the aged bone marrow microenvironment, leading to decreased Bmi-1 expression in MSCs and consequently, MSC senescence.

Senescent Cell Depletion Through Targeting BCL-Family Proteins and Mitochondria.

Senescent cells with replicative arrest can be generated during genotoxic, oxidative, and oncogenic stress. Long-term retention of senescent cells in the body, which is attributed to highly expressed BCL-family proteins, chronically damages tissues mainly through a senescence-associated secretory phenotype (SASP). It has been documented that accumulation of senescent cells contributes to chronic diseases and aging-related diseases. Despite the fact that no unique marker is available to identify senescent cells, increased p16INK4a expression has long been used as an in vitro and in vivo marker of senescent cells. We reviewed five existing p16INK4a reporter mouse models to detect, isolate, and deplete senescent cells. Senescent cells express high levels of anti-apoptotic and pro-apoptotic genes compared to normal cells. Thus, disrupting the balance between anti-apoptotic and pro-apoptotic gene expression, such as ABT-263 and ABT-737, can activate the apoptotic signaling pathway and remove senescent cells. Mitochondrial abnormalities in senescent cells were also discussed, for example mitochondrial DNA mutation accumulation, dysfunctional mitophagy, and mitochondrial unfolded protein response (mtUPR). The mitochondrial-targeted tamoxifen, MitoTam, can efficiently remove senescent cells due to its inhibition of respiratory complex I and low expression of adenine nucleotide translocase-2 (ANT2) in senescent cells. Therefore, senescent cells can be removed by various strategies, which delays chronic and aging-related diseases and enhances lifespan and healthy conditions in the body.

Abstract 5833: Senescence is a central response to chemotherapy in ovarian cancer

Abstract High-grade serous ovarian carcinoma (HGSOC) commonly responds to initial therapy, but this response is rarely durable. Understanding the cell fate decisions taken by HGSOC cells in response to treatment could guide new therapeutic opportunities. Here, we find that more than 90% of tissue-derived primary HGSOC cultures, reflecting the original disease, retain the capacity to undergo stress-induced cellular senescence and primarily undergo therapy-induced senescence (TIS) in response to first-line carboplatin/taxol chemotherapy. HGSOC-TIS displays senescence-associated hallmarks, including a stable proliferation arrest, increased p16INK4A expression, persistent DNA damage, an inflammatory secretome, and senolytic sensitivity, suggesting new avenues for selective pharmacological manipulation of these cells. Comparison of pre- and post-chemotherapy patient HGSOC tissue samples revealed changes in physio-pathological senescence biomarkers supporting the occurrence of post-treatment TIS. Whether cell senescence induced by cancer therapy is beneficial or detrimental to treatment outcomes remains unknown. We find that patients with stronger TIS biomarkers in post-chemotherapy tissues have a more favorale 5-year survival, suggesting that the induction of senescence in HGSOC cells accounts, at least in part, for beneficial responses to treatment. Given that HGSOC cells almost universally retain the capacity to undergo senescence and that senescence appears beneficial in this context, senescence-centric therapeutic avenues should be further explored. Citation Format: Michael Skulimowski, Llilians Calvo-Gonzales, Shuofei Cheng, Isabelle Clément, Lise Portelance, Yu Zhan, Euridice Carmona, Manon de Ladurantaye, Julie Lafontaine, Kurosh Rahimi, Diane Provencher, Anne-Marie Mes-Masson, Francis Rodier. Senescence is a central response to chemotherapy in ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5833.

Hallmarks of cellular senescence more abundant in damaged compared to intact cartilage in osteoarthritis

Purpose: Cellular senescence is a consequence of aging, which is associated with cell cycle arrest and has recently been identified as a major factor in the pathogenesis of osteoarthritis. Senescence could contribute to OA by several mechanisms including joint tissue increase in pro-inflammatory senescence-associated secretory proteins (SASPs). Our goal was to quantify the extent and characteristics of cellular senescence of human OA cartilage. To this end, we developed a short enzymatic digestion protocol for primary chondrocyte isolation and compared intact and damaged regions of cartilage within each patient to minimize potential confounding by cell stress that could induce senescence phenotypes during the cell isolation and manipulation procedures. We hypothesized that differences between damaged and intact regions of cartilage from each patient will more reliably identify disease associated senescence than evaluation of damaged regions alone. We characterized senescence based on β-galactosidase (β-gal) activity and p16 INK4a protein expression (two accepted markers for senescent cell identification), quantification of MMP3 protein expression (a SASP) and characterization of cell cycle phases. Methods: Human knee joints were obtained as surgical waste tissue under IRB approval from 4 OA patients during total knee arthroplasty. To isolate primary chondrocytes from macroscopically damaged vs intact regions, human articular cartilage from selected regions was finely diced and digested in 0.3% type II collagenase solution for three hours at 37 °C. Chondrocytes were isolated by centrifugation (300G) and directly stored as cell pellets in -80oC for β-gal activity (fluorimetric quantification) or fixed in 80% methanol and stored in -20oC for flow-cytometric analysis of p16 INK4a(ROCHE #9517 ), MMP3 (Thermo #701285) and cell cycle phase (by propidium iodide, Miltenyi Biotec). β-gal was assessed by SensoLyte ® MUG β - Galactosidase Assay Kit (Anaspec AS-72132) and normalized to DNA content (Biotium #31008). Results: Our protocol yielded a mean of 2.78 x 106 (range: 1.94-3.65x106) chondrocytes from 1 gram of human cartilage. Compared to intact cartilage, β-gal activity and p16INK4a protein expression were higher in damaged cartilage (Figure 1A, N=4, P=0.034; Figure 1B, N=4, P=0.031). The proportion of p16INK4a+/MMP3+ cells was 2.8-fold higher in damaged cartilage compared with intact cartilage (Figure 1C, N=4, P=0.082). Overall, β-gal activity was positively correlated with p16INK4aprotein expression. (Figure 1D, N=8, r=0.57, p=0.072). Compared with chondrocytes from intact cartilage, a higher proportion of chondrocytes from damaged cartilage were in the tetraploid G2 cell cycle phase (P=0.04) and a lower proportion were in the diploid G1 phase (P=0.02) (Figure 2A, N=4). Compared to diploid (G1 phase) chondrocytes,p16INK4a protein was much more highly expressed in tetraploid (G2 phase) chondrocytes (Figure 2B, N=8, P=0.001). Conclusions: We successfully isolated primary human chondrocytes using a short enzymatic digestion protocol to minimize phenotypic changes from cell manipulation, a stressor that might artifactually increase senescence cell markers in chondrocytes. β-gal activity was positively correlated with p16 INK4a expression and both markers were higher in damaged cartilage, which suggested an association of senescent chondrocytes with OA. Moreover, MMP3 expression was positively correlated with p16 INK4a expression. These results suggest at least one means, namely increased cartilage degradation by MMP3, by which senescent chondrocytes could contribute to the pathogenesis of OA. A previous study reported a higher percentage of tetraploid chondrocytes within damaged cartilage; we further identified increased p16INK4a expression in tetraploid chondrocytes. In conclusion, this study provides supportive evidence for the role of cellular senescence in the pathogenesis of human OA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Abstract B018: Restoration of p16INK4A expression in circulating tumor cells in patients with advanced solid tumors treated with deoxycytidine analogs and associated dynamic EMT phenotypic changes

Abstract Background. In vitro experiments from multiple laboratories have shown that the methylation-induced silencing of the p16INK4A gene, a tumor suppressor gene, can be reversed by DNA methyltransferase (DNMT1) inhibitor-based epigenetic modulation. However, it remains unclear whether DNMT1 inhibitors induce re-expression of p16INK4A in patients treated with demethylation promoting agents. Here we report p16INK4A expression status in human circulating tumor cells (CTCs) and its dynamic changes during DNMT1 inhibitor therapy in patients with advanced solid tumors, by using a novel CTC-based p16INK4A immunofluorescence assay. Methods. We demonstrated in vitro re-expression of p16 by Western blot and whole cell immunofluorescence in EJ-6 cells treated with decitabine, 4-thio-2’-deoxycytidine (TdCyd) and 5-fluoro-2’-deoxyctidine (FdC) but not in the p16-deleted line A549. We analyzed p16 re-expression in epithelial /mesenchymal CTC subpopulations as a clinical pharmacodynamic (PD) marker to assess the effects of DNMT1 inhibitors (analyzed using CellSearch® and fluorescence microscopy). Results. Over 300 specimens from 70 patients were analyzed from patients enrolled in both Phase 1 and Phase 2 clinical trials of the DNMT1 inhibitors TdCyd or FdC in combination with tetrahydrouridine (THU). Circulating tumor cells were characterized as DAPI+/CD45-/CEA or MUC1 positive, then classified as epithelial or mesenchymal based on expression of (pan-)cytokeratin (CK) or vimentin (VIM), respectively. The p16INK4A-positive CTC baseline level ranged from 0 to 15% in 57 of 58 patients with assessable CK+CTC numbers and in 46 of 53 patients with assessable VIM+CTC numbers. There were no patient specimens with CTCs that were positive for only VIM, but most specimens had higher numbers of VIM+ CTC compared to CK+ CTC, and not all patients had both CTC phenotypes present at baseline. During treatment with DNMT1 inhibitors, p16INK4A-positive CTCs increased in 46 of 58 patients with assessable CK+ CTCs (epithelial phenotype), but only 6 patients with VIM+ CTCs (mesenchymal phenotype) had p16 re-expression, and these patients had both CTC phenotypes present. The restoration of p16INK4A expression in CTC, not the CTC number, was correlated with evidence of drug clinical activity in patients. Drug treatment longer than 2 cycles was associated with both EMT phenotypic conversion and obvious morphological changes in CTCs that accompanied increased p16INK4A expression. Conclusions. Our studies indicate that monitoring dynamic changes of CTC-based p16INK4A expression is a sensitive PD biomarker for assessing DNMT1-targeted epigenetic anticancer therapeutics in clinical development. CTCs were predominantly mesenchymal phenotype. In addition, the CTC phenotype (epithelial vs mesenchymal) appeared to be quite dynamic in treated patients. Funded by NCI Contract No. HHSN261200800001E, and Cooperative Agreements U01CA062505 and UM1CA186717. Citation Format: Lihua Wang, Brandon Miller, Sonny Khin, Geraldine O’Sullivan-Coyne, Alice P. Chen, Shivaani Kummar, Naoko Takebe, Kate Ferry-Galow, Ralph E. Parchment, Larry Rubinstein, Richard Piekarz, Edward M. Newman, James H. Doroshow, Robert J. Kinders. Restoration of p16INK4A expression in circulating tumor cells in patients with advanced solid tumors treated with deoxycytidine analogs and associated dynamic EMT phenotypic changes [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B018. doi:10.1158/1535-7163.TARG-19-B018

Knockout of Canopy 2 activates p16INK4a pathway to impair cardiac repair

BackgroundCardiac repair depends on angiogenesis and cell proliferation. Previously we identified Canopy 2 (CNPY2) as a secreted angiogenic growth factor which promotes neovascularization. We investigated the role of CNPY2 in cardiac repair following myocardial infarction (MI) and the possible mediators involved using Cnpy2 knockout (KO) mice and human cardiac tissue. Methods and resultsCardiac tissue from patients with end-stage heart failure had significantly lower endogenous CNPY2 expression compared to samples from control patients. CNPY2 expression in mouse hearts significantly decreased following MI. Significantly less leukocyte and endothelial cell proliferation was found in Cnpy2 KO than wild-type (WT) mice post MI which contributed to impaired angiogenesis, tissue repair, and decreased cardiac function (fractional shortening: WT: 21.1 ± 2.1% vs. KO: 16.4 ± 1.6%, p < .01 at day 28 post MI). RT-qPCR revealed significantly increased p16INK4a expression in Cnpy2 KO mouse hearts (WT: 1.0 ± 0.04 vs. KO: 2.33 ± 0.11 [relative expression of p16 INK4a], p < .01) which was confirmed by immunostaining (WT: 8.47 ± 1.22 vs. KO: 12.9 ± 1.22 [% total cells], p < .05) for the p16INK4a protein. Expression of cell cycle-related proteins, cyclin D1, cyclin-dependent kinases 4 and 6, and phosphorylated retinoblastoma protein (pRb) was significantly decreased in Cnpy2 KO mouse hearts. The up-regulation of the p16INK4a/cyclin D1/Rb pathway by knockout of Cnpy2 was accompanied by attenuation of PDK1/Akt phosphorylation. MI exacerbated the detrimental effects of p16INK4a on tissue repair in Cnpy2 KO mice. Overexpression of CNPY2 in the cardiac tissue of transgenic mice reversed the inhibition of cell proliferation through suppression of the p16INK4a pathway. ConclusionsCardiac injury and progressive heart failure were associated with decreased CNPY2 levels in both humans and mice. Knockout of Cnpy2 resulted in up-regulation of p16INK4a which impaired cardiac function and tissue repair. These data suggest that CNPY2 is an important regulator of p16INK4a and promotes cell proliferation and tissue repair through inhibition of the p16INK4a pathway. CNPY2 treatment may offer a new approach to restore cardiac function after an MI.

Expression of p16INK4a is a biomarker of chondrocyte aging but does not cause osteoarthritis.

SummaryCellular senescence drives a functional decline of numerous tissues with aging by limiting regenerative proliferation and/or by producing pro‐inflammatory molecules known as the senescence‐associated secretory phenotype (SASP). The senescence biomarker p16 INK 4a is a potent inhibitor of the cell cycle but is not essential for SASP production. Thus, it is unclear whether p16 INK 4a identifies senescence in hyporeplicative cells such as articular chondrocytes and whether p16 INK 4a contributes to pathologic characteristics of cartilage aging. To address these questions, we examined the role of p16 INK 4a in murine and human models of chondrocyte aging. We observed that p16 INK 4a mRNA expression was significantly upregulated with chronological aging in murine cartilage (~50‐fold from 4 to 18 months of age) and in primary human chondrocytes from 57 cadaveric donors (r 2 = .27, p < .0001). Human chondrocytes exhibited substantial replicative potential in vitro that depended on the activity of cyclin‐dependent kinases 4 or 6 (CDK4/6), and proliferation was reduced in cells from older donors with increased p16 INK 4a expression. Moreover, increased chondrocyte p16 INK 4a expression correlated with several SASP transcripts. Despite the relationship between p16 INK 4a expression and these features of senescence, somatic inactivation of p16 INK 4a in chondrocytes of adult mice did not mitigate SASP expression and did not alter the rate of osteoarthritis (OA) with physiological aging or after destabilization of the medial meniscus. These results establish that p16 INK 4a expression is a biomarker of dysfunctional chondrocytes, but that the effects of chondrocyte senescence on OA are more likely driven by production of SASP molecules than by loss of chondrocyte replicative function.

5-Aza-2'-deoxycytidine protects against emphysema in mice via suppressing p16Ink4a expression in lung tissue.

BackgroundThere is a growing realization that COPD, or at least emphysema, involves several processes presenting in aging and cellular senescence. Endothelial progenitor cells (EPCs) contribute to neovascularization and play an important role in the development of COPD. The gene for p16Ink4a is a major dominant senescence one. The aim of the present study was to observe changes in lung function, histomorphology of lung tissue, and expression of p16Ink4a in lung tissue and bone marrow-derived EPCs in emphysematous mice induced by cigarette-smoke extract (CSE), and further to search for a potential candidate agent protecting against emphysema induced by CSE.Materials and methodsAn animal emphysema model was induced by intraperitoneal injection of CSE. 5-Aza-2′-deoxycytidine (5-Aza-CdR) was administered to the emphysematous mice. Lung function and histomorphology of lung tissue were measured. The p16Ink4a protein and mRNA in EPCs and lung tissues were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction, respectively.ResultsCSE induced emphysema with increased p16Ink4a expression in lung tissue and bone marrow-derived EPCs. 5-Aza-CdR partly protected against emphysema, especially in the lung-morphology profile, and partly protest against the overexpression of p16Ink4a in EPCs and lung tissue induced by CSE.Conclusion5-Aza-CdR partly protected against emphysema in mice via suppressing p16Ink4a expression in EPCs and lung tissue.

Sustained p16INK4a expression is required to prevent IR-induced tumorigenesis in mice.

Exposure of murine and human tissues to ionizing radiation (IR) induces the expression of p16INK4a, a tumor suppressor gene and senescence/aging biomarker. Increased p16INK4a expression is often delayed several weeks post exposure to IR. In this context, it remains unclear if it occurs to suppress aberrant cellular growth of potentially transformed cells or is simply a result of IR-induced loss of tissue homeostasis. To address this question, we used a conditional p16INK4a null mouse model and determined the impact of p16INK4a inactivation long-term post exposure to IR. We found that, in vitro, bone marrow stromal cells exposed to IR enter DNA replication following p16INK4a inactivation. However, these cells did not resume growth; instead, they mostly underwent cell cycle arrest in G2. Similarly, delayed inactivation of p16INK4a in mice several weeks post exposure to IR resulted in increased BrdU incorporation and cancer incidence. In fact, we found that the onset of tumorigenesis was similar whether p16INK4a was inactivated before or after exposure to IR. Overall, our results suggest that IR-induced p16INK4a dependent growth arrest is reversible in mice and that sustained p16INK4a expression is necessary to protect against tumorigenesis.

Chronic sleep fragmentation induces endothelial dysfunction and structural vascular changes in mice.

Sleep fragmentation (SF) is a common occurrence and constitutes a major characteristic of obstructive sleep apnea (OSA). SF has been implicated in multiple OSA-related morbidities, but it is unclear whether SF underlies any of the cardiovascular morbidities of OSA. We hypothesized that long-term SF exposures may lead to endothelial dysfunction and altered vessel wall structure. Adult male C57BL/6J mice were fed normal chow and exposed to daylight SF or control sleep (CTL) for 20 weeks. Telemetric blood pressure and endothelial function were assessed weekly using a modified laser-Doppler hyperemic test. Atherosclerotic plaques, elastic fiber disruption, lumen area, wall thickness, foam cells, and macrophage recruitment, as well as expression of senescence-associated markers were examined in excised aortas. Increased latencies to reach baseline perfusion levels during the post-occlusive period emerged in SF mice with increased systemic BP values starting at 8 weeks of SF and persisting thereafter. No obvious atherosclerotic plaques emerged, but marked elastic fiber disruption and fiber disorganization were apparent in SF-exposed mice, along with increases in the number of foam cells and macrophages in the aorta wall. Senescence markers showed reduced TERT and cyclin A and increased p16INK4a expression, with higher IL-6 plasma levels in SF-exposed mice. Long-term sleep fragmentation induces vascular endothelial dysfunction and mild blood pressure increases. Sleep fragmentation also leads to morphologic vessel changes characterized by elastic fiber disruption and disorganization, increased recruitment of inflammatory cells, and altered expression of senescence markers, thereby supporting a role for sleep fragmentation in the cardiovascular morbidity of OSA.

Improvement of biological age by physical activity

While physical inactivity has been associated with greater mortality [ [1] Huffman M.D. Lloyd-Jones D.M. Ning H. et al. Quantifying options for reducing coronary heart disease mortality by 2020. Circulation. 2013; 127: 2477-2484 Crossref PubMed Scopus (53) Google Scholar ], physical activity leads to a reduction in mortality even when exercise starts relatively late during lifetime [ [2] Byberg L. Melhus H. Gedeborg R. et al. Total mortality after changes in leisure time physical activity in 50 year old men: 35 year follow-up of population based cohort. BMJ. 2009; 338: b688 Crossref PubMed Scopus (183) Google Scholar ]. Telomere shortening and increased p16INK4a expression are markers of biological age and indicate the loss of regenerative capacity. Shorter telomeres are associated with an increased cardiovascular risk [ 3 Krauss J. Farzaneh-Far R. Puterman E. et al. Physical fitness and telomere length in patients with coronary heart disease: findings from the Heart and Soul Study. PLoS One. 2011; 6: e26983 Crossref PubMed Scopus (53) Google Scholar , 4 Willeit P. Willeit J. Brandstatter A. et al. Cellular aging reflected by leukocyte telomere length predicts advanced atherosclerosis and cardiovascular disease risk. Arterioscler Thromb Vasc Biol. 2010; 30: 1649-1656 Crossref PubMed Scopus (221) Google Scholar ] and cardiovascular mortality [ [5] Cawthon R.M. Smith K.R. O'Brien E. Sivatchenko A. Kerber R.A. Association between telomere length in blood and mortality in people aged 60 years or older. Lancet. 2003; 361: 393-395 Abstract Full Text Full Text PDF PubMed Scopus (1347) Google Scholar ]. Cross sectional data suggested that regular physical exercise in male athletes is associated with maintaining longer telomeres [ [6] Werner C. Furster T. Widmann T. et al. Physical exercise prevents cellular senescence in circulating leukocytes and in the vessel wall. Circulation. 2009; 120: 2438-2447 Crossref PubMed Scopus (251) Google Scholar ]. We addressed the question whether physical activity affects telomere length (TL) and p16INK4a expression by investigating 59 healthy middle-aged men (45–65 years old; mean age: 53 ± 6 years) with former sedentary life-style in a prospective longitudinal study. A questionnaire confirmed sedentary lifestyle with an average of 5.1 (±10.2) metabolic equivalent of task (MET)-weighted hours per week for physical activity. Subjects were encouraged to perform 210 min of endurance training per week.

Sustained p16INK4a expression is required to protect against IR-induced lymphoma in mice

We recently showed that mice and human tissues exposed to ionizing radiation (IR) have an increased expression of p16INK4a, a tumor suppressor gene and proven senescence marker. Expression of p16INK4a prevents adequate tissue renewal and is hypothesized to be an underlying mechanism by which most childhood cancer survivors develop IR treatment-related health conditions. Intriguingly, increased p16INK4a expression is delayed several weeks post exposure to IR. Hence, it is unclear if delayed expression occurs as an orchestred tumor suppressive pathway or simply as a bystandard effect to IR-induced loss of tissue homeostasis.

Accelerated senescence of cord blood endothelial progenitor cells in premature neonates is driven by SIRT1 decreased expression

AbstractEpidemiological and experimental studies indicate that early vascular dysfunction occurs in low-birth-weight subjects, especially preterm (PT) infants. We recently reported impaired angiogenic activity of endothelial colony-forming cells (ECFCs) in this condition. We hypothesized that ECFC dysfunction in PT might result from premature senescence and investigated the underlying mechanisms. Compared with ECFCs from term neonates (n = 18), ECFCs isolated from PT (n = 29) display an accelerated senescence sustained by growth arrest and increased senescence-associated β-galactosidase activity. Increased p16INK4a expression, in the absence of telomere shortening, indicates that premature PT-ECFC aging results from stress-induced senescence. SIRT1 level, a nicotinamide adenine dinucleotide-dependent deacetylase with anti-aging activities, is dramatically decreased in PT-ECFCs and correlated with gestational age. SIRT1 deficiency is subsequent to epigenetic silencing of its promoter. Transient SIRT1 overexpression or chemical induction by resveratrol treatment reverses senescence phenotype, and rescues in vitro PT-ECFC angiogenic defect in a SIRT1-dependent manner. SIRT1 overexpression also restores PT-ECFC capacity for neovessel formation in vivo. We thus demonstrate that decreased expression of SIRT1 drives accelerated senescence of PT-ECFCs, and acts as a critical determinant of the PT-ECFC angiogenic defect. These findings lay new grounds for understanding the increased cardiovascular risk in individuals born prematurely and open perspectives for therapeutic strategy.

P16INK4a in cellular senescence

p16INK4a in cellular senescence

Abstract 2607: BMI1 and dominant negative p53 cooperate to suppress AKT-mediated oncogene-induced senescence and promote transformation in human neural stem cells.

Abstract Purpose: BMI-1 is required for the maintenance of normal brain stem cells and is associated with aggressive brain tumors such as glioblastoma multiforme (GBM). BMI1 is a polycomb group protein that can modify chromatin to silence the pro-senescence tumor suppressor p16INK4a. The PI3K/AKT pathway is also associated with clinically aggressive brain tumors. Interestingly, persistent activation of AKT can cause oncogene-induced senescence. Dominant negative R248W p53 mutation blocks induction of the tumor suppressor p21CIP1 and is found in aggressive brain tumors such as GBM. Telomere maintenance through telomerase enzyme activity is also found in aggressive brain tumors. We investigated if constitutively active AKT, dominant negative p53, BMI1, and hTERT can cooperate to bring about the transformation of human neural stem and progenitor cells, and in particular if BMI1 can suppress AKT induced senescence. Experimental Design: We used lentivirus to introduce singly and in combination BMI1, R248W p53, hTERT and constitutively active AKT into human cortex derived stem and progenitor cells. We assayed the effects of one, two, three or four oncogenes on the proliferation and induction of cellular senescence. We measured proliferation using bromodeoxyurine (BrdU) and senescence by western blotting for p16INK4a and p21 CIP1. Results: Increased expression of BMI1 alone did not have a significant effect on human neural stem cell proliferation, but did lead to the suppression of p16INK4a. AKT alone transduced cells showed no increased proliferation compared to control cells, but did show upregulation of p16INK4a. AKT alone expressing cells stopped growing after several passages in culture, in association with increased p16 INK4a expression. Cells transduced with BMI1 and AKT initially grew at a similar rate to control cells, but after several passages, similar to AKT alone transduced cells, BMI1/AKT cells stopped proliferating and expressed p21CIP1and p16INK4a. Control cells continued to proliferate for multiple additional passages. Cells transduced with R248W p53/hTERT/BMI1/AKT proliferated at an increased rate compared to control cells (17% BrdU positive for pBABE control cells compared to 37% for R248W p53/hTERT/BMI1/AKT p=0.0003, t-test). These cells did not undergo senescence, did not induce p16INK4a or p21CIP1 and continued proliferating more than control or single oncogene transduced cells long after other cells had senesced. Human neural stem cells transduced with R248W p53/hTERT/BMI1/AKT formed aggressive orthotopic xenograft glial tumors, while control cells did not form tumors. Conclusions: BMI1 alone cannot suppress AKT induced senescence in human cortex derived neural stem cells. But BMI1 combined with R248W p53 and hTERT caused suppression of AKT induced senescence, and resulting cells showed signs of oncogenic transformation Citation Format: Dacheng Ding, Ulf Kahlert, Jarek Maciaczyk, Guido Nikkhah, Charles Eberhart, Eric Raabe. BMI1 and dominant negative p53 cooperate to suppress AKT-mediated oncogene-induced senescence and promote transformation in human neural stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2607. doi:10.1158/1538-7445.AM2013-2607

Human nevi lack distinguishing senescence traits

Oncogene-induced senescence is a program initiated by the aberrant activation of oncogenes. Once engaged, this program irreversibly limits the proliferative capacity of cells and may potently prevent tumor formation in vivo. Human nevi (moles) display many features of oncogene-induced senescence; they remain growth arrested for decades, display increased p16INK4a expression, stain positive for senescence-associated-s-galactosidase (SA-s-Gal) [1] and carry oncogenic mutations in the BRAF kinase [2]. Evidence demonstrating that human nevi may have undergone senescence is based on the accumulation of a few predictive markers (p16INK4a, SA-s-gal and Ki67) that are not exclusive to the senescence program [1]. Thus, we sought to clarify whether human nevi display a consistent senescence signature by examining an expanded panel of senescence-associated markers. These markers were used to evaluate DNA damage (γ-H2AX and p53), chromatin remodelling (senescence-associated heterochromatin foci, promyelocytic leukemia protein and histone H3 lysine 9 methylation), proliferation (Ki67, p16INK4a), morphology (nuclear size) and SA-s-Gal activity [3]. Importantly, to ensure the specificity of this senescence signature, the expression of these markers was evaluated in a panel of human nevi, epidermal melanocytes, primary and metastatic melanomas. Only Ki67 distinguished nevi from melanomas. The commonly evaluated p16INK4a was abundant in both nevi and primary melanomas and the most widely accepted senescence marker, SA-s-Gal, was detected in both nevi (5/7) and metastatic melanomas (3/7) [3]. Thus, there is not sufficient evidence to define human nevi as senescent lesions. There are also several important clinical observations that challenge the view that nevi have undergone oncogene-induced senescence. Nevus cells can be induced to proliferate in vivo (e.g. after partial resection or during pregnancy) and growing nevi are more likely to carry mutant BRAF ([4]; reviewed in [3]). The identification of nevi in contiguity with up to 40% of melanomas also suggests that senescence is either a poor suppressor of melanomagenesis or that a proportion of nevus cells retain proliferative potential. This is consistent with BRAF-induced benign nevi in mice. These murine nevi expressed SA-s-Gal and p16INK4a, but also contained rare mitotically active cells, and were occasionally associated with melanoma tumors [5]. Our work underscores the fact that human nevi are capable of occasional, induced proliferation and transformation. This is in accordance with the strong relationship between nevus number and melanoma risk [6] and the observation that most melanomas in younger patients (<30 years) are associated with a nevus precursor [7]. Identifying more-specific senescence-associated markers may help clarify the role of senescence in arresting human naevi, but the focus on senescence has also framed current thinking and may narrow research into other pathways of arrest. Defining the tumour suppressor mechanisms that restrict nevus cell proliferation and the drivers (genetic and non-genetic) that permit escape from this proliferative arrest remains an important research priority. This is particularly relevant as rapidly changing nevi, the emergence of new nevi (with wild-type BRAF) and newly developing melanomas have been reported in melanoma patients treated with the mutant BRAF inhibitors vemurafenib and dabrafenib [8].

P16INK4A overexpression is frequently detected in tumour‐free tonsil tissue without association with HPV

Oncogenic human papillomavirus (HPV) type 16 has been strongly associated with tonsillar squamous cell carcinoma (TSCC) and appears to be of prognostic significance. Because HPV+ TSCC also accumulates p16(INK4A), this cyclin-dependent kinase inhibitor has been proposed as a potential biomarker for HPV in clinical diagnosis. The aim of this study was to determine the prevalence of HPV in tumour-free tonsillar tissue and the value of p16(INK4A) overexpression in predicting its presence. p16(INK4A) overexpression was detected by immunohistochemistry in tissue sections of tumour-free tonsils of 262 patients. They were treated for non-oncological reasons (snoring or chronic/recurrent tonsillitis) consisting of tonsillectomy. Genomic DNA isolated from these tissues was subjected to HPV-specific polymerase chain reaction (PCR) analysis. p16(INK4A) immunoreactivity was detected in 28% of samples in both crypt epithelium (49/177) and lymphoid germinal centres (52/187), which correlated with each other (P < 0.0001). No reactivity was observed in superficial squamous cell epithelium. HPV16 and 18 were detected by PCR analysis in 2/195 cases (1%), which, however, were negative on fluorescence in situ hybridization analysis and discrepant on p16(INK4A) immunostaining. No proof was found for the presence of HPV in tumour-free tonsil tissue, despite increased p16(INK4A) expression in a quarter of tonsil cases. Other mechanisms than HPV infection are therefore implicated in p16(INK4A) up-regulation.

Ionizing radiation-induced long-term expression of senescence markers in mice is independent of p53 and immune status.

Exposure to IR has been shown to induce the formation of senescence markers, a phenotype that coincides with lifelong delayed repair and regeneration of irradiated tissues. We hypothesized that IR-induced senescence markers could persist long-term in vivo, possibly contributing to the permanent reduction in tissue functionality. Here, we show that mouse tissues exposed to a sublethal dose of IR display persistent (up to 45 weeks, the maximum time analyzed) DNA damage foci and increased p16(INK4a) expression, two hallmarks of cellular senescence and aging. BrdU-labeling experiments revealed that IR-induced damaged cells are preferentially eliminated, at least partially, in a tissue-dependent manner. Unexpectedly, the accumulation of damaged cells was found to occur independent from the DNA damage response modulator p53, and from an intact immune system, as their levels were similar in wild-type and Rag2(-/-) gammaC(-/-) mice, the latter being deficient in T, B, and NK cells. Together, our results provide compelling evidence that exposure to IR induces long-term expression of senescence markers in vivo, an effect that may contribute to the reduced tissue functionality observed in cancer survivors.