Markers Of Cellular Senescence Research Articles

Absence of AMPKα2 accelerates cellular senescence via p16 induction in mouse embryonic fibroblasts

Emerging evidence suggests that activation of adenosine monophosphate-activated protein kinase (AMPK), an energy gauge and redox sensor, delays aging process. However, the molecular mechanisms by which AMPKα isoform regulates cellular senescence remain largely unknown. The aim of this study was to determine if AMPKα deletion contributes to the accelerated cell senescence by inducing p16INK4A (p16) expression thereby arresting cell cycle. The markers of cellular senescence, cell cycle proteins, and reactive oxygen species (ROS) were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J), AMPKα1, or AMPKα2 homozygous deficient (AMPKα1−/−, AMPKα2−/−) mice by Western blot and cellular immunofluorescence staining, as well as immunohistochemistry (IHC) in skin tissue of young and aged mice. Deletion of AMPKα2, the minor isoform of AMPKα, but not AMPKα1 in high-passaged MEFs led to spontaneous cell senescence demonstrated by accumulation of senescence-associated-β-galactosidase (SA-β-gal) staining and foci formation of heterochromatin protein 1 homolog gamma (HP1γ). It was shown here that AMPKα2 deletion upregulates cyclin-dependent kinase (CDK) inhibitor, p16, which arrests cell cycle. Furthermore, AMPKα2 null cells exhibited elevated ROS production. Interestingly, knockdown of HMG box-containing protein 1 (HBP1) partially blocked the cellular senescence of AMPKα2-deleted MEFs via the reduction of p16. Finally, dermal cells senescence, including fibroblasts senescence evidenced by the staining of p16, HBP1, and Ki-67, in the skin of aged AMPKα2−/− mice was enhanced when compared with that in wild type mice. Taken together, our results suggest that AMPKα2 isoform plays a fundamental role in anti-oxidant stress and anti-senescence.

Geriatric Assessment Metrics Are Associated with Hospital Length of Stay in Pre-Bone Marrow Transplant Myeloma Patients

A geriatric assessment (GA) is a global approach to improve healthy aging, wherein occult problems are assessed and intervened upon using a multidisciplinary method. A GA is feasible and can predict chemotherapy-induced toxicity and overall survival in cancer patients. Biomarkers of aging are also being explored as objective and reproducible measures of health and fitness. p16INK4a (p16) is a marker of cellular senescence that rises exponentially with chronologic age and is influenced by factors such as physical activity, smoking, and solid tumor chemotherapy. Here, we investigated the relationship of both the GA and molecular (i.e. p16) metrics in pre-bone marrow transplant (BMT) multiple myeloma (MM) patients. We selected this group for our studies as BMT patients are a vulnerable cohort in which transplant eligibility is subjective and age related. BMT patients are also at high risk for adverse events and treatment toxicity. In this preliminary analysis, we explored the predictive value of GA metrics and p16 with inpatient length of stay (LOS) during autologous BMT.Methods: We performed a pilot prospective cohort study on 55 MM patients during their pre-transplant evaluation. MM patients >18 years completed GA assessments related to physical function, distress, comorbidities, social support, and cognition. Patients completed surveys using the Brief Fatigue Inventory (BFI) (scale 1-10; moderate fatigue 4-6, severe fatigue 7+); Hospital Anxiety and Depression (HADS) (borderline case 8-10, definite case 11+); medical outcome study-social support survey (MOS-SSS) (scale 0-100, higher scores indicated greater support), Human Activity Profile (HAP) maximum activity score (MAS) and HAP-adjusted activity score (AAS), a 94-item questionnaire ranking tasks according to energy use validated in the BMT population, with higher scores indicating higher activity (Herzberg BBMT 2010). Objective measures of physical activity were measured using the Short Physical Performance Battery (SPPB) (range 0-12; impairment <9) and cognition was evaluated using the Modified Mini Mental Status exam (3MS). At the pre-transplant evaluation, p16 mRNA was measured in peripheral blood T-cells using established laboratory techniques (Liu Aging Cell 2009). The association between GA metrics and p16 were evaluated using Spearman's correlation coefficient. Univariable generalized linear models were fit to model LOS as a function of GA metrics or p16.Results: The median patient age was 61 (range 42-76). Most patients exhibited early stage disease (ISS Stage 1 53%) with minimal comorbidities (HCT-CI median 1; range 0-8) and a median of 2 prior lines of treatment (range 1-11). Pre-transplant Karnofsky Performance Status (KPS) was reported as 70% (n=10), 80% (n=10), 90% (n=15) and 100% (n=12). 7 patients did not proceed with BMT, 1 inpatient for BMT. Patients reported moderate fatigue by BFI (median 4.3; range 0-9.8), with minimal anxiety or depression as measured by the HADS. Self-reported physical activity by HAP-MAS was 73 (range 30-94) and HAP-AAS was 64 (range 20-94). Patients reported high levels of social support (median 86.7%; range 18.2-100) by MOS-SSS. Objective measures of physical function were also high as measured by the SPPB (median 10; range 4-12) and no cognitive impairment was identified by the 3MS. p16 expression was adjusted for age and did not correlate with GA tools including BFI, HADS, HAP-AAS, HAP-MAS, MOS-SSS, SPPB or 3MS. The median length LOS during transplant was 16 days (range 12-36). Univariate analysis revealed that SPPB score was significantly associated with LOS, where each one unit increase in physical performance corresponded to an average LOS decrease of 0.63 days (p=0.04). Self-reported activity by HAP-AAS also correlated with LOS (p=0.05). LOS was not influenced by p16, age, KPS or HCT-CI. Age and HCT-CI had no relationship with SPPB scores, but KPS did (p=0.03727).Conclusions: A comprehensive GA can be used to identify factors that contribute to BMT outcomes. Physical function appears to be most predictive of hospital LOS as measured by SPPB or a detailed self-report of physical function. Baseline p16 levels had no relationship with GA metrics in this selected population. A standardized approach for determining patient fitness including SPPB and HAP-AAS assessments may improve treatment tolerance, reduce hospital LOS, and decrease the risk for adverse outcomes in BMT populations. DisclosuresJaglowski:Immunomedics: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Seattle Genetics: Consultancy.

Polychlorinated Biphenyls and Cancer: Are Telomeres to Blame?

The accumulating epidemiological evidence of elevated cancer risk and mortality in individuals exposed to polychlorinated biphenyls (PCBs) led to their recent classification as human carcinogen by the International Agency for Research on Cancer (IARC) (International Agency for Research on Cancer (IARC), 2015). However, the mechanisms by which PCBs are linked to cancer are still unclear. In this issue, Scinicariello and Buser (Scinicariello, 2015) conducted a study of the association between leukocyte telomere length (LTL) and PCB blood levels in a nationally representative sample of the civilian US adult population using data from the National Health and Nutrition Examination Survey (NHANES). The authors showed that higher PCB blood levels were associated with longer LTL, and hypothesized that there could be a link to PCB-related carcinogenesis. A separate study reported similar relationships between LTL and PCB blood levels in a subset of NHANES participants (Mitro et al., 2015). However, a small study of healthy Koreans (Shin et al., 2010) showed that this relationship was only present at low PCB levels. In contrast, short LTL has been associated with a number of environmental or occupational exposures including particulate matter, black carbon, benzene, toluene, polycyclic aromatic hydrocarbons, N-nitrosamines, pesticides, and lead (reviewed in Zhang et al., 2013), all of which may contribute to PCB blood levels. Telomere shortening in response to chemical exposures may be explained, at least in part, by the induction of an oxidative stress DNA damage response (Zhang et al., 2013). Telomeres, the long (TTAGGG)n nucleotide repeats and an associated protein complex at chromosome ends, are essential for maintaining chromosomal stability. They shorten with each cell division and therefore are markers for cellular replicative capacity, cellular senescence, and aging. Telomere shortening and chromosomal instability has been described at early stages of carcinogenesis, suggesting a role of telomere dysfunction in cancer initiation (Ferguson et al., 2015). Cancer epidemiology studies have shown relationships with short LTL, but differences by cancer site and study design were noted. In a meta-analysis of 27 reports (Wentzensen et al., 2011), short LTL was associated with cancer risk, primarily in case–control studies (OR = 2.9 in case–control studies vs. 1.2 in prospective studies). This suggests that short LTL is a disease marker rather than a risk factor. Recently, a large study linked long LTL to melanoma risk (Iles et al., 2014). The spectrum of cancers associated with long LTL is not clear yet, but suggested to include lung and Non-Hodgkin's lymphoma (NHL). It is of interest that cutaneous melanoma and possibly NHL are the main cancers associated with excess risk in PCBs exposed individuals (International Agency for Research on Cancer (IARC), 2015), suggesting a possible contribution of LTL in PCB-related carcinogenesis. The large sample size, the national representation, and the wealth of collected information NHANES offers are among the strengths of this study. On the other hand, the cross-sectional design restricted its ability to establish a causal inference between LTL, PCB exposure, and cancer risk. The one-time measurement of PCB blood level and LTL may not reflect the full spectrum of the association. Information on the duration and dose of exposure to PCBs would have been useful (reviewed in Zhang et al., 2013) Short-term exposure to ambient particulate matter was associated with long LTL, while longer exposures were associated with short LTL. Exposure dose in relation to LTL was noted in other environmental exposures. For example, exposure to low dose of arsenic resulted in telomerase overexpression and telomere elongation in cord blood cells, while exposure at large doses suppressed telomerase, shortened telomeres, and induced apoptosis. A recent study (Andreotti et al., 2015) evaluating the association between LTL and pesticide exposure suggested a differential effect on LTL in long-term versus recent exposures for certain pesticides. While LTL may present a good surrogate for telomere length in other tissues, it is not clear how good of a surrogate it is for tissues affected by chemical exposures. Measuring telomere length in organs directly affected by PCB exposure, and comparing this to LTL may explain the role telomeres may play in PCB-related carcinogenesis. Also, LTL measurement obtained by quantitative polymerase chain reaction (qPCR) assay may be affected by the cell composition of the sample since telomere length differs by peripheral blood cell subtype. Using a leukocyte cell-type specific TL measurement assay such as flow FISH may provide better insights into such association. Important considerations in epidemiological studies of telomere length are discussed in details elsewhere (Bodelon et al., 2014). In conclusion, the mechanism behind PCB-related carcinogenesis is still unknown. The similarity between cancer sites associated with long LTL, and cancers linked with PCBs provides an interesting hypothesis to test. A large prospective study with serial LTL and PCBs level measurements is required to better understand this relationship.

Cellular Senescence Markers p16INK4a and p21CIP1/WAF Are Predictors of Hodgkin Lymphoma Outcome.

There is evidence that Hodgkin Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) could display some molecular and morphologic markers of cellular senescence (CS). We hypothesized that CS mechanisms may have potential prognostic relevance in cHL and investigated whether the expression of the well-established CS biomarkers p21(CIP1/WAF1) and p16(INK4a) by HRS cells might be predictive of the probability of event-free survival (EFS). The study analyzed a retrospective cohort of 147 patients and the results were validated on a cohort of 91 patients independently diagnosed and treated in a different institution. p16(INK4a) and p21(CIP1/WAF1) were categorized as dichotomous variables (< or ≥ 30% of HRS cells at diagnosis) and evaluated in univariate and multivariate analysis. Both molecules were independent prognostic factors. A positive staining of one of the two molecules in more than 30% HRS cells predicted a better EFS (P < 0.01). p16(INK4a)/p21(CIP1/WAF1) together as a unique categorical variable (both <30%, either <30%, both ≥ 30%) sorted out three prognostic groups with better, intermediate, or worse outcome either overall or within I-II, bulky and advanced stages. The presence or the lack of the robust expression of p21(CIP1/WAF1) and/or p16(INK4a) defined the prognosis in our series. These findings point to (i) the relevance of CS-related mechanisms in cHL, and to (ii) the prognostic value of a simple, reproducible, and low-cost immunohistochemical evaluation of p16(INK4a) and p21(CIP1/WAF1) expression.

Phosphodiesterase 1 regulation is a key mechanism in vascular aging.

Reduced nitric oxide (NO)/cGMP signalling is observed in age-related vascular disease. We hypothesize that this disturbed signalling involves effects of genomic instability, a primary causal factor in aging, on vascular smooth muscle cells (VSMCs) and that the underlying mechanism plays a role in human age-related vascular disease. To test our hypothesis, we combined experiments in mice with genomic instability resulting from the defective nucleotide excision repair gene ERCC1 (Ercc1(d/-) mice), human VSMC cultures and population genome-wide association studies (GWAS). Aortic rings of Ercc1(d/-) mice showed 43% reduced responses to the soluble guanylate cyclase (sGC) stimulator sodium nitroprusside (SNP). Inhibition of phosphodiesterase (PDE) 1 and 5 normalized SNP-relaxing effects in Ercc1(d/-) to wild-type (WT) levels. PDE1C levels were increased in lung and aorta. cGMP hydrolysis by PDE in lungs was higher in Ercc1(d/-) mice. No differences in activity or levels of cGMP-dependent protein kinase 1 or sGC were observed in Ercc1(d/-) mice compared with WT. Senescent human VSMC showed elevated PDE1A and PDE1C and PDE5 mRNA levels (11.6-, 9- and 2.3-fold respectively), which associated with markers of cellular senescence. Conversely, PDE1 inhibition lowered expression of these markers. Human genetic studies revealed significant associations of PDE1A single nucleotide polymorphisms with diastolic blood pressure (DBP; β=0.28, P=2.47×10(-5)) and carotid intima-media thickness (cIMT; β=-0.0061, P=2.89×10(-5)). In summary, these results show that genomic instability and cellular senescence in VSMCs increase PDE1 expression. This might play a role in aging-related loss of vasodilator function, VSMC senescence, increased blood pressure and vascular hypertrophy.

Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment

Ca(2+)/Calmodulin-dependent protein kinase II (CaMKII) signaling in the heart regulates cardiomyocyte contractility and growth in response to elevated intracellular Ca(2+). The δB isoform of CaMKII is the predominant nuclear splice variant in the adult heart and regulates cardiomyocyte hypertrophic gene expression by signaling to the histone deacetylase HDAC4. However, the role of CaMKIIδ in cardiac progenitor cells (CPCs) has not been previously explored. During post-natal growth endogenous CPCs display primarily cytosolic CaMKIIδ, which localizes to the nuclear compartment of CPCs after myocardial infarction injury. CPCs undergoing early differentiation in vitro increase levels of CaMKIIδB in the nuclear compartment where the kinase may contribute to the regulation of CPC commitment. CPCs modified with lentiviral-based constructs to overexpress CaMKIIδB (CPCeδB) have reduced proliferative rate compared with CPCs expressing eGFP alone (CPCe). Additionally, stable expression of CaMKIIδB promotes distinct morphological changes such as increased cell surface area and length of cells compared with CPCe. CPCeδB are resistant to oxidative stress induced by hydrogen peroxide (H2O2) relative to CPCe, whereas knockdown of CaMKIIδB resulted in an up-regulation of cell death and cellular senescence markers compared with scrambled treated controls. Dexamethasone (Dex) treatment increased mRNA and protein expression of cardiomyogenic markers cardiac troponin T and α-smooth muscle actin in CPCeδB compared with CPCe, suggesting increased differentiation. Therefore, CaMKIIδB may serve as a novel modulatory protein to enhance CPC survival and commitment into the cardiac and smooth muscle lineages.

Selective coexpression of VEGF receptor 2 in EGFRvIII-positive glioblastoma cells prevents cellular senescence and contributes to their aggressive nature.

In glioblastoma (GBM), the gene for epidermal growth factor receptor (EGFR) is frequently amplified. EGFR mutations also are common, including a truncation mutation that yields a constitutively active variant called EGFR variant (v)III. EGFRvIII-positive GBM progresses rapidly; however, the reason for this is not clear because the activity of EGFRvIII is attenuated compared with EGF-ligated wild-type EGFR. We hypothesized that EGFRvIII-expressing GBM cells selectively express other oncogenic receptors that support tumor progression. Mining of The Cancer Genome Atlas prompted us to test whether GBM cells in culture, which express EGFRvIII, selectively express vascular endothelial growth factor receptor (VEGFR)2. We also studied human GBM propagated as xenografts. We then applied multiple approaches to test the effects of VEGFR2 on GBM cell growth, apoptosis, and cellular senescence. In human GBM, EGFR overexpression and EGFRvIII positivity were associated with increased VEGFR2 expression. In GBM cells in culture, EGFRvIII-initiated cell signaling increased expression of VEGFR2, which prevented cellular senescence and promoted cell cycle progression. The VEGFR-selective tyrosine kinase inhibitor cediranib decreased tumor DNA synthesis, increased staining for senescence-associated β-galactosidase, reduced retinoblastoma phosphorylation, and increased p27(Kip1), all markers of cellular senescence. Similar results were obtained when VEGFR2 was silenced. VEGFR2 expression by GBM cells supports cell cycle progression and prevents cellular senescence. Coexpression of VEGFR2 by GBM cells in which EGFR signaling is activated may contribute to the aggressive nature of these cells.

Areca nut alkaloids induce irreparable DNA damage and senescence in fibroblasts and may create a favourable environment for tumour progression

Oral submucous fibrosis (OSMF) is a pre-malignant condition that is strongly associated with the areca nut alkaloids, arecoline (ARC) and arecaidine (ARD). The condition is characterised by the presence of senescent fibroblasts in the subepithelial mesenchyme which have the potential to promote malignancy in the neighbouring epithelial cells. We tested the hypothesis that areca nut alkaloids induce senescence in oral fibroblasts and promote the secretion of invasion-promoting transforming growth factor β (TGF-β) and matrix metalloproteinase-2 (MMP-2). Two oral fibroblast lines were treated for 48h with ARC and ARD. Senescence-associated β-galactosidase (SA-βGal) activity, Ki67 (cycling cells), large 53BP1 foci (irreparable DNA strand breaks) and p16(INK) (4A) (late senescence) were used as markers of cellular senescence and were quantified using indirect immunofluorescence and the ImageJ program. TGF-β and MMP-2 levels were measured using ELISA. Statistical analyses were performed with the two-tailed unpaired t-test where n = 3 and the Wilcoxon-Mann-Whitney test where n = 6. ARC (100 and 300 μM) and ARD (30 and 100 μM) significantly (P < 0.05) induced fibroblast senescence, as determined by the increased expression of SA-βGal, 53BP1 staining and CDKN2A/p16(INK) (4A) ; there was also a non-significant reduction in Ki67 staining. Treated cells also showed a three- fivefold increase in TGF-β and a small non-significant increase in MMP-2. Areca nut alkaloids induce senescence in oral fibroblasts and promote increased secretion of TGF-β and perhaps MMP-2 that may create a tissue environment thought to be critical in the progression of OSMF to malignancy.

PP39 - Indole- and Chromane-type heterocycles: Playing with molecules to defeat ageing

Heterocyclic compounds with chromane- and indole-type skeleton represent two fairly large groups of substances with proved beneficial antioxidant effects even beyond the nutritional base. Although these compounds have usual origin in the nature, a rational chemical synthesis might offer an efficient tool for “playing” with their physico-chemical properties so as to optimize their biological effects. In the present study, on using the cellular models, we tried to evaluate the potential of chemically modified flavonoids and synthetic hexahydropyridoindoles to ameliorate ageing-related degenerative processes. Introduction of electron-accepting quinonoid substituent into quercetin increased its prooxidant capacity and toxicity, however, at lower concentration, the effects encompassing hormesis were observed. These properties involved enhanced protection of human fibroblast-like cells from oxidative insult as well as anti-inflammatory effects. 3´-O-Acylation of quercetin (ensuring its higher lipophilicity) yielded in enhanced protection of adult rat microglial cells based on suppression of their protein oxidation, downregulation of the cellular senescence markers and induction of a resting, ramified morphology. On the other hand, weakly basic piperidine moiety in pyridoindoles appeared to steer these substances into lysosomal compartments via a proton-trap mechanism, this being followed by their enhanced accumulation. This mechanism yielded in increased staining of the cells, induction of osmotic processes and enhanced protection of the macrophageous cells from oxidative damage. Thus, rational synthetic variations of indole- and chromane-type compounds can facilitate the search for the substances showing enhanced interference in ageing-related pathologies. This is to say, there are numerous modifications susceptible to optimize either specific targeting, bioavailability or electron properties of these hopeful biological molecules [VEGA2/0031/12].

Aging-associated subpopulations of human CD8+ T-lymphocytes identified by their CD28 and CD57 phenotypes

BackgroundDuring organismal aging, human T-cells shift towards less functional phenotypes, often called senescent cells. As these cells have not been well characterized, we aimed to relate surface markers of human T-cell senescence with characteristics of in vitro cellular aging and to further characterize these cells. MethodsWe identified, by flow cytometry, subpopulations of CD8+ T-cells based on CD57 and CD28 expression, and tested them for some markers of cellular senescence, apoptosis, differentiation and homing. ResultsElderly persons presented significantly higher proportions not only of CD28−CD57+, but also of CD28+CD57+ cells. CD28+CD57+ cells had the highest expression of p16, p21, Bcl-2, CD95, CD45RO, CCR5 and PD-1, thereby arguing in favor of a senescent phenotype. ConclusionAmong CD8+ T-lymphocytes, CD28+CD57+ cells represent a subset with some senescent features that are distinct from the CD28−CD57+ cells.

Abstract 110: The role of the p53 target Wig-1 in senescence and cancer

Abstract The wild type p53-induced gene 1, Wig-1 (also known as Zmat3 or PAG608) binds to AU-rich elements in mRNAs and thereby regulates important tumor associated factors including p53, FAS, and N-Myc. Focusing on normal human diploid fibroblasts (HDF), we are now exploring the role of Wig-1 in senescence. SiRNA-mediated Wig-1 depletion increases senescence markers such as Beta-galactosidase staining, H3K9me3, H4K20me3, and p21. Also, Wig-1 is spontaneously downregulated in cells undergoing replicative senescence. The trimethylation of lysine 9 on histone 3 (H3K9me3) is an established marker of cellular senescence and increases upon siRNA-mediated Wig-1 knockdown in HDF cells. It is also increased in late passage (senescent) fibroblasts that express low Wig-1 levels. Our RNA-immunoprecipitation sequencing (RIP-seq) data of tumor cells shows that Wig-1 binds histone-modifying enzymes SUV39H2 (which trimethylates H3K9), and subunits of PP1 (protein phosphatase 1, which dephosphorylates H3T10). These mRNAs have AU-rich elements in the 3´UTR. Importantly, we have observed that both SUV39H2 and its corresponding methylation (H3K9me3) is increased upon Wig-1 silencing. In a cohort of 350 lung cancer patients, we found Wig-1 gene amplification in a large portion of squamous cell carcinomas, whereas in lung adenocarcinomas the Wig-1 gene was unchanged. Interestingly, strong Wig-1 staining in this cohort correlates to worse prognosis. We think Wig-1 attenuates senescence via downregulating important senescence markers in both tumor and normal cells. Evidence point to Wig-1 playing an inhibitory role on the mRNAs of histone modifying enzymes, thereby acting on well established senescence markers of gene expression and chromatin regulation. Citation Format: Fredrik Jerhammar, Cinzia Bersani, Dijana Djureinovic, Patrick Micke, Klas G. Wiman. The role of the p53 target Wig-1 in senescence and cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 110. doi:10.1158/1538-7445.AM2015-110

Senescent cells talk frankly with their neighbors

Communication between cells is a vital process that governs cellular organization and coordination in an organism. Thus, multiple mechanisms of cell communication have evolved to respond to all the needs of an organism. The modes of cell-cell communication range from the ones that require direct contact between cells, such as cytoplasmic bridges (CBs) and gap junctions, to others that may take place over long distances throughout the organism, such as hormone signaling. Precise integration of data acquired from all these different modes of communication maintains tissue homeostasis and allows optimal adaptation of an organism to various stresses. Cellular senescence represents one of the outcomes of cellular response to stress.1 Senescent cells execute essential functions in different physiological and pathophysiological conditions. They are present in pre-malignant lesions, sites of tissue damage, aging tissues and even during embryonic development. In all these places senescent cells communicate with cells in their surroundings and modulate the function of these cells.2-5 The effect of senescent cells on nearby cells is commonly attributed to the secretion of cytokines, chemokines and matrix metalloproteinases.4,6 We have recently demonstrated that in addition to secretion, senescent cells affect neighboring cells by direct intercellular protein transfer (IPT).2 Proteins from senescent cells are directly transferred to recipient neighboring cells, such as immune and cancer cells, triggering activation of signaling pathways in these cells, ultimately leading to changes in cellular behavior. We have detected IPT from senescent cells and demonstrated that these cells form CBs with other cells, including immune cells, cancer and non-cancer epithelial cells.2 CBs, which in some conditions are called tunneling nanotubes, are open on both edges and allow transfer of cytoplasmic content to neighboring cells. Interestingly, we also identified mitochondria and lysosomes in CBs, implying that organelles might be transferred between the cells. Transfer of these organelles was indeed identified in several other systems. Since dysfunctional mitochondria are thought to play a role in cellular senescence, it can be speculated that transfer of the dysfunctional mitochondria to healthy neighboring cells may induce senescence in these cells. Transfer of cytoplasmic content or whole organelles might serve therefore, for communication of cellular stress. The functional consequences of cell communication of senescent cells through IPT are not completely understood and could be cell type dependent. For instance, we have reported that IPT facilitates elimination of senescent cells by NK cells since transfer of proteins to NK cells correlated with NK cell activation and cytotoxicity. Remarkably, inhibition of CDC42 in senescent cells resulted in decreased IPT and impaired elimination of the senescent cells by NK cells.2 In addition to interaction with NK cells senescent cells also communicate with other components of the innate and adaptive immune systems.2,5,6 Thus, it is possible that senescent cells transfer proteins to other types of immune cells and alter their behavior. To fulfill their function immune cells respond to cytokines and chemokines using receptor-ligand interactions to activate cellular responses (Fig. 1). IPT can serve as an additional mechanism regulating activity of immune cells following contact mediated interaction with resident cells and thereby fine-tune the immune response. Figure 1. Senescent cells communicate with NK cells by multiple mechanisms. Senescent cells influence NK cells by (i) senescent associated secretory phenotype (SASP); (ii) specific receptor ligand interactions; (iii) cytoplasmic bridges. These diverse mechanisms ... Senescent cells can impact tumorigenesis, mainly by secretion of pro-inflammatory cytokines.3,5 Our data demonstrate that senescent cells form CBs with cancer cells, thereby implementing IPT in interaction of senescent and cancer cells. Since the interaction of these cells is long lasting, in contrast to the interaction with NK cells, higher amount and number of proteins could potentially be transferred. The functional impact of IPT from senescent cells to cancer cells is unknown, as it could potentially promote or restrain cancer cells. Senescent cells could transfer cell cycle inhibitors (i.e. p16, p15 and p21) to cancer cells, thereby inhibiting their proliferation. Conversely, cancer cells may also receive organelles and proteins to support their rapid proliferation. In addition to pathological conditions, senescent cells modulate physiological processes, including embryonic development.4 During embryonic development human placental syncytiotrophoblast exhibit features and molecular markers of cellular senescence.7 The syncytiotrophoblast is a multinucleate epithelium which supports fetal growth by creating an interface between maternal and fetal circulation. The syncytiotrophoblast cells communicate with subjacent layer of mono-nucleated cytotrophoblasts to promote division and fusion of cytotrophoblasts into the syncytiotrophoblast. These cells also communicate with immune cells to control immune tolerance at the maternal-fetal interface. Therefore, senescent syncytiotrophoblast cells might also form CBs to facilitate these non-cell autonomous effects which are essential for embryonic development. Overall, IPT from senescent cells represents a pivotal mode of cellular communication with possible physiological outcomes on cancer progression, tissue repair, embryonic development, aging and immune modulation.

Cholangiocyte senescence caused by lysophosphatidylcholine as a potential implication in carcinogenesis.

The incidence of biliary tract cancer in patients with pancreaticobiliary maljunction or intrahepatic cholelithiasis is markedly high with undefined mechanism. In these diseases, biliary lysophosphatidylcholine (LPC) level is reportedly increased. This study investigated the influence of LPC on cholangiocytes focusing on cellular senescence and its potential contribution to carcinogenesis. Cultured MMNK-1, an immortalized human cholangiocyte was treated with LPC in vitro and its effect was evaluated. Lysophosphatidylcholine demonstrated cytotoxicity with generation of intracellular reactive oxygen species. Accordingly, LPC provoked oxidative DNA injury, whereas the gene expressions of DNA repair enzyme (OGG1, MUTYH, MTH1) remained unchanged. Interestingly, LPC caused global DNA hypomethylation, which is frequently observed in cancer tissues. Microarray analysis identified differentially regulated genes in response to LPC, which included the components of senescence-associated secretory phenotype (SASP) including interleukin-8 (IL-8), IL-6, transforming growth factor-β and plasminogen activator inhibitor-1. Significant induction of these genes was further confirmed by quantitative real-time polymerase chain reaction. In addition to upregulation of p21 gene expression, senescence-associated beta-galactosidase activity, a widely used marker of cellular senescence was significantly induced by the treatment of LPC. Based on these data, cholangiocyte senescence and SASP caused by LPC are potential pathogenic mechanisms in the development of biliary tract cancer.

Intermittent high glucose implements stress-induced senescence in human vascular endothelial cells: role of superoxide production by NADPH oxidase.

Impaired glucose tolerance characterized by postprandial hyperglycemia, which occurs frequently in elderly persons and represents an important preliminary step in diabetes mellitus, poses an independent risk factor for the development of atherosclerosis. Endothelial cellular senescence is reported to precede atherosclerosis. We reported that continuous high glucose stimulus causes endothelial senescence more markedly than hypertension or dyslipidemia stimulus. In the present study, we evaluated the effect of fluctuating glucose levels on human endothelial senescence. Constant high glucose increased senescence-associated-β-galactosidase(SA-β-gal) activity, a widely used marker for cellular senescence. Interestingly, in intermittent high glucose, this effect was more pronounced as well as increase of p21 and p16INK4a , senescence related proteins with DNA damage. However, telomerase was not activated and telomere length was not shortened, thus stress-induced senescence was shown. However, constant high glucose activated telomerase and shortened telomere length, which suggested replicative senescence. Intermittent but not constant high glucose strikingly up-regulated the expression of p22phox, an NADPH oxidase component, increasing superoxide. The small interfering RNA of p22phox undermined the increase in SA-β-gal activity induced by intermittent high glucose. Conclusively, intermittent high glucose can promote vascular endothelial senescence more than constant high glucose, which is in partially dependent on superoxide overproduction.

Overexpression of MnSOD Does Not Improve Endothelial Function in Hypercholesterolemic Mice

We previously reported that MnSOD‐deficiency does not cause overt vasomotor dysfunction in aged or hypercholesterolemic mice. To determine whether increasing MnSOD protects against vasomotor dysfunction and deleterious molecular changes in atherosclerosis, we crossed hypercholesterolemic mice with mice overexpressing MnSOD to test the hypothesis that lifelong increases in MnSOD protect against vasomotor dysfunction. We used Ldlr‐/‐/ApoB100/100 mice that were wild‐type (MnSOD0/0) or transgenic (MnSODTg/0) and fed a western diet for 6 months. We used organ bath chambers to measure aortic endothelium‐dependent relaxation (relaxation to acetylcholine, Ach), and mRNA from aortic arch was used for qRT‐PCR. Surprisingly, relaxation to Ach was unchanged between MnSOD0/0 and MnSODTg/0 mice, as was relaxation to sodium nitroprusside. As expected, gene expression of MnSOD was significantly increased in the MnSODTg/0 mice compared to MnSOD0/0 mice. Unlike our findings in MnSOD+/‐ mice, however (where Nox2 was increased), Nox2 expression was unchanged between MnSOD0/0 and MnSODTg/0 mice. Importantly, markers of osteogenesis such as Runx2 and BMP2 were unchanged between genotypes. Furthermore, MnSOD overexpression did not alter cellular senescence markers. Collectively, our data support the surprising conclusion that physiologically relevant increases in MnSOD do not significantly affect endothelial function or molecular regulators of histopathological changes in advanced atherosclerosis in hypercholesterolemic mice.

Wnt7a is a novel inducer of β-catenin-independent tumor-suppressive cellular senescence in lung cancer.

Cellular senescence is an initial barrier for carcinogenesis. However, the signaling mechanisms that trigger cellular senescence are incompletely understood, particularly in vivo. Here we identify Wnt7a as a novel upstream inducer of cellular senescence. In two different mouse strains (C57Bl/6J and FVB/NJ), we show that the loss of Wnt7a is a major contributing factor for increased lung tumorigenesis owing to reduced cellular senescence, and not reduced apoptosis, or autophagy. Wnt7a-null mice under de novo conditions and in both the strains display E-cadherin-to-N-cadherin switch, reduced expression of cellular senescence markers and reduced expression of senescence-associated secretory phenotype, indicating a genetic predisposition of these mice to increased carcinogen-induced lung tumorigenesis. Interestingly, Wnt7a induced an alternate senescence pathway, which was independent of β-catenin, and distinct from that of classical oncogene-induced senescence mediated by the well-known p16INK4a and p19ARF pathways. Mechanistically, Wnt7a induced cellular senescence via inactivation of S-phase kinase-associated protein 2, an important alternate regulator of cellular senescence. Additionally, we identified Iloprost, a prostacyclin analog, which initiates downstream signaling cascades similar to that of Wnt7a, as a novel inducer of cellular senescence, presenting potential future clinical translational strategies. Thus pro-senescence therapies using either Wnt7a or its mimic, Iloprost, might represent a new class of therapeutic treatments for lung cancer.

Telomere Length in Hematopoietic Stem Cell Transplantation for Severe Aplastic Anemia

Severe aplastic anemia is a heterogeneous bone marrow failure syndrome, characterized by peripheral blood pancytopenia and bone marrow aplasia. The disease affects both children and young adults. The incidence is estimated at 2per 1millionannally.1-3Early treatmentmaydeterminepatientoutcomes, thusdevelopingbetter treatments for this condition could have an important clinical implications. Immunosuppressionanduseof somenewerdrugshavebeen shown tobeeffective.4-6However, allogeneichematopoietic stemcell transplant (HSCT) canbecurative for severeaplastic anemia.7-9 Although the survival results of HLA-matched related donor andHLA-matchedunrelateddonorHSCT in leukemiaare comparable, this is not the case in severe aplastic anemia.10 The results of matched unrelated donor HSCT for severe aplastic anemiahavebeensuboptimal.Comparedwith80%to90%expected long-term survival withmatched related donorHSCT, that ofmatchedunrelateddonorHSCThasbeen39%to64%.10 Thus, further improvement of the survival results inmatched unrelateddonorHSCT insevereaplastic anemia is critically important; thus, risk factors associatedwith improved outcome need to be identified. The article by Gadalla et al11 in this issue of JAMA evaluated the association between leukocyte telomere length and outcomes inmatchedunrelateddonorHSCT for severe aplastic anemia. A telomere is a noncoding region of repetitive nucleotide at each end of the chromatid that becomes progressively shorterwith serial cellularmitosis, thus protecting telomeric (terminally located) genes during mitosis. Telomeres become shorter with cellular aging (genomic senescence).12 Based on donor telomere length distribution, the authors classified recipient and donor leukocyte telomere length as long (third tertile) and short (first and second tertiles combined) and examined the associationwith overall survival, neutrophil recovery, and acute and chronic graft-vshostdiseaseduring follow-up toMarch2013.Longerdonor leukocyte telomere lengthwasassociatedwithhigheroverall survivalaftermatchedunrelateddonorHSCT;amongpatientswho received HCT from donors with longer telomeres (third tertile,n = 113)vs shorter telomeres (first andsecond tertiles combined, n = 217), the survival probabilities were 60%vs 50% at 1 year, 58% vs 44% at 3 years, and 56% vs 40% at 5 years. Although the outcomeofmatchedunrelateddonorHSCT has been shown to be betterwith younger donors,13 the study by Gadalla et al showed that shorter donor leukocyte telomere length is an independent risk factor fromdonor age. Importantly, the authors found that recipient leukocyte telomere length was not associated with outcome after matched unrelated donor HSCT, although recipient leukocyte telomere lengthhas been shown tobe associatedwith survival after immunosuppressive therapy alone.14 Although the findings reported by Gadalla et al may help in choosing optimal donors for HSCT, many questions remainunanswered.For instance,whywassurvivalbetteramong patients who received grafts from donors with longer leukocyte telomere length?Asnotedby the authors, leukocyte telomere length is a marker of cellular replicative capacity, cellular senescence, and aging. If leukocyte telomere length were associatedwithengraftmentstatus, itwouldprovideastraightforward explanation. However, leukocyte telomere length is not associated with any engraftment characteristics such as neutrophil andplatelet engraftment or graft failure rate nor is leukocyte telomere length associated with acute or chronic graft-vs-host disease. The authors cited a report showingpossible effect of leukocyte telomere lengthonengraftment in the pediatric population.15However, in the studybyGadalla et al, the engraftment patterns seemed to overlap in the longer and shorter leukocyte telomere length cohorts and did not show any difference. In addition, if engraftment is not the reason for the survival difference,whichmechanisms account for the apparent association between leukocyte telomere length and survival? It thenstands toreasonthat thedifferencemustbeduetotransplant-relatedmortality, although the authors didnot find any relationshipbetweendonor leukocyte telomere lengthandspecific causes of death in this study. Were there less infections or less regimen-related toxicities? If there were fewer infections, is longer leukocyte telomere lengthassociatedwithmore rapid immunereconstitution?Furtherdataareneededtoclarify these issues. This observational report by Gadalla et al11 provides potentially important data and may mark the beginning of other new findings to come. Another question iswhether leukocyte telomere length is an independent risk factor for transplant outcome, particularly in any other transplant populations. The authors analyzed the outcome of matched unrelated donor HSCT for severeaplastic anemiaamongrelativelyyoungpatientsprimarily using bone marrow graft. Would similar findings occur with different type of grafts, such as peripheral blood stem cells or Related article page 594 Opinion

The Effect of Age on Osteogenic and Adipogenic Differentiation Potential of Human Adipose Derived Stromal Stem Cells (hASCs) and the Impact of Stress Factors in the Course of the Differentiation Process

Human adipose tissue is a great source of autologous mesenchymal stem cells (hASCs), which are recognized for their vast therapeutic applications. Their ability to self-renew and differentiate into several lineages makes them a promising tool for cell-based therapies in different types of degenerative diseases. Thus it is crucial to evaluate age-related changes in hASCs, as the elderly are a group that will benefit most from their considerable potential. In this study we investigated the effect of donor age on growth kinetics, cellular senescence marker levels, and osteogenic and adipogenic potential of hASCs. It also has been known that, during life, organisms accumulate oxidative damage that negatively affects cell metabolism. Taking this into consideration, we evaluated the levels of nitric oxide, reactive oxygen species, and superoxide dismutase activity. We observed that ROS and NO increase with aging, while SOD activity is significantly reduced. Moreover cells obtained from older patients displayed senescence associated features, for example, β-galactosidase activity, enlarged morphology, and p53 protein upregulation. All of those characteristics seem to contribute to decreased proliferation potential of those cells. Our results suggest that due to aging some cellular modification may be required before applying aged cells efficiently in therapies such as tissue engineering and regenerative medicine.

Identification of Markers for Cellular Stress and Senescence in Laminopathic Cells by Proteomic Analysis

Lamins are components of the structural network in the nucleus and play a key role in nuclear or ganization and function. Mutations in the human lamin A gene cause a spectrum of genetic diseases that include muscular dystrophies, cardiomyopathy, lipodystrophy and premature ageing syndromes. Laminopathic cells display several abnormalities in nuclear morphology and function. We have carried out a comprehensive analysis of alterations in the protein profiles of HEK293T cells expressing a mutation in lamin A (Q294P) that causes Emery-Dreifuss muscular dystrophy, in comparison with cells expressing wild-type lamin A. Initially a total of 27 dif ferentially expressed proteins were identified by quantitative two-dimensional gel electrophoresis followed by mass spectral analysis. Importantly , a 5-fold decrease in lamin B1 levels in mutant cells was observed by quantitative two-dimensional gel electrophoresis, which was supported by immunofluorescence analysis. Changes in levels of specific heat shock proteins, hnRNPs, DNA damage response proteins, metabolic enzymes and cytoskeletal proteins were also observed. Further detailed analysis of cell lysates by one-dimensional gel electrophoresis followed by high throughput mass spectrometry revealed 650 unique proteins from wild-type cells and 1494 unique proteins from mutant cells (with two or more than two peptide hits). Alterations in a number of proteins that are involved in chromatin structure, chromosome condensation, nucleosome assembly, epigenetic modifications, centromere organization, telomere length and DNA repair were observed. These data suggest that laminopathic cells exhibit characteristics of cellular stress and senescence, and provide new insights into the cellular basis of pathologies caused by laminopathic mutations.

T-Cell p16INK4A Expression Increases Post-Transplant in Patients with Multiple Myeloma

Multiple myeloma (MM) is an incurable hematologic malignancy primarily of older adults. MM treatment decisions are based partially upon chronologic age, a measure that may not reflect the patients’ physiologic age or ability to tolerate therapy. Recently, investigators have established methodology to better quantify biologic age using the molecular marker, p16INK4a (p16). p16 expression is a marker of cellular senescence and is strongly associated with gerontogenic models. p16 rises exponentially with chronologic aging and is influenced by external factors such as physical activity, tobacco use, and solid tumor chemotherapy. Additionally, single nucleotide polymorphisms located near the p16-encoding INK4/ARF locus are linked to age-related diseases (e.g. cardiovascular disease, diabetes, glaucoma) and decreased physical function. p16 mRNA can be measured in peripheral blood T-cells thus providing a practical means of quantifying this molecular marker of age. To determine the effects of MM therapy and autologous hematopoietic stem cell transplant (AHSCT) on biologic aging, we serially measured T-cell p16 expression in MM cohorts and aged matched controls.Methods: 23 MM patients were divided into cohorts of newly diagnosed (ND; n=11) and prior treatment (T; n=12). T-cell isolation was performed using techniques described by Liu and colleagues (Liu et al. Aging Cell 2009); p16 expression was measured using quantitative RT-PCR. 16 MM patients underwent sequential testing, with a median 42 days between each measurement. During this time, 8 patients had no intervention and 8 received immune modulatory (IMiD) therapy. Healthy age matched control T-cells were analyzed for comparison (n=17).Results: MM patients with a median age of 62 were evaluated; 12 with early stage ISS disease, 21 with an ECOG performance status of 0-1, and 6 with high risk FISH abnormalities. 12 MM patients had prior treatment, most included AHSCT (n=11). MM treated patients had several lines of therapy; half with 1-2 lines and half with 3+ lines. Median time from AHSCT to p16 analysis was 1003 days (range 49-3630) and transplant date was unrelated to p16 expression (p=0.9577). The healthy control median age was 60 (SD 11.77) with a range of 35-82 years. T-cell yield among MM patients and healthy controls was not significantly different. Using univariate linear regression, age correlates significantly with p16 expression in healthy controls (p=0.0039) wherein p16 is estimated to increase by 1.035 fold each year [0.05 Cts per year].Using multivariable regression analysis, age and MM status (ND and T), were both independently associated with p16 expression. When controlling for age, patients previously treated for MM had p16 expression that was ~3.68-fold higher than healthy controls (p=0.0003). There was no significant difference in p16 expression between ND MM patients and healthy controls (fold-change=1.15, p=0.70). MM p16 levels were significantly higher in patients with prior therapy (p=0.0439) and for those who had underwent AHSCT (p=0.0129). However, p16 was unrelated to other clinical factors such as cardiovascular disease, tobacco use, radiation, stage, absolute lymphocyte count, or renal function. 8 MM patients where p16 expression was measured pre and post-IMiD treatment, a significant change in expression was not observed (median percent change: 1.16%, p=0.11); in another 8 MM patients who were untreated, p16 levels were also unchanged over time (median percent change: -0.91%, p=0.38).Conclusions: AHSCT significantly impacts p16 expression in T-cells from MM patients. Specifically, MM patients post-AHSCT show a 3.68 fold increase in p16 expression compared to age-matched controls. Time from transplant to assessment does not impact p16 measurements, suggesting that cellular senescence of the T-cell compartment following AHSCT is persistent. MM patients exhibit known T-cell defects including variable T-cell frequency, abnormal signal transduction, and impaired cytokine secretion. Moreover, MM AHSCT patients also show impaired immune reconstitution with changes in T-cell frequency and function. To our knowledge, this is the first report describing cellular senescence post-transplant. Health status and function are affected by AHSCT; our work is important in understanding how biologic markers of age change with intensive therapy, such as AHSCT, to better predict outcomes in future studies. DisclosuresHofmeister:Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millenium: Honoraria, Research Funding; ARNO Therapeutics: Research Funding; Onyx: Membership on an entity’s Board of Directors or advisory committees.