Guidelines for minimal information on cellular senescence experimentation in vivo.

Cellular senescence is a cell fate triggered in response to stress and is characterized by stable cell-cycle arrest and a hypersecretory state. It has diverse biological roles, ranging from tissue repair to chronic disease. The development of new tools to study senescence in vivo has paved the way for uncovering its physiological and pathological roles and testing senescent cells as a therapeutic target. However, the lack of specific and broadly applicable markers makes it difficult to identify and characterize senescent cells in tissues and living organisms. To address this, we provide practical guidelines called "minimum information for cellular senescence experimentation in vivo" (MICSE). It presents an overview of senescence markers in rodent tissues, transgenic models, non-mammalian systems, human tissues, and tumors and their use in the identification and specification of senescent cells. These guidelines provide a uniform, state-of-the-art, and accessible toolset to improve our understanding of cellular senescence in vivo.

Therapy-induced polyploidization and senescence: Coincidence or interconnection?

Polyploid somatic cells have 'programmed' roles in normal development and stress responses. Transient polyploidy states have been observed in several tumor types at early stages of tumorigenesis. They can give rise to the aneuploidy state which is a common feature of human cancer cells. Similarly, to cancer development, cancer treatment can lead to transient polyploidy. Polyploid giant cells (PGCCs) in cancer are often associated with poor prognosis and disease relapse. Cancer cell senescence- a proliferation arrest accompanied by a set of characteristic markers- induced by therapy is also associated with transient polyploidy formation and cancer relapse. The question is whether therapy-induced senescence (TIS) and therapy induced polyploidy (TIP) are mechanistically or coincidentally connected. This problem needs to be solved rather urgently, because TIS appears to be more common phenomena than originally believed. Another arising question concerns reversibility of cancer cell senescence as a consequence of atypical divisions of polyploid cells. In our review we will try to answer this fundamental question by referring to published literature and to our own studies.

Association of Torquetenovirus Viremia with Physical Frailty and Cognitive Impairment in Three Independent European Cohorts.

INTRODUCTION: Immunosenescence and inflammaging have been implicated in the pathophysiology of frailty. Torquetenovirus (TTV), a single-stranded DNA anellovirus, the major component of the human blood virome, shows an increased replication rate with advancing age. An elevated TTV viremia has been associated with an impaired immune function and an increased risk of mortality in the older population. The objective of this study was to analyze the relation between TTV viremia, physical frailty, and cognitive impairment. METHODS: TTV viremia was measured in 1,131 nonfrail, 45 physically frail, and 113 cognitively impaired older adults recruited in the MARK-AGE study (overall mean age 64.7 ± 5.9 years), and then the results were checked in two other independent cohorts from Spain and Portugal, including 126 frail, 252 prefrail, and 141 nonfrail individuals (overall mean age: 77.5 ± 8.3 years). RESULTS: TTV viremia ≥4log was associated with physical frailty (OR: 4.69; 95% CI: 2.06-10.67, p < 0.0001) and cognitive impairment (OR: 3.49, 95% CI: 2.14-5.69, p < 0.0001) in the MARK-AGE population. The association between TTV DNA load and frailty status was confirmed in the Spanish cohort, while a slight association with cognitive impairment was observed (OR: 1.33; 95% CI: 1.000-1.773), only in the unadjusted model. No association between TTV load and frailty or cognitive impairment was found in the Portuguese sample, although a negative association between TTV viremia and MMSE score was observed in Spanish and Portuguese females. CONCLUSIONS: These findings demonstrate an association between TTV viremia and physical frailty, while the association with cognitive impairment was observed only in the younger population from the MARK-AGE study. Further research is necessary to clarify TTV's clinical relevance in the onset and progression of frailty and cognitive decline in older individuals.

Association between fat-soluble vitamins and self-reported health status: a cross-sectional analysis of the MARK-AGE cohort.

Self-rated health (SRH) is associated with higher risk of death. Since low plasma levels of fat-soluble vitamins are related to mortality, we aimed to assess whether plasma concentrations of vitamins A, D and E were associated with SRH in the MARK-AGE study. We included 3158 participants (52 % female) aged between 35 and 75 years. Cross-sectional data were collected via questionnaires. An enzyme immunoassay quantified 25-hydroxyvitamin D and HPLC determined α-tocopherol and retinol plasma concentrations. The median 25-hydroxyvitamin D and retinol concentrations differed significantly (P < 0·001) between SRH categories and were lower in the combined fair/poor category v. the excellent, very good and good categories (25-hydroxvitamin D: 40·8 v. 51·9, 49·3, 46·7 nmol/l, respectively; retinol: 1·67 v. 1·75, 1·74, 1·70 µmol/l, respectively). Both vitamin D and retinol status were independently associated with fair/poor SRH in multiple regression analyses: adjusted OR (95 % CI) for the vitamin D insufficiency, deficiency and severe deficiency categories were 1·33 (1·06-1·68), 1·50 (1·17-1·93) and 1·83 (1·34-2·50), respectively; P = 0·015, P = 0·001 and P < 0·001, and for the second/third/fourth retinol quartiles: 1·44 (1·18-1·75), 1·57 (1·28-1·93) and 1·49 (1·20-1·84); all P < 0·001. No significant associations were reported for α-tocopherol quartiles. Lower vitamin A and D status emerged as independent markers for fair/poor SRH. Further insights into the long-term implications of these modifiable nutrients on health status are warranted.

Therapy-Induced Senescent/Polyploid Cancer Cells Undergo Atypical Divisions Associated with Altered Expression of Meiosis, Spermatogenesis and EMT Genes.

Upon anticancer treatment, cancer cells can undergo cellular senescence, i.e., the temporal arrest of cell division, accompanied by polyploidization and subsequent amitotic divisions, giving rise to mitotically dividing progeny. In this study, we sought to further characterize the cells undergoing senescence/polyploidization and their propensity for atypical divisions. We used p53-wild type MCF-7 cells treated with irinotecan (IRI), which we have previously shown undergo senescence/polyploidization. The propensity of cells to divide was measured by a BrdU incorporation assay, Ki67 protein level (cell cycle marker) and a time-lapse technique. Advanced electron microscopy-based cell visualization and bioinformatics for gene transcription analysis were also used. We found that after IRI-treatment of MCF-7 cells, the DNA replication and Ki67 level decreased temporally. Eventually, polyploid cells divided by budding. With the use of transmission electron microscopy, we showed the presence of mononuclear small cells inside senescent/polyploid ones. A comparison of the transcriptome of senescent cells at day three with day eight (when cells just start to escape senescence) revealed an altered expression of gene sets related to meiotic cell cycles, spermatogenesis and epithelial-mesenchymal transition. Although chemotherapy (DNA damage)-induced senescence is indispensable for temporary proliferation arrest of cancer cells, this response can be followed by their polyploidization and reprogramming, leading to more fit offspring.

Do low molecular weight antioxidants contribute to the Protection against oxidative damage? The interrelation between oxidative stress and low molecular weight antioxidants based on data from the MARK-AGE study.

A redox steady state is important in maintaining vital cellular functions and is therefore homeostatically controlled by a number of antioxidative agents, the most important of which are enzymes. Oxidative Stress (OS) is associated with (or/and caused by) excessive production of damaging reactive oxygen and/or nitrogen species (ROS, RNS), which play a role in many pathologies. Because OS is a risk factor for many diseases, much effort (and money) is devoted to early diagnosis and treatment of OS. The desired benefit of the "identify (OS) and treat (by low molecular weight antioxidants, LMWA)" approach is to enable selective treatment of patients under OS. The present work aims at gaining understanding of the benefit of the antioxidants based on interrelationship between the concentration of different OS biomarkers and LMWA. Both the concentrations of a variety of biomarkers and of LMWA were previously determined and some analyses have been published by the MARK-AGE team. For the sake of simplicity, we assume that the concentration of an OS biomarker is a linear function of the concentration of a LMWA (if the association is due to causal relationship). A negative slope of this dependence (and sign of the correlation coefficient) can be intuitively expected for an antioxidant, a positive slope indicates that the LMWA is pro-oxidative, whereas extrapolation of the OS biomarker to [LMWA] = 0 is an approximation of the concentration of the OS biomarker in the absence of the LMWA. Using this strategy, we studied the effects of 12 LMWA (including tocopherols, carotenoids and ascorbic acid) on the OS status, as observed with 8 biomarkers of oxidative damage (including malondialdehyde, protein carbonyls, 3-nitrotyrosine). The results of this communication show that in a cross-sectional study the LMWA contribute little to the redox state and that different "antioxidants" are very different, so that single LMWA treatment of OS is not scientifically justified assuming our simple model. In view of the difficulty of quantitating the OS and the very different effects of various LMWA, the use of the "identify and treat" approach is questionable.

Improved Autophagic Flux in Escapers from Doxorubicin-Induced Senescence/Polyploidy of Breast Cancer Cells.

The induction of senescence/polyploidization and their role in cancer recurrence is still a poorly explored issue. We showed that MDA-MB-231 and MCF-7 breast cancer cells underwent reversible senescence/polyploidization upon pulse treatment with doxorubicin (dox). Subsequently, senescent/polyploid cells produced progeny (escapers) that possessed the same amount of DNA as parental cells. In a dox-induced senescence/polyploidization state, the accumulation of autophagy protein markers, such as LC3B II and p62/SQSTM1, was observed. However, the senescent cells were characterized by a very low rate of new autophagosome formation and degradation, estimated by autophagic index. In contrast to senescent cells, escapers had a substantially increased autophagic index and transcription factor EB activation, but a decreased level of an autophagy inhibitor, Rubicon, and autophagic vesicles with non-degraded cargo. These results strongly suggested that autophagy in escapers was improved, especially in MDA-MB-231 cells. The escapers of both cell lines were also susceptible to dox-induced senescence. However, MDA-MB-231 cells which escaped from senescence were characterized by a lower number of γH2AX foci and a different pattern of interleukin synthesis than senescent cells. Thus, our studies showed that breast cancer cells can undergo senescence uncoupled from autophagy status, but autophagic flux resumption may be indispensable in cancer cell escape from senescence/polyploidy.

"Mitotic Slippage" and Extranuclear DNA in Cancer Chemoresistance: A Focus on Telomeres.

Mitotic slippage (MS), the incomplete mitosis that results in a doubled genome in interphase, is a typical response of TP53-mutant tumors resistant to genotoxic therapy. These polyploidized cells display premature senescence and sort the damaged DNA into the cytoplasm. In this study, we explored MS in the MDA-MB-231 cell line treated with doxorubicin (DOX). We found selective release into the cytoplasm of telomere fragments enriched in telomerase reverse transcriptase (hTERT), telomere capping protein TRF2, and DNA double-strand breaks marked by γH2AX, in association with ubiquitin-binding protein SQSTM1/p62. This occurs along with the alternative lengthening of telomeres (ALT) and DNA repair by homologous recombination (HR) in the nuclear promyelocytic leukemia (PML) bodies. The cells in repeated MS cycles activate meiotic genes and display holocentric chromosomes characteristic for inverted meiosis (IM). These giant cells acquire an amoeboid phenotype and finally bud the depolyploidized progeny, restarting the mitotic cycling. We suggest the reversible conversion of the telomerase-driven telomere maintenance into ALT coupled with IM at the sub-telomere breakage sites introduced by meiotic nuclease SPO11. All three MS mechanisms converging at telomeres recapitulate the amoeba-like agamic life-cycle, decreasing the mutagenic load and enabling the recovery of recombined, reduced progeny for return into the mitotic cycle.

Curcumin induces multiple signaling pathways leading to vascular smooth muscle cell senescence.

Curcumin, a phytochemical present in the spice named turmeric, and one of the promising anti-aging factors, is itself able to induce cellular senescence. We have recently shown that cells building the vasculature senesced as a result of curcumin treatment. Curcumin-induced senescence was DNA damage-independent; however, activation of ATM was observed. Moreover, neither increased ROS production, nor even ATM were indispensable for senescence progression. In this paper we tried to elucidate the mechanism of curcumin-induced senescence. We analyzed the time-dependence of the level and activity of numerous proteins involved in senescence progression in vascular smooth muscle cells and how inhibition p38 or p38 together with ATM, two proteins involved in canonical signaling pathways, influenced cell senescence. We showed that curcumin was able to influence many signaling pathways of which probably none was dominant and sufficient to induce senescence by itself. However, we cannot exclude that the switch between initiation and progression of senescence is the result of the impact of curcumin on signaling pathways engaging AMPK, ATM, sirtuin 1 and p300 and on their reciprocal interplay. Cytostatic concentration of curcumin induced cellular stress, which exceeded the adaptive response and, in consequence, led to cellular senescence, which is triggered by time dependent activation of several signaling pathways playing diverse roles in different phases of senescence progression. We also showed that activity of ß-glucuronidase, the enzyme involved in deconjugation of the main metabolites of curcumin, glucuronides, increased in senescent cells. It suggests a possible local elevation of curcumin concentration in the organism.

The Role of Curcumin in the Modulation of Ageing.

It is believed that postponing ageing is more effective and less expensive than the treatment of particular age-related diseases. Compounds which could delay symptoms of ageing, especially natural products present in a daily diet, are intensively studied. One of them is curcumin. It causes the elongation of the lifespan of model organisms, alleviates ageing symptoms and postpones the progression of age-related diseases in which cellular senescence is directly involved. It has been demonstrated that the elimination of senescent cells significantly improves the quality of life of mice. There is a continuous search for compounds, named senolytic drugs, that selectively eliminate senescent cells from organisms. In this paper, we endeavor to review the current knowledge about the anti-ageing role of curcumin and discuss its senolytic potential.

What is and what is not cell senescence.

Cell senescence is a process that occurs due to telomere erosion or can be induced by various stresses. Senescent cells cease to divide but remain alive, metabolically active and able to secrete many molecules. They also show many hallmarks of senescence, such as enlarged size, increased granularity, increased activity of SA-ß-galactosidase, increased level of cyclin-dependent kinase inhibitors, p16 and p21, and DNA damage foci. Originally, cell senescence was attributed to proliferating normal cells, in contrast to cancer cells, which were considered as those endowed with indefinite growth ability. Recently, it has become evident that anticancer treatment induces senescence in cancer cells. Moreover, certain hallmarks of senescence were detected in non-proliferating post-mitotic cells. There are many signalling pathways involved in cell senescence, but the most prevalent is the DNA damage response pathway. In this review we have summarized our long lasting input in the global study of the mechanisms of senescence of normal and cancer cells and discussed the diversity of the concept of cell senescence.

Sirtuins, a promising target in slowing down the ageing process.

Ageing is a plastic process and can be successfully modulated by some biomedical approaches or pharmaceutics. In this manner it is possible to delay or even prevent some age-related pathologies. There are some defined interventions, which give promising results in animal models or even in human studies, resulting in lifespan elongation or healthspan improvement. One of the most promising targets for anti-ageing approaches are proteins belonging to the sirtuin family. Sirtuins were originally discovered as transcription repressors in yeast, however, nowadays they are known to occur in bacteria and eukaryotes (including mammals). In humans the family consists of seven members (SIRT1-7) that possess either mono-ADP ribosyltransferase or deacetylase activity. It is believed that sirtuins play key role during cell response to a variety of stresses, such as oxidative or genotoxic stress and are crucial for cell metabolism. Although some data put in question direct involvement of sirtuins in extending human lifespan, it was documented that proper lifestyle including physical activity and diet can influence healthspan via increasing the level of sirtuins. The search for an activator of sirtuins is one of the most extensive and robust topic of research. Some hopes are put on natural compounds, including curcumin. In this review we summarize the involvement and usefulness of sirtuins in anti-ageing interventions and discuss the potential role of curcumin in sirtuins regulation.

The role of gender and labour status in immunosenescence of 65+ Polish population.

Women are living longer than men and it seems that one of the reasons could be better immune system of females. In Poland, contrary to many European countries, women retire earlier than men, namely at 60 and 65, respectively. We asked the question how the gender and labour status were interconnected with some immunological parameters included in the so called immune risk profile (IRP), such as CD4+/CD8+ ratio, percentage of CD8+CD28-, and NK, and the level of circulating cytokines. A total of 92 men and 100 women past the retirement age, namely 65-74 years old, still working or not, were examined. We have found statistically significant lower percentage of CD8+CD28- cells and non-statistically significant higher CD4+/CD8+ ratio in women, whereas the percentage of NK was higher in men. Moreover, the percentage of CD8+CD28- cells was negatively correlated with the CD4+/CD8+ ratio and the concentration of IL8 was positively correlated with the concentration of IL10 both in men and women. In men, the level of IL10 was higher than in women. Altogether, we found that gender, but not labour status, influences immunosenescence of the examined population of 65-74 years old Polish people.

A comparison of replicative senescence and doxorubicin-induced premature senescence of vascular smooth muscle cells isolated from human aorta.

Senescence of vascular smooth muscle cells (VSMCs) contributes to aging as well as age-related diseases of the cardiovascular system. Senescent VSMCs have been shown to be present in atherosclerotic plaques. Both replicative (RS) and stress-induced premature senescence (SIPS) accompany cardiovascular diseases. We aimed to establish the signature of RS and SIPS of VSMCs, induced by a common anticancer drug, doxorubicin, and to discover the so far undisclosed features of senescent cells that are potentially harmful to the organism. Most of the senescence hallmarks were common for both RS and SIPS; however, some differences were observed. 32 % of doxorubicin-treated cells were arrested in the G2/M phase of the cell cycle, while 73 % of replicatively senescing cells were arrested in the G1 phase. Moreover, on the basis of alkaline phosphatase activity measurements, we show that a 7-day treatment with doxorubicin (dox), does not cause precocious cell calcification, which is a characteristic feature of RS. We did not observe calcification even though after 7 days of dox-treatment many other markers characteristic for senescent cells were present. It can suggest that dox-induced SIPS does not accelerate the mineralization of vessels. We consider that detailed characterization of the two types of cellular senescence can be useful in in vitro studies of potential anti-aging factors.

[Aging and longevity]. / Starzenie i dlugowiecznosc.

During the last three centuries the expected lifespan in civilized countries has increased several times. The fastest growing groups are seniors (65+) and centenarians. Increased lifespan results in postponing of aging and age-related diseases. On the other hand, an increase in the number of people suffering from age-related diseases can be observed. Studies concerning longevity and aging help to elucidate the mechanisms responsible for these processes and give hope for finding the recipe for a healthy and long lifespan. Aging and longevity are modulated by genetic, epigenetic and stochastic factors. Already some variants of genes which correlate with longevity are known. Products of these genes are involved in lipid metabolism and in nutrient sensing signaling pathways such as: insulin/IGF-1 and TOR. Good indicators for human polymorphism study are results obtained using model organisms such as S. cerevisiae, C. elegans, D. melanogaster and laboratory mice. Aging and longevity are evolutionary conserved. Evolutionary theories concerning aging can be divided into two general categories: programmed and non-programmed ones. According to programmed theories aging is adaptive and can lead to altruistic death of kins. Non-programmed theories predict that organisms only have a limited amount of energy that has to be divided between reproductive activities and the maintenance of the non-reproductive aspects of the organism. Aging is the effect of natural degrading processes that result in the accumulation of damage. Accumulation of damaged DNA and proteins can lead to cellular senescence, inflammaging and age-related diseases. Strategies for postponing aging mainly rely on protecting and/or eliminating these lesions.

2-Bromopalmitate treatment attenuates senescence phenotype in human adult cells - possible role of palmitoylation.

Cells may undergo senescence in response to DNA damage, which is associated with cell cycle arrest, altered gene expression and altered cell morphology. Protein palmitoylation is one of the mechanisms by which the DNA damage response is regulated. Therefore, we hypothesized that protein palmitoylation played a role in regulation of the senescent phenotype. Here, we showed that treatment of senescent human vascular smooth muscle cells (VSMCs) with 2-bromopalmitate (2-BP), an inhibitor of protein acyltransferases, is associated with changes in different aspects of the senescent phenotype, including the resumption of cell proliferation, a decrease in DNA damage markers and the downregulation of senescence-associated ß-galactosidase activity. The effects were dose dependent and associated with significantly decreased total protein palmitoylation level. We also showed that the senescence-modifying properties of 2-BP were at least partially mediated by the downregulation of elements of DNA damage-related molecular pathways, such as phosphorylated p53. Our data suggest that cell senescence may be regulated by palmitoylation, which provides a new perspective on the role of this posttranslational modification in age-related diseases.

Associations of circulating GDF15 with combined cognitive frailty and depression in older adults of the MARK-AGE study.

Growth differentiation factor-15 (GDF15) might be involved in the development of cognitive frailty and depression. Therefore, we evaluated cross-sectional associations of plasma GDF15 with combined cognitive-frailty-and-depression in older (i.e. ≥ 55 years) and younger adults of the MARK-AGE study. In the present work, samples and data of MARK-AGE ("European study to establish bioMARKers of human AGEing") participants (N = 2736) were analyzed. Cognitive frailty was determined by the global cognitive functioning score (GCF) and depression by the Self-Rating Depression Scale (SDS score). Adults were classified into three groups: (I) neither-cognitive-frailty-nor-depression, (II) either-cognitive-frailty-or-depression or (III) both-cognitive-frailty-and-depression. Cross-sectional associations were determined by unadjusted and by age, BMI, sex, comorbidities and hsCRP-adjusted linear and logistic regression analyses. Cognitive frailty, depression, age and GDF15 were significantly related within the whole study sample. High GDF15 levels were significantly associated with both-cognitive-frailty-and-depression (adjusted ß = 0.177 [0.044 - 0.310], p = 0.009), and with low GCF scores and high SDS scores. High GDF15 concentrations and quartiles were significantly associated with higher odds to have both-cognitive-frailty-and-depression (adjusted odds ratio = 2.353 [1.267 - 4.372], p = 0.007; and adjusted odds ratio = 1.414 [1.025 - 1.951], p = 0.035, respectively) independent of age, BMI, sex, comorbidities and hsCRP. These associations remained significant when evaluating older adults. We conclude that plasma GDF15 concentrations are significantly associated with combined cognitive-frailty-and-depression status and, with cognitive frailty and depressive symptoms separately in old as well as young community-dwelling adults.

Association between bilirubin and biomarkers of metabolic health and oxidative stress in the MARK-AGE cohort.

Recent studies have shown that elevated concentrations of unconjugated bilirubin (UCB) may be a protective host factor against the development of noncommunicable diseases (NCDs), whereas low levels of UCB are associated with the opposite effect. The results of this European study, in which 2,489 samples were tested for their UCB concentration using high-performance liquid chromatography (HPLC) and additional data from the MARK-AGE database were used for analysis, provide further evidence that elevated UCB concentrations are linked to a lower risk of developing NCDs and may act as a predictive marker of biological aging as individuals with elevated UCB concentrations showed favorable outcomes in metabolic health and oxidative-stress-related biomarkers. These findings underline the significance of studying individuals with moderate hyperbilirubinemia and investigate UCB routinely, also in the setting of aging, since this condition affects millions of people worldwide but has been underrepresented in clinical research and practice until now.

Development and validation of cardiometabolic risk predictive models based on LDL oxidation and candidate geromarkers from the MARK-AGE data.

The predictive value of the susceptibility to oxidation of LDL particles (LDLox) in cardiometabolic risk assessment is incompletely understood. The main objective of the current study was to assess its relationship with other relevant biomarkers and cardiometabolic risk factors from MARK-AGE data. A cross-sectional observational study was carried out on 1089 subjects (528 men and 561 women), aged 40-75 years old, randomly recruited age- and sex-stratified individuals from the general population. A correlation analysis exploring the relationships between LDLox and relevant biomarkers was undertaken, as well as the development and validation of several machine learning algorithms, for estimating the risk of the combined status of high blood pressure and obesity for the MARK-AGE subjects. The machine learning models yielded Area Under the Receiver Operating Characteristic Curve Score ranging 0.783-0.839 for the internal validation, while the external validation resulted in an Under the Receiver Operating Characteristic Curve Score between 0.648 and 0.787, with the variables based on LDLox reaching significant importance within the obtained predictions. The current study offers novel insights regarding the combined effects of LDL oxidation and other ageing markers on cardiometabolic risk. Future studies might be extended on larger patient cohorts, in order to obtain reproducible clinical assessment models.