Expansion and Cell-Cycle Arrest: Common Denominators of Cellular Senescence

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Opinion

Expansion and Cell-Cycle Arrest: Common Denominators of Cellular Senescence Mikolaj Ogrodnik,1,* Hanna Salmonowicz,2 Diana Jurk,1,2 and João F. Passos1,2,* Highlights

Cellular senescence is a major driver of age-related diseases, and senotherapies are being tested in clinical trials. Despite its popularity, cellular senescence is weakly defined and is frequently referred to as irreversible cell-cycle arrest. In this article we hypothesize that cellular senescence is a phenotype that results from the coordination of two processes: cell expansion and cell-cycle arrest. We provide evidence for the compatibility of the proposed model with recent findings showing senescence in postmitotic tissues, wound healing, obesity, and development. We believe our model also explains why some characteristics of senescence can be found in non-senescent cells. Finally, we propose new avenues for research from our model.

Senescence markers are the result of cell-cycle arrest and expansionstimulation signals. Senescent cells increase in vivo when cell-cycle arrest and expansion stimulation are present. Our model explains senescent markers in postmitotic and non-permanently arrested cells.

In Search of a Common Denominator of Senescence Induction Since the seminal discovery of the limited replicative capacity of primary cells [1], which has been interpreted as aging at the cellular level and termed cellular senescence, researchers have been trying to answer two fundamental questions: (i) what are the molecular markers of senescent cells, and (ii) why and how do cells acquire the phenotype of cellular senescence? Regarding the first question, researchers have identified a long list of molecular phenotypes that are characteristic of senescent cells. The most widely used markers include senescenceassociated β-galactosidase activity (SA-β-Gal) at pH 6 and the expression of cell-cycle kinase inhibitors p16 and p21 [2,3]. The full list of proposed markers of senescence is long because induction of senescence alters almost every aspect of cell biology, from epigenetic remodeling of chromatin [4], through changes in the quantity and functionality of organelles [5,6], to the enhanced secretion of proinflammatory molecules that is commonly known as the senescence-associated secretory phenotype (SASP) [7]. Given that a wide range of senescence markers are available, it is thus surprising that the majority of markers show low specificity because their presence has been documented in cells that are not senescent. For example, high SA-β-Gal activity is detected in vitro in confluent cells [8,9] and in activated macrophages [10,11], and p16 and p21 can be induced in a reversible manner in some physiological contexts [10–14]. It is generally accepted in the field that senescence should be determined by utilizing a combination of markers, but it is not well understood why markers of senescence have low specificity in the first place. Regarding the second question, different stressors, such as oncogene activation, telomere shortening, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, and DNA damage, can result in senescence [2,3,15,16]. However, recent findings have questioned whether irreversible cell-cycle arrest (see Glossary) is necessary for the induction of senescence markers. Markers of senescence have been detected in postmitotic cells such as neurons [17–19] and cardiomyocytes [20], among others [21–23]. Although one can question whether these cells are truly senescent, the data indicate that the induction of this phenotype in vivo Trends in Biochemical Sciences, Month 2019, Vol. xx, No. xx

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Department of Physiology and Biochemical Engineering, Mayo Clinic, Rochester, MN, USA 2 Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK

*Correspondence: [email protected] (M. Ogrodnik) and [email protected] (J.F. Passos).

https://doi.org/10.1016/j.tibs.2019.06.011 © 2019 Elsevier Ltd. All rights reserved.

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can lead to consequences similar to those produced by the induction of senescence in proliferation-competent cells (discussed below). Furthermore, senescence, which originally was considered to be an aging-specific phenomenon, is now known to occur early in life during embryogenesis [24–28], in wound healing and regeneration [29,30], and in obesity [31–35]. Currently, the field accepts that all these conditions lead to the induction of senescence; however, the common stimuli that contribute to the initiation of cellular senescence under different physiological contexts are not well characterized. In this article we propose a model to unify the complexity of the cellular senescence phenotype in vitro and in vivo. In so doing, we hypothesize that two types of signals, expansion and cell-cycle arrest signals, induce and maintain senescence in different physiological contexts.

Cell Senescence Requires both Expansion and Cell-Cycle Arrest Signals In Vitro The specific environment under which cells are maintained in vitro contains factors (mostly from fetal bovine serum, FBS) with predominantly mitogenic (e.g., EGF and PDGF) and growthstimulating (e.g., FGF2, IGF1, and IGF2) effects [36]. Because the activities of growth and mitogenic factors overlap, we herein use the term expansion to define any activity that results in either or both increased proliferation or cellular growth (Box 1). On the other hand, factors such as DNA damage, telomere dysfunction, and activation of tumor-suppressor pathways have been shown to contribute to the induction of cell-cycle arrest, and are termed here cellcycle arrest factors. Cells proliferating in vitro are constantly exposed to expansion factors, and cells can only acquire a senescent phenotype when a sufficiently potent cell-cycle arrest stimulus is provided (Figure 1A). Conditions inhibiting expansion signals, such as contact inhibition and serum starvation, lead to quiescence (Figure 1B). Cells maintained in quiescence accumulate lipofuscin [37] as well as DNA double- [38] and single-strand breaks [39], which are known inducers of cell-cycle arrest signals. However, in contrast to proliferating cells, stimulation of quiescent cells with cell-cycle arrest factors was found to be insufficient to induce senescence [40,41]. We use the term senile cells to refer here to quiescent cells exposed to high levels of cell-cycle arrest factors but that are unable to become senescent without expansion stimulation (Figure 1B). Therefore, we hypothesize that a combination of expansion and cell-cycle arrest factors is necessary to induce cellular senescence (Figure 1A). In support, several studies have shown that these senile cells acquire a senescent phenotype only after resumption of the cell cycle (Figure 1B) [37–39,41]. For instance, Marthandan and colleagues observed that cells, which reached senescence upon dividing in culture for 100–150 days, showed similar levels of markers of senescence (SA-β-gal, p21 and p16) to cells stimulated to expand after a 100– 150 day period of quiescence [38]. This finding is potentially relevant in vivo because cells in tissues are predominantly quiescent [8].

Box 1. Causes and Consequences of Cellular Expansion An increase in the volume of a cell culture (expansion) can be achieved by an increase in the number of cells via stimulation of mitogenesis/proliferation and/or by an increase in volume of individual cells (i.e., growth) [36]. Excessive activity of these processes is termed hyperplasia and hypertrophy, respectively. The former term is often associated with tumor growth, and hypertrophy with age-related dysfunction of organs such as muscle, heart, and pancreas. Factors that stimulate growth and proliferation are called growth factors and mitogenic factors, respectively [36]. Although there have been cases of growth and mitogenic factors driving explicitly one type of expansion (e.g., in Schwann cells [110]), in the majority of cases these factors have highly overlapping signaling pathways, and an overall increase in size of a cell population in culture is coordinated by both growth and proliferation.

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Glossary Cell-cycle arrest: an active process which prevents cell-cycle progression. Cell-cycle arrest can be temporary or permanent. Arrested cells show features of the cell-cycle phase in which they were arrested. Cell-cycle arrest factors: factors which, upon reaching a threshold, lead to the induction of cell-cycle arrest: for example DNA damage, oncogene activity, telomere shortening, or the expression of checkpoint genes. Cellular growth: an increase in the volume of an individual cell. Expansion: a process which results in an increase in volume of a cell population. Expansion can be driven by an increase in the volume of individual cells (growth/trophia) and/or by an increase in cell number (driven by proliferation/plasia). Expansion factors: factors which contribute to increased cell growth or number. For example: epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF2), and insulin-like growth factors 1 and 2 (IGF1 and IGF2), among many others. False-positive senescence: a cellular phenotype characterized by the presence of senescence markers without permanent cell-cycle arrest. Hypertrophy: an abnormal increase in cell volume. Lipofuscin: a non-degradable aggregate of oxidized lipids, covalently crosslinked proteins, oligosaccharides, and transition metals. Mitogenic factors: factors which stimulate mitogenesis/proliferation. Postmitotic cells: cells which exit irreversibly from the cell cycle and are in G0 phase. Quiescence: quiescent cells temporarily exit from the cell cycle and are in G0 phase. Senile cells: damaged, quiescent, or differentiated cells which are likely to become senescent upon exposure to expansion factors.

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Figure 1. Concomitant Stimulation of Expansion and Cell-Cycle Arrest Can Explain the Phenotype of Cellular Senescence. (A) Phenotypic features of cellular senescence often require a combination of expansion and cell-cycle arrest factors. This model proposes that exposure to expansion factors explains why cells which are already arrested (whether permanently or temporarily) can acquire a senescent-like phenotype. We propose that cells (under conditions of expansion stimulation) can only be considered to be senescent when they are in an irreversible state of cell-cycle arrest or are non-dividing and are stimulated towards cell-cycle arrest. (B) A combination of expansion (growth and proliferation) and cell-cycle arrest signals (e.g., short telomeres, overexpression of oncogenes, DNA damage) is necessary to induce senescence. Proliferating cells can be induced to senesce by an increase in cell-cycle arrest signals, whereas damaged quiescent or differentiated cells become senescent upon expansion stimulation. Thus, in cells maintained in vitro (green) (i.e., in continuous expansion stimulation), induction of permanent cell-cycle arrest is used to trigger senescence.

Markers of Senescence Are the Result of Continuous Expansion Signals in Arrested Cells When a cell divides, its organelles, cytoplasm, and nuclear content are invariably halved. To compensate for this loss of intracellular structures upon cell division, expansion signals are activated to increase the mass and quantity of cellular organelles [42–44]. An increase in cell size that is not followed by cell division leads to hypertrophy (Box 1). We hypothesize that expansion signals after the induction of cell-cycle arrest are responsible for the increase in cellular and organellar size that is characteristic of senescent cells (Figure 2). In fact, several studies indicate that an increase in the size and quantity of organelles such as the nucleus, lysosomes, and mitochondria represents a set of markers of senescence, including SA-β-Gal activity, which correlate with lysosomal mass [45,46], karyomegaly [32,47], and mitochondrial mass increase [5,48–50], respectively. Similarly, interventions to reduce the expansion activity of cells, such as rapamycin, metformin, or JAK inhibitor treatments, have been shown to alleviate many of the phenotypes of senescent cells [5,51–54]. It should be noted, however, that some of these features may not be universally found in all senescent cells. Although recent studies have shown that senescent cells positive for SA-β-Gal in multiple tissues from aged mice are characterized by an increase in cell size [55], only a limited number of tissues have been analyzed. In addition, although increased mitochondrial mass occurs in different senescent cell types such as fibroblasts [5] and pancreatic β-cells [56], senescent CD8 T cells for instance exhibit a decrease in mitochondrial mass [57]. On the other hand, cell-cycle arrest factors underlie the phenotypic manifestations of processes aimed at reinforcing the arrest; these processes include epigenetic remodeling, that includes senescence markers such as the formation of senescence-associated heterochromatin foci Trends in Biochemical Sciences, Month 2019, Vol. xx, No. xx

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Figure 2. Commonly Used Markers of Senescence Derive from Expansion and Cell-Cycle Arrest Stimuli. We propose that the spectrum of senescence markers can be essentially divided into those predominantly derived from expansion signals (green), others predominantly derived from cell-cycle arrest signals (orange), or a combination of the two (pink). Expansion signals result in increased cell and organelle size, which is associated with senescence-associated-βgalactosidase (SA-β-Gal), karyomegaly, and mitochondrial mass increase. Features originating from cell-cycle arrest signals include expression of cell-cycle inhibitors p16 and p21, specific epigenetic modifications (such as senescenceassociated decondensation of satellites, SADS), and nuclear exclusion of high mobility group B1 (HMGB1) protein. Finally, features originating from both types of signals include mitochondrial dysfunction, the senescence-associated secretory phenotype (SASP), accumulation of lipids in senescence (ALISE), and lipofuscin accumulation.

(SAHF) [4] and senescence-associated decondensation of satellites (SADS) [58], as well as p53and p16-dependent nuclear exclusion of high mobility group protein B1 (HMGB1) [59]. Finally, some phenotypes of senescence have been shown to be dependent on both expansion and cell-cycle arrest signaling. One such example is mitochondrial dysfunction, a characteristic of senescent cells [49,50,60]. Mitochondria in senescent cells are characterized by increased levels of reactive oxygen species (ROS), decreased mitochondrial membrane potential, and increased proton leakage [50,51]. Interestingly, there is evidence that mitochondrial dysfunction may be a consequence of expansion signaling-related hypertrophic growth of senescent cells. A series of studies from the field of allometry showed that mitochondrial function declines with increasing cell size [61–63]. It has been reported that, in larger cells, a higher cytoplasmic volume causes alterations in mitochondrial dynamics [64], as well as affecting the trafficking of mitochondrial metabolites including oxygen and nutrients [63]. Thus, it is possible that the increased cellular size observed in senescent cells contributes to the reported mitochondrial dysfunction. In accord, alleviation of mitochondrial dysfunction during senescence can be achieved via inhibition of expansion signals, for instance via mTOR [5] and p38 MAPK [49]. On the other hand, cell-cycle arrest signals have also been shown to play a role in mitochondrial dysfunction. In oncogene-induced senescence, mitochondrial dysfunction has been shown to be dependent on either p53 or Rb [50]. Moreover, activation of either p53 or p21 has been shown to induce senescence in a ROS-dependent manner, presumably of mitochondrial origin [65,66]. Finally, the DNA damage response has been shown to directly contribute to 4

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mitochondrial dysfunction during senescence via the activation of Akt and mTORC1 [5]. Thus, the data consistently suggest that cell-cycle arrest signals interact with expansion signals to contribute to mitochondrial dysfunction during senescence. Another example of a senescent phenotype, which is dependent on both cell-cycle and expansion signals, is the SASP [7]. This phenotype has been shown to be dependent on expansion signals such as mTOR [51,52] and p38 MAPK signaling [67]. However, some components of the SASP such as HMGB1 secretion are dependent on cell-cycle arrest signals such as p53 [59], and the SASP has been shown to be dependent on the DNA damage response [68]. These examples further highlight the intricate interplay between cell-cycle arrest and expansion during the establishment of the senescent phenotype.

Accumulation of Damage Is Necessary but Not Sufficient To Trigger Senescence during Aging The frequency of senescent cells has been reported to increase during aging and contributes to the pathologies of age-related diseases [69,70] and cancer [70–72]. An increase in intracellular damage occurs in almost every chronologically aged cell [18,20,32,73]; however, other markers of senescence, such as SA-β-Gal, are present in only a very small proportion of cells [20,74]. For example, we have observed that, in the hearts of old mice, ~80% of cardiomyocytes showed telomere-associated damage and p21 positivity, whereas the frequency of SA-β-Gal-positive cells was below 5% [20]. These results are consistent with another recent study which quantified SA-β-Gal in different mice tissues using flow cytometry combined with high-content image analysis [55]. Using this method, the authors reported relatively low frequencies of SA-β-Gal-positive cells in different tissues of aged mice, in contrast to the much higher frequencies of markers of intracellular damage [18,20,32,73,75]. We hypothesize that intracellular damage, functioning as a driver of cell-cycle arrest signals, is not sufficient by itself to trigger some features of the senescent phenotype, and thus requires an additional contribution of expansion factors. We believe that including expansion signaling as a necessary element of senescence induction allows a better understanding of the interaction between senescence and age-related diseases (Box 2) and cancer (Box 3).

Box 2. The Impact of Cellular Senescence on Aging and Age-Related Diseases The length of life (lifespan) can be measured as average lifespan and maximum lifespan (determined by rate of aging). In practical terms, average lifespan is usually increased by alleviating age-related diseases, whereas maximum lifespan or the rate of aging is increased by slowing down the basic mechanisms of aging [3]. Do we have evidence that cellular senescence causes age-related diseases? Cells bearing senescent markers have been shown to accumulate during aging in multiple tissues from mice, baboons, and humans [2,32,74,111,112], as well as in several age-related diseases [32,69,74,107,113–116]. Clearance of senescent cells has been reported by several groups to alleviate age-related diseases [32,69,74,107,113–116] and extend average lifespan in mice [74]. More specifically, alleviation of age-related conditions, such as frailty [74,113], atherosclerosis [116], osteoporosis [107], liver steatosis [32], and osteoarthritis [117], has been described in several studies where mice were treated with compounds which are thought to specifically eliminate senescent cells (named senolytics). The data suggest that the detrimental effects of senescent cells in tissues occur mostly through chronic exposure to the SASP. Do we have evidence that cellular senescence determines the rate of aging? On the one hand, published studies did not find evidence that elimination of senescent cells greatly affects the maximum lifespan of mice [69,74,118]. These findings suggest that cellular senescence is predominantly responsible for the pathologies of aging but does not necessarily affect the rate of aging [3]. On the other hand, it should be pointed out that additional lifespan experiments should be performed by independent laboratories to effectively test this hypothesis. Furthermore, there are several limitations to the mouse models used for clearance of senescent cells in terms of their efficiency to clear senescent cells – which is variable between different tissues, as in the case of the INK-ATTAC mouse model where clearance is restricted to p16-positive senescent cells and might not distinguish between senescent cells and false-positive senescent cells.

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Box 3. Senescence and Risk of Cancer Development Senescence has been hypothesized to have evolved as an anticancer mechanism [70–72]. Numerous mouse studies have shown that impairment of senescence induction increases the risk of cancer development [119–121]. The curve of the incidence of cancer diagnosis as a function of age [122] (Figure I) shows that the all-cause risk of cancer development increases during aging; however, it also shows a temporary increase during childhood and a tendency to plateau later in life. Is there any association between cancer incidence and the kinetics of senescence burden during aging? We hypothesize that if the total quantity of senescent cells is determined by the superimposed concentrations of expansion and cell-cycle arrest signals, one could then speculate that the kinetics of senescence during aging would bear a high similarity to the cancer diagnosis curve (Figure I). The lowest concentration of these senescence-driving factors would be found in young organisms, but only after the cessation of the growth. Earlier in life and during embryogenesis, the demand on rapid expansion is likely to generate senescent cells despite a limited impact of cell-cycle arrest factors derived from damage. After development, the availability of expansion factors slowly declines during aging. However, we hypothesize that age-dependent damage accumulation contributes to the activation of cell-cycle arrest signals and can explain the increase in senescent cell burden. Finally, we predict that, later in life, as the availability of growth factors declines to its minimum level [123], the rate of senescent cell accumulation may plateau, paralleling the plateau of cancer incidence. Careful kinetic studies of changes in senescence taking place during aging will need to be conducted to answer these questions.

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Figure I. Association between the Risk of Cancer and the Kinetics of Senescent Cell Accumulation during Aging. The curve of incidence of all-cause cancer diagnosis as a function of age [122] in humans increases temporarily early in life and starts to increase exponentially after sexual maturity and growth cessation. However, the risk of cancer development levels plateaus later in life. Markers of senescence have been reported in early life events, including embryogenesis and in post-birth developmental stages, and to increase during aging. We hypothesize that the levels of expansion factors (pink background) are highest early in life and decrease linearly during aging. By contrast, intracellular macromolecular damage and other factors known to induce cell-cycle arrest (green background) have been reported to increase during aging. Similarly to cancer, we propose that the induction of cellular senescence is influenced by the presence of both expansion factors and cell-cycle inhibitory factors. It remains to be determined experimentally if the frequencies of senescent cells also plateau at later stages of life, consistent with low exposure to expansion signals.

There is evidence that expansion signals contribute to the accumulation of senescent cells during aging in vivo. For instance, dietary restriction (DR), an intervention which has been shown to extend lifespan in a variety of species [76], inhibits expansion signals. Consistently, DR has been shown to reduce the rate of wound healing [77] and, owing to reduced proliferation rate 6

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of immune cells, increases susceptibility to bacterial and viral infections [78]. Consistent with the idea that expansion signals are necessary for senescence in vivo, several studies indicate that DR greatly reduces the rate of accumulation of senescent cells in aging mice [18,32,79–81] and humans [81]. Moreover, growth hormone signaling-deficient Snell dwarf and growth hormone receptor (GHR)-knockout mice, which are known for their exceptional longevity [82], show a reduction in senescence markers [83]. By contrast, overexpression or injection of growth hormone has been shown to increase senescent cell burden [83]. Finally, the mTOR inhibitor rapamycin, which increases the lifespan of mice [84] and inhibits expansion signals, has been shown to reduce the frequency of some senescent markers during aging [5,60]. The different degrees of expansion and cell-cycle arrest factors could also explain why the fraction of senescent cells differs between organs of aging animals and why certain regions in tissues are more prone to senescence accumulation. For example, it was documented, in aging mice, that senescent hepatocytes are found more often in regions proximal to the central vein rather than to the portal tract [80], which corresponds to a higher proliferative capacity of the central vein region [85,86]. Similarly, cells in the periventricular area of the murine brain, a region of high expansion potential [87], are particularly susceptible to the induction of senescence during obesity [33]. Together, these data have led us to hypothesize that a combination of cell-cycle arrest and expansion signals is necessary for the accumulation of senescent cells in vivo.

Hyperexpansion Conditions Explain the Presence of Senescence Outside Aging Cells bearing senescent markers occur very early as a response to a cutaneous wound [30]. In a similar manner, there is a relationship between markers of senescence and tissue regeneration [29,30,41]. Why do senescent cells arise under these conditions? We hypothesize that the wound-healing process shares many of the factors required for senescence, both in terms of expansion and cell-cycle arrest signals. Tissue damage drives the activity of expansion signals in the traumatized area to promote cell proliferation and growth, migration of immune cells into the wound area, angiogenesis, and matrix remodeling [88]. Secondly, wound healing is also characterized by inflammation and increased production of ROS [88,89] that have been shown to drive cell-cycle arrest signals [90]. Thus, it is unsurprising that markers of senescence have been detected in activated fibroblasts [30] and macrophages [10,11,29] during wound healing, regeneration, and in conditions of increased inflammation. Liver regeneration provides an example that well illustrates the dependency of costimulation of cell-cycle arrest and expansion factors in organ injury [41]. Liver has a high regenerative capacity, and, even following major injury or removal of up to 70% of the liver, the growth and proliferation of hepatocytes is able to completely restore liver mass and function. Satyanarayana and colleagues [41] observed that the presence of critically short telomeres, as is found in the third generation of telomerase-deficient Terc−/− mice, is insufficient to significantly increase the frequency of senescent hepatocytes in the liver. However, this study showed that stimulation of expansion signals following partial hepatectomy (PH) highly increases the frequency of senescence markers SA-β-Gal and p21 in late-generation Terc−/− but not wild-type mice [41]. At the same time, p16 and p19 were not significantly changed following PH [41]. Overall, these data indicate that neither growth stimulation (as during PH in wild-type mice) nor cell-cycle arrest stimulation (as in late-generation Terc−/− mice) is sufficient, and that only the combination of both drives some features of hepatocyte senescence [41]. Trends in Biochemical Sciences, Month 2019, Vol. xx, No. xx

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A similar phenomenon was observed in the context of muscle aging. It was shown that muscle satellite cells (which are quiescent) show increased levels of the cell-cycle inhibitor p16 in old mice, and are incapable of regenerating the muscle of an injured young host following transplantation [91]. Importantly, it was found that satellite cells only start to express senescent markers such as SA-β-Gal, DNA damage, p19, and components of the SASP upon transplantation to young mice [91]. This suggests that exposure to expansion signals is required for the establishment of the senescent phenotype. Recently, senescence has also been shown to play an important role in obesity. Markers of senescence have been observed in liver [32], fat [34,35], and brain [33] of obese mice, and clearance of senescent cells has been shown to impact positively on organ function [32,33]. Why, however, does senescence occur during obesity? A robust body of evidence demonstrates that obesity is associated with increased concentrations of expansion factors, including insulin and IGF1, but is concomitantly associated with reduced concentrations of negative regulators of IGF1, such as IGFBP1 and IGFBP2 [92,93]. The function of tissues susceptible to obesity-induced senescence, such as visceral fat [34,35] and liver [32], is also highly dependent on signaling by expansion factors, including IGFs and insulin. Interestingly, administration of recombinant IGF1 was sufficient to induce senescence in the liver of a mouse model of nonalcoholic steatohepatitis (NASH) [94]. On the other hand, treatments counteracting obesity and downregulating levels of circulating expansion factors, such as diet-induced weight loss [95] and physical exercise [35], are sufficient to reduce senescent cell burden. Apart from expansion factors, obesity is also associated with an increased availability of nutrients, the induction of chronic low-grade inflammation [92,93], and increased generation of ROS [96], which can further feed into the expansion and cell-cycle arrest signals that are necessary for senescence. Furthermore, an increase in cells showing features of senescence has been observed in the developing embryo [24,25], fetal membranes, placenta, and decidua/uterus [26–28]. Interestingly, it was suggested that senescent cells, detected under such conditions, share signatures with oncogene-induced senescence [24] which, in light of the framework presented here, could be explained by a predominant role of expansion factors in the induction of cellular senescence.

Senescent Markers Can Be Found in Cells Which Are False Positive for Senescence In this article we propose that a combination of expansion and cell-cycle inhibitory signals is required for the induction and establishment of cellular senescence. We further hypothesize that, under circumstances that resemble senescence-inducing conditions, cells can exhibit senescent markers but still retain the potential to proliferate, a phenotype we have named false-positive senescence. For example, elevated SA-β-Gal was observed in confluent fibroblast cultures, as well as in immortal fibroblast cultures at high cell density [8,9]. In in vitro settings, confluent cells are maintained under continuous expansion signaling simultaneously with cellcycle arrest signals (contact inhibition); this is insufficient, however, to induce permanent cell-cycle arrest [36]. Contact inhibition-mediated cell-cycle arrest and senescence-mediated cell-cycle arrest share overlapping features such as increased expression of p16 [97] and p27 [98], as well as activation of p38-MAPK [99]. Regarding expansion signaling, on the one hand, it has been suggested that some expansion pathways, such as mTOR, are disabled in confluent cells [100]. On the other, some studies have shown that contact inhibition does not prevent the activation of upstream elements of mitogenic pathways [101,102]. The incomplete shutdown of expansion signaling in confluent cells could be due to the monolayer nature of in vitro growth conditions. Accordingly, cells maintained in 3D cell cultures are characterized by a more physiological 8

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response to contact inhibition and by more specific inhibition of expansion signaling [103,104]. It remains to be determined whether 3D cell cultures display false-positive markers of senescence. Another example of the unspecificity of senescence markers has been observed during macrophage activation [10,11,105]. Tissue damage, phagocytosis, and inflammation, that are known to induce polarization of macrophages towards M2 phenotypes, result in the simultaneous expression of numerous markers of senescence including p16 and SA-β-Gal [10,11,105]. The increased expression of p16 in M2 macrophages is highly problematic for studies using p16 expression-driven killing switches to clear senescent cells because it raises the question of whether the reported effects result from the elimination of senescent cells or M2 macrophages. Based on this evidence, we propose that the major factor differentiating between senescence and false-positive senescence is the irreversibility of the cell-cycle arrest (Box 4). However, it remains a challenge to test this idea experimentally owing to the lack of methods to examine the kinetics of cell division in vivo. Measuring changes which have been associated with the irreversibility of cell-cycle arrest, such as SAHF [4] and SADS [58], may be more informative than assessment of the expression of cell-cycle arrest signals. In addition, increased cell size may also be a useful marker, particularly because it has been recently suggested to be the driver of irreversible cell-cycle arrest [105] (Box 4). Most senescent cell types are characterized by increased cell size in vivo [55]; however, although macrophages change shape following M2 activation [106], they do not change cell size despite expressing SA-β-Gal.

Expansion Stimulation of Damaged, Non-Dividing Cells Explains Postmitotic Senescence Recent work by several groups has indicated that postmitotic cells such as neurons, adipocytes, osteocytes, osteoblasts, retinal ganglion cells, and cardiomyocytes can acquire a multitude of senescent markers during aging [17–23]. This led several investigators to suggest that senescence may not be a phenomenon exclusive to proliferation-competent cells, and can occur in postmitotic cells [106]. Although it may be argued that the activation of senescent pathways Box 4. Hypertrophy and the Irreversibility of Cell-Cycle Arrest in Senescence What defines the irreversibility of cell-cycle arrest in senescence? An answer to this question is not simple because the two pathways responsible for the induction of cell-cycle arrest, p21 and p16, have been shown to be reversible under some conditions [10,124,125]. One possibility is that a specific threshold of p16/p21 expression level must be reached to produce irreversible cell-cycle arrest. Another possibility involves a hypertrophic increase in senescent cell size. A recently published article has provided evidence that an overall increase in cell size may prevent resumption of proliferation [126]. In this study it was shown that, upon induction of temporary cell-cycle arrest (via pharmacological inhibition of Cdk4/6), cells in culture do not stop increasing their volume, which leads to continuous, hypertrophic growth. This temporary cell-cycle arrest resulted in only a twofold increase in cell volume (compared to ~eightfold increase in senescence), but was coupled with an inability of 95% to resume proliferation upon release from arrest. Similarly, cells with a knockout or a knockdown of Cdk1, when exposed to expansion-permissive conditions (cell culture or partial hepatectomy), increase in size and express markers of senescence [127,128]. The researchers stated that cytoplasm dilution and an increase in the cytoplasm/DNA ratio disrupts progression through later phases of the cell cycle [126]. An alternative mechanism for hypertrophy-driven irreversibility of cell-cycle arrest is a cell-size threshold for G1 exit, as suggested by several studies [42,129–131]. Although it is not known whether hypertrophy contributes to the irreversibility of the cell-cycle arrest in vivo, there is evidence for a relationship between cell size and the phenotype of cellular senescence. For example, pancreatic β cells positive for markers of senescence including p16 and SA-β-Gal were found to be significantly larger than cells negative for markers of senescence [56]. We found that hepatocytes with enlarged nuclei (karyomegalic) show increased levels of senescence markers, including oxidative lipid damage (4-HNE), telomere-associated foci (TAF), and centromere decondensation [32]. Similarly, a direct relationship between cell area and TAF frequency was observed in aging cardiomyocytes [20]. Finally, Biran and colleagues have shown that SA-β-Gal-positive cells isolated from fat, spleen, intestine, lymph nodes, and lungs show a 30–70% increase in cell size [55]. Nevertheless, the impact of cell size on the irreversibility of the senescence arrest in vivo has not been demonstrated.

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is irrelevant in terminally differentiated cells, it is possible that postmitotic senescence has important roles in vivo. For example, genetic or pharmacological clearance of p16-positive cells has been shown to result in a decrease in markers of senescence in postmitotic osteocytes, adipocytes, cardiomyocytes, and neurons, which is accompanied by beneficial effects on healthspan [20,33,107]. The mechanisms by which senescence occurs in postmitotic cells are less clear; however, evidence points to their dependence on the activation of cell-cycle arrest signals. For instance, telomere dysfunction has been associated with neuronal senescence in a p21dependent manner [18]. In addition, senescence of adipocytes, cardiomyocytes, neurons, and osteocytes is characterized by increased expression of cyclin-dependent kinase inhibitors and the presence of DNA damage [20,21,33,54,107]. Consistent with our hypothesis, the data argue that induction of postmitotic senescence also requires expansion signals. For instance, in a study where mice were exposed to whole-brain X-ray irradiation, senescence markers associated with cell-cycle arrest, including p21 expression and an increase in DNA damage, were observed in neurons coupled with reentry into the cell-cycle, a process that itself requires expansion factors [108]. Although there is currently no evidence to state the causality of cell-cycle reentry processes in the induction of postmitotic senescence, we hypothesize that the stimulation of these processes may be an important determinant of the senescent phenotype. Further supporting this hypothesis, DR, which inhibits expansion signals, has been shown to reduce the number of senescent markers in postmitotic cells such as Purkinje neurons [18], whereas obesity, which enhances expansion signals, has been shown to increase senescence in neurons in the amygdala [33]. Furthermore, obesity has been shown to induce senescence characterized by SA-β-Gal, p53, and p21 expression, as well as by SASP, in postmitotic adipocytes [21]. Importantly, adipocyte senescence was shown to be dependent on p53 expression, suggesting again that an interplay between expansion signals and cell-cycle inhibitory signals is necessary for this phenotype to occur [21]. Cardiomyocytes provide another example of a postmitotic cell type that has been shown to senesce as a result of the interaction between expansion and cell-cycle arrest factors. Cardiomyocytes, despite being essentially postmitotic, show increased DNA damage with age as well as downstream activation of cyclin-dependent kinase inhibitors p21, p15, and p16 [20]. Importantly, we found that increased cellular size and the expression of hypertrophy-related genes are directly correlated with markers of senescence [20]. Consistently, genetic and pharmacologic elimination of senescent cells [20] and mTOR inhibition [60] reduced cardiomyocyte hypertrophy in aged [20] and hyperinflammatory mice [60], further illustrating the dependency of senescence on expansion signaling.

Concluding Remarks Given recent evidence that elimination of senescent cells leads to an extension in average lifespan [74], and ongoing clinical trials of senotherapies [109], interest in cellular senescence has increased exponentially. At the same time, senescent cells have been shown to be involved in an increasing number of processes in vivo, and multiple studies indicate that senescence involves complex molecular pathways across different cell types and physiological contexts. Therefore, we propose here a model in which the induction of cellular senescence requires signals of both expansion and cell-cycle arrest. This, we propose, can explain why senescence occurs in such varied processes such as aging, wound healing, development, and obesity. However, we acknowledge that this model may not explain all the features of senescence and that further experimental validation is required (see Outstanding Questions). 10

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Outstanding Questions Can molecular markers enable us to distinguish between classic senescent cells and false-positive senescent cells in vivo? What is the rate of senescent cell accumulation during aging? Further research will be necessary to establish how the processes of senescence induction and elimination impact on the frequency of senescent cells in different tissues during aging. To test our hypothesis that reduced availability of expansion factors later in life may result in a plateau in senescent cell accumulation, the detailed kinetics of senescent cell accumulation during the lifetime of an organism needs to be established. The development of reporter mouse models where other senescent markers, beyond p16positive cells, can be detected would greatly improve our understanding of the kinetics of senescence in vivo. Does expansion stimulation of senile cells induce senescence in vivo? We hypothesize that, upon expansion stimulation, stem cells may acquire senescent-like phenotypes. This hypothesis can be tested by molecular characterization of stem cells isolated from aged mice in which expansion signals are stimulated or repressed. In addition, genetic lineage-tracing studies combined with characterization of senescence markers should allow us to investigate the impact of these signals on stem cell senescence. What is the relevance of senescence in postmitotic cells? Health benefits have been observed in postmitotic tissues of mice following systemic clearance of senescent cells. However, it is not clear if these effects are due to the clearance of postmitotic senescent cells or to the clearance of classic senescent cells located within the tissue or elsewhere in the body. The development of mouse models which allow temporal and cell type-specific clearance of senescent cells should allow us to answer some of these questions. What are the long-term consequences of clearance of postmitotic senescent cells? Such clearance has been associated with improvements in tissue function during aging; however, it is

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Acknowledgments M.O. is supported by the Glenn Foundation for Medical Research Postdoctoral Fellowship PD19217. J.F.P and H.S. are supported by the Biotechnology and Biological Sciences Research Council (BBSRC) grants (BB/H022384/1 and BB/K017314/1) and the Ted Nash foundation. D.J. was funded by the Academy of Medical Sciences (SBF003_1179). We thank Professor Ewa Sikora for valuable comments.

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