The senescent phenotype and promiscuous gene expression

Senescent cells are often associated with changes in gene expression that appear to occur independent of the regulated gene expression linked to aspects of the senescent phenotype such as cell cycle arrest, the secretory response and apoptosis resistance. This phenomenon has been termed promiscuous gene expression (pGE) (Burton and Krizhanovsky, 2014) and can be more specifically defined as gene expression that is uncoupled from tissue or developmental regulation. 

pGE can be observed in microarray analysis by comparing the gene expression profiles of different senescent cell types and lines. Zhang et al (2003) has demonstrated that the up-regulation of genes in senescent fibroblasts was associated with gene clustering (150 of the 376 gene up-regulated), whereas the down-regulation of genes (313) was not. 48.1% of the up-regulated genes were designated as membrane-associated proteins, 10.5% related to apoptosis and 15.8% to transport, whereas 17.9% of the down-regulated genes are involved in cell cycle regulation. Gene expression changes in senescent human mammary epithelial cells (HMECs) were shown to be drastically different than that of the fibroblasts, despite both undergoing senescence induced by telomere attrition. Only five genes up-regulated and seven genes down-regulated in HMECs showed similar regulation in fibroblasts. However, like senescent fibroblasts, HMECs also demonstrated gene clustering associated with up-regulated genes only. Zhang et al postulated at the time, that if senescence is a response to DNA damage, then the observed differences in gene expression between senescent fibroblasts and HMECs imply that the effects of DNA damage must vary according to cell type and line. This study also suggested that processes occurring during senescence may lead to localized alteration in chromatin and the consequent up-regulation of groups of genes within “opened” domains. 

Shelton et al (1999) also demonstrated that senescence-mediated gene expression between different cell lineages varies greatly. BJ fibroblasts, HUVECs and retinal pigment epithelial cells (RPE340) that underwent replicative senescence demonstrated substantial variation in gene expression. A genomic comparison of three different senescent fibroblasts strains also demonstrated significant differences in gene expression, but also shared trends were apparent. If indeed pGE is uncoupled from tissue or developmental regulation, then stochastic processes that alter chromatin structure could be at play and the different response between cell types and cell strains could reflect differences in cell-specific chromatin architecture important for cell-specific gene expression. Elevated levels of oxidative stress, a feature of senescent cells could be one such stochastic process. 

Bahar et al demonstrated that although gene expression levels varied among cardiomyocytes taken from hearts of young mice, the heterogeneity is elevated with age (Bahar et al. 2006). This increased stochastic gene expression with age was suggested to be the result of genomic damage, as mouse embryonic fibroblasts treated with hydrogen peroxide in culture resulted in significant cell-cell variation in gene expression in conjunction with these cells showing morphological signs of cellular senescence (Bahar et al. 2006). 

So how could DNA damage induced by oxidative stress result in stochastic changes in gene expression? When cells sustain DNA damage, chromatin undergoes remodeling to facilitate DNA repair (Price and D’Andrea, 2013, House et al. 2014). This remodeling or “opening” of tightly packed DNA could allow transcription factors access to previously inaccessible genes. Therefore, persistent DNA damage and consequently continuous chromatin remodeling may facilitate pGE. While the induction of DNA damage is likely a stochastic process, the sites of DNA damage may not be completely random, as certain areas of the genome may be more or less prone to genomic insults (Ma et al. 2012). The clustering phenomenon reported by Zhang et al may be the result of these DNA damage prone sites (Zhang et al. 2003). If this were indeed the case, while there may be substantial differences in gene expression at a cell-cell comparison, an overall comparison between cell cultures would likely demonstrate consistent gene alterations resulting from an average expression of all cells within a culture. 

In addition to oxidative stress, a number of other possible mechanisms may exist for generating pGE. Senescent fibroblasts are known to undergo methylation changes (Cruickshanks et al. 2013) and these alterations may lead to epigenetic alterations that promote stochastic changes in gene expression. Alternatively, it has been suggested that DNA damage may modulate gene expression by altering the binding capacity of transcription factors (Rose et al. 2012). 

Interestingly, the reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs) via the addition of OCT4, SOX2, KLF4 and MYC (OSKM) requires a long stochastic phase of gene activation associated with changes in histone modifications at somatic genes and activation of DNA repair and RNA processing (Buganim et al. 2013). This stochastic gene expression may be the result of “promiscuous binding” by OCT4, SOX2 and KLF4, where they occupy accessible chromatin and bind to promoters of genes that are active or repressed (Buganim et al. 2013). It is possible that pGE in senescent cells partly mimics stochastic gene activation associated with cellular reprogramming. However, whether pGE in senescent cells is associated with factors that can undergo “promiscuous binding” has yet to be determined. 

Whether pGE plays a functional role in cell senescence has yet to be determined. However, it can be speculated that pGE may function to generate an array of tissue-restricted proteins that can subsequently be processed into peptides by autophagic proteases for presentation on MHC molecules (Dengjel et al. 2005). Similar to the presentation of tumour-associated antigens (Reuschenbach et al. 2009), senescent cells may also present antigens that can be recognized by immune cells, thereby becoming antigen-presenting cells (APCs). Although the up-regulation of MHC molecules on senescent cells have yet to be fully evaluated, the up-regulation of MHC class I but not MHC class II in response to DNA damage in fibroblasts has been reported (Tang et al. 2014). It remains to be determined whether pGE is a component of immunogenic conversion.

Taken from: Cellular Senescence: From Growth Arrest to Immunogenic Conversion  

Immune Ligand Expression in Senescent Cells

In addition to secreting soluble factors for the attraction of immune cells, senescent cells can also become immunogenic through the up-regulation of ligands that can specifically be recognized by immune cells.  While research into the recognition and interaction of immune cells with senescent cells is at its infancy, a number of studies have reported the up-regulation of the Natural Killer Group 2D (NKG2D) ligands in senescent cells that can be recognized by receptors on Natural Killer (NK) cells and CD8+ T-cells.  Since NKG2D ligands are not widely expressed on healthy cells, this would allow for specific recognition, interaction and elimination of senescent cells by immune cells.  As with the senescent secretome, this response is likely not exclusive to cell senescence as the same mechanism functions in immunosurveillance of tumour cells (López-Soto et al. 2014).  The human NKG2D ligands primarily consist of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6.  The transcriptional up-regulation of MICA and ULBP2 during cell senescence have been reported in senescent activated hepatic stellate cells, replicative senescent fibroblasts and HUVECs, etoposide-induced senescent fibroblasts, fusion-induced senescent fibroblasts and chemotherapy-induced senescent multiple myeloma cells (Krizhanovsky, et al. 2008, Kim et al. 2008 Chuprin et al. 2013, Soriani et al. 2014, Lackner et al, 2014).  In addition to MICA and ULBP2, microarray analysis of replicative senescent fibroblasts demonstrated an increase in the expression of ULBP1 (2.75 fold) compared to growing cells, in addition to the up-regulation of HLA-E (2 fold) (Lackner et al. 2014).  HLA-E is a non-classical MHC class I molecule that plays a role in cell recognition by NK cells. However, replicative senescent vascular smooth muscle cells do not appear to up-regulate MICA, ULBP2 or ULBP1, at least not greater than 2 fold as assessed by microarray analysis (Burton et al. 2009).  Therefore, it should not be assumed that all senescent cell types up regulate NKG2D ligands and this should be evaluated in underexplored senescent cell types. Mechanisms involved in the interaction of senescent cells with T-cells is less understood, but it appears that major histocompatibility complex class II (MHCII) expression is required for killing of pre-malignant senescent hepatocytes by T-cells (Kang et al. 2011).  Mice with liver specific MHCII deficiency resulted in impaired immunosurveillance of senescent cells.

At the mechanistic level, little is currently known about the regulation of NKG2D ligand expression in senescent cells.  Nonetheless, some extrapolation from others models is possible.  For example, MICA and MICB have been reported to be regulated by endogenous miRNAs in tumours and as a result of infection with cytomegalovirus (Stern-Ginossar et al. 2008).  Since miRNAs appear to play a role in regulating cellular senescence (Feliciano et al. 2011, Liu et al. 2012 Benhamad et al. 2012) and their expression is altered in response to DNA damage (Dolezalova et al. 2012, Wang and Taniguchi, 2013), it is possible that changes in miRNA expression also regulate the expression of immune ligands in senescent cells. 

Soriani et al demonstrated that the up-regulation of MICA in senescent multiple myeloma cells was dependent upon the DDR (Soriani et al. 2014).   In other systems, NKG2D ligands have also been shown to be up-regulated in response to DNA damage and Ras activation via ATM and ATR (Gasser et al. 2005, Cerboni et al. 2014).  Inhibition of the ATM or ATR pathways prevented the up-regulation of immune ligands. 

It is also possible that the up-regulation of immune ligands on senescent cells is mediated via the secretory response.  In addition to activating and attracting immune cells, the senescent secretome may serve to up-regulate immune ligands in an autocrine or paracrine manner.  It has been shown for example, that TNFα can up-regulate MICA on human endothelial cells and that the addition of exogenous MICA seems to induce senescence in HUVECs (Lin et al. 2011), but the extent to which this occurs under more physiologically reflective situations remains unclear. 

Immune ligands can also be up-regulated in response to various other forms of cell stress such as heat shock, metabolic stress and endoplasmic reticulum (ER) stress (Cerwenka, 2009, Valés-Gómez et al. 2008).  Thus, as with the secretory response, mechanisms exists that can up-regulate immune ligands independent of DNA damage.  Given that this is an important aspect of senescent cell clearance and the number of cell types in which the up-regulation of immune ligands has been shown is limited, a more detailed study of this aspect of immunogenic conversion seems warranted.

While senescent cells are likely eliminated by the immune system during normal physiological processes, it has been speculated that the accumulation of senescent cells with age could be due to inefficient elimination by an ageing immune system (Burton, 2009).  In fact, immune cells may themselves undergo cellular senescence, a process that requires further investigations (Effros et al. 2005, Rajagopalan et al. 2012). As such, induction of cell senescence in immune cells may represent one aspect of immunosenescence, the gradual deterioration of the immune system, which consequently leads to impaired immunosurveillance of non-immune senescent cells.  It can be speculated that impaired immunosurveillance may result from altered expression of surface receptors on immune cells that impair recognition and interaction with target senescent cells (and cancer cells).  In addition, it is possible that aged or senescent immune cells do not respond as efficiently to chemoattractants secreted by senescent cells.  In order to understand the mechanisms associated with age-related changes resulting in impaired immunosurveillance of senescent cells, we must first fully understand the normal processes governing immune clearance of senescent cells.  However, evaluating the hypothesis that aged or senescent immune cells display a reduced capacity to target senescent cells and the physiological impact of this decline can still be assessed.  If this were indeed found to be the case, the rejuvenation of an ageing immune system would represent an attractive approach for promoting health span.

Taken from: Cellular Senescence: From Growth Arrest to Immunogenic Conversion

Cellular senescence: from growth arrest to immunogenic conversion

Abstract

Cellular senescence was first reported in human fibroblasts as a state of stable in vitro growth arrest following extended culture. Since that initial observation, a variety of other phenotypic characteristics have been shown to co-associate with irreversible cell cycle exit in senescent fibroblasts. These include (1) a pro-inflammatory secretory response, (2) the up-regulation of immune ligands, (3) altered responses to apoptotic stimuli and (4) promiscuous gene expression (stochastic activation of genes possibly as a result of chromatin remodeling). Many features associated with senescent fibroblasts appear to promote conversion to an immunogenic phenotype that facilitates self-elimination by the immune system. Pro-inflammatory cytokines can attract and activate immune cells, the presentation of membrane bound immune ligands allows for specific recognition and promiscuous gene expression may function to generate an array of tissue restricted proteins that could subsequently be processed into peptides for presentation via MHC molecules. However, the phenotypes of senescent cells from different tissues and species are often assumed to be broadly similar to those seen in senescent human fibroblasts, but the data show a more complex picture in which the growth arrest mechanism, tissue of origin and species can all radically modulate this basic pattern. Furthermore, well-established triggers of cell senescence are often associated with a DNA damage response (DDR), but this may not be a universal feature of senescent cells. As such, we discuss the role of DNA damage in regulating an immunogenic response in senescent cells, in addition to discussing less established “atypical” senescent states that may occur independent of DNA damage.

Link: Burton and Faragher (2015) Cellular senescence: from growth arrest to immunogenic conversion. AGE. DOI 10.1007/s11357-015-9764-2

Cellular Senescence in Placental Development.

In addition to providing a protective role in tumour suppression and tissue damage, senescent cells may also function in embryonic development. It was suggested that cell- cell fusion induced senescence might play a physiological function in the placenta, thereby aiding embryonic development. ERVWE1, a fusion protein involved in the formation of the syncytiotrophoblast of the placenta causes cell fusion and induction of cell senescence in both cancer cells and normal fibroblasts. Fusion induced senescence (FIS) in vitro and in vivo is accompanied by activation of a DDR, p53 and p16(INK4a) dependent pathways. ERVWE1 mediated physiological cell fusion during embryonic development forms the syncytiotrophoblast that serves as the maternal/fetal interface at the placenta. The question of why the senescence program may be useful in normal placental function remains to be answered. However, it can be suggested that the resistance of senescent cells to apoptosis is necessary to maintain the viability of the syncytiotrophoblast. In addition, secretion of proteases, that are normally associated with senescent cells, may function to maintain feto-placental homeostasis. Placental proteases are required for the metabolism of vasoactive and immunomodulating peptides, thereby controlling the exchange of peptide hormones across the placenta and metabolic breakdown of maternal nutrients. Cytokine production is another feature of senescent cells that may play important roles within the placenta. IL-8, one of the main cytokines secreted by senescent cells, is necessary for normal placental function . Cytokine secretion may help regulate placental growth during pregnancy in addition to protecting the foetus from pathological organisms and facilitating interaction with immune cells. Further research is necessary in order to understand the functional significance of the senescence program in the placenta.

LINKS:

Cell fusion induced by ERVWE1 or measles virus causes cellular senescence

Physiological and pathological consequences of cellular senescence

Quiescent to Senescent Conversion

The presence of DNA damage and subsequent up-regulation of p16(INK4a) in quiescent cells in vivo may induce a pre-senescent state that converts to a full senescent state when cells are stimulated to proliferate. This suggests that DNA replication is required to induce a persistent DDR associated with cell senescence.   For example, damage to skeletal muscle in normal young mice causes the activation of quiescent satellite cells (adult stem cells), which proliferate and undergo myogenic differentiation required for muscle repair. However, a recent study has shown that in geriatric mice (28-32 months of age), satellite cell activation is impaired and satellite cells instead convert from a pre-senescent state (quiescent cells with high p16(INK4a) expression) to a full senescent state (including a DDR) when stimulated to proliferate in response to injury. As such, the induction of senescent satellite cells with age can impair satellite muscle regeneration. This study suggests that senescent cells may accumulate in late life due to a conversion from quiescence to senescence (termed geroconversion) in response to a requirement for cells to replicate over time to regenerate tissue. In this model, more and more quiescent cells are likely to accumulate DNA damage over the life-time of an organism and are therefore more likely to become senescent when induced to proliferate later in life. Therefore, if quiescent cells inflicted with DNA damage convert to senescence when stimulated to proliferate, then eliminating such damage may prevent this conversion.

Geriatric muscle stem cells switch reversible quiescence into senescence

Physiological and pathological consequences of cellular senescence

Immune surveillance of senescent cells

Taken from: Physiological and Pathological Consequences of Cellular Senescence

The ability of senescent cells to trigger an innate immune response via the up-regulation of pro-inflammatory cytokines was first suggested to play a role in limiting tumourigenesis. This immune response was later shown to be important in the elimination of senescent stellate cells during liver damage. In natural killer (NK) cell mediated cytotoxicity, NK cells identify senescent cells by the presence of NKG2D ligands on the membrane of senescent cells. The presentation of these ligands on senescent cells might be mediated by a DDR, which was previously shown to induce their expression. In particular, it appears that the ATM-ATR pathway is important for the up-regulation of NKG2D ligands in response to stress. NK cell induced cytotoxicity of senescent cells is mediated by granule exocytosis and perforin-mediated death rather than death-receptor-induced apoptosis. The perforin mediated cytotoxicity decreases in humans with age, and might therefore contribute to accumulation of senescent cells in the organism during ageing and in age-related diseases. As discussed, senescent cells are known to accumulate with age and in disease states, suggesting that senescent cells may be evading immune surveillance or their rate of accumulation is greater than the rate of removal or both. It has been advocated that the accumulation of senescent cells with age might be the consequence of an impaired ageing immune system. In fact, immune cells can also become senescent and these changes may contribute to impaired elimination of senescent cells. Therefore, strategies to restore an ageing immune system are a compelling approach for the elimination of senescent cells and for promoting an increased health-span.

A recent study has shown that senescent HSCs can be eliminated by another component of the innate immune system, the M1-like macrophages during liver damage and tumorigenesis in the liver. Secretory factors from senescent HSCs were shown to aid the elimination of these cells by macrophages. In contrast, cells that could not become senescent due to deletion of p53 and were not targeted by macrophages. Therefore, the innate immune system appears to be an initial early barrier that regulates the presence of senescent cells in physiological conditions such as in wound healing. 

The elimination of senescent cells by the adaptive immune system has also been demonstrated. OIS hepatocytes were shown to secrete cytokines to evoke an immune response leading to the elimination of senescent cells by CD4(+) T-cells, a process which required the action of macrophages. The elimination of senescent hepatocytes was required to prevent the development of liver cancer. This study mentions the attraction of T-cells by soluble factors but not the mechanism of senescent cell recognition, an area of research that still needs to be explored. However, there is some indication that RS cells may up-regulate MHC1 expression, possibly via p53. It can be speculated that MHC1 proteins in senescent cells may function to display senescence-associated antigens similar to cancer cells, allowing recognition and elimination by cytotoxic T-cells. Further research will provide multiple insights into the mechanisms and consequences of the interaction of senescent cells with the immune system.

Detecting Senescent Cells: Biomarkers

The standard SA-beta-gal staining, while indicative of the presence of senescent cells, is not an absolute marker for senescent cell and indicates increased lysosmal b-galactosidase activity. The use of several molecular markers that represent different characteristics of senescent cells is necessary (see figure). Such molecular markers can represent the cell cycle arrest machinery (e.g. p53, p21, p16), lack of cellular proliferation (e.g. lack of BrdU incorporation, Ki67), activation of the DDR (e.g. gamamH2AX or p53BP1 foci), expression of secretory factors (e.g. IL-6 and IL-8), the activation of the pathways that regulate the secretory phenotype (e.g. p-p65 or p-p38), the activation of immune surveillance-related genes and possible regulators for their pro-survival response (DCR2, p-Akt, p-Erk).

Link: Physiological and pathological consequences of cellular senescence

Physiological and pathological consequences of cellular senescence

Abstract

Cellular senescence, a permanent state of cell cycle arrest accompanied by a complex phenotype, is an essential mechanism that limits tumorigenesis and tissue damage. In physiological conditions, senescent cells can be removed by the immune system, facilitating tumor suppression and wound healing. However, as we age, senescent cells accumulate in tissues, either because an aging immune system fails to remove them, the rate of senescent cell formation is elevated, or both. If senescent cells persist in tissues, they have the potential to paradoxically promote pathological conditions. Cellular senescence is associated with an enhanced pro-survival phenotype, which most likely promotes persistence of senescent cells in vivo. This phenotype may have evolved to favor facilitation of a short-term wound healing, followed by the elimination of senescent cells by the immune system. In this review, we provide a perspective on the triggers, mechanisms and physiological as well as pathological consequences of senescent cells.

LINK: Burton and Krizhanovsky (2014) Physiological and pathological consequences of cellular senescence

Reclassifying Cellular Senescence into Two Main Types

Persistent activation of the DNA-damage response (DDR) can trigger cells to undergo cellular senescence, a state of irreversible, immune evoking, growth arrest.  In such a way, cellular senescence can prevent tumourigenesis firstly by blocking cells from replicating and producing abnormal and potentially cancerous daughter cells and secondly by coordinating their removal by immune cells.  Additionally, senescent cells can aid tissue repair by preventing extensive cellular proliferation leading to fibrosis, possibly triggered by replication stressed-induced DNA damage.  However, if this orchestrated removal of senescent cells becomes dysregulated, then persistent senescent cells can promote tumourigenesis and tissue damage.

An aspect of the DDR in senescent cells is the induction of an array of secretory factors, including cytokines/chemokine’s, which are important in attracting/activating immune cells to their vicinity.  When immune cells reach the locality of senescent cells, they can then specifically recognize them by the expression of immune ligands on the cell membrane, a process that may also be regulated primarily by the DDR.  The specific ligands recognized and the mechanism of senescent cell death will then be dependent upon the type of immune cell interacting with the senescent cell. 

However, cells induced to undergo permanent growth arrest in vitro by the overexpression of the cyclin dependent kinase inhibitor p16ink4a, do not develop an immune evoking secretory phenotype until the addition of DNA damage.   If cells in physiological or pathological conditions can indeed undergo permanent cell cycle arrest in a p16 dependent, DDR-independent manner, then these cells are unlikely to evoke an immune response for their clearance.  In support of this, a recent study demonstrated that cells overexpressing p14(ARF) in the epidermis of mice remained present for weeks after transgene silencing (Tokarsky-Amiel et al 2013).  Even if p16-induced senescent cells do not display a pro-inflammatory phenotype, they can still cause physiological problems simply by their inability to proliferate, an essential feature required for tissue regeneration and maintenance.  In this regard, the growth arrest and pro-inflammatory phenotype of senescent cells can be investigated separately to determine which feature is important in different physiological contexts. 

Until the phenotype of p16-induced senescent cells in vivo have been researched more extensively, cellular senescence could be divided into two separate types.  Firstly, immunogenic senescence related to a DNA damage response, consisting of a pro-inflammatory phenotype and the presence of immune ligands, triggered by telomere shortening, oncogene-activation, and chemical stressors.  Secondly, sterile senescence which lacks a pro-inflammatory phenotype and the inability to evoke an immune response. 

If this distinction is made, then studies focused on the effect of cellular senescence on ageing, disease and cancer development can better design their experiments and avoid confusion between conflicting results due to differences in the types of senescence used.  

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