Mammalian cells mostly rely on extracellular molecules to transfer signals to other cells. However, in stress conditions, more robust mechanisms might be necessary to facilitate cell–cell communications. Cellular senescence, a stress response associated with permanent exit from the cell cycle and the development of an immunogenic phenotype, limits both tumorigenesis and tissue damage. Paradoxically, the long-term presence of senescent cells can promote tissue damage and aging within their microenvironment. Soluble factors secreted from senescent cells mediate some of these cell-nonautonomous effects. However, it is unknown whether senescent cells impact neighboring cells by other mechanisms. Here we show that senescent cells directly transfer proteins to neighboring cells and that this process facilitates immune surveillance of senescent cells by natural killer (NK) cells. We found that transfer of proteins to NK and T cells is increased in the murine preneoplastic pancreas, a site where senescent cells are present in vivo. Proteomic analysis and functional studies of the transferred proteins revealed that the transfer is strictly dependent on cell–cell contact and CDC42-regulated actin polymerization and is mediated at least partially by cytoplasmic bridges. These findings reveal a novel mode of intercellular communication by which senescent cells regulate their immune surveillance and might impact tumorigenesis and tissue aging.
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.
For many researchers, irreversible cell cycle
arrest is the canonical trait of senescent cells. Such growth arrest can be induced
experimentally by the up-regulation or over-expression of cyclin dependent
kinase inhibitors (CDKi). Thus valuable
models are, at least potentially, available in which to study the physiological
effect of growth arrest distinct from the DDR or any other upstream response. Unfortunately there has been little
characterization of the phenotype of cells rendered ‘senescent’ by this means.
Blagosklonny and co-workers (Korotchkina et al. 2009) used an
isopropyl-thio-galactosidase (IPTG)-inducible p21 expression construct to
induce a senescence-like state in an HT1080-derived cell line (HT-p21-9). Characterisation of the phenotype of these
cells does not appear to have been attempted beyond observing irreversible
growth arrest and the presence of increased SA-β-Gal
activity. Given that HT1080 is a highly
tumorigenic fibrosarcoma carrying an activated N-ras oncogene (Benedict et al. 1984), it probably represents a
poor genetic background in which to assess whether markers of immunogenic
conversion or resistance to cell death can be induced by CDKi overexpression
alone. However, the basic principle of using
such a construct for that purpose is sound.
Tokarsky-Amiel et al (2013) showed that overexpression of p14ARF in the
epidermis of the skin of mice (using a tetracyclin-inducible construct) resulted
in mass apoptosis and cell cycle arrest.
As measured by SA-β-Gal
activity, the p14ARF transgene drove senescence in up to 8% of the
surviving cells in the epithelium by a p53-dependent mechanism (demonstrated by
ablation of p53 through co-expression of a specific shRNA directed against it). These senescent cells were viable within the
epidermis for several weeks consistent with lack of clearance. Unfortunately, minimal analysis of their
phenotype was conducted (beyond assessment of the message levels for the
senescence-associated genes Pai-1 and Dcr2).
Thus, the immune state of the p14ARF-senescent cells is currently
unclear and the picture is complicated by the fact that senescent rodent cells
do not display a senescent secretome under some conditions. However, given that alopecia and follical
stem cell dysfunction were observed in the animals, it is clear that cells
rendered ‘senescent’ in this manner can exert phenotypic effects. Thus, there is some evidence that cell cycle
arrest alone may be sufficient to cause problems in highly mitotic tissues such
as the epidermis, but large amounts of work remain to be done.
CDKi overexpression systems clearly have the
potential to be valuable tools. However the
extent to which these are physiologically reflective can legitimately be challenged. This can be understood in two ways (i) the
mechanism by which the growth arrest is induced has not been reported in vivo and (ii) cells do not become
senescent en mass but gradually as a
result of tissue turnover throughout life.
Thus, findings made with these systems could be considered ‘artefactual’
By way of addressing these concerns, it is worth
remembering that for many years replicative senescence was dismissed as a
‘tissue culture artefact’ because senescent cells had not been observed in vivo (evidence for their existence in
tissue remained severely limited until the late 1990s). By the same token, elevation of CDKi alone in
cells in vivo is not impossible. Absence of evidence is never evidence of
absence. Similarly, many
over-expression systems model systems can be said to be non-physiological. However, valuable data is routinely gathered
using them and in this instance could allow researchers to gage the maximum
physiological impact that irreversible growth arrest can have on tissue
function. Thus, if these limits are
recognized, such models are potentially utile, especially when combined with
detailed analysis of phenotypes known to exist in other ‘senescent cells’ (e.g.
apoptosis resistance, immune ligand presentation and the secretory response)
Endoplasmic reticulum (ER) stress may also
promote a senescent-like response. The
accumulation of unfolded proteins in the ER triggers a stress-signaling pathway
that can result in cell cycle arrest mediated by p27 (Han et al. 2013) and the p53/47 isoform (Bourougaa et al. 2010). Furthermore,
ER stress has also been shown to induce an inflammatory response via NFkB
activation (Garg et al. 2012) and
induce cytokines such as MCP-1, IL-6 and IL-8 (Schroder, 2008), which are
capable of attracting and activating immune cells (Sagiv and Krizhanovsky, 2013).
ER stress has also been shown to promote cell survival, another feature of cell
senescence (Raciti et al. 2012). Interestingly, a senescent state via
activation of ER stress-dependent p21 signaling has been reported in proximal
tubular epithelial cells, triggered by receptors for advanced glycation
end-products (RAGE) (Liu et al. 2014). Although, ER stress-induced senescence has
the potential induce an immunogenic phenotype in the absence of DNA damage, a full
evaluation of the phenotype is required to determine if this is so.
Metabolic stress, defined here as a combination
of aerobic glycolysis and mitochondria dysfunction can potentially trigger a senescent
state. All organisms that use aerobic
glycolysis form reactive acyclic α-oxoaldehydes
(e.g. methylglyoxal and glyoxal) spontaneously from triosephosphates and by a
wide variety of other routes (Thornalley, 2009). These dicarbonyl compounds are highly
reactive and damage proteins through non-enzymatic modification producing a
wide variety of covalent adducts (AGEs).
Elevated levels of methylglyoxal and glyoxal are known to be cytotoxic
and although the mechanism of action remains imprecisely defined, it can be
blocked by ROS scavengers, suggesting that oxidative stress mediates at least
some of the deleterious effects (Shangari and O’Brian, 2004).
Cytosolic and mitochondrial protection from dicarbonly
damage is primarily mediated through the action of the glyoxalase system that
consists of two enzymes, glyoxalase I and II.
However, in cultures of WI38 fibroblasts a significant reduction in the
activity of glyoxalase-I occurs with serial passage (Ahmed et al. 2010). Treatment of cultures
of ASF2 human adult dermal fibroblasts with micro or millimolar concentrations of
glyoxal or methylglyoxal renders them senescent within 72 hours. This was defined by the presence of typical
senescent morphology, irreversible growth arrest and increased SA-β-Gal
activity (Sejersen & Rattan, 2009). Further
studies (Larsen et al. 2012) extended
these observations to immortalized human mesenchymal stem cells (MSCs) and demonstrated
that treatment with physiologically reflective (Han et al. 2007) concentrations of glyoxal for 72 hours led to
senescence without significant cell death (although massive cell death occurred
at higher glyoxal concentrations). Elevated
levels of SA-β-Gal, p16 and DNA damage (as measured by
COMET) accompanied the growth arrest. Interestingly,
a profound reduction in the ability of these senescent MSCs to differentiate
into functional osteoblasts (as determined by alkaline phosphatase and
mineralization assays) was also observed.
Given the imbalances in glucose metabolism that accompany mammalian
ageing (and diabetes), the authors proposed that this type of metabolic stress
might underlie age-related changes in bone function. Unfortunately, no markers of immunogenic
conversion have yet been measured in this system and whilst the presence of DNA
damage could indicate the likelihood of a secretory response, this cannot be
assumed. Thus, the propensity of
senescence human MSCs to be cleared by the immune system remains unknown and is
of considerable physiological significance.
TGFβ-induced senescence: A growing body of evidence suggests that the members of the transforming growth factor beta (TGF- β) family can induce a senescence-like state. Experimentally, senescence has been predominantly, but not exclusively, characterized by the presence of senescence-associated beta galactosidase (SA-β-Gal) staining and the up-regulation of cyclin dependent kinase inhibitors (CDKi) (see below). Human prostate basal cells treated with TGF-β1/2/3 show increased SA-β-Gal activity, which is associated with the flattened, and enlarged cell morphology typical of adherent senescent cells in vitro (Untergasser et al. 2003). Similarly TGF-β1 has been reported to induce a senescent state in bone marrow mesenchymal stem cells as a result of increased mitochondria ROS production (Wu et al. 2014). These cells also showed SA-β-Gal staining and an increased expression of p16. Yu et al. (2010) demonstrated that TGF-β2 could induce a senescent-like state in human trabecular meshwork cells. Again, this was associated with SA-β-Gal staining, increased levels of p16 at both the message and protein level and a reduction in the level of pRB protein. No impact on p21 mRNA or protein expression was observed in response to TGF-β2 exposure. Other groups have also reported a role for TGF-β signaling in inducing a senescent state (Senturk et al. 2010, Minagawa et al. 2011, Acosta et al. 2013).
It is generally accepted that SA-β-Gal staining should be used in conjunction with several other senescent markers, as it does not appear to detect senescent cells specifically (Severino et al, 2000). However, other than the expression of CDKi, it appears that the phenotypes of cells induced to enter senescence by exposure to TGF-βs have been poorly characterized, especially in regard to immunogenic conversion. Some cell types that become senescent via this route may be cleared by the immune system in a manner analogous to those undergoing developmentally programmed senescence. Others may not and this area represents a fruitful field for further investigation.
Developmentally programmed senescence: Cells sharing features of senescence have been reported within the mesonephros and the endolymphatic sac of the inner ear in human and mouse embryos; as well as the neural roof plate and apical ectodermal ridge in rodents (Munoz-Espin et al. 2013, Storer et al. 2013). The authors hypothesize that this “developmental senescence” (DS) is a programmed part of normal embryonic development. DS was demonstrated experimentally by the presence of SA-β-Gal activity and senescence associated heterochromatin (Munoz-Espin et al. 2013). These cells seem to lack detectable DNA damage and appear to have become senescent independent of p53 and p16 and have gene expression patterns that significantly overlap with those of IMR90 fibroblasts in a state of oncogene-induced senescence. Arrest in this instance is dependent instead upon p21, regulated via the TGF-β/SMAD and PI3K/FOXO pathways (thus showing some affinity with other TGF-β induced senescent states). Interestingly, DS cells are removed during normal embryonic development by macrophages in a manner related to immune clearance of senescent cells in the mature organism (or by apoptosis should senescence fail) contributing to the formation of normal tissue architecture. Thus, the long-recognized distinction between programmed cell death in development and apoptosis in the mature organism appears to be mirrored in DS. Given that the expression of p21 in developing embryos is often attributed to ‘terminal differentiation’ (Vasey et al. 2011), it will be interesting to determine how many of these p21 positive cells are senescent cells and have undergone immunogenic conversion. Taken from:Cellular Senescence: From Growth Arrest to Immunogenic Conversion