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  

Post-mitotic tissue

Damage to proteins

Damage from ROS is also thought to have an age-related impact on proteins. Such damage is thought to result in amino acid modifications, fragmentation of peptide chains, altered electrical charge and protein aggregation (Davies, 1987). Since the structure of proteins is pivotal for performing its functions then any structural changes would therefore result in an impairment of function. As discussed previously, the turn-over rate of proteins is an important factor in determining whether any protein damage is going to have a significant affect. Since the majority of intra-cellular proteins have high turn-over rates, damage proteins are not going to persist long enough to cause any problems. Therefore, long-lived proteins which may be affected by AGE formation may also be affected by oxidative damage. Examination of extracellular tissues in the lens, skin collagen and articular cartilage (low turnover proteins) of humans ranging in age from infancy to 80 years showed an increase in oxidative markers with age (Linton et al, 2001). The same study also looked at intracellular proteins with high turnover rates and found no evidence to suggest that intracellular proteins accumulate oxidative damage with age.

Damage to Lipids

Lipids which make up the membranes of cells are also potential targets for ROS which may consequently result in biologically significant alterations to membrane proteins. ROS are thought to attack membrane phospholipids and act on unsaturated fatty acids to produce lipid peroxidation. The consequent of this may be alterations in membrane fluidity, increased permeability and loss of membrane integrity. Experimental evidence suggests that altered membrane fluidity might affect permeability, transport systems, receptor functions or enzyme activities (Stark, 2005). The functionality of proteins in the membrane is critically dependant on membrane fluidity, especially when proteins have to collide with other molecules to exert their effects (such as G-proteins). This is seen in many receptor mediated pathways. For example, cardiac membranes from rats with cirrhotic cardiomyopathy are rigid and associated with diminished cAMP production. When the fluidity of these membranes are restored to control values cAMP production was significantly increased (Ma et al, 1997).

Biological impact of ROS

The above discussion on ageing of post-mitotic cells reviews the mechanisms thought to result in an overall increase in cellular damage by ROS, but little evidence is provided for the age-related biological consequence of such damage. If accumulative damage from ROS is an ageing mechanism of post-mitotic tissue, then such damage may result in three possible outcomes:

1) Post-mitotic cells become damaged and are subsequently removed. Post-mitotic-cells cannot be replaced, thereby resulting in a decrease in overall cell number and a decrease in tissue function.

2) Damage to post-mitotic cells results in an impairment of cellular function but such damage is not extensive enough for the removal of cells and so they persist.

3) Cells are removed due to damage and are subsequently replaced (i.e. by mitotic cells). However, the ageing mechanism in this case may therefore be a decline in the capacity to replace cells due to an ageing mechanism specific for mitotic tissues which will be discussed later. This may result in an observation similar to point 1).

The literature was reviewed to identify whether these three possible outcomes are present in ageing of post-mitotic tissues. One study observed a substantial increase in the amount of DNA single strand breaks in hippocampal pyramidal and granule cells as well as cerebellar granule cells but not in cerebellar Purkinje cells in the brain of ageing rats (Rutten et al, 2007). However, a reverse pattern was found for age-related reductions in total numbers of neurons. Cerebellar Purkinje cells were found to be significantly reduced during ageing (point 1 above) whereas the total number of hippocampal pyramidal and granule cells as well as cerebellar granule cells were not (points 2 or 3 above). This may seem as a confusing result since those cells which have undergone the most DNA damage may be expected to decline in numbers. This result may be explained if there are processes in place that replace damaged pyramidal and granule cells but do not, or cannot, replace damaged Purkinje cells. Cells that may be more prone to damage may have a higher potential capacity to be replaced than cells that are less prone to damage. It is also possible that the damage inflicted on hippocampal pyramidal and granule cells is not severe enough to warrant their removal. These findings suggest that if ROS does cause detrimental damage leading to an ageing phenotype, such damage is not tissue specific damage, but rather, cell type specific. All post-mitotic cells cannot therefore be treated equally.

A decline in cell numbers may not be related to damage from ROS but instead due to the loss or dysfunction of mitotic cells such as glial cells and astrocytes which are known to provide support and protection for neurons. Discussed later in more detail in ageing of mitotic tissue, mitotic cells can undergo an irreversible cell cycle growth-arrest known as replicative senescence. Senescent cells display a radically altered phenotype with potentially detrimental consequences on other cells if they accumulate in tissue. For example, a recent study has demonstrated that alterations in astrocyte function with ageing may not only affect its neuroprotective capacity, but may also contribute to neuronal injury in age-related neurodegenerative processes (Pertusa et al, 2007). Also, microglial, cells involved in immunological surveillance and neuroprotection have been shown to be subject to replicative senescence and it has been suggested that the dysfunction of these cells may contribute to the development of neurodegenerative disease by diminishing glial neuroprotection (Streit, 2006).

Myocytes, also known as muscle fibres are post-mitotic cells found in skeletal, smooth and cardiac muscle. Studies have shown that ageing muscle results in a decline in myocyte numbers, signs of atrophy and increased susceptibility to contraction-induced injury (Alnaqeeb and Goldspink 1986, Musaro and Rosenthal, 1999, McArdle et al 2002,). When myocytes become damaged and need to be repaired, mitotic satellite cells are able to differentiate and fuse to augment existing muscle fibres and to form new fibres. Decline in myocyte numbers and increases in damage with age may therefore, at least in part, be due to a reduction in number or the impairment of satellite cells (point 3). Such a reduction in satellite cell numbers would result in a decrease in cellular maintenance and an increase in cellular damage, possibly leading to the removal of the cll altogether. One study has found that the abundance of satellite cells does appear to decline with age, however, the myogenic potential of these cells does not diminish with age (Shefer et al, 2006).

Post-mitotic tissue

The predominant ageing mechanism of post-mitotic cells (i.e. neurons, myoblasts and osteocytes) is often thought to be due to the accumulation of damage to DNA, protein and lipids caused by reactive oxygen species (ROS). Since these cells do not divide in tissues and can potentially persist for a lifetime, they are more likely to accumulate damage which may subsequently result in an age-related phenotype. Also, since mitotic cells are mostly found in a non-dividing state in vivo known as quiescence, with very little cell turnover, they could also in some regard be treated similar to post-mitotic cells.

ROS are thought to cause accumulative damage to DNA, protein and lipids, but how and if such damage results in an ageing phenotype still remains unclear. Damage from ROS is thought to be imposed on tissues as we age due to three possible factors:

1) An increase in ROS production.
2) A decrease in antioxidant defences.
3) A failure to repair oxidative damage.

Increase in ROS

Increases in ROS production is thought to be the result of mitochondrial dysfunction which leads to an increase in electrons leaking out of the respiratory chain within the mitochondria as we age (Wei et al, 2001). This increase in ROS is thought to increase damage to genomic DNA, mitochondrial DNA, proteins and lipids. Despite this theory, there appears to be little evidence to suggest that the levels of oxidative stress increases with age. One study however investigated age-induced ROS generation in healthy subjects ranging in age from 20-80 years quantifying ROS production using a chemiluminescence assay (Chaves et al, 2002). Results demonstrated a significant increase of ROS production from 40 years of age suggesting ROS does increase with age. However, little is known about the source of ROS. As mentioned, respiratory chain dysfunction leading to electron leakage is one suggestion.

With this in mind, a mutator mouse was created to investigate whether the mutations which were introduced in the mitochondrial resulted in mutant mitochondrial proteins that are defective in coupling of oxygen metabolism with ATP causing increased ROS production (Trifunovic et al, 2004, Trifunovic et al, 2005). Results indicated that despite the presence of severe respiratory chain dysfunction, the amount of ROS produced in these mice was normal, no increased sensitivity to oxidative stress-induced cell death was observed and no difference in oxidative damage to protein was seen. This data thus suggests that if ROS does increase with age, it may not be due to respiratory chain dysfunction.

An alternative source of ROS may come from age-dependent up-regulation of the inflammatory response. Inflammation has been implicated in many age-related diseases such as rheumatoid arthritis, osteoarthritis and cardiovascular disease (Licastro et al, 2005). The presence of inflammation is associated with increased ROS production, promoting the destruction of normal tissue (Winrow et al, 1993). This production of ROS during an inflammatory response may also initiate and/or amplify inflammation via the up-regulation of several genes involved in the pro-inflammatory response (Conner and Grisham, 1996). In some instances however, inflammation associated with ageing may result from direct damage from ROS (Chung et al, 2001). It is therefore difficult to determine to determine what come first, the inflammation leading ROS production or ROS production leading to damage leading to an inflammatory response and further ROS production.

One source of inflammation, as discussed later, may come from the presence of senescent cells within tissues which adopt a proinflammatory phenotype. It is hypothesised that the accumulation of senescent cells within tissues contributes to ageing (Hayflick, 1965). As the number of senescent cells increases, so does the intensity of the inflammatory response. In part, this proinflammatory phenotype is thought to damage tissues by the production of ROS.

Another alternative for increase in ROS with age may be due to a reduction in antioxidant defences and/or a decrease in the failure to repair oxidative damage.