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Human ageing can be defined as the collection of changes that render human beings progressively more likely to die hence a decline in physiological efficiency and ability to respond to stress and an increase in homeostatic imbalance and an increased risk of ageing-associated diseases. It is a syndrome of changes that are deleterious, progressive, universal and thus far irreversible. Ageing damage occurs to molecules (DNA, proteins, lipids), to cells and to organs thus affecting the whole organism in a whole assortment of ways. The duration of ageing in humans makes it almost impossible to perform in vivo studies and so the mechanisms involved remain largely mystery. Ageing can also be defined as organsimal senescence whereas cellular or replicative senescence is characterised by the inability of cells to proliferate in vitro after prolonged division in culture despite the presence of abundant nutrients and mitogens causing the cell cycle to irreversibly arrest. It was first observed by Hayflick and Moorfield in 1961. The majority of senescent cells assume a distinguishing flattened and enlarged morphology, increased reactive oxygen species and the accumulation of resultant reactive oxygen species damage products and more recently a number of molecular phenotypes have been described, such as changes in gene expression, protein processing and chromatin organization. Senescent cells maintain metabolic activity and have the ability to remain viable indefinitely. Importantly senescent cells can resist apoptosis. In addition only a few cells such as stem cells, haematopoietic cells and a few other cell lines that do not undergo senescence but it affects the majority of cells such as fibroblasts (the cells that Hayflick did his research on), chondrocytes, epidermal cells, smooth muscle cells, hepatocytes and many more. 1, 2, 3,6
In essence there are two ways a cell can senesce; there are two broad categories of replicative cellular senescence. The first is telomere dysfunction or other forms of genomic stress thus inducing a DNA damage response mediated primarily by the p53 and tumour suppressor pathway. The other has not yet been very well researched and does not involve DNA damage or telomeres and is described as the upregulation of the cyclin-dependent kinase inhibitor p16 which in turn causes the pRB pathway to become disrupted.1, 2, 3, 6
From Hayflick's research it was clear that the onset of senescence in vitro would cause proposals that senescence could lead to in vivo organismal ageing phenotypes thus accumulating evidence that cell senescence is behind many of the symptoms of normal ageing such as immune failure age related diseases, poor wound healing and skin deterioration. This shows that the senescent response has two sides to it; first is promotes life by decreasing chances of cancer and allowing cells to become damaged but eventually it leads to limiting the durability of the human body. The gradual attrition of the telomeres provided grounding as the molecular mechanism for the cell division clock. The cell division clock can be considerably affected by extrinsic factors, such as reactive oxygen species, which speed up the rate of telomere shortening.1, 3, 4, 6
Due to the fact that senescence has the consequence of cell cycle arrest, it has been thought some time ago that it has a potential to stop cancer development and more recently it has been proved that senescence could be an important tumour suppressor mechanism. This claim provides us with a logical explanation as to the role of why senescence evolved.
In this review I shall focus on the relationship between ageing and senescence albeit that the proof is unsubstantial but in due time it will advance our knowledge of the mechanisms involved in ageing associated diseases as well as demonstrate the typical ageing process.
Molecular Mechanisms of Ageing and Senescence
At the molecular level, evidence suggests that there are several mechanisms but a few are more important than others.
Telomere Loss Theory
Here I shall briefly write about what is known about telomeres, the proteins that associate with them and how dysfunctional telomeres can contribute to ageing. Telomeres are distinctive DNA-protein structures at the ends of linear chromosomes. Most commonly composes of non coding tandemly repeated sequences. In humans that sequence is TTAGGG and is normally 15-20 kb long and highly repetitive. This extends from the double stranded DNA sequence to a single stranded region in a 5' to 3' direction. A t- loop structure is formed from the 3'G-rich overhang and telomere-binding proteins. The telomeres along with therir associated proteins serve to protect the chromosome ends from the DNA repair machinery that would otherwise distinguish them as double strand breaks, and from exonucleases that could treat them as substrates. DNA double strand breaks are potentially disastrous lesions. If not repaired they are subject degradation and even if they are repaired they can lead to a loss of heterozygosity or chromosomal deletions or translocations. Telomeres thus provide a protective mechanism to preserve chromosomal integrity and without it chromosome ends are at risk for degradation, recombination or random fusion by different DNA repair systems. Degradation inevitably results with deletion of genes and thus causes cell death. Telomeric recombination can lead to an increase or decrease in telomere length. Fusion with the DNA repair systems can create additional double stranded breaks causing genetic instability. 7, 8,9,10
Telomerase, senescence and ageing, Mechanisms of Ageing and Development, Volume 129, Issues 1-2, January-February 2008, Pages 3-10
There are two types of protein that associate themselves with telomeres, those that bind entirely to the telomeres whereas others localise to subnuclear or subcellar sites. The group of proteins that associate exclusively to the telomeres seem to have an important role in regulating the length and/or structure of the telomere. Recently it has been recognised that several proteins that associate with telomeres also play a part in DNA repair processes. From this research two main findings have come into sight; first, many telomere-associated proteins are needed to act as a unit to establish and sustain the telomeric organization and second, telomere structure could be even more significant than telomere length in determining the phenotype of cells. 7,8,9,11,13
Germ cells and early embryonic cells have their telomeres maintained by the balanced action of the enzyme telomerase and a variety of proteins associated with the telomere. Telomerase adds the repeat sequence straight on to the 3' G rich overhang. Most somatic cells do not express telomerase unlike the germ and embryonic cells and this causes a dilemma for them. This is due to DNA replication. The ability of DNA polymerase to synthesize DNA only in the 5' to 3' direction and its need for the short RNA primers during conventional DNA replication result in the incomplete replication of the lagging strand. Thus, the biochemistry of DNA replication results in failure to replicate part of the telomere with each cell division. This is now termed the end-replication problem. There is a 3' overhang at both ends of mammalian chromosomes therefore suggesting that a 5' to 3' exonuclease specifically trims back the telomere. Due to the exhibition of this end-replication problem the telomeres of the majority of somatic cells i.e. those with no telomerase cause the telomeres to become progressively shorter with each cycle. 7, 9,11,13,14
The telomeres then reach a critical length and this causes replicative senescence to be triggered in the cell. This response probably came about to suppress cancer growth thus acting as a failsafe mechanism to prevent the proliferation of cells that could possibly have any chance of introduction of oncogenic damage or stimuli. The critical length of the telomere is recognised as something similar to a DNA break by a DNA damage complex, which can be visualised the presence of nuclear foci containing the phosphorylated form of the histone variant H2AX. These foci contain many proteins involved in the recognition, signalling and repair of DNA damage and are referred to as telomere dysfunction induced foci. This results in the recruitment of a tumour suppressor which signals growth arrest thus inducing senescence thus rendering the cell incapable of forming a tumour. 7, 8, 10, 12
As we know telomeres end in a large t-loop, which is thought to may protect the G rich 3' overhang by forming a triplex or other unusual, protective DNA structure. T-loop formation depends on the telomeric proteins (one that binds to the telomeric DNA exclusively) known as TRF2. Loss of the t-loop, owing to expression of dominant-negative TRF2, causes tumour cell death by a mechanism that requires both p53 and ATM. Thus, ATM very likely transmits telomere dysfunction signals through p53. 7,8,9,10,14
Mechanisms of p53 and pRB
There are a vast range of stimuli that can induce senescence but they all seem to trigger one of two pathways present. These pathways are known as the p53 and pRB pathways which are both tumour suppressor proteins. The full picture to how they are triggered and how exactly they maintain the senescent state is still tenuous but as time goes one it is becoming clearer.
P53 is a vital mediator in response to DNA damage, including that response of senescence. It is transcription factor encoded by the TP53 gene. It is responsible for regulating the cell cycle and thus functions as a tumour suppressor. Due to its role in preventing gene mutation it has been described as the "guardian of the genome". It works through several different mechanisms. It can initiate apoptosis, if DNA damage is deemed irreversible, it can activate DNA repair machinery when DNA has become damaged and it can also induce senescence in that it induces growth arrest by holding the cell cycle at the G1/S regulation point. P53 is a tight controller of the cyclin-dependent kinase inhibitor p21. This binds to CDK-4 and -2 complexes with them and thus inhibits them causing cell cycle to be arrested. P53 activates transcription of p21 and in theory anything that inhibits the function of p53 will down-regulate p21. It is now well known that a loss of p53 function can delay replicative senescence. This is what caused the theory that the dysfunctional telomeres are recognised as something similar to DNA damage because so as to trigger a p53 - dependent DNA damage response. What can also happen is a tumour suppressor gene known as p14ARF (member of the ADP ribosylation factor family) inhibits mdm2 (oncogenic negative regulator of p53), thus promoting p53, which promotes p21 activation, which then binds and inactivates certain cyclin-CDK complexes, which as I have previously mentioned would otherwise encourage transcription of genes that would carry the cell through the G1/S checkpoint of the cell cycle. Loss of p14ARF by a homozygous mutation in the CDKN2A (INK4A) gene will lead to elevated levels in mdm2 and, therefore, loss of p53 function and cell cycle control and is the cause of some cancers. 3,4,5,7,15.,16,17
In the case of senescence caused by oxidative damage, p53 levels are momentarily induced, but subsequently fall to levels that are normal before senescence. p53 levels also increase in response to RAS- and E2F-induced senescence but it is not known whether the levels eventually decline. It is possible that p53 is transiently induced during replicative senescence, but the induction is undetectable owing to the fact that replicative senescence is asynchronous. Cellular senescence does not necessarily mean that the levels of tumour suppressor p53 need to be elevated. As I have just said the p53 pathway is also involved in the senescence response to overexpressed oncogenes such as RAS. 3,4,5,7,15.,16,17
If p53 is inactivated it can cause the senescent response to be completely reversed. . For example in postmitotic senescent cells that have had their p53 function eliminated, even though telomeres have reached their critical short length the cell will continue to proliferate until they are driven into 'crisis' which is a state of severe genomic instability. Another example being where p21 which is vital for the actions of p53 is inactivated the once senescent cells become active again and reach crisis in a similar fashion to the other cells. This shows why p53 is necessary to maintain a senescent state and although the senescent state cannot be reversed by any physiological means, in senescence can be reversed if there is a lack of p53. 3,5,16,17
However, p53 does not reverse the senescence in all cells. This is because there is another tumour suppressor gene at work. It has now been revealed that this tumour suppressor is p16 and the cells that remain senescent show greater levels of p16. P16 is a positive regulator of pRB. 3,4,5,16,17
There is now research which reveals that p53 can have two effects depending on the strength of p53 activity. Two different mice strains were given different types of p53. One expressed constitutively active mutant p53 and one had an increased level of p53 than normal by carrying an extra copy of the whole p53 gene. The first type displayed greater protection against cancer but they also showed signs of premature ageing. Whilst in the other type they also displayed increased cancer protection without the premature ageing and displayed typical longevity and it was also found that the amount of there was a decrease in the number of cells with critically damaged telomeres. Increased function of p53 also showed that there was a delay in ageing and in a decrease in the reactive oxygen species. These findings suggest that there may be a cost to enhanced protection from cancer3,4,5,15.,16,17
These indications show that the main role of p53 is to eliminate DNA damaged cells, either by inducing apoptosis or by inducing senescence. During normal physiological ageing p53 could have an antiageing effect that competes with main role of p53 i.e. inducing senescence. But when there is critical DNA damage i.e. telomere loss then p53 causes excessive elimination of cells thus exhausting the capacity of tissue regeneration leading to ageing symptoms. p53 clearly plays a pivotal role in controlling the senescence response to various stimuli. This is consistent with the idea that cellular senescence is a tumour suppressive mechanism and the known role of p53 as a tumour suppressor. 3,4,5,7,15.,16,17
K. Itahana, G. Dimri and J. Campisi, Regulation of cellular senescence by p53, Eur. J. Biochem. 268 (2001), pp. 2784-2791
The cyclin dependent kinase inhibitor p16 inactivates CDK4 and CDK6 thus making sure that pRB (retinoblastoma protein) remains in its active state. pRB prevents the cell through its progression of the cell cycle through G1 into S phase. pRB interacts with transcription factors of E2F and inhibits them. When pRB is bound to E2F, the complex acts as a growth suppressor and prevents progression through the cell cycle. pRB does this in its hypophosphorylated state i.e. it is active in its role of tumour suppressing and inhibiting cell cycle progression. During the start of cell cycle phosphorylation inactivates pRB but as the cell cycle progresses it is PP1 dephosphorylates thus making it active. Once G1 has ended at the cell needs to enter the S phase a host of cyclin dependent kinases phosphorylate pRB thus inhibiting its actions. It allows E2F pRB complex to dissociate so that E2F can progress the cell cycle further. 3,4,5,7,15.,16
P16 is can be up-regulated by a variety of stimuli and it is thought that it could be involved in all types of senescence so recently it has been used as a valuable biomarker because its levels were found to be in concordance to that of Î²-galactosidase which up until now has been the major biomarker of the senescent state. P16's expression is very low in young organisms but it increases with age. It was found in ageing human skin and in human kidneys. It is now thought that induction of p16 could have something to do with rejection of graft procedures. 3,4,5,7,15.,16
Although specific mechanisms are as yet unknown, a cell seems to undergo senescence because of chromatin remodelling which require pRB activity. These senescent cells develop dense foci of heterochromatin which corresponds to what cells look like when pRB is functional. Once the pRB pathway of senescence is engaged the growth arrest cannot be stopped or reversed by inactivation of p53, silencing of p16 or pRB. This means that once pRB is establishes itself to make repressive genes which are normally part of the E2F family the maintenance of the senescent state no longer requires p16 or pRB. This shows that the pRB pathway is relatively important in ensuring that the growth arrest in senescence is practically irreversible. 3,4,5,7,15.,16,19
It is thought that there is a link between the p53 and pRB pathways. For example p21 is a global inhibitor of CDKs more so than p16 and so should also cause pRB to become active. However, pRB activation by p21 and p16 differ. P53 inactivation can induce proliferation in senescent cells that express p21 but those that are expressing p16. Also p16 is more effective at inducing senescence than p21. Senescent cells normally only express either p21 or p16 but scarcely both. This could be because they both require slightly different signals to induce senescence; p53 mediating senescence largely due to telomere dysfunction and DNA damage and the pRB pathway provoking senescence due to oncogenes, chromatin disruption mainly nuclear stresses.
At the moment even with all this research there are still huge gaps in knowledge about how the different pathways interact to induce a senescent response. It is thought that senescence brought on by p53 is due to telomere dysfunction of DNA damage but in some cells extensive telomere uncapping due to mutations can induce p16 to become up-regulated, which corresponds with the fact that p16 acts in response to a variety of stimuli. In addition some cells that senesce in response to oncogenic stimuli induce senescence with p16. This shows that there is clearly a relationship between the two pathways but they act independently and asynchronously. It also tells us that the network which decides which pathway will induce senescence is extremely complex and a lot of research is yet to be carried out to find out exactly how they interact. 3,4,5,7,15.,16,18
Campisi, 2005 J. Campisi, Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors, Cell 120 (2005), pp. 513-522
As I previously mentioned that p16 could prove to be a valuable biomarker to the senescent phenotype and that it is elevated in a parallel fashion to other biomarkers such as ARF and Î²-galactosidase as an organism ages. In one experiment where rodents were their intake of calories was restricted levels p16 were gradually lost and the basis for this study was that a dietary regimen of this type slows down the ageing process in these animals. In essence, because p16 activates the pRB pathway thus inducing senescence, studies such as this suggest that just like the p53 pathway the p16/pRB pathway can promote the ageing phenotype by inducing senescence. 20
After having discussed these pathways I have come to the conclusion that a lot more research needs to be carried out because there are very few reviews or papers discussing the exact mechanism of senescence through these pathways. Any information that is gathered is unsubstantial and although some mechanisms have been described the full picture is not quite fully understood yet.
Different causes of Senescence
It is now apparent that many kinds of oncogenic or stressful stimuli can induce a cell to undergo senescence and not just telomere dysfunction. The chief stimuli being certain types of DNA damage, including DNA breaks and oxidative lesions. When cell damage is irreparable or the DNA repair mechanisms are insignificant that is when cells seem to undergo senescence. Sometimes cells senesce when they are seen to overexpress oncogenes such as RAS. There are also external stimuli such as chemotoxic agents, ionising radiation and genotoxic medicines that can cause sufficient DNA damage for senescence to occur. DNA damage can also occur due to DNA damaging agents such as reactive oxygen species. 3,4,5, 23
Key sources of cellular reactive oxygen species are the mitochondria, peroxisomes, cytochromes and the superoxide burst of phagocytes. These species can cause damage to many macromolecules and cause several different lesions to DNA. As normal byproducts of metabolism, Reactive Oxygen Species are a potential source of chronic, persistent DNA damage in all cells and may contribute to aging. They are produced due to oxidative phosphorylation which uses many reactive electrons to end up with the goal of producing ATP. Mitochondria pose another problem as one ages mitochondrial DNA accumulates many mutations thus causing mitochondrial dysfunction and can cause either an increase or decrease in the reactive oxygen species. 5, 22
The accumulation of reactive oxygen species means that a cell has not got the capability to deal with the constant damage that occurs to DNA meaning that an ageing phenotype will occur as cells undergo apoptosis or senescence. From what I have read it what directs a cell to either undergo senescence or apoptosis is not fully understood yet. But from some research it has been put forward that senescent cells display protein signals prevent it from undergoing apoptosis. This means that there will be an accumulation of senescent cells as an organism ages which is another hypothesis to the ageing phenotype. 3
Another hypothesis towards a cause of senescence is that as we age DNA repair machinery become less functional and more cells have more damaged DNA such as double strand breaks, which I mentioned earlier and has the consequence of senescence. The mechanism involved for inducing senescence due to DNA damage is slightly different. Ataxia telangiectasia mutated is recruited by the DNA break and phosphorylates key proteins that initiate the DNA damage checkpoint. In this case it is p53 which as we know is a key regulator or senescence is then active and can cause cell cycle arrest and thus senescence. 3,4,5
The link between Senescence and ageing
The association between cellular senescence and organismal aging is highly suggestive of a causal link between these two processes, although establishing a direct causative relationship is challenging. To start with, the factors that trigger senescence also trigger apoptosis and quiescence, making it difficult to dissect the contribution of each of these responses to aging. 3,4,5
Senescent cells are known to release several components that can lead to tissue disruption which is one of the features of the ageing phenotype. Components such as pro-inflammatory cytokines, enzymes that cause the 'wear and tear' of tissues, and growth factors that can aggregate the ageing phenotype. It is also known that in an aged body the number of senescent cells is much higher that of one which is much younger proving that more cellular senescence occurs as we age. There is in vivo research which shows that the symptoms produced from atherosclerosis is due to the release of components from senescent cells. It has also been proved that reactive oxygen species lead to the decrease in levels of nitric oxide in vascular smooth muscle due to senescent cells. This shows that there is a lot of research in favour of the idea that replicative senescence is one of the causes of ageing and that the symptoms of ageing on the outside are in accordance with what is happening inside a senescent cell. 3,4,5, 21
Ageing is a process of the accumulation of changes that occur to an organism over time that lead to death ultimately. I have discussed the major mechanisms that lead to ageing and senescence and I shall stress again that the key ones are the p53 and pRB pathway. But as yet, more research needs to be done as there could be more pathways and the p53 and pRB pathway could be linked as one. There are still many questions that need answering. P16 has only recently emerged as a potential biomarker for ageing and senescence so its exact function is still yet to be studied.
There are many causes that lead to the phenotype of ageing such as excess free radicals and the decline in anti-oxidant defense, glycations, telomere shortening, declining and inadequate DNA repair, defective cell cycle control, inflammatory cytokines, protein cross-linking, lipofuscin and many more. From the research that I have read about I can conclude the majority have a link with senescent cells to cause these age related pathologies.
Finally, as gerontology has not been researched that much, a great deal amount of studies need to be undertaken so we can fully comprehend what happens in an ageing body and what signals are required to trigger the ageing response. For example, a method needs to be created so that we can tell why a cell chooses to either undergo apoptosis or senesce as it is still very unclear as to how a cell chooses which pathway to undertake.