Biological aging or Senescence is the change in organism biology as it ages after its maturity. These changes range from those affecting its cells and their function to that of the whole organism. In fact, there are a number of theories why senescence takes place including those that it is programmed by gene expression changes and that it is the accumulative damage of biological processes (Lodish et al., 2000)
In simple words, when the normal diploid cells lose their ability to divide, this call cellular senescence phenomenon. This usually occurs after around 50 cell division in vitro. As a result of toxins, DNA double strand breaks etc. Some cells become senescent after fewer replications cycles. The other name of this phenomenon is "replicative senescence", the "Hayflick limit ", or the Hayflick phenomenon. In 1965 one scientist called Dr. Leonard Hayflick (Figure 1.1) published this information for the first time and therefore this phenomenon took his name as honour for this great man (Woodring et al., 2002)
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However, in response to DNA damage cells either self-destruct (apoptosis, programmed cell death) or age and this is in case the damage cannot be repaired. In this 'cellular suicide', the death of one, or more, cells may benefit the organism as a whole (William et al., 2003)
Figure 1.1 Dr Leonard Hayflick in 1988.
Hayflick limit and replicative senescence
Hayflick and Moorhead (1961) stated that "The Hayflick limit is the number of times a normal cell population will divide before it stops, presumably because the telomeres reach a critical length"
Hayflick described three phases in the cell life. At the beginning of his practical experiment, he called phase one to the primary culture, phase two to that stage when the cells were proliferating and finally phase three when the cells growth is diminished and eventually stopped and this is obviously occurred after many months of doubling (Hayflick and Moorhead ,1961).
He found this limit is correlated with the length of the telomeres at the end of a strand of DNA. However, during the DNA replication process, small segments of DNA at each end of the DNA strand (telomeres) are unable to be copied and are lost after each time DNA is duplicated (Watson,1972).
Basically, telomeres are a region of DNA that code for no proteins; they are simply a repeated code on the end region of DNA that is lost. Ultimately, after so many divisions, the telomeres become depleted and then the cell commences apoptosis. This is considering as a defence mechanism of the cell to prevent any replicating error that could occurs which might cause mutations in DNA. Olovnikov (1996) stated that once the telomeres are depleted due to the cell dividing many times, the cell will no longer divide and the Hayflick limit has been reached.
However, this correlation is only true for normal cells. Cancer cells possess an enzyme called telomerase which is able to restore telomere length. This gives cancer cells their infinite replicative potential and explains why cancer cells are not restricted to Hayflick's limit because their telomere length is never depleted (Wright, 2000).
A telomerase inhibitor is being proposed as a treatment for cancer; so this way cancer cells would not have the ability to maintain telomere length and therefore would die just like normal cells (Wright, 2000).
The end replication problem
Basically, the end-replication problem (Figure 3.1) is a very essential problem which is associated with linear DNA replicating. As it is well known, that DNA is a molecule made up of two strands of nucleic acid subunits. The direction of a strand of DNA is specified by how these nucleic acid subunits are attached. The nucleic acid structure has as its backbone a ribose, or 5 carbon sugar and nucleic acids attach to one another between two of these carbons, the 5' carbon and the 3' carbon. The two strands of a DNA molecule are anti-parallel to one another. which means that one strand runs 5'-3' while the complimentary strand runs 3'-5' (Russell and Peter, 2001).
Telomeres and telomerase:
Figure 3.1 explains "End replication problem "
Always on Time
Marked to Standard
Telomeres and telomerase:
As it is well known that, a telomere is a repeating DNA sequence (for example, TTAGGG) at the end of the body's chromosomes (Griffith et al., 1999). The telomere length can reach up to 15,000 base pairs. Telomeres function by preventing chromosomes from losing base pair sequences at their ends. In the same time, they also stop chromosomes from fusing to each other. However, each time a cell divides, some of the telomere is lost. An therefore, when the telomere becomes too short, the chromosome reaches a critical length and can no longer replicate, which means that a cell becomes old and dies by a process called apoptosis. The activity of telomere is controlled by two mechanisms: erosion and addition. Erosion takes place each time a cell divides. On the other hand, addition is determined by the activity of telomerase (Ben-Porath and Weinberg, 2005).
On the other hand, Telomerase, also called telomere terminal transferase, is basically an enzyme which is made of protein and RNA subunits that elongates chromosomes by adding TTAGGG sequences to the end of existing chromosomes. Telomerase is found in fatal tissues, adult germ cells, and also in tumour cells. Telomerase activity is regulated during development and has a very low, almost undetectable activity in somatic (body) cells. Because these somatic cells do not regularly use telomerase, they age. The result of aging cells is an aging body. If telomerase is activated in a cell, the cell will continue to grow and divide (Cohen et al., 2007)
Figure 4.1 Telomeres and Telomerase
Adopted from: http://stemcells.nih.gov/info/2001report/appendixC.asp
Oxidative stress is caused by the presence of any of a number of reactive oxygen species (ROS) which the cell is unable to counterbalance. The result is damage to one or more biomolecules including DNA, RNA, proteins and lipids (Krohn et al., 2007). In humans, oxidative stress is involved in many diseases, such as atherosclerosis, Parkinson's disease, heart failure, myocardial infarction, Alzheimer's disease.etc. (Diego et al., 2009), but short-term oxidative stress may also be important in aging prevention by induction of a process called mitohormesis (Gems and Partridge, 2008).
Reactive oxygen species can be beneficial, since they are used by the immune system as a way to attack or kill pathogens. In addition to that these species are also used in cell signalling. This is dubbed redox signalling (Schafer and Buettner, 2001).
Role of mitochondria in apoptosis
0A1s it is well known, Mitochondria play an important role in the regulation of cell death. They contain many pro-apoptotic proteins such as Apoptosis Inducing Factor (AIF), Smac/DIABLO and cytochrome C. These factors are released from the mitochondria following the formation of a pore in the mitochondrial membrane called the Permeability Transition pore, or PT pore. These pores are thought to form through the action of the pro-apoptotic members of the bcl-2 family of proteins, which in turn are activated by apoptotic signals such as cell stress, free radical damage or growth factor deprivation. Mitochondria also play an important role in amplifying the apoptotic signalling from the death receptors, with receptor recruited caspase 8 activating the pro-apoptotic bcl-2 protein, Bid (Gulbins et al., 2003)
Figure 1.6 shows Role of mitochondria in apoptosis
Several pathways can trigger senescence in various cell types and under a variety of different conditions. The most common pathways described in relation to senescence are the p16/Rb and p53/p21 pathways.
7.1 - The p16/Rb pathway
Rb mediates regulation of the cell cycle at the transition from first gap phase (G1) to DNA synthesis phase (S phase). Rb is hypophosphorylated during G1/G0 and is bound to E2F whereby the activity of E2F is inhibited. When Rb is phosphorylated it releases E2F and this occurs before the G1/S transition and through S-phase. E2F mediates transcription of a variety of genes necessary for G1 to S progression and replication including cyclin-E, cyclin-A and thymidine kinase (Sherr and McCormick, 2002). Phosphorylation of Rb is mediated by cyclin dependent kinases (CDK) bound to cyclins (cyclin-D1/CDK4-6 and cyclin-E/CDK2). CDK4/cyclin-D is activated by mitogenic signaling through the RAS pathway by transcriptional induction of cyclin-D (Sherr and McCormick, 2002). There are proteins called cyclin dependent kinase inhibitors that can inhibit the CDKs. One of them is p16 which inhibits phosphorylation of Rb and thereby G1 to S progression by inhibiting CDK4/cyclin-D (Sherr and McCormick, 2002). p16 can in turn be regulated transcriptionally by several proteins and seems to be a sensor for cellular stress .
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There is extensive evidence for an important role for the p16/Rb pathway during the induction of senescence. Overexpression of p16 induces features of senescence including growth arrest (McConnell et al, 1998) while knock-down of p16 using short interfering RNAs (siRNAs) inhibited RAS-induced senescence in epithelial cells (Bond et al, 2004). Re-expression of Rb in a cancer cell line (Xu et al, 1997) or inhibition of E2F (Maehara et al, 2005) also induces senescence, indicating that the p16/Rb pathway can induce senescence under several conditions (Figure 7.1)
7.2 - The p53/p21 pathway
p53 has been named the "guardian of the genome" and is mutated in 50% of all tumors. It acts as an integrator for various signals and can mediate cell cycle arrest, apoptosis and differentiation. There are several mechanisms that regulate the activity of p53. The DNA-damage-ATM/ATR-Chk1/Chk2 pathway activate p53 by phosphorylation (Sancar et al, 2004) leading to displacement of the cellular protein MDM2, which relocates p53 from the nucleus to the cytoplasm and targets it for degradation (Sherr and McCormick, 2002). MDM2 can also be regulated by p19ARF, which inactivates MDM2 leading to an increased activity of p53 (Weber et al, 1999). Many other proteins e.g. SUMO-1 and Parc can modulate p53 activity and the p53 activity can further be modulated by protein modifications (e.g. acetylation) (Sancar et al, 2004; Sherr and McCormick, 2002). Once activated, p53 induces transcription of many genes involved with cell cycle arrest and apoptosis (Giaccia and Kastan, 1998; Zhao et al, 2000). One of the activated proteins that mediate the cell cycle arrest downstream of p53 is p21. p21 is a member of the "Cip/Kip" family of cyclin-dependent kinase inhibitors (CDKI) that inhibits CDK2/cyclin-E and to a lesser extent CDK4/cyclin-D (Giaccia and Kastan, 1998). p21 is believed to be the main target for cell cycle arrest downstream of p53(Figure 7.1)
Figure 7.1 Pathways to senescence
Reactive oxygen species
Reactive oxygen species (ROS) are reactive molecules that contain the oxygen atom. They are very small molecules that include oxygen ions and peroxides and can be either inorganic or organic (Shouval et al., 2007). They are highly reactive due to the presence of unpaired valence shell electrons. ROS form as a natural by product of the normal metabolism of oxygen and have important roles in cell signalling. However, during times of environmental stress (e.g. UV or heat exposure) ROS levels can increase dramatically, which can result in significant damage to cell structures. This cumulates into a situation known as oxidative stress. ROS are also generated by exogenous sources such as ionizing radiation (Zhang and Chen, 2004).
The G1/S control in cellular senescence
pRb is a crucial gatekeeper of cell cycle progression in higher eukaryotes. The pRb activity is tightly regulated by various post-translational modifications, like phosphorylation, acetylation and ubiquitination, and is thought to impose a block on G1 progression which is alleviated by phosphorylation (Sherr and McCormick, 2002). Especially, a series of cyclin-dependent kinases (CDKs), CDK2, CDK4 and CDK6, play an important role in the phosphorylation of pRb . When pRb is phosphorylated by these CDKs, pRb loses its ability to bind E2F/DP transcription factor complexes resulting in entry into S-phase of the cell cycle. However in senescent cells, the activity of CDKs is blocked by elevated expression of CDK inhibitors, p21Cip1/Waf1/Sdi1 and p16INK4a (Malumbres and Barbacid, 2007).
p21Cip1/Waf1/Sdi1 is a founding member of the mammalian CDK inhibitor family and is one of the best characterized transcriptional targets of the p53 tumor suppressor protein (Sherr and McCormick, 2002). Thefore, p21Waf1/Cip1 links the p53- pathway to the Rb- pathway, providing a tight security network towards tumor suppression. Indeed, the role of p21Cip1/Waf1/Sdi1 expression is well documented in various cell culture studies; up-regulation of p21Cip1/Waf1/Sdi1 expression participates in processes such as DNA damage-induced cell cycle arrest, cellular senescence and terminal differentiation that may prevent tumor formation (Malumbres and Barbacid, 2007). And since mutations in the p21Waf1/Cip1/Sdi1 gene are rarely observed in human cancers and mice lacking p21Waf1/Cip1/Sdi1 gene do not exhibit any predisposition to spontaneous tumor formation (Sherr and McCormick, 2002), it remains unclear whether p21Cip1/Waf1/Sdi1 indeed plays a key role in tumor suppression in vivo.
The INK4a gene encodes another type of CDK inhibitor, p16INK4a, which specifically binds to and inactivates D-type CDKs, CDK4 and CDK6. The binding of p16INK4a to CDK4/6 also induces redistribution of Cip/Kip family CDK inhibitors, p21Cip1/Waf1/Sdi1 and p27Kip1, from cyclinD-CDK4/6 to cyclinE-CDK2 complexes resulting in the inactivation of CDK2-kinase (Shouval et al., 2007). Thus, induction of p16INK4a collaborates with p21Cip1/Waf1/Sdi1 to prevent phosphorylation of pRb, leading to a stable G1 arrest in senescent cells. Importantly, the p16INK4a gene is frequently inactivated in a wide range of human cancers and is therefore recognized as a tumor suppressor gene. This may also be because the coding region of the p16INK4a gene is partly shared with another tumor suppressor gene called p14ARF (Zhang and Chen, 2004).
In man cancer, quite large number of the point mutations within this region only affects p16INK4a activity but not p14ARF activity, indicating that p16INK4a /Rb-pathway, in itself, also play key roles in the suppression of tumour.
Basically, p16INK4a is known to exert its effects through pRb, subsequent inactivation of pRb stimulates DNA synthesis but not cell proliferation if p16INK4a is ectopically expressed prior to inactivation of pRb in human cells (Beausejour et al., 2003). By contrast, inactivation of pRb is sufficient to override the p16INK4a effect if pRb is inactivated prior to p16INK4a expression. It is therefore likely that once pRb is fully activated by p16INK4a, pRb activates yet another mechanism that irreversibly causes cell cycle arrest either in G2 or M phase. Indeed, a dramatic increase of poly-nucleated cells is observed when pRb and p53 were subsequently inactivated in human cells expressing high level of p16INK4a , suggesting that this mechanism may target cytokinesis (Beausejour et al., 2003).
Although ROS are required for the physiological function of the cells, excessive ROS cause anti-proliferative effects such as apoptosis and/or cellular senescence. During low stress condition, mitogenic signals inactivate pRb and therefore activate E2F/DP complexes to stimulate S-phase entry (Finkel, 2003) . Moreover, E2F/DP activation decrease ROS levels by regulating genes involved in ROS production. Thus, although mitogenic signals have the potential to stimulate ROS production, this effect appears to be counterbalanced by E2F/DP activity in proliferating normal human cells. In condition of high cellular stress, however, the activity of E2F/DP is blocked by p16INK4a/Rb-pathway. In this setting, mitogenic signaling, in turn, increases the ROS production, thereby activating PKCÎ´, a critical downstream mediator of the ROS signaling pathway (Wheaton and Riabowol, 2004). Moreover, once, activated by ROS, PKCÎ´, promotes further generation of ROS, thus establishing a positive feedback loop to sustain ROS- PKCÎ´ signaling . Sustained activation of ROS- PKCÎ´ signaling irreversibly blocks cytokinesis, at least partly through reducing the level of WARTS (also known as LATS1), a mitotic exit network (MEN) kinase required for cytokinesis , in human senescent cells (Finkel, 2003) .Thus, elevated levels of p16INK4a establish an autonomous activation of ROS- PKCÎ´ signaling, leading to an irrevocable block to cytokinesis in human senescent cells This system may serve as a fail-safe mechanism, especially in case of the accidental inactivation of pRb and p53 in human senescent cells It is noteworthy that we were unable to see activation of PKCÎ´ during replicative senescence in MEFs (Finkel, 2003) .This difference may account for the reversibility of murine cell senescence.