Cellular senescence is the barriers that protecting our cells from undergoing abnormal proliferation. However, there are many factors that can induce the cellular senescence such as, DNA damage and telomere shortening. (Campisi J, 2005) Study by Dr. Leonard Hayflick and Dr. Paul Moorhead, showed that human cells have a limited capacity to grow in culture by dividing. They showed that, human cells derived from newborn tissue, embryonic and fetal can divide from 40 to 60 times, but then can't divide more than that. However, the number of cell divisions is called Hayflick Limit. (Maity and Koumenis, 2006)
The mechanism of cellular Senescence was first explained in seminal cells, this study was done by Hayflick and Moorhead in 1961 when they indicated that normal human fibroblasts were entered a condition of irreversible growth arrest after sequential growth in vitro. Whereas cancer cells can't enter in this process. They hypothesized that, the survival of cellular factors, which consumed through cell divisions limited the proliferation of normal cells. They want to explain that this stopwatch may play a key role in the process of aging.
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Nowadays, scientists believe that the determination of the Hayflick Limit is the length of human telomeres. However, scientists considered that a cellular senescence is a stress response activated by several counting mechanism factors such as telomere shortening. Telomeres are the end part of chromosomes. When the cell divides, it must to double its chromosomes. Results in each daughter cell have a full complement of genetic materials. When the cells double the chromosomes, in each time the chromosomes lose a little bit of its telomeres. After 40 to 60 times of cells divisions, the telomeres have reached a critically short length, the replication of chromosomes will stop and no longer cell dividing. So, the cells that have shortened telomeres and can't divide called senescent.
However, they are three main factors of molecular biology of cellular senescence, the accumulation of DNA damage, derepression of the INK4a/ARF locus and telomere loss.
One of the important signals factors that stimulate senescence response is dysfunctional telomeres. However, there are many signals factors that may induce senescence response; such as: strong mitogenic signals, non-telemetric damage DNA, and chromatin perturbations.
Cells arrest growth in association to phenotype are not identifiable as different from replicatively senescent cells, when they provide medium high level of DNA damage, occurs. However, when they deal with agent or expose to mutation, which may damage normal chromatin complex. In addition, strong mitogenic or stress signals can enhance the cell to become senescent. There are many oncogenes that may induce the cell to become senescent such as activation of growth factor signalling like RAS or RAF.
All these persuaders of cellular senescence have a power to cause cancer. As result, chromatin perturbation, DNA damage, and release of oncogenes don't cause any change in normal cells, but enhance cells to arrest growth in association with a senescent phenotype. This may lead to cellular senescence.
Cell Divisions by Telomere Loss:
Telomere works to protect the DNA ends from degradation and recombination. ( de Lange, 2005) Because of the intrinsic inability of the DNA replication mechanism to replicate the ends of linear molecules, telomeres become consequently shorter in each time of cell division. (Blasco, 2005) By times, telomeres become critically short length. Then, telomeres act as double-stranded DNA breaks, which stimulate the p53 tumour suppressor leading to telomere-iniated senescence or apotosis. (de Lange,2005)
The end replication problem:
Several studies shows that, the process of semi-conservative replication of DNA works only in the 5' to 3' direction, and the DNA polymerase needs binding with an RNA primer. (Smeal et al., 2007) Scientists predicated the characteristic of the consequences of DNA replication and they termed it the end-replication problem. However, the loss of a small 5' nucleotide segment because of the DNA synthesis took place and with repeated replication the telomere become shorter.
Telomeres and telomerase:
Telomeres are located at the end of liner chromosomes, and are composed of repetitive DNA sequences. There are 92 telomeres in humans because 23 chromosomes and in each end of each chromosome one telomeres. Hence, each human telomere has thousands of repeats of the six nucleotide sequence, TTAGGG. The hypothesis of aging and cancer based on the telomere-telomerase shows that most human somatic cells don't have telomerase activity, whereas most human tumours have telomerase activity. However, the balance between the end-replication problem and telomerase maintained the telomere length. Telomerase can be defining as a cellular reverse transcriptase (a ribonucleoprotein enzyme complex) which is referred to as a cellular immortalizing enzyme. It maintains the telomere length by adding hexameric (TTAGGG) repeats onto the end of the chromosomes. Telomerase has ability to reinstate lost telomeri DNA- repeat sequence, which can help cells to bypass replicative senescence and may confer cellular immortality. (Beitzinger et al., 2006) There is evidence that replicative senescence of human cells is due to telomere shortening. (Vijji et al., 2007)
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The two crucial constituents of telomerase consist of two enzymes, a human telomerase reverse transcriptase (hTERT) and an RNA template molecule which has a complimentary to the human telomeric DNA (hTR). However, hTERT is the primary rate-limiting component of telomerase, and hTR can be limiting for telomere maintenance. Telomerase is protecting germ line cells against telomere shortening. Studies show that, telomeres of human germ line cells are maintained at about 15 kbp. In contrast, in most somatic cells that undergo more than 40-80 times of cellular replicative senescence, telomerase is not expressed in these cells, with the considering of telomere shorting. Whereas, the expression and activity of telomerase in tumours cells and transformed cells is very high. (Vincent et al., 1992; Koji et al., 2001)
Telomeres are the regulator that regulates the number of cell division. However, normal somatic human cells have a limited capacity to divide, while the tumour cells can divide forever. Studies show that, the telomeric sequences become shorter after each DNA replicates. So, the cells stop dividing (senescent) when the telomere reaches a critically short length. However, when the telomerase introduced into human cells result in maintain a normal chromosome complement.
Loss of telomere integrity:
Telomerase is an enzyme whose synthesised Telomeric DNA and this enzyme play a key role in detecting the starting of replicative senescence. However, telomeric DNA become shorter in case of absence or low expressed of telomerase during cell division. This phenomenon called telomere integrity, which is triggering senescence by trigger DNA damage checkpoints and then stop cell multiplication (Kurz et al., 2004). As mention above, the significant progressive oxidative damage of DNA resulting from the frequent contact of cellular contents to oxidative stress is one of the important processes that related to aging and age-related pathologies. However, studies on fibroblasts shows that oxidative stress may activate the growth of replicative senescence (Kurz et al., 2004). This process called stress-induces premature senescence (Toussaint et al., 2000). Several studies show that, the starting point of senescence has been caused by accelerated telomere attrition, most likely resulting from the formation of single strand breaks in the telomere (von Zglinicki, 2002). Some other studies illustrated that; telomere damage may not attributed by stress-induced premature senescence (Chen et al., 2001; Toussaint et al., 2002). However, it is not clear why these differences are their between the studies. It may be of cell type, the type or level of oxidative used in the studies, the level of antioxidant protection and may be for some other experimental differences.
The Telomere clock in Cancer protection:
The telomere clock works to limit the process of normal cells as well as those cells are already on the road to neoplastic transformation. However, cancer cells release high level of telomerase to maintain telomere. (Stewart and Weinberg, 2006) Exceptionally, telomeres in immortal human cells and cancer cells can maintain in the absence of telomerase by a process called alternative lenghthening of telomerase (ALT). (Muntoni and Reddel, 2005) This finding suggests that, telomerase is a tumorgenic factor that plays a vital role in cancer progression. Studies show that, the cellular outcome responsible for telomeres protection against tumour involves apoptosis and senescence. (Kelland, 2005; Shammas et al., 2005) Furthermore, preneoplastic cells are exposed to dysfunctional short telomeres and may also expose to other potential triggers of apoptosis or senescence.
The Telomere Clock in Aging:
Several studies demonstrated the relationship between the telomere length and age as well as telomere length and diseases related to aging. (Canela et al., 2007; Cawthon et al., 2003) Moreover, syndromes related to the aging such as dyskeratosis congenita (DC) and aplastic anemia, are associated to mutations in the telomerase or proteins that involved in telomerase activity, which are characterised by a faster rate of telomere attrition with age. (Mason and Bessler, 2004) On the other hand, aging syndromes that are produced by the mutations in DNA repair such as BLM (Bloom syndrome), FANC ( Fanconi anemia), and NBS1 (Nijmegen breakage syndrome), are also characterized by the rate of telomere attrition and chromosomal instability. (Blasco, 2005) Furthermore, the rate of telomere shorting is activated by oxidative damage. (von Zglinicki ) many studies suggested that, telomere biology such as rate of attrition , telomere capping and telomere length has a strong effect on longevity both in humans and in mice. (Garcia-Cao et al., 2006; Gonzalez-Suarez et al., 2005)
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However, telomere shorting in stem cell may lead to telomere-driven aging, which may lead to a high rate of senescence, as well as stem cells loss its function. Consequently, the combine of these two factors may lead to impaired tissue function. On the one hand, telomerase deficiency causing short telomeres may lead to decreased tumorigenesis, impaired stem cell functionality, and defective tissue regeneration, on the other hand, the overproduction of telomerase may lead to the opposite effects. (Flores et al., 2005) These findings may explain that telomerase activity and telomere length play a vital role in the stem cells fitness and it may stimulate cancer and aging.
The INK4a/ARF Locus:
p16INK4 and ARF are a tumour suppressors encoded by the INK4a/ARF locus, which in addition to their function in cancer, also activate cellular senescence. However, these two protein sharing the same exons but they have different reading frames, and they possess different molecular functions. On the on hand, p16INK4a is work as an inhibitor of the cyclin-dependent kinases CDK4 and CDK6 and works to impose a G1 cell cycle arrest, while, ARF works to regulates p53 atability by inactivation of the p53-degrading ubiquitin ligase MDM2 . ( Gil and Peters, 2006) The expression of INK4a/ARF locus in young organisms is very low, but starts to reduce with the aging. ( Krishnamurthy et al., 2004)
Role of p53 and p16/pRB Tumor-suppressior pathways:
Factors like, activated oncogenes and ionizing radition have been known to transform cells. These agents are involved after cell mutation, which may stimulate the senescence response. However, mutation by senescence leads to dysfunction of the genes encoding contents of the p53 or PRB tumor-suppressor pathways. Both, p53 and PRB are involved in the production of many cellular genes. However, P53 and PRB, each one of them has his pathway to stimulate senescence. The p53 pathways, results to DNA damage, even it may also response to nongenctoxic stress, and temprarly stop cell cycle progression by expression of p21. While p16 and PRB are basically induced by non genetic stress.
The G1/S control in cellular senescence:
pRb is a vital protector of cell cycle progression in eukaryotes. Ther are several factors that are regulate the activity of pRb, such as acetylation, ubiqitination and phosphorylation. However, the activity of pRb may creat a block on G1 progression that is developed by phosphorylation (Sherr and McCormick, 2002). Furthermore, a series of cyclin -dependent kinases (CDKs), CDK2, CDK4 and CDK6, play a vital function in the phosphorylation of pRb (Rowland and Bernards, 2006). When CDKs phosphorylated pRb , pRb lose its ability to join E2F/Dp transcription factor complexes, which may result in entry into S-phase of the cell cycle. The action of CDKs is inactivated in senescent cells due to release of CDK inhibitors. There are two main CDK inhibitors that are released by senescent cells such as, p21Cip1/Waf1/Sdi1 and P16NK4a. However, p21Cip1/Waf1/Sdi1 work to links between p53 pathway and Rb-pathway to provide a tight security network in the direction of tumour suppression (Gil, Peters G, 2006). Several studies indicated that, the regulation of p21Cip1/Waf1/Sdi1 expression has a vital role in processes like cellular senescence and DNA damage-induced cell cycle, which may prevent the cells to become carcinoma cell (Sherr and Roberts, 1999). p16INK4a is wok to inhibit the activation of D-type CDKs, CDK4 and CDK6. When the p16INK4a is binding to CDK4 and CDK6, it stimulates the redistribution of Cip/Kip family CDK inhibitors, P27Kip1 and P21Cip1/Waf1/Sid1, from cyclinD-CD4/6 to cyclinE-CDK2 complexes, causing inactivation of CDK2-kinase. Thereafter, the association between p16INK4a and p21Cip1/Waf1/Sdi1 prevent the phosphorylation of pRb, which result in stable G1 arrest in senescent cells (Gil and Peters, 2006) . Interestingly, the p16INK4a gene is recognized as a tumour suppressor gene because it is often inactivated in a wide range of human cancers. In addition, it may also participate with another tumour suppressor gene called p14ARF. Studies show that the mutation within this region only affects p16INK4a activity but not p14ARF activity. This finding suggests that, p16INK4a /Rb-pathway play a vital function in tumour suppression (Gil and Peters, 2006).
However, it is known that, the p16INK4a inactivate the pRb pathway, following the inactivation of pRb pathway induce the DNA synthesis but not cell proliferation, this happened if p16INK4a is ectopically released prior to inactivation of pRb in human cells. Whereas, inactivation of pRb is adequate to inactivate the p16INK4a effect, this is happened if pRb pathway is inactivated prior to p16INK4a expression (Gil and Peters, 2006). However, when p16INK4a activated pRb so pRb enhance another pathway which is irreversibly causes cell cycle arrest either in M or G2 phase. Moreover, poly-nucleated cells is gradually increase when the human cells inactivated by pRb and P53 and release high level of p16INK4a (Takahashi et al., 2006). This finding may explain that this mechanism may target cytokinesis. Studies using SVts8 cells indicated that, p16INK4a/ Rb pathway associate with mitogenic signals to stimulate irreversible cytokinetic block during the generation of reactive oxygen species (ROS) (Takahashi et al., 2006). It has been mention before that, the physiological function of the cells required ROS, but overproduction of ROS may lead to apoptosis or cellular senescence. In case of low stress, mitogenic signals inactivate pRb , which is then stimulate E2F/DP complexes to induce S-phase entry. E2F/DP activation reduces the amount of ROS (Takahashi et al., 2006). These finding shows that, the E2F/DP activity manage this reaction in proliferating normal human cells, even mitogenic signals have the power to generate ROS. Whereas, in case of high cellular stress, p16INK4a/Rb -pathway inactivate E2F/DP. So in this case, mitogenic signals stimulate the ROS production by activating PKCδ, which is a significant downstream mediator of the ROS signalling pathway.
Interestingly, when ROS and PKCδ activate ROS signalling pathway lead to the over generation of ROS, which may create a positive feedback loop to mainatain ROS- PKCδ signalling. In human senescent cells, maintained activation of ROS- PKCδ signaling permanently blocks cytokinesis by decreasing WARTS and a mitotic exit network (MEN) kinase, which are required for cytokinesis (Takahashi et al., 2006). This may lead to increase the level of p16INK4a and create an autonomous stimulation of ROS- PKCδ signalling, result in an irreversible block to cytokinesis in human senescent cells. However, this mechanism may act as a fail-safe mechanism, practically in case of the sudden inactivation of p53 and pRb in human senescent cells (Ramsey and Sharpless, 2006).
It is well know that, DNA damage may lead to the development of cancer. However, study by using in vitro cultured cells illustrated that, the infliction of different forms of DNA damage stimulate senescence. (Parrinello et al., 2003) Recently, studies shows that DNA damage could be the most common causative agent underlying various forms of cellular senescence, including telomere dysfunction and oncogene-induced senescence. (Di Micco et al., 2006; Bartkova et al., 2006) There are several factors that may contribute to cellular senescence and aging including; significant increase of DNA mutations, DNA oxidation, chromosomal losses, and telomere- independent É£H2AX foci. (Vijg, 2000; Weaver et al., 2007; Seldelnikova et al., 2004) However, DNA damage may lead to the accumulation of Senescent cells in aged tissues.
Senescence provoked by oxidative stress. Several studies shows that, the reactive oxygen species in endothelial cells produced from intracellular or extracellular origin, which can stimulate the growth of senescence by acting at multiply sub-cellular levels. As mentioned above, telomeres are mainly damaged by oxidation. Furthermore, telomerase is inhibited by oxidative damage which lead to effect directly or indirectly in telomeres. However, ROS can stimulate senescence by telomere-independent mechanisms. Finlay, this reaction will damage the DNA and mitochondria. In addition, it may also activate sytosolic response kinases or other redox-sensitive signalling proteins, which are produced in senescence responses.
However, over mutagenic stimulation by acceleration of activated oncogenes has been shown to stimulate senescence. This finding has been observed during the overproduction of Akt, Ras, or Racl activation. However, in this stage the stimulation of senescence is assumed as a result of dysfunction of the cellular redox-balance causing the overproduction of ROS, which may lead to induce p53 activity.
The Role of mitochondria:
Reaction oxygen species (ROS) may destroy the DNA and mitochondria, which may cause dysfunction of mitochondria. However, during normal respiration ROS is generated by mitochondria. Several studies illustrated that; ROS may affect the electron transport chain leading to increase the oxidative burden of the cell. Recently, the significance of mitochondria-derived ROS in the induction of cell senescence has been focused by a study investigating the function of prohibitin-1 (PHB1) in this process. PHB1 is a part of the inner mitochondrial membrane, and assume to be the vital part that maintains the normal mitochondrial function. This can explain that, the function of PHB1 in the cells stimulate mitochondria to generate ROS. Moreover, over production of ROS in mitochondria lead to oxidative destruction of cell components such as proteins, nucleic acids, and lipids.
The senescent phenotype:
Cellular senescent involve several changes in gene expression. However, a few of which are essential for the growth arrest. Interestingly, senescent cells need two or three features that show senescent phenotypes, such as; irreversible of cell division, and in other cell types, resistance to signals that may lead to apoptotic cell death. It may also affect the cell functions.
However, the induction of senescence phenotype may lead to impediment to normal mitosis creating a mutation in the genetic material such as; genotoxin-induced premature senescence, cumulative DNA damage induced senescence, intrinsic ageing induced senescence (M1), and proliferate history dependent telomere attrition induced mitotic crisis (M2). (Roninoson et al., 2003)
B-Galactosidase is a common feature of senescent cells that is expressed as a natural (pH 6) and called the senescence-associated B-Galactosidase (SA-Bgal). SA-gal expressed in different senescent human cells types, such as: keratinocytes, fibrobalsts, mammary epithelial cells and adult melanocytes. Furthermore, SA-B gal induced by diverse senescence- inducing stimuli such as short telomeres. However, the assay of SA-gal is simple and commonly used as a marker for the senescent phenotype.
Senescence and Cancer:
Senescence works as antiproliferative against tumour suppressor mechanism. However, senescence was found to be mediated by ARF/p53 and INK4a/RB pathways, which are tumour suppressor (Sharpless, 2005). However, loss of these tumour suppressors may lead to oncogenic transformation of human cells in vitro (Han and Weinberg, 2002). Studies show that, a genetic defect in neurofibromas may lead to high levels of Ras activity, benign lesions in prostate and bening lesions of the skin carrying oncogenic mutant BRAF (Chen et al., 2005; Michaloglou et al., 2005). Moreover, chemotherapy (anti-cancer) may cause severe DNA damage leading to trigger cellular senescence cell (Roberson et al., 2005).
Senescence and Aging:
It has been know that when the telomere become shorter at the end cells called senescence cells, which mean the cell can't divide more than this limit ( Hayflick limit). However, accumulation of senescent cells in tissues lead to aging of the tissue and also senescence may limit the regenerative potential of stem cells. On the one hand, aging could result of the accumulation of senescent cells in the tissue; on the other hand, the fatigue of the regenerative potential of stem cell may result in aging.
Senescence and Apoptosis:
The final fate of senescent cells has pathophysiological disorders, due to altered phenotype. Several studies investigated the relation between senescence and apoptosis in endothelial cells. Study by Wagner and his group, shows that, apoptosis is the final fate of senescent cells. (Wagner et al., 2001) Whereas, other studies show that, senescent cells don't undergo apoptosis, but it may induce the sensitivity of these cells to apoptotic stimuli such as TNF-α and oxidized LDL. (Hoffmann et al., 2001; Spyridopoulos et al., 2002) However, this effect may attribute to, p53, p21, and p16 expression on the induction of apoptosis and senescence. (Sharpless and DePinho, 2004) The p53/p21 pathway is involved in apoptosis and senescence (Napolitano et al., 2007), while p16 pathway is only involved in senescence.
Cellular Senescence can be triggered by several mechanisms such as; DNA damage, telomere shortening and derepression of INK4a/ARF locus. These mechanisms control the excessive of cellular proliferation, which lead to protect the cell to become cancer cells. Recently, several studies suggest that cellular senescence also play a key role of aging. Further studies are needed to address and clarify the effect of senescence in cancer and aging.