Dendritic cells are antigen-presenting cells found in all tissues of the body and are crucial for stimulating a naïve T-lymphocyte response in the removal of tumour cells.
In the presence of tumour cells, dendritic cells capture (by engulfing portions of the tumour cell) and process tumour-specific molecules (antigens) so that they become presented on their cell surface. Dendritic cells start to mature as they migrate to the lymph nodes, a process which enables dendritic cells to present the tumour information. The maturation process is needed as additional co-stimulatory molecules are required so that they can be recognised by other immune cells. When mature dendritic cells reach the lymph nodes, they interact with cytotoxic T-lymphocytes (CTLs), and pass on the tumour information, causing CTLs to become activated and consequently proliferate. The large numbers of CTLs then circulate the body, recognising the tumour-specific antigens, binding to them and destroying tumour cells by the release of enzymes.
If a cancer cell (or a senescent cell) is not removed by the immune system, then something isn’t working as it should. From the brief overview above, there are a number of points between cell recognition and removal that could have failed. These are:
(1) Dendritic cells did not recognise the cancer/senescent cell.
(2) The dendritic cells did not display cancer/senescent specific markers on it’s surface.
(3) Lymphocytes were not activated in response to dendritic cells.
The changes which occur as we age which may have an impact on the removal of tumour cells/senescent cells will be discussed next.
If the accumulation of senescent cells are so detrimental to the tissues in which they reside, why haven’t we evolved mechanisms to remove them? The answer is that we probably have, but the mechanism which removes them from the tissues becomes impaired as with age.
To understand how this removal system may work, we need to look at the phenotype of senescent cells. Although a large number of the changes which occur during cellular senescence may be cell specific, there appears to be features which are common to the majority of senescent cell types. These include the secretion of growth factors, matrix degrading proteins (MMPs) and the production of cytokines. Since these factors are a common feature, it is likely that they have a common function and are not just a random consequence of the changes which occur during senescence.
One possibility is that senescent cells are removed by the immune system. Senescent cells secrete cytokines to attract immune cells to their location (for their removal), secrete matrix degrading proteins to allow the immune cells easy access and secrete growth factors to stimulate the proliferation of surrounding cells for its replacement once the cell is removed. However, since the immune system itself is governed by ageing mechanisms, its ability to remove senescent cells gradually decreases, therefore the accumulation of senescent cells gradually increases.
The majority of the work on the immune clearance of unwanted cells has been carried out in cancer research. The prevalence of cancer as we all know increases with age, and this may be due to an ageing immune system, consequently resulting in an impaired ability to remove cancer cells as they appear. Over the past several years it has become clear that the immune system plays a crucial role in preventing cancer. As a consequence, there has been a great deal of interest in using our bodies own immune system to recognise and destroy cancer cells (FDA, cancer research uk), a process which could potentially be used to target and destroy senescent cells in ageing tissues.
Richard Faragher presented a light-hearted and often amusing talk on the question “Would it be a disaster to find a cure for ageing and death by natural causes?” This talk was part of the Big Questions series held at the University of Derby (UK) which allows scientists to discuss various science-based issues with people of faith (and of no faith). This talk was recorded for a podcast for all to enjoy.
Richard begins his talk with a brief background, explaining what ageing is, the theories behind why we age and a discussion of the current theories of how we age. He then goes on to talk about whether immortality is actually possible, finishing with religious perspectives on ageing research.
During his talk, Richard discusses data which demonstrates that life expectancy is increasing, but healthy life expectancy is not increasing as fast. With this in mine, he talks about how research into ageing will help increase the rate of healthy life expectancy. This he does by mentioning research using animal models which alter the rate of ageing and the resulting decreases in both the rate at which pathology appear and their severity once present. He goes on to talk about promising research by a team headed by Janet Lord at the University of Birmingham (UK), and how such research has the potential to save tens of thousands of lives a year, thus providing an excellent example of how research into the biology of ageing can help people.
Not often discussed, but a much needed mention in regard to ageing research, especially to those new to the area, are those people who are detrimental to the reputation of ageing research. Richard puts these into three classes of people:
(1) Scientists who say off-the-cuff statements which consequently get blown out of proportion by the media (i.e. “Scientists find cure for ageing”). Ageing is not cured.
(2) Those who say ageing is cured, for the purpose of making money. i.e. from immortality devices and anti-ageing pills.
(3) Visionaries (or “persuasive prophets”) which tell of things to come (i.e. Living to 1000), gaining valuable media attention which should be spent for those doing proper ageing research.
The final discussion on religious perspectives of ageing research is something I’ve never encountered before and definitely worth a listen.
So what you waiting for?...get listening (click here)
Blog entries over the coming weeks is going to be sparse as I finally need to concentrate my efforts on my Phd Thesis…..but stay tuned.
"Our meeting takes a multidisciplinary approach to one of the key problems in ageing today: the concept of frailty, says Richard Faragher, BSRA Secretary. The speakers programme combines established leaders in the fields of calorie restriction, muscle biology and cellular senescence with some of the best and brightest newcomers to ageing research.
Confirmed speakers include:
• Paul Thornalley, University of Warwick (the chemistry of ageing)
• Chris Moulin, University of Leeds (the ageing mind)
• Calvin Harley, Geron Corporation (replicative senescence)
• David Gems, University College London (ageing in invertebrates)
• Arlan Richardson, University of Texas San Antonio (rodent models of ageing)
• Steve Allen, Bournemouth (respiratory frailty and ageing)
The meeting hopes to capture the international quality and reach of British ageing research. It is also designed to promote networking and collaboration for attendees from around the world. Certificates of attendance will be available for CPD purposes.
At present, the most commonly used method to detect senescent cells is a modified beta-galactosidase assay (Dimri et al, 1995). Detectable β-galactosidase at pH 6 was found to increase during replicative senescence of fibroblast cultures in vitro and in vivo and was absent in immortal cell cultures. This was termed senescent-associated β-galactosidase or SA-β-Gal. However, since this first report, there have been numerous studies that have demonstrated SA-β-Gal staining in non-senescent cells.
For example, it has been reported that SA-β-Gal activity is detectable in quiescent cultures of Swiss 3T3 as well as some types of human cancer cells that were chemically stimulated to differentiate (Yegorov et al, 1998). After 21 days in culture, Swiss 3T3 cells in low serum displayed 40-50% SA-β-Gal positive cells and cells treated to differentiate after 13 days displayed as high as 75% staining. Another study looked at the expression of SA-β-Gal in human ovarian surface epithelial cells (HOSE 6-3) undergoing immortalisation by the human papilloma viral oncogene E6 and E7 (Litaker et al, 1998). They found that HOSE 6-3 cells expressing SA-β-Gal was highest (39%) when cells were at crisis. After this stage when cells achieved immortalisation status SA-β-Gal activity sharply decreased (1.3%).
Severino et al (2000) specifically focused on determining the robustness of SA-β-Gal activity as a marker of replicative senescence . This study characterised changes in SA-β-Gal staining in a variety of different conditions. SA-β-Gal activity was found to be elevated in confluent non-transformed fibroblast cultures, in immortal fibroblast cultures that had reached a high cell density and in low-density young, normal cultures oxidatively challenged by treatment with H2O2. They concluded that although SA-β-Gal staining is increased under a variety of different conditions, the interpretation of increased staining remains unclear.
SA-β-Gal staining has also been shown to be a marker for differentiation of human prostate epithelial cells (HPEC) (Untergasser et al, 2003). HPEC cells stimulated with transforming growth factor beta (TGF-β), resulted in an increase in SA-β-Gal activity but showed no terminal growth arrest nor induction of important senescent-associated genes such as p16. It was therefore suggested that TGF-β could contribute to the increased number of SA-β-Gal positive epithelial cells observed in benign prostatic hyperplasia (BPH).
A recent report demonstrated that fibroblasts from patients with autosomal recessive G(M1)-gangliosidosis, which have defective lysosomal beta-galactosidase did not express SA-β-Gal at late passage even though they underwent replicative senescence (Lee et al, 2006). It was also demonstrated that cells depleted of GLB1 (the gene encoding lysosomal beta-D-galactosidase) mRNA underwent senescence but failed to express SA-β-Gal. SA-β-Gal activity is therefore dependent upon lysosomal mass rather than growth state. If this is indeed the case, SA-β-GAL staining would most likely underestimate the percentage of senescent cells in a sample.
Data suggesting that cellular senescence plays a role in vascular calcification was first presented by myself (as far as I am aware, please let me know otherwise) at the Integrative Physiology Post-Graduate Conference, Aberdeen (2007). The abstract was as follows:
A transcriptomic analysis of vascular smooth muscle cells
The senescence of mitotic cells is thought to play a role in ageing and age-related disease. To investigate this further, RNA was extracted from growing and senescent cultures of vascular smooth muscle cells (VSMCs) and subjected to microarray analysis. A literature search of genes involved in atherosclerosis and vascular calcification (an age-related vascular disease) was undertaken and the expression of those genes investigated using the microarray data of senescent VSMCs. Results show that genes known to be involved in atherosclerosis and vascular calcification are significantly up or down regulated in senescent VSMC. ELISA and Western blot analysis was used to validate the microarray data. These results suggest that senescent VSMCs play a role in the development and/or the progression of atherosclerosis and for the first time suggest a role in vascular calcification.
Data from this study (soon to be published) shows that those proteins which are either up or down-regulated at sites of calcification are also transcriptionally up or down-regulated in cultures of senescent vascular smooth muscle cells (VSMCs). The main two culprits involved in calcification appear to be matrix gla protein (MGP) and bone morphogenic proteins (BMP). MGP is normally expressed in endothelial cells and has been identified as a calcification inhibitor of the arterial wall and is thought to neutralise the known effects of BMPs (Zebboudj et al, 2002). In contrast, BMPs are important anabolic factors in bone formation and determinant of bone mineral content (Garrett et al, 2007).
In cultures of senescent VSMC, MGP expression is down-regulated 24-fold (the largest down-regulation of any gene on the chip (affymetrix)), whereas BMP2 is up-regulated more than 4-fold. Since control of BMP activity is important for normal bone formation, the up-regulation of BMPs in senescent VSMC (and the down-regulation of its inhibitor, MGP), suggests senescent VSMC play an important role in the pathophysiology of vascular calcification. BMP2 may be responsible for inducing osteoblastic differentiation of vascular smooth muscle cells, a process thought critical in the initiation of vascular calcification (Hruska et al 2005).
Although, much more work is required to validate the microarray data and investigate these findings in living tissues, this preliminary work suggests for the first time that senescent VSMC may play a role in the development/progression of vascular calcification.
Inflamed endothelial cells in the lining of arteries release pro-inflammatory cytokines which provide a chemotactic stimulus to adhere leukocytes and monocytes, directing their migration into the intima (Boisvert, 2004). These inflammatory cells release pro-inflammatory mediators responsible for differentiating monocytes to lipid-laden macrophages, foam cells (Frostegard et al 1999). These foam cells also secrete proinflammatory cytokines that amplify the local inflammatory response in the lesion (Libby, 2002). The secretion of cytokines and growth factors stimulate the migration and proliferation of SMC. These cytokines also stimulate the secretion of matrix degrading proteins from SMC which permits the penetration of SMC through the elastic laminae and extracellular matrix (ECM) of the growing plaque. Inflammatory mediators can inhibit ECM protein synthesis and increase expression of matrix degrading proteins by foam cells within the intimal lesion (Libby, 2002). Since the strength of the plaques fibrous cap is due to the extracellular matrix, its degradation would result in loss of strength and increased chance of rupture.
Early histological studies of advanced human atherosclerotic lesions suggested the presence of senescent endothelial cells (Burrig et al, 1991). Endothelial cells exhibiting the morphological features of senescence were frequently found on the plaque surface. The presence of senescent cells within plaques was also found in studies of vascular cells in culture, derived from human atherosclerotic plaques (Bennett et al 1998). VSMC derived from atherosclerotic plaques were shown to have lower rates of proliferation and underwent senescence earlier than cells derived from normal vessels. With the emergence of a biomarker (SA-β-Gal) which could detect senescent cells in vivo, a more direct approach for investigating cellular senescence in diseased tissue was undertaken (Fenton et al, 2001). This study sought to detect the presence of senescent cells in injured rabbit carotid arteries. Results indicated the accumulation of senescent cells in the neointima and media of all injured vessels, in contrast to the near absence of such cells in control vessels. Similar investigations have also been carried out on human atherosclerotic plaques (Vasile et al 2001, Minamino et al 2002). Both these studies demonstrated the presence of senescent vascular endothelial cells in vivo at sites of atherosclerotic plaque formation as detected by SA- β-Gal.
More recently due to advances in molecular biology, there have been numerous investigations involving the biology of telomeres in atherosclerosis. One such study examined telomere length in cells from atherosclerotic plaques and normal vessels and demonstrated that VSMC from plaques had markedly shorter telomeres compared with normal VSMC (Matthews et al 2006). This shortening was found to be closely associated with increasing severity of atherosclerosis. As with previously mentioned studies, these VSMC demonstrate morphological features of senescence when cultured in vitro. A similar study investigated telomere lengths of endothelial cells (EC) from coronary artery disease (CAD) and also found that telomeres were significantly shorter in CAD compared with normal arteries (Ogami et al, 2004). Since both VSMC and EC of atherosclerotic plaques have been shown to have senescent cells present and cellular senescence is generally attributed to the attrition of telomeres, the presence of cells with shorter telomeres in these tissues is therefore not surprising.
The above studies provide evidence for the presence of senescent cells in atherosclerotic plaques, but provide little explanation for their occurrence. This is less true for senescent SMC, since their presence can be explained by stimulated proliferation and migration observed in atherosclerosis. SMC have a finite replicative capacity, most likely as a result of telomere shortening, therefore, constant rounds of cell division would eventually result in the cell becoming senescent. The presence of senescent SMC would therefore only be observed in late stage plaque development, since this is when SMC are stimulated to migrate and proliferate.
Since the initiation of plaque development begins at EC, an explanation for why there may be senescent cells present is harder to explain. One possibility is that senescent EC cells in atherosclerotic plaques is most likely due to proliferative exhaustion as a result of replacing lost and damaged cells. As previously discussed, plaque formation is commonly seen within arteries at areas of high shear stress. It is therefore possible that such high shear stress could lead to the loss of ECs in these areas, which subsequently need to be replaced. This would result in an increase in cell turnover at those sites and consequently the occurrence of senescent EC. Since senescent cells in general can be classed as dysfunctional, it may be the presence of senescent EC within the endothelium which is the initiating factor in plaque formation. Further evidence for this may be provided if the expression profile of senescent vascular ECs were compared with vascular ECs of lesion-prone sites within arteries.
Since the recruitment and accumulation of leukocytes and monocytes is an important step in the development of atherosclerosis, the expression of proteins such as intracellular adhesion molecule-1 (ICAM1) and vascular cell adhesion molecular-1 (VCAM-1), important mediators of leukocyte and monocyte adherence have been investigated. One study looked at the expression of VCAM-1 and ICAM-1 at lesion-prone sites on the endothelium in ApoE-deficient mice (which are more prone to lesion development) (Nakashima et al, 1998). Staining for VCAM-1 showed localised staining at lesion-prone sites in ApoE-/- mice and only weak staining limited to sites of altered blood flow in control mice. ICAM-1 was the most prominent adhesion molecule in lesion prone sites and was up-regulated in ApoE -/- mice and control mice. If ICAM-1 is being up-regulated as a result of senescent cell formation, it is not surprising that it is found up-regulated in both ApoE -/- and control mice, since the same high shear stress is most likely occurring in both mice. Another study specifically investigated whether endothelial dysfunction was the result of endothelial cell senescence by inducing senescence in human aortic endothelial cells (HAECs) and examining the expression of ICAM-1 and endothelial nitric oxide synthase activity (eNOS) (Minamino et al, 2002). Results showed that ICAM-1 expression was increased and eNOS activity decreased in senescent HAECs. There are numerous studies that have shown eNOS activity to be decreased during endothelial dysfunction and this decrease is thought to play a critical role in the development and progression of atherosclerosis (Yang et al, 2006). Up-regulation of ICAM-1 and a decrease in eNOS activity in both senescent endothelial cells and at lesion-prone sites, strongly suggests that senescent cells are a significant contributing factor in initiation of atherosclerotic plaque formation.
To better understand the pathological processes that occur with atherosclerosis, an understanding of the structure of arteries is required. Human arteries are composed of three layers, the intima, the media and the adventitia. The intima is the innermost layer of the artery, composed of EC’s, SMC’s, macrophages and extracellular matrix (ECM) components. An internal elastic lamina separates the intima from the media, which is made up mainly of SMC. The adventitia is separated from the media by external elastic lamina and is mainly composed of fibroblasts and connective tissue.
The initiation and progression of atherosclerotic plaques generally takes place over many years during which the affected individual remains symptom free. Therefore, when a patient becomes symptomatic, the disease is already well established. These plaques occur at specific sites within arteries and these sites are dictated by fluid shear stress, the frictional force generated by blood flow over the vascular endothelium (Hwang et al, 2003). Regions of branched and curved arteries experience the greatest disturbed blood flow and it is at these sites that high incidences of plaque formation is found (VanderLaan et al, 2004). Relatively straight arteries however, experience the least shear stress and are usually protected from plaque development. Explanations for why high fluid stress sites are more “lesion-prone” is currently speculative.
The initial factors which result in the initiation of plaque formation are currently unknown. A common hypothesis is that plaque formation occurs as a result of EC damage leading to cellular dysfunction (Shimokawa, 1999, Davignon and Ganz, 2004). The source of the initial damage to EC’s is also currently unknown, but hypertension, viruses, toxins, smoking have all been suggested. Cellular dysfunction results in subsequent recruitment and accumulation of leukocytes and monocytes which would otherwise have resisted any adhesive interactions (Bobryshev et al, 2005). These adhered monocytes then differentiate into macrophages, engulf lipids, become foam cells and form fatty streaks. As the progression of the plaque continues, SMC’s migrate from the intima and synthesis extracellular matrix proteins in the intima (Boyle et al, 1997). Progressive macrophage accumulation, SMC migration and proliferation and extracellular matrix protein synthesis result in the formation of an advanced lesion.
A schematic representation of the structure of an artery, showing the intima, media and adventitia (commons.wikimedia.org)
Impairment in cell mobility, secretion of matrix degrading proteins, secretion of growth factors and pro-inflammatory cytokines are considered as significant changes associated with cellular senescence. All these factors have the potential to cause detrimental damage to tissues.
A number of papers have reported that the ability of senescent cells to migrate is severely reduced (Schneider and Mitsui, 1976; Sandeman et al, 2000; Reed et al, 2001). This decline in the ability to migrate may be related to changes which occur to the cytoskeleton during cellular senescence (Nishio and Inoue, 2005). Actin is an important component of the cytoskeleton required for cellular migration. However, in senescent fibroblasts for example it has been shown that vimentin is produced in place of actin which is down-regulated. This migration deficit has important implications during wound healing since cells are stimulated to migrate into the wound, proliferate and construct the new extra-cellular matrix (ECM). Also, since senescent cells tend to secrete proteins which degrade the matrix, wound repair would be impaired.
Matrix metalloproteases (MMPs) are also commonly up-regulated in senescent cells (Sandeman et al, 2001, Campisi, 2005). In normal tissue processes, MMPs are required for fertilization, cellular adhesion, development, neurogenesis, and metastasis (Page-McCaw et al, 2007). However, MMP secretion by senescent cells has also been suggested to play a role in the progression of disease such as in the pathogenesis of coronary heart disease (CAD) (Nanni et al, 2007). MMPs have also been implicated in the progression of osteoporosis, since MMPs play important roles in bone resorption (Logar et al, 2007). One study has also shown that the secretion of MMPs by senescent chondrocytes may contribute to the development or progression of osteoarthritis (Price et al, 2002).
Abnormal secretion of some growth factors has been shown to be another general characteristic of senescent cells. Work on human fibroblasts found that vascular endothelial growth factor (VEGF) secretion is elevated in senescent cell cultures (Coppe et al, 2006). Since growth factors are capable of stimulating cellular proliferation it has been suggested that while initially cellular senescence may be a mechanism to suppress tumourigenesis early in life it may promote cancer in aged organisms (Campisi, 1997). Human senescent fibroblasts for example have been shown to stimulate premalignant and malignant, but not normal epithelial cells to proliferate in culture and form tumours in mice (Krtolica et al, 2001, Krtolica and Campisi 2002). Another study sought to characterise the molecular alterations that occur during prostate fibroblast senescence to identify factors which may be capable of promoting the proliferation and potentially the neoplastic progression of prostate epithelium (Bavik et al, 2006). Fibroblast growth factor 7 (FGF7), hepatocyte growth factor and amphiregulin (AREG) were found to be elevated in the extracellular environment of senescent prostate fibroblasts. Direct co-culture and conditioned medium from senescent prostate fibroblasts stimulated epithelial cell proliferation 3-fold and 2-fold respectively. These results suggest that senescent cells may contribute to the progression of prostate neoplasia by altering the prostate microenvironment.
Probably the most potentially detrimental changes which can occur when cells become senescent is that of secreted cytokines since they not only effect local tissue but can have much wider impacts throughout the organism. Enhanced inflammation during ageing is thought to contribute to many of the diseases of ageing.
Vascular smooth muscle cells (VSMC) that have become senescent due to the activation of ras have been shown to drastically increase the expression of pro-inflammatory cytokines (Minamino et al, 2003). IL1α was shown to be up-regulated 11-fold, IL1β 50-fold, IL-6 12-fold and IL-8 77-fold. With such dramatic changes, it was suggested that this proinflammatory phenotype may contribute to the progression of atherosclerosis.
Senescent T-cells in vivo have been shown to produce high levels of two cytokines, IL6 and TNFα (Effros, 2004). Interestingly, the up-regulation of TNFα by T-cells in the bone marrow has been implicated as a causal mechanism in bone loss (Roggia et al, 2001).
Replicative senescence of human hepatic stellate cells (a major cell type involved in liver fibrosis) in culture also display a higher expression of inflammation genes (Schnabl et al, 2003). Interleukin-8 is among the cytokines up-regulated in senescent stellate cells (SC) which correlates with increase expression observed with disease activity in human alcoholic liver fibrosis (Sheron et al, 1993). Interleukin-6 is a known fibrogenic cytokine which was also shown to be up-regulated in SC senescent cultures. In normal conditions, chronic tissue damage results in the activation of SC characterised by proliferation, motility, contractility and synthesis of extracellular matrix (Gutiérrez-Ru, 2002). Since SC are stimulated to proliferate in response to tissue damage, the replicative capacity of these cells will be reduced and the accumulation of senescent cells accelerated. This activation of SC in response to tissue damage is regulated by cytokines and growth factors. Therefore, unregulated secretion of pro-inflammatory cytokines and growth factors from senescent SC within the liver may cause further damage. One study has shown for example that replicative senescence does have a significant impact in the long-term progression of fibrosis (Trak-Smayra et al, 2004).
This pro-inflammatory phenotype may partly be due to the up-regulation of intercellular adhesion molecule-1 (ICAM-1), a molecule known to be involved in inflammatory response and is over-expressed in senescent cells and aged tissues (Gorgoulis et al, 2005). One study has shown that p53 can directly activate the expression of ICAM-1 (Kletsas et al, 2004) and since p53 is activated and up-regulated during cellular senescence, it may activate ICAM-1, thereby contributing to the pro-inflammatory phenotype of senescent cells.
A recent study has provided strong evidence to suggest that intervertebral disc degeneration, a major cause of low back pain, is due to accelerated cellular senescence (Le Maitre et al, 2007). Cells isolated from normal and degenerate human tissue were assessed for mean telomere length, SA-β-Gal, and replicative potential. Mean telomere length decreased with age in cells from non-degenerate tissue and also decreased with progressive stages of degeneration. SA-β-Gal staining was not observed in non-degenerative patients unlike cells from degenerative discs which did exhibit 10-12% SA-β-Gal staining and decrease in replicative potential. However, the factors which may have led to accelerated senescence in this instance was not discussed. There are three possible reasons why cellular senescence was accelerated in this instance; (1) Unknown factors resulted in the damage and removal of cells, resulting in cell turnover for replacement, (2) ROS were involved causing stress induced premature senescence (SIPS) or (3) telomeres in these cells for some unknown reason started off shorter than normal, meaning less cell turnover is required for the appearance of senescent cells.
Other studies have shown a correlation between disease states and the presence of senescent cells in vivo. SA-β-Gal staining was used to detect senescent cells in normal liver, liver with chronic hepatitis C and hepatocellular carcinoma (HCC) (Paradis et al, 2001). They found senescent cells present in 3 of 15 (20%) normal livers tested, 16 of 32 (50%) in livers with chronic hepatitis C and in 6 of 10 (60%) livers with HCC. The presence of senescent cells in normal livers was found to be associated with old age. Interestingly, the presence of senescent cells in non-tumoural tissues was strongly correlated with the presence of HCC in the surrounding liver. This demonstrates not only that the ageing of one tissue can have a direct impact on another but also as suggested by Judith Campisi, senescent cells may contribute to carcinogenesis (Campisi, 1997).
Another study looked at cellular senescence in human benign prostatic hyperplasia (BPH) specimens (Choi et al, 2000). BPH is a disease associated with an abnormal growth of the adult prostate that begins mid to late life. Results from this study found that 40% of the analysed samples showed positive staining for SA-β-Gal and only in the epithelial cells. A high prostate weight (> 55g) was found to correlate strongly with the expression of SA-β-Gal. Prostates weighing less than 55g tended to lack senescent epithelial cells. It was suggested that the accumulation of senescent epithelial cells may play a role in the development of prostatic enlargement associated with BPH. However, the accumulation of senescent cells in this case is likely to be a consequence of the disease, which may lead further to its progression. The enlargement of the prostate may be the result of unregulated stimulated proliferation, increasing cell turnover and consequently the appearance of senescent cells. This may explain why a stronger expression of SA-β-Gal is detected in prostates weighing more than 55g since they may have undergone more cellular divisions.
During the pathogenesis of type 2 diabetes, insulin resistance causes compensatory proliferation of pancreatic beta cells. This compensatory proliferation might accelerate cellular senescence contributing further to the progression of diabetes. To investigate this, one group used nutrient-induced diabetic mice to analyse beta cells for SA-β-Gal and the proliferation marker Ki67 (Sone and Kagawa, 2005). At 4 months, the proliferation of beta cells was 2.2 fold higher than in the control group. At 12 months, the frequency of Ki67 decreased to one-third that of the control and SA-β-Gal positive cells increased to 4.7 fold that of the control group. This increase in the senescent beta cell fraction correlated with insufficient insulin release, suggesting cellular senescence may contribute to diet-induced diabetes. In this instance it is difficult to determine whether senescence is the cause or the consequence of insulin resistance. It later appears to be a contributor but whether it is also the initiating factor is unknown.
Another study used senescent associated p16 instead of SA-β-Gal to detect senescent cells in kidneys with glomerular disease (GD) (Sis et al, 2007). Glomerular diseases include many conditions which fall into two major categories; Glomerulonephritis describes the inflammation of the membrane tissue in the kidney that serves as a filter, separating wastes and extra fluid from the blood. Glomerulosclerosis is the scarring or hardening of the tiny blood vessels within the kidney. This study found an increased expression of the nuclear p16 in samples with GD compared with normal. Independently, older age and interstitial inflammation was associated with increased expression of nuclear p16. Since senescent cells adopt a pro-inflammatory phenotype, their presence may be a contributing factor in inflammation observed in glomerulonephritis.
All these examples demonstrate the presence of senescent cells not only in vivo, but more specifically in disease states. This suggests that the accumulation of senescent cells in normal tissues is the result of injuries to those tissues or in some cases unregulated stimulation of proliferation. This suggests that if no injuries occurred as a consequence of disease, environmental factors or by normal biological/mechanical wear and tear, ageing of mitotic tissues would be greatly reduced.
Whether cellular senescence is the cause or the consequence of some diseases is yet to be answered.
At the time of Hayflicks proposal there was no way of detecting senescent cells in vivo. It wasn’t until over 30 years later that a marker was used to demonstrate the presence of senescent cells in human dermis in vivo (Dimri et al, 1995). This detection system is based on a modified beta-galactosidase assay and is termed senescence-associated beta-galactosidase (SA-β-Gal). Skin samples were taken from 20 human donors aged 20-90 years, sectioned and stained for SA-β-Gal. Results showed an age-dependent increase in SA-β-Gal staining in dermal fibroblasts and epidermal keratinocytes. None of the young donors (<40yr)>69 yr) donor did display positive staining. About half of the young donors showed some epidermal staining whereas positive staining was always observed in the epidermis of old donors. Using the same technique to identify senescence cells, another group looked at senescence of the retinal pigment epithelium (RPE) of Rhesus monkeys (Hjelmeland et al, 1999). Results also showed an accumulation of SA-β-Gal positive cells in the eyes of older monkeys. Another study also found little or no SA-β-Gal staining in HCECs of corneas from young donors but was easily detected in corneas from older donors (Mimura and Joyce, 2006).
The proposal that oxidative stress may cause CCIS comes from studies that have treated cells with concentrations of H2O2 and found that those cells enter a long-term growth arrest resembling replicative senescence; often called stress induced premature senescence or SIPS. One such study treated human fibroblasts with sub-lethal concentrations of hydrogen peroxide which induced cell cycle arrest, with an initial 2-3 fold increase in the level of p53 protein and subsequently an increase in the level of p21 protein (Chen et al, 1998). One study has shown that the up-regulation of Caveolin-1 by oxidative stress is required to induce premature senescence (Volonte et al, 2002). Caveolin-1 is thought to function as a “transformation suppressor” protein. For example Caveolin-1 mRNA and protein expression are lost or reduced during cell transformations by activated oncogenes.
Oxidative stress is thought to result in damage to DNA and DNA mutations. The most severe type of DNA damage is double-strand breaks (DSBs). Studies have shown that cells treated with agents that cause DSBs lead to an increase in p16 expression and premature senescence (Robles and Adami, 1998). Induction of DNA damage resulted in the induction of p53 and p21. The concentration of p21 protein increases 100-fold and then begins to drop, followed by an increase in p16. This is similar to what occurs during replicative senescence.
Oxidative stress is also thought to result in DNA becoming mutated and this may impact oncogenes such as RAS. Studies have shown that mutated oncogenic Ras can result in a permanent G1 arrest (Serrano et al, 1997). Oncogenic Ras is commonly associated with the transformation of primary cells to an immortal state. It seems, however, that this transformation can only occur with either the co-operation of another oncogene or the inactivation of tumour suppressers such as p53 or p16. Serrano and co-workers also found that Ras induced cell senescence is accompanied by the accumulation of p53 and p16 and that the inactivation of either one of these prevents Ras induced arrest in rodent cells. More recent studies have also shown that Ras can induce cell senescence in vascular smooth muscle cells (Minamino et al, 2003) and endothelial cells (Spyridopoulos et al, 2002). In the first study, an activated ras allele was introduced into human vascular smooth muscle cells using retroviral infection. This resulted in growth arrest with phenotypic characteristics of cell senescence. In the second study, bovine aortic endothelial cells were infected using the adenovirus containing the activated ras gene. Over-expression of ras in this case led to G1 and G2/M-cell cycle arrest after 72 hours due to induction of p21. p21 induction again appears to be the initiating factor of Ras induced senescence and induction of p16 is required to maintain the senescent state. One group for example found that Ras was capable of causing growth arrest in both p21 and p53 negative human fibroblasts, suggesting Ras is activating the p16 pathway (Wei et al, 2001).
Like p21, p16 is a CDK inhibitor involved in cell cycle arrest. Over expression of p16 causes G1 arrest in early passage cells by inhibiting the phosphorylation of the retinoblastoma protein (Kato et al, 1998). However, the removal of p16 activity results in only minimal lifespan extension that was terminated by senescence (Wei et al, 2003). p16 plays a role in maintaining senescence triggered by a short telomere. It appears that p21 is the initiating factor in the growth arrest observed in senescent cells, but p16 is required to maintain that state. p21 has been shown to initially increase in fibroblast cultures but later gradually decrease (Alcorta et al, 1996). During the decline of p21, p16 protein levels gradually increased in senescent cultures, reaching nearly 40-fold higher than in early cultures.
P16 is thought to trigger senescence independently of telomere length. For example, the inactivated p53 in two different fibroblast strains: WI-38 (from foetal lung) and BJ (from neonatal foreskin) resulted in the continuation of growth without Rb inactivation in BJ cells, whereas WI-38 cells fail to grow even when both Rb and p53 are inactivated. It was suggested that these two outcomes was due to the intrinsic differences in the ability to induce p16 at senescence. This proposal was based on the understanding that like WI-38, other human cells appear to undergo replicative senescence prior to telomere shortening by the induction of p16 (Itahana et al, 2003). The ability of p16 to cause growth arrest even after Rb inactivation also suggests that p16 can function independently of Rb activity. Further support for a telomere-independent mechanism was provided from studies which have shown that telomerase activity alone was insufficient to extend the replicative potential of human keratinocytes or mammary epithelial cells (Kiyono et al, 1998). It was shown that down-regulation of p16 in combination with telomerase activity did lead to replicative lifespan extension. Another study found that cyclin D1 over-expression in primary oral keratinocytes extend the lifespan, whereas the combination of cyclin D1 over-expression and p53 inactivation led to their immortalisation (Opitz et al, 2001). Cyclin D1 forms complexes with CDK4 which subsequently phosphorylates and inactivates retinoblastoma (Rb) growth repression (Connell-Crowley et al, 1997). P16 inhibits cell cycle progression by inhibiting CDK4. It appears that the over-expression of cyclin D1 bypasses p16-TIS resulting in an extension of lifespan. In this instance the p16 mechanism has become redundant and the cells continue to divide until TDS mechanism is activated. This may be why these cells are immortalised by p53 inactivation.
This telomere-independent mechanism may be the primary trigger of cellular senescence in mice and rats. Mice and rats have telomeres which are 5-10 times longer than those of human cells (Shay and Wright, 2001). Despite this increase in length, rodent cells engineered to lack telomerase show telomere shortening with no effect on replicative potential. These cells senesce in the presence of long telomeres. Since mouse and rat cells repair DNA damage far less efficiently than do human cells, it has been suggested that such damage may be the trigger for p16 induction.
Human telomeres consist of short tandem GC-rich repeats (TTAGGG/AATCCC). These telomeric repeats are predominantly double-stranded with a 3’ single-stranded overhang terminating with a G-rich tail (Collins, 2000). The proposed mechanism by which telomeres and their associated proteins protect the chromosome ends is by creating large terminal loops known as a t-loop (Greider, 1999). In this structure, the double-stranded telomeric DNA is looped around and the 3’ single-strand overhang invades the duplex of telomeric repeats and forms a displacement loop (D-loop) structure is important for maintaining chromosomal stability. Without this protection, it is thought that the ends of chromosomes would become exposed, resembling double-strand breaks which can trigger cell-cycle arrest and senescence (Shore, 2001).
The majority of short-telomere growth arrested cells is thought to occur in the G1 phase of the cell cycle, a process regulated by proteins such as p21. The appearance of DNA damage results in the activation of p53, a transcription factor whose activation results numerous downstream responses including the up-regulation of p21 and activation of DNA repair proteins. P21 is a cyclin dependent kinase (CDK) inhibitor which halts cell cycle progression by directly interacting and inhibiting cyclin-CDK complexes and consequently preventing the phosphorylation of Rb.
Experiments in which p21 was inactivated within senescent cells resulted in the ability of these cells to re-enter the S phase of the cell cycle, but were still unable to divide (Ma et al, 1999). It was concluded that p21 plays an important role in the maintenance of senescence and in the inhibition of S-phase progression, but inhibition of p21 is insufficient to permit cells to complete the cell cycle. Since p21 expression has been shown to be dependent upon p53, the impact of p53 inactivation on cell cycle progression was also investigated (Bond et al, 1999). Fibroblasts lacking p53 function bypassed replicative senescence and continued to proliferate for another 20 CPD before entering growth arrest. The point at which growth arrest occurs due to replicative senescence is also known as M1, whereas the second growth arrest point if replicative senescence is bypassed is known as M2 or crisis. Cells which enter crisis result in apoptosis rather than senescence. This suggests that when a cell becomes senescent at M1 it may not be due to the disruption of the telomere-end structure. Since there appears to be a reserved replicative capacity, it may only be at M2 that the telomere end-structure becomes disrupted, and this may be the reason why apoptosis is common at this point. The idea that a cell becomes senescent long before the telomere-end structure is disrupted suggests that telomere length may not be the trigger for replicative senescence and instead is due to another mechanism.
One such mechanism may be alterations or exposure of the telomere 3’ single-stranded overhang. One study reported that exposure of telomere 3’ overhang sequences induces senescence (Li et al, 2003). In this study, cells were exposed to oligonucleotides homologous to the telomere 3’ overhang sequence TTAGGG for 1 week. As a result, a senescent phenotype in cultured fibroblasts was observed. The team concluded that exposure the 3’ overhang by t-loop disruption or possibly DNA damage leads replicative senescence. Telomere shortening may result in the exposure of the 3’ overhangs, but the telomere end-structure is still maintained efficiently to prevent complete disruption. Interestingly, this 3’ overhang has been found to be eroded at replicative senescence indicating it as a possible trigger (Stewart et al, 2003). It was indicated that overhang erosion is the result of continuous cell division and not a consequence of senescence. Therefore it was concluded that this alteration in telomere structure and not overall telomere length serves as a trigger for replicative senescence.
Throughout the cell cycle there are a number of checkpoints which regulate cell progression from one phase to another. There is a G1 checkpoint that ensures everything is ready for DNA synthesis, a G2 checkpoint to determine whether the cell can proceed to M phase and a checkpoint within M phase to ensure the cell is ready to complete cell division.
Entry into each of the phases of the cell cycle are controlled by two classes of molecules, cyclins and cyclin dependent kinases (CDKs). Cyclins form the regulatory subunits and CDKs the catalytic subunits of an activated heterodimer; cyclins have no catalytic activity and CDKs are inactive in the absence of a partner cyclin. When activated by a bound cyclin, CDKs perform a biochemical reaction called phosphorylation that activates or inactivates target proteins to orchestrate coordinated entry into the next phase of the cell cycle. Different cyclin-CDK combinations determine which downstream proteins are targeted.
For example, during the latter stages of the G1 phase cyclin D1 forms a complex with CDK4, which subsequently phosphorylates and inactivates retinoblastoma (Rb) growth repression (Connell-Crowley et al, 1997). Conversely, growth arrest caused by DNA damage for example is the result of an up-regulation of CDK inhibitors such as p21 and p16 which bind to and inhibit the activity of CDK thereby preventing the phosphorylation of Rb (Aprelikova et al, 1995).
Figure 1: Basic overview of the eukaryotic cell cycle, showing each of the different phases
Another group investigating atherosclerosis took vascular smooth muscle cells (VSMC) from human atherosclerotic plaques and grew them in culture (Bennett et al, 1998). Results showed that VSMCs taken from plaques have lower rates of proliferation and underwent senescence earlier than cells derived from normal vessels.
A more recent study looked at the replicative capacity of osteoblasts in Rheumatoid arthritis (RA) compared with Osteoarthritis (OA) (Yudoh et al, 2000). The results indicated that the replicative capacity of osteoblasts decreased gradually with donor age and this decrease was higher in RA patients than with OA patients at any donor age. They also reported an increase in senescent osteoblastic cells with age in both groups in which the rate of expression of senescent cells was higher in RA patients than with age-matched OA patients.
Tesco et al (1993) looked at the replicative capacity of fibroblasts in patients with familial Alzheimer’s disease (FAD) to examine whether features compatible with a systemic premature aging were present. Data showed that there was no significant difference in replicative capacity of fibroblasts between FAD patients and controls. This is not a surprising result, since the fibroblasts studied are unrelated to the development of FAD and if features of premature ageing were present they would have most likely manifested themselves as other diseases other than just Alzheimer’s. For example, Werner’s syndrome is a premature ageing disorder which displays a multitude of age-related afflictions including diabetes and heart disease (Kipling and Faragher, 1997). When fibroblasts were taken from patients with Werner’s syndrome and grown in culture, the number of population doublings achieved was smaller compared with normal cells of a similar chronological age (Martin et al, 1970)
These studies suggest that disease is an important factor contributing to the exhaustion of the replicative capacity of cells. However, it is possible that some diseases arise as a result of the gradual increase in senescent cells with time. It is also possible that unknown factors result in accelerated senescence, which subsequently manifests itself as a biological impairment or disease.
The first of these studies showed an inverse relationship between donor age and the number of population doublings achieved in vitro (Martin et al, 1970). This study looked at the replicative lifespan of fibroblasts taken from 100 subjects with an age range from foetal to 90 years. These cells were cultured and the number of population doublings before entering senescence was recorded. The results showed that the replicative potential decreased as donor age increased. A later study showed similar results (Schneider, 1979). This study looked at the ability of fibroblasts taken from young (20-35 years) and old (65+ years) to proliferate in culture. It was reported that cell cultures from old human donors have a reduction in their proliferative capacity. A more recent study looked at the replicative capacity of human adrenocortical cells to proliferate as a function of donor age (Yang et al, 2001). Again, it was found that younger cells have a higher proliferative capability than the old. In this instance, population doubling fell from 50 for foetal cells to almost a total lack of division in culture from older cells.
To investigate the possible link between replicative lifespan and organismal ageing, a few studies compared replicative capacity with longevity in animals. One such study investigated the relationship between longevity of eight mammalian species (mouse, rat, rat-kangaroo, mink, rabbit, bat, horse and human) and the lifespan of normal fibroblasts in vitro (Röhme, 1981). It was reported that there was a direct relationship found between the longevity of the eight mammalian species and the replicative capacity of their cultured fibroblasts. A much later, but similar study, compared animal life spans and in vitro replicative capacity of skin fibroblasts in groupings of small, middle, large, and very large breeds of dogs of specific ages (Li et al, 1996). It was found that the life spans were inversely correlated to the frame sizes of the breeds. It was shown that all the small breeds studied have a longer life span than that of the large breeds. The replicative capacity of fibroblasts from the large dogs (Great Dane and Irish Wolfhound) was significantly decreased compared with that of the small dogs. The reasoning behind these observations may again be due to varying degrees of cell turnover between the species. Large dogs consist of more cells than small dogs and as a result more cell turnover was initially required in their development compared to small dogs. This increase in cell turnover would subsequently lead to a decrease in replicative potential and an increase in the rate of senescent cell formation.
Interestingly, a recent study looked at the replicative capacity of 124 skin fibroblast cell lines from donors of different ages which were medically examined and declared “healthy” (Cristofalo et al, 1998). Healthy people were used specifically as previous studies, discussed later, have shown that disease states may accelerate the reduction in replicative capacity. Results indicated that there was no significant correlation between the replicative capacity of the cell lines and donor age. In the same study, a comparison of multiple cell lines established from the same donors of different ages also failed to show any significant differences. It was concluded that the replicative capacity of fibroblasts in vitro does not correlate with donor age. However, differences in replicative capacity with age may only be observed as a result of increased cell turnover in response to disease and cellular injury. Therefore, a healthy old person who has had little or no cellular injuries or disease would have had little cell turnover and therefore have cells which may have a replicative capacity similar to someone much younger. Thus, this study supports the notion that replicative capacity is an indicator of biological age.
The results also show that the replicative capacity of the same tissues between individuals of the same age also differs. This difference may again be due to the same differences which effect proliferative variability between different tissues of the same individual. For example, one individual may have a shorter replicative capacity in a particular tissue than another of the same age due to increases in cell turnover, maybe in response to disease or injury, or maybe differences in initial telomere lengths. Cultured human embryonic fibroblasts were found to senesce at 50±10 cPD (Hayflick and Moorehead, 1961). This meant that some cultures were senescent only after 40 cPD while others at 60 cPD. These differences in replicative lifespan may be a consequence of the stochastic mechanism which triggers a cell to senesce. Therefore, the difference in replicative capacities of the same tissues between individuals of the same age may also be due to the stochastic events which govern a cell becoming senescent. Thus, the replicative capacity of a tissue measures biological age and not chronological age. Unfortunately there have been few studies looking at the replicative capacity of different tissues from the same individuals. This would have given a better insight into the relationship between chronological and biological age.
Dynamics of normal cell populations.
The early observation by Hayflick and Moorehead (1961) that cultured cells have a maximum limit on the number of divisions before entering senescence lead to the assumption that all cells in a given culture divide roughly the same number of times before entering senescence. Hayflick considered the senescence of cultures was marked by three distinct phases. In Phase 1 the initial culture, was considered to terminate with the formation of the first confluent sheet of cells. Phase 2 was characterised by vigorous growth requiring repeated subculture. In phase 3, the senescence of the culture was characterised by the cessation of mitosis. In this model it was assumed that cultures were composed of a homogenous population of cells which were either all growing (Phase 1 or 2) or all non-growing (Phase 3) and that failure to grow was due to cell death (Kalashnik et al, 2000). The notion that senescence was cell death was soon disproved with the demonstration that RNA synthesis occurred in these cells (Macieira-Coelho et al, 1966). Evidence against the idea that cultures were composed of homogenous populations was provided by a number of different studies. Cristofalo and Scharf (1973) demonstrated the presence of senescent cells in early passage cultures using long pulse-labelling experiments on embryonic fibroblasts. 3H-thymidine labels those cells which have entered S-phase (dividing cells), and since senescence cells are halted in G1 they cannot enter S-phase, and so the percentage of unlabelled cells can be calculated. It was shown that senescent cells are present in early passage cultures and that the percentage of senescent cells gradually increases with each serial passage of the culture. This observation was explained by experiments demonstrating that cultured fibroblasts are composed of cells which display variation in proliferative potential (Smith and Whitney, 1980). Related experiments also showed that two cells arising from a single mitosis differed in their ability to proliferate by as many as eight doublings (Jones et al, 1985). Using the miniclone technique the replicative capacity of individual cells growing in bulk culture can be measured as well as the sizes of colonies generated by dividing cells (Ponton et al, 1983). Results showed that the percentage of glial cells capable of dividing gradually decreases with every new passage. This data is based on the broad distribution of colony sizes which showed a shift from many large colonies to more small colonies as population doublings increased.
Modern techniques for measuring the dynamics of normal cell populations involve measuring not only the senescent fraction of cells, but also the proliferating and apoptotic fraction. The most commonly used method to visualise senescent cells both in culture and in vivo is the senescence-associated beta-galactosidase assay (SA-β-Gal) (Dimri et al, 1995). Although this is a safer method than using 3H-thymidine labels, its robustness as a biomarker is questionable since the assay is dependent upon lysosomal mass (and cell size) rather than growth state (Lee et al, 2006). Cellular proliferation markers such as bromodeoxyuridine (BrdU) and Ki67 are commonly used to label and calculate the proliferating fraction. For measuring the apoptotic fraction, terminal transferase dUTP nick end labelling (TUNEL) is a commonly used method. This assay can detect DNA fragmentation that results from apoptotic signaling cascades.
An example of these methods being used for determining the growth dynamics of human umbilical vein endothelial cells (HUVEC) can be observed in a paper by Kalashnik et al (2000). Results show a gradual decline in the growth fraction as measured by Ki67, an increase in the senescent fraction and the apoptotic fraction remaining unchanged with each serial passage.