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Although Figure 3 and the discussion above provide an overview of the current knowledge of cell cycle regulation, it is likely other players exist. For instance, p53 itself may be upregulated. Although the issue is controversial, some evidence indicates that the ATM gene, or other players involved in DNA damage response, may be the "sensor" that detects telomere dysfunction and then regulates p53 (Vaziri et al., 1997; Rouse and Jackson, 2002). Other results suggest that a novel transcriptional element regulates cyclin D1, and possibly other senescence-associated genes, in senescence cells (Berardi et al., 2003).
Immortalization with viral proteins is not as simple as it may seem at first. Infection of human fibroblasts with viral oncogenes results in an extended replicative lifespan after which cells enter a stage called crisis (reviewed in Goldstein, 1990; McCormick and Campisi, 1991; Wei and Sedivy, 1999). During crisis, cells proliferate but the proportion of cells entering apoptosis gradually increases and thus cell numbers eventually diminish (Macera-Bloch et al., 2002). Since both p53 and pRb/p16INK4a pathways are inactive and chromosomal instability and fusions are abundant, crisis is thought to emerge due to extremely short telomeres. Occasionally, immortal cells emerge from crisis with stabilized telomeres, normally involving telomerase activation (reviewed in Stewart and Weinberg, 2000; Mathon and Lloyd, 2001). In a sense, crisis can be seen as the ultimate consequence of telomere dysfunction since it occurs when the mechanisms that respond to short telomeres, like p53 and pRb, are inactive.
One last point is that even assuming that the p53 and pRb/p16INK4a pathways explain RS, they do not entirely explain the gradual aging of cells in culture. One hypothesis is that cell populations become more heterogeneous as they age. For example, since the percentage of cells actively dividing decreases with CPD, it is normal that the cell population as a whole ages, without changes other than more cells entering RS.
Overall, whatever changes occur during telomere dysfunction, the mechanisms triggering growth arrest appear to involve DNA damage pathways. As such, the most likely explanation is that dysfunctional telomeres are recognized as DNA damage and repairing the short telomeres leads to chromosome fusions. Although unidentified genes may also be involved (e.g., Blasco and Hahn, 2003; Yawata et al., 2003), the most widely accepted hypothesis is that the p53 and pRb/p16INK4a pathways collaborate to stop cellular proliferation derived from telomere shortening in normal human fibroblasts (Fig. 3). Probably, the p53 pathway involving p21WAF1 is activated beforehand, while p16INK4a prevails under strong physiological stimuli or stress and to maintain cells growth arrested, a state also called quiescence.
Aging, Cancer, and the Telomeres
The role of telomeres in RS has led to suggestions that telomerase can be used as an anti-aging therapy (reviewed in Fossel, 1996; Blasco, 2005; Shawi and Autexier, 2008). As mentioned before, however, the relation between RS and organismal aging is controversial. Whether telomere shortening plays a role in human aging is a hotly-debated issue, as reviewed below.
Most, not all, human somatic tissues have no detectable telomerase activity (reviewed in Collins and Mitchell, 2002). In the bone marrow, hematopoietic cells express telomerase. Telomerase activity is higher in primitive progenitor cells and then downregulated during proliferation and differentiation (Chiu et al., 1996). Other reports associate, normally low, levels of telomerase activity with human stem cells (Sugihara et al., 1999), though probably not mesenchymal stem cells (Zimmermann et al., 2003). Telomerase activity has been detected in some highly proliferating normal human somatic cells; for instance, in skin cells (Harle-Bachor and Boukamp, 1996; Taylor et al., 1996), immune system cells (Counter et al., 1995; Morrison et al., 1996), and colorectal tissues (Tahara et al., 1999). A decline in telomerase activity was reported in blood mononuclear cells with age (Iwama et al., 1998). Human germ cells have been found to express hTERT (Kilian et al., 1997).
As with replicative potential, telomere length in vivo is very heterogeneous (Serra and von Zglinicki, 2002; Takubo et al., 2002). Telomere shortening in vivo has been reported in liver cells (Aikata et al., 2000), lymphocytes (Pan et al., 1997), skin cells (Lindsey et al., 1991), blood (Iwama et al., 1998), and colon mucosa (Hastie et al., 1990). For example, telomere shortening appears to impact on the function of immune T cells, and telomerase activators can restore a more youthful functional profile (reviewed in Effros, 2009). Other studies found weak correlations between donor age and telomere length (Allsopp et al., 1992; Kammori et al., 2002; Njajou et al., 2007), while some studies found no correlation at all (Mondello et al., 1999; Renault et al., 2002; Serra and von Zglinicki, 2002; Takubo et al., 2002; Nwosu et al., 2005). Long telomeres have been found in cells from centenarians (Franceschi et al., 1999). Taken as a whole, these results indicate that telomere length varies widely between individuals and between different tissues, and that telomere shortening may occur in some tissues in vivo in association with certain pathologies and with age; this is similar to what is observed for senescent cells. An association between telomere length and mortality has been reported in people aged 60 and over (Cawthon et al., 2003), and telomere shortening appears to be accelerated in people living more stressful lives (Epel et al., 2004). While these results support the idea that telomere shortening is a marker of stress and age-related pathology, they do not prove that telomere shortening is a causal factor in aging. Lastly, although telomerase may prevent the accelerated clonal senescence of Werner's syndrome cells (Wyllie et al., 2000), it does not appear to fully reverse the WS phenotype (Choi et al., 2001).
No connection appears to exist between the mean telomere length of cells and the longevity of mammalian species. Of all studied primates, humans appear to have the shortest telomeres and the longest lifespan (Kakuo et al., 1999; Steinert et al., 2002). Mice also have long telomeres and feature high telomerase activity in many organs, in contrast to humans (Prowse and Greider, 1995). Interestingly, inbred mice have long (Kipling and Cooke, 1990) while wild mice have short telomeres, suggesting telomere length does not affect organismal longevity (Hemann and Greider, 2000). In rodents, telomerase activity correlates negatively with lifespan but does not correlate with longevity (Seluanov et al., 2007). The largest comparative study of telomeres and telomerase, involving over 60 mammalian species, found that smaller, short-lived species tend to have long telomeres and high levels of telomerase. This suggests that short telomeres and suppression of telomerase are necessary for the evolution of large body sizes and longevity, presumably by suppressing cancer (Gomes et al., 2011).
Though mean telomere length at birth does not correlate with longevity in birds, rate of telomere shortening in erythrocytes was reported to inversely correlate with bird longevity. Telomere shortening in a variety of tissues was also reported to correlate, though to a lesser extent, with mammalian longevity (Haussmann et al., 2003; Vleck et al., 2003). In fact, a correlation between erythrocyte longevity and organismal longevity was previously reported, suggesting that cells, in this case erythrocyte stem cells, from long-lived animals divide fewer times (Rohme, 1981). In zebra finches telomere length early in life was a good predictor of lifespan (Heidinger et al., 2012). One study in rodents, however, failed to find evidence of a correlation between rate of telomere shortening in vitro and longevity (Seluanov et al., 2008).
Mice overexpressing telomerase have a higher cancer incidence and hence a shorter lifespan (Artandi et al., 2002). But mice lacking telomerase were viable up to six generations. Telomeres gradually shortened and cells from animals of generation four displayed aneuploidy and other chromosomal aberrations. Abnormalities were observed as early as in the third generation and late-generation animals showed a few signs of accelerated aging (Blasco et al., 1997; Rudolph et al., 1999); it is controversial whether these animals are aging faster or merely developing a variety of pathologies. All in all, these results suggest that telomerase activity could be crucial for the normal functioning of highly proliferative organs in mice (Lee et al., 1998). Nonetheless, telomere length and/or telomerase activity do not explain why humans age slower than other primates and live so much longer than mice. They may help explain, however, why mice have a much higher cancer incidence than humans (Blasco, 2005).
Telomerase expression has been found in lobsters, a species in which aging remains undetected (Klapper et al., 1998), though it could be due to molting. On the other hand, in the frog Xenopus laevis, another animal with a slow rate of aging (Brocas and Verzar, 1961), not only a great variation in telomere length has been observed (Bassham et al., 1998), but telomere length can diminish from parents to offspring with no detectable consequences and despite telomerase activity in germ cells (Mantell and Greider, 1994). The way telomere length does not impact on the life history of cloned animals is also in contradiction with a role of telomeres in aging. For instance, scientists took cells from a 17-year old bull and allowed them to divide (Kubota et al., 2000); they then used cells at different stages of their replicative lifespan to create clones and, surprisingly, it appears that the older cells with shorter telomeres are more efficient for generating clones. It would be interesting to know the longevity of these clones as well as that of cloned calves with extended telomeres (Lanza et al., 2000). Overall, maybe telomeres are the cellular clock, but judging from these results telomere length is not a major determinant of the aging process.
As with RS, telomere shortening is a tumor suppressor mechanism (de Magalhaes, 2004; Campisi, 2005; Deng et al., 2008; de Magalhaes, 2013). Tumor development is dependent on telomere stabilization, normally by telomerase (Chen et al., 2000). For example, telomerase activation has been associated with skin malignancy as a result of exposure to ultraviolet radiation (Ueda et al., 1997). In contrast, telomerase inhibition can induce senescence in some cancer cells (Shammas et al., 1999). Knocking-out telomerase in mice through deletion of its RNA component, while not preventing cancer (Blasco et al., 1997; Rudolph et al., 1999), appears to increase cancer resistance (Gonzalez-Suarez et al., 2000; Rudolph et al., 2001). On the other hand, telomerase overexpression in mice promoted cancer development (Gonzalez-Suarez et al., 2001; Artandi et al., 2002). In addition, the connection between telomere signaling pathways and cancer is obvious (reviewed in Fearon, 1997. The human Li-Fraumeni syndrome has been associated with mutations in p53 and is characterized by increased cancer incidence (reviewed in Varley et al., 1997). Human germline mutations in p53 are also associated with a major cancer risk (Hwang et al., 2003). Retinoblastoma is also recognized as hereditary cancer (Murphree and Benedict, 1984; Goodrich and Lee, 1993). Germline mutations in p16INK4a have too been implicated in familial melanoma (Hussussian et al., 1994).
More debatable is the role of telomeres in animal aging (de Magalhaes and Toussaint, 2004a). As mentioned elsewhere, senescent cells likely accumulate in some tissues and may contribute to organ disfunction yet telomere-independent mechanisms may play a more prominent role. Some genetic interventions that alter aging appear to influence tissue homeostasis by affecting senescence, cell proliferation, and cell death, yet such evidence is circumstantial (reviewed in de Magalhaes and Faragher, 2008). Evidence from genetic manipulation experiments of players involved in telomeric signal transduction (Fig. 3) is mixed (reviewed in de Magalhaes, 2004). Increasing the dosage in mice of INK4a/ARF (the gene coding the mouse homolog of p16INK4a) offers resistance against cancer but does not affect aging (Matheu et al., 2004). Another study found that INK4a/ARF induction in mice results in premature senescence and inhibits cell proliferation but does not induce cell senescence (Boquoi et al., 2015). There is some evidence that p53 may influence aging in mice (Donehower, 2002), as debated elsewhere, but it is not clear the same is true for humans. Likewise, disruption of p63, a homologue of p53, appears to accelerate aging (Keyes et al., 2005), yet human defects in p63 do not (Celli et al., 1999). Mouse strains with increased levels of p53 and INK4a/ARF are long-lived (Matheu et al., 2007), though it is unclear whether their aging process is altered--as defined before. Arguably the strongest evidence for a role of telomerase in aging comes from telomerase overexpressing mice also engineered to resist cancer via enhanced expression of p53 and INK4a/ARF as these are long-lived (Tomas-Loba et al., 2008). Even though it is not clear whether aging is delayed in these animals or the exact mechanisms, these findings do point towards some level of protection from age-related degeneration via optimization of pathways associated with telomeres and RS. Telomerase gene therapy in old mice also modestly increased lifespan (Bernardes de Jesus et al., 2012). It should be noted, however, that telomerase may have functions independent of telomere elongation, such as in protecting mitochondria from stress (Ahmed et al., 2008). Another study showed that telomerase reactivation reverses degeneration in mice (Jaskelioff et al., 2011). However, this study was conducted in animals that have no telomerase to begin with and thus develop a number of pathologies. Benefits from reactivating telomerase in mice that become sick for lack of telomerase are hardly surprising or noteworthy.
Dyskeratosis congenita is an inherited disease involving skin and bone marrow failure (reviewed in Marrone and Mason, 2003). It is caused by a mutation in the DKC1 gene. Intriguingly, the protein encoded by DKC1, dyskerin, is a component of telomerase. Mutations in the RNA component of telomerase are associated with an autosomal dominant form of dyskeratosis congenita (Vulliamy et al., 2001). Families with this form of the disease are more severely affected in later generations, suggesting telomere shortening could be involved. Features of dyskeratosis congenita include bone marrow failure, which is the most usual cause of death, abnormal skin pigmentation, leukoplakia and nail dystrophy (Knight et al., 1998). The role of stem cells has also been suggested (Mason, 2003). As judged from the phenotype of dyskeratosis congenita, telomeres are crucial in rapidly proliferating tissues but it is unclear whether telomere shortening is involved in human aging. One study measured changes in telomere length in over 4,000 people over 10 years but found no association with mortality or morbidity (Weischer et al., 2014). Human epidemiological data suggest a causal role of both short and very long telomeres in cancer, heart disease and other age-related diseases (Codd et al., 2013), so some impact of telomere shortening in age-related diseases besides cancer cannot be excluded.
In conclusion, it is unquestionable that cellular senescence and telomere biology are important in cancer and may be suitable to develop anti-cancer treatments (reviewed in Campisi et al., 2001; Blasco and Hahn, 2003; Hahn, 2003; Lee and Schmitt, 2003; Wang et al., 2003; de Magalhaes, 2013). Whether these can aid in understanding human aging is unknown. Hopefully readers can make their own mind from the aforementioned discussion. My personal opinion is that cellular senescence, primarily caused by stress but to some degree perhaps also by telomere shortening, can contribute to aging and age-related diseases. Indeed, a genetic variant of telomerase has been associated with longer telomeres and exceptional human longevity (Atzmon et al., 2010). Having said that, I am not convinced by the empirical evidence that telomere shortening and cell senescence are causes of aging. They may be contributors or intermediaries, for example by enhancing the effects of other types of molecular damage, but I see little evidence that targeting the telomeres and/or telomerase by itself will have much effect on human aging even if it might be helpful in the case of some specific pathologies. I also think that cellular studies are crucial to gerontology, yet so much focus on measuring cellular proliferation does not appear to me to be the best approach, as mentioned before. Other methodologies are desperately needed to assess the role of cellular changes in organismal aging.
Note 1:
Telomeres and Telomere Length: A General OverviewNote 2:
Physical Activity on Telomere Length as a Biomarker for Aging: A Systematic ReviewNote 4:
Canonical and extra-telomeric functions of telomerase: Implications for healthy ageing conferred by endurance trainingNote 5:
Associations of meditation with telomere dynamics: a case-control study in healthy adultsNote 6:
Effects of Physical Exercise on Telomere Length in Healthy Adults: Systematic Review, Meta-Analysis, and Meta-Regression