Cells are the fundamental structure composing our bodies, and so it makes sense that cellular decline contributes to the aging process. In this essay I review the methods used to study cellular aging in vitro and debate whether these findings could be related to organismic aging.
Keywords: ageing, cellular immortality, cytogerontology, Hayflick's phenomenon, irreversible growth arrest, life span, terminal restriction fragments, TRF
Hayflick's Limit
In 1961, and in contradiction to what was thought at the time, Leonard Hayflick and Paul Moorhead discovered that human cells derived from embryonic tissues can only divide a finite number of times in culture (Hayflick and Moorhead, 1961). They devided the stages of cell culture in three phases: Phase I is the primary culture, when cells from the explant simply multiply to cover the surface of the culture flask. Phase II represents the period when cells divide in culture. Briefly, once cells cover a flask's surface, they stop multiplying. For cell growth to continue, the cells must be subcultivated. To do so, one removes the culture's medium and adds a digestive enzyme called trypsin that dissolves the substances keeping cells together. If you add growth medium afterwards, you obtain the cells in suspension that can then be divided by two--or more--new flasks. Later, cells attach to the flask's floor and start dividing once again until a new subcultivation is required. Cells divide vigorously and can often be subcultivated in a matter of a few days. Eventually, however, cells start dividing slower, which marks the beginning of Phase III. Eventually they stop dividing at all and may or not die (reviewed in Hayflick, 1985; Hayflick, 1994). Hayflick and Moorhead noticed that cultures stopped dividing after an average of fifty cumulative population doublings (CPDs). This phenomenon is known as Hayflick's limit, Phase III phenomenon, or, as it will be called herein, replicative senescence (RS).
Hayflick and Moorhead worked with fibroblasts, a cell type found in connective tissue, but RS has been found in other cell types: keratinocytes, endothelial cells, lymphocytes, adrenocortical cells, vascular smooth muscle cells, chondrocytes, etc. In addition, RS is observed in cells derived from embryonic tissues, in cells from adults of all ages, and in cells taken from many animals: mice, chickens, Galapagos tortoises, etc. (reviewed in Hayflick, 1994). Early results suggested a relation between the number of CPDs cells undergo in culture and the longevity of the species from which the cells were derived. For example, cells from the Galapagos tortoise, which--as described--can live over a century, divide about 110 times (Goldstein, 1974), while mouse cells divide roughly 15 times (Stanley et al., 1975; Rohme, 1981). In addition, cells taken from patients with progeroid syndromes such as Werner syndrome (WS)--described before--endure far less CPDs than normal cells (Salk et al., 1981). Exceptions exist and certain cell lines never reach RS. These are said to be "immortal" and include embryonic germ cells and most cell lines derived from tumors, such as HeLa cells (Brunmark et al., 1986; Chen and Yu, 1994; Pera et al., 2000). Some types of rat cells have also been claimed as capable of evading RS (Mathon et al., 2001; Tang et al., 2001).
Biomarkers of RS
The phenotype of RS in human diploid fibroblasts (HDFs) is characterized by a series of features, termed "biomarkers" (reviewed in Campisi, 1999). The most obvious biomarker is growth arrest, i.e., cells stop dividing, which can be detected by different methods. Even vigorously dividing cultures are heterogeneous and contain a percentage of senescent and growth-arrested cells; this percentage progressively increases until all cells in the population are quiescent, that is, they have stopped dividing (Cristofalo and Sharf, 1973; Smith and Whitney, 1980); this percentage is higher in WS cells when compared with normal cells at the same CPD (Kill et al., 1994). Senescent cells are growth arrested in the transition from phase G1 to phase S of the cell cycle (Sherwood et al., 1988). The growth arrest in RS is irreversible in the sense that growth factors cannot stimulate the cells to divide (reviewed in Cristofalo and Pignolo, 1993), even though senescent cells can remain metabolically active for long periods of time (reviewed in Goldstein, 1990).
Another important biomarker is cellular morphology (Fig. 1). The progressive morphological changes cells endure while they age in vitro were particularly well-studied by Klaus Bayreuther and colleagues (e.g., Bayreuther et al., 1988). In brief, senescent cells are bigger and a senescent population has more diverse morphotypes than cells at earlier CPDs. In fact, a confluent senescent culture has a smaller cellular density than a confluent young culture, though this also occurs because senescent cells are more sensitive to cell-cell contact inhibition.
Figure 1: Normal human fibroblasts (left) and fibroblasts showing a senescent morphology (three cells on the right). Notice the common elongated morphology of senescent cells.
In 1995, Judith Campisi and her team discovered that the enzyme β-galactosidase has an abnormal behavior associated with senescent cells, which is termed senescence-associated β-galactosidase (SA β-gal) activity. β-galactosidase, a lysosomal hydrolase, is normally active at pH 4, but in senescent cells it often happens for β-galactosidase to be active at pH 6. Both in vitro and in vivo, the percentage of cells positive for SA β-gal increases with, respectively, CPDs and age (Dimri et al., 1995). On the contrary, in immortal cell lines, such as HeLa tumor cells, the percentage of cells positive for SA β-gal does not correlate with CPDs. In addition, it is possible to find a correlation between the increase in SA β-gal and the appearance of the senescent morphotypes (Toussaint et al., 2000). Early reports showed that lysosomes increase in number and size in senescent cells (Robbins et al., 1970; Brunk et al., 1973). SA β-gal appears to be a result of increased lysosomal activity at a suboptimal pH, which becomes detectable in senescent cells due to an increase in lysosomal content (Kurz et al., 2000). Other results also suggest that during in vitro aging increased autophagy--i.e., digestion of the cell's organelles--may be associated with an increase of lysosomal mass and SA β-gal (Gerland et al., 2003).
Normal human cells are diploid, that means they have two copies of each chromosome. Yet with each subcultivation, the percentage of polyploid cells--i.e., with three or more copies of chromosomes--increases (Matsumura, 1980). Mutations to the mitochondrial DNA (mtDNA) also appear to increase with age in vivo, though at low levels (e.g., Tanhauser and Laipis, 1995). For example, the first identified mutation was a deletion of 4,977 base pairs (bp) in the 16,569 bp mtDNA. This deletion is observed both in vivo (Corral-Debrinski et al., 1992; Yang et al., 1994; Liu et al., 1998) and in vitro (Dumont et al., 2000a).
Senescent cells also have a decreased ability to express heat shock proteins both in vivo (Blake et al., 1991; Fargnoli et al., 1990) and in vitro (Choi et al., 1990; Bonelli et al., 1999). In addition, in vitro aging makes HDFs lose c-fos inducibility by serum (Seshadri and Campisi, 1990).
The expression levels of several genes change during in vitro cellular aging (reviewed in Cristofalo et al., 1998a). Some of the most commonly used biomarkers are: osteonectin, fibronectin, apolipoprotein J, smooth muscle cells 22 (SM22), and type II (1)-procollagen, whose expression increases in senescent HDFs (Kumazaki et al., 1991; Gonos et al., 1998; Dumont et al., 2000a). Lastly, senescent cells also display an increased activity of metalloproteinases, which degrade the extracellular matrix (reviewed in Campisi, 1999).
Telomeres are non-coding regions at the tips of chromosomes. In vertebrates, they are composed of repeated sequences of TTAGGG (Moyzis et al., 1988; Meyne et al., 1989). During in vitro aging, the telomeres shorten gradually in each subcultivation (Harley et al., 1990). The same process might occur in vivo too (Hastie et al., 1990; Lindsey et al., 1991; Allsopp et al., 1992). Since the telomeres are one of the main players in RS, I will focus on them again in another essay.
Stress-Induced Premature Senescence
Normally, cell culture conditions include 20% oxygen (O2) and these were the conditions initially used by Hayflick and Moorhead and most subsequent studies. When HDFs are cultured at 3% O2, which is closer to physiological conditions, they achieve a further 20 CPDs (Chen et al., 1995). In contrast, different types of human cells cultured above 20% O2 display a reduced growth rate and endure fewer CPDs (Horikoshi et al., 1986 & 1991; von Zglinicki et al., 1995). Interestingly, the same effect is not witnessed in tumor cell lines (Saito et al., 1995). In normal human cells, O2 has been shown to accelerate growth arrest (Alaluf et al., 2000). If O2 is above 50%, it becomes cytotoxic (Horikoshi et al., 1991). The way subcytotoxic stress can accelerate the appearance of the senescent phenotype in cells has been deemed as another form of cellular senescence (Fig. 2). In 1999, at the EMBO workshop of Molecular and Cellular Gerontology, Olivone, Switzerland, the term stress-induced premature senescence (SIPS) was coined (Brack et al., 2000).
Figure 2: Schematic drawing of SIPS. Source: Ageing and stress group, University of Namur, Belgium.
Depending on the dose of stressor used, a cell population will react in different ways. For instance, a high, cytotoxic dosage of a stressor causes such an amount of damage that cellular biochemical activities decrease leading to cellular death by necrosis. The level of damage sustained by cells determines whether programmed cell death--apoptosis--can unfold or, if the damage is even lower, senescence. Since a cellular population is not homogeneous, the dose of the stressor will shift the percentage of cells executing each of the possible programs depending, respectively, on the amoung of stress: cellular proliferation, senescence, apoptosis, and necrosis (reviewed in Toussaint et al., 2002a). In order for SIPS to occur, a precise subcytotoxic dose must be determined for each cell population.
In addition to O2, other sources of oxidative damage, such as H2O2 and tert-butylhydroperoxide, and other stressors--e.g., ethanol, ionizing radiations, and mitomycin C--can induce SIPS in many types of proliferative cells such as lung and skin fibroblasts, endothelial cells, melanocytes, and retinal pigment epithelial cells (reviewed in Toussaint et al., 2002b; Dierick et al., 2003). The list of stressors that can cause SIPS is constantly growing. Instead of chronic stress, SIPS can also be induced based on a single or repeated short exposure(s) to stressors.
The Cell Cycle and Its Influence on Aging
The connection between aging and RS is not obvious. At least post partum, there is no relation between the number of CPDs cells can endure and the age of the donor (Cristofalo et al., 1998b). Chances are previous studies showing otherwise were biased (Cristofalo, 2001). Studies in centenarians failed to find differences in the CPDs cells taken from centenarians could endure (Tesco et al., 1998). In addition, they raised doubts on whether telomere shortening occurs in vivo and whether senescence-associated genes in vitro are also differentially expressed in vivo (Mondello et al., 1999). In fact, gene expression patterns show differences between in vitro senescent cells and cells from old donors (Takeda et al., 1992). Lastly, the relation between SA β-gal and in vivo aging has also been attacked (Going et al., 2002).
Cell senescence in vivo can be found without telomere shortening (Melk et al., 2003), suggesting that RS may not be the prevailing mechanism in vivo--as detailed elsewhere, telomeres are crucial for RS. Since cells taken from old donors do not endure less CPDs, one hypothesis is that in vivo RS does not widely occur and that cellular senescence in vivo is dependent on stress factors. When cell lines are derived from people, the selected cells are those that grow because people, even very old people, never run out of proliferating cells (Tesco et al., 1998; Cristofalo, 2001). In fact, from a simple mathematical perspective, it appears unlikely that RS occurs in vivo. Assuming HDFs endure 50 CPDs, 250 is more than enough cells for several lifetimes (Hayflick, 1994). Therefore, only a minority of cells may reach RS in vivo (Toussaint et al., 2002b). Of course that even a small percentage of senescent cells may interfere with tissue homeostasis and function (Shay and Wright, 2000), but RS such as defined by the phase III phenomenon appears unlikely to be widely present in vivo.
Although the relation between a species' longevity and the CPDs its cells can endure in vitro exists, it is also debatable if this is related to aging since optimal culture conditions vary from species to species. For instance, O2 partial pressure can affect cellular proliferation and recent results show that O2 limits the replicative capacity of mouse fibroblasts (Parrinello et al., 2003). These results show that comparisons between different species may be biased due to intra-species differences in O2 sensitivity; instead of showing maximum cellular proliferate capacity, these results show O2 sensitivity (Toussaint et al., 2002b). In addition, due to the positive correlation between body size and longevity, as mentioned before, perhaps cells taken from long-lived animals endure more CPDs because of the difference in size, not due to the difference in longevity, as supported by some results (Lorenzini et al., 2005).
As mentioned above, cells at birth from persons with certain progeroid syndromes have fewer divisions than cells from normal persons. This, however, might be a result of increased cell death or exit from the cell cycle for reasons unrelated to RS (Johnson et al., 1999). In fact, senescent cells from persons with Werner's syndrome have different patterns of gene expression (Oshima et al., 1995; Toda et al., 1998) and biomarkers of senescence (Schulz et al., 1996), as happens in progeria (Park et al., 2001).
It was initially reported that cells from older donors have a slower proliferative capacity (Waters and Walford, 1970; Hayflick, 1994). This effect, known as the latent period, occurs because fewer cells are in the replication cycle, not because they take longer to divide (Ponten et al., 1983; Karatza et al., 1984), but it has also been under attack (Cristofalo et al., 1998b).
Senescent cells and senescence-associated biomarkers can be found in vivo (Paradis et al., 2001; Going et al., 2002). Interestingly, stress-prone tissues appear to be the most affected. HDFs cultured from distal lower extremities of patients with venous reflux, which precedes the development of venous ulcers, display characteristics of senescent cells (Mendez et al., 1998). Similar results also relate cellular senescence to atherosclerosis (Minamino et al., 2002) and benign prostatic hyperplasia, a common age-related male pathology (Castro et al., 2003). Senescence and inflammatory processes may also be related to age-related pathologies such as osteoarthritis (Martin and Buckwalter, 2002; Price et al., 2002) and skin aging (Giacomoni et al., 2000). Indeed, repeated stimulation of WI-38 HDFs with pro-inflammatory cytokines interleukin-1a or tumor necrosis factor-a induces SIPS (Dumont et al., 2000b). These cytokines' circulating levels increase in vivo (reviewed in Lio et al., 2003), favoring inflammation and SIPS, so in vivo senescence may be a result of SIPS rather than RS. Some data indicate that chronic stressors may accelerate risk of a host of age-related diseases by prematurely aging the immune response (Kiecolt-Glaser et al., 2003). One study argued that cells ceasing division is not relevant to aging. Instead, altered gene expression, resulting from quality control defects that allow errors to accumulate as cells divide, leads to cells with diminished function (Ly et al., 2000). Lastly, as hinted by the above mentioned results on the impact of O2 in cell proliferation, RS for many cell lines in vitro and in vivo may instead be better defined as SIPS resulting from oxidative stress. Not surprisingly, certain cell lines such as hemopoietic cells and exfoliating epithelial cells endure many more population doublings in vivo than they do in vitro (Strehler, 1999, p. 46).
A relation appears to exist between stress resistance and aging. In fact, in model organisms, extended longevity is often associated with increased stress resistance (reviewed in Longo, 1999). Concisely, manipulations in C. elegans that extend longevity show a strong correlation with resistance to stress (Murakami et al., 2000). In Drosophila too some mutations can increase longevity and augment stress resistance (Lin et al., 1998). Cell lines from long-lived mouse strains are also stress resistant (Salmon et al., 2005). Stress resistance in vitro also correlates with mammalian longevity (Kapahi et al., 1999), though it is not clear other sources of bias exist. Finally, cells taken from patients with progeroid syndromes are more susceptible to stress (Gebhart et al., 1988), as are senescent fibroblasts (Yuan et al., 1996). Of course, it is not known whether stress-resistance is related to rate of aging. Nevertheless, aging and stress-resistance appear to be inversely related. Importantly, an association between cellular stress resistance and organismal aging appears to exist. Whether SIPS is a precise simulation of in vivo degeneration, however, remains a point of discord.
Is it possible that changes occur with age at a cellular level? Of course it is. Some genetic interventions regulating aging appear to influence tissue homeostasis by affecting senescence, cell proliferation, and cell death, as detailed in the context of telomeres and regarding the endocrine theory of aging. Results from mice suggest that systemic factors can influence aging, but only to some degree, showing that intrinsic cellular mechanisms likely play a role in aging (Conboy et al., 2005). In some tissues, such as the immune system, decreased proliferative ability may play a role in age-related degeneration (reviewed in Effros, 1996). Successive transplants of spleen and bone marrow yielded far from conclusive results but it appears that a slight decrease in proliferative ability does occur in vivo in spite the cells having to divide much more than 50 times (Strehler, 1999, p. 53). Therefore, mechanisms of aging intrinsic to cells likely exist (reviewed in de Magalhaes, 2004).
Nevertheless, RS does not appear to be a faithful model of changes occurring in vivo (Gershon and Gershon, 2000a for arguments). The purpose of cellular senescence is unclear but a strong hypothesis is that it is an anti-cancer mechanism (Wynford-Thomas, 1999), as also debated ahead. Though cellular studies could be useful for studying long-lived species whose study as organisms would be practically impossible (Austad, 2001), RS and SIPS are not accurate representations of human aging. Maybe SIPS reflects certain pathological changes whose incidence increases with age, but there is little evidence to suggest that cells running out of divisions, due to RS or SIPS, are a major factor in aging. This topic is further debated ahead.
Germ and Stem Cells
Stem cells are found in different places throughout the body and participate in tissue homeostasis by replacing differentiated cells that died; due to their high place in each "tissue's hierarchy," they are interesting subjects for study in the context of aging. Human stem cells can express telomerase (Chiu et al., 1996; Sugihara et al., 1999) indicating that the most actively dividing cell lines in the body overcome telomere shortening--though somatic stem cells can show senescence in vitro (Smith and Schofield, 1994) and in vivo (Martin et al., 2000). Interestingly, a correlation between mean telomeres and age is found in the first two decades for muscle satellite cells but not afterwards (Decary et al., 1997). So, one hypothesis is that somatic cells can only divide a limited amount of times but are constantly being replenished by stem cells. Interestingly, one study found a correlation between stem cell turnover and mice lifespan (de Haan and Van Zant, 1999), meaning that perhaps stem cell senescence influences organismic senescence (Snyder and Loring, 2005). At present it is speculative to consider this hypothesis but certainly much work will be done in the future attempting to relate stem cells to aging.
As previously mentioned, the doctrine of the immortal germplasm claims that germ cells are immortal and can divide forever (Weismann, 1891; Kirkwood, 1977). A prediction of such hypothesis is that the germ cells should have increased stress resistance and repair mechanisms (Kirkwood, 1977). Experimental evidence, however, is contradictory: the soma of Drosophila has been reported to be more sensitive to mutagens (Vogel and Zijlstra, 1987); increased DNA repair has been documented in male mice germ cell (Walter et al., 1994), but using ionizing radiation no difference in sensibility was found between mice male germ cells and bone marrow (van Loon et al., 1993). It has also been proposed that meiosis and gametogenesis can have recombinational and other genetic events that contribute to a rejuvenation not possible in differentiated somatic cells (Medvedev, 1981; Holliday, 1984). Yet little or no evidence exists to support such claims. Furthermore, the common notion that germ cells have improved DNA repair mechanisms and thus avoid aging is itself debatable (reviewed in Walter et al., 2003).
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