When cells get stressed: an integrative view of cellular senescence (original) (raw)
The initial discovery of the replicative senescence of human fibroblasts has led to the view that senescence serves as a mechanism whose purpose is to limit the proliferative capacity of normal cells (11, 14). According to this thinking, it is undesirable for cells to be capable of dividing beyond what is required for their participation in normal development and tissue maintenance. The capacity of cells to divide is therefore limited by an intrinsic mechanism that counts the number of divisions through which cell lineages have undergone, and triggers senescence when the predetermined limit for division is reached. While the senescence of cells might contribute to the aging of tissues, the breakdown of this division-restricting mechanism can lead to cancer.
The finding that telomeres, the nucleoprotein structures protecting chromosome ends, shorten with every cycle of cell growth and division suggested a molecular mechanism that could record the number of divisions that a lineage of cells has undergone. Erosion of telomeres to a critical length could serve to activate the senescence program (15). In accord with this mechanistic model, ectopic expression of the catalytic subunit of the telomerase enzyme, hTERT (human telomerase reverse transcriptase), halts the erosion of telomeres in human cells; in some cell types the expression of this gene prevents the entrance into replicative senescence, suggesting that indeed telomere shortening is the cause for senescence (16, 17).
As mentioned above, yet other work has demonstrated that normal cells that are exposed to various physiologic stresses rapidly enter into a state of senescence, doing so within a period as short as several days. Such stresses include DNA-damaging agents, oxidative stress, “oncogenic stress” (due to oncogene overexpression), and other metabolic perturbations (5, 18–22). Typically, these forms of senescence do not involve significant telomere shortening and cannot be prevented by ectopic hTERT expression (23, 24). Accordingly, the hypothesized telomere-based mechanism of division counting could not be invoked to explain these acute responses. Moreover, these situations of stress-induced senescence could not be accommodated by a model proposing that senescence functions exclusively as a barrier to extended growth-and-division cycles.
These observations raised the question of whether replicative senescence and stress-induced senescence serve the same biological function, and which of these mechanisms operates in vivo (10, 11, 25). In fact, the conditions that induce these two responses are not as distinct as the above description would suggest. The onset of replicative senescence exhibited by some types of normal cells is dependent on the conditions in which they are propagated. This suggests that certain culture conditions are physiologically stressful to the cells, and that cells can undergo replicative senescence due to the cumulative effect of this stress, rather than the progressive erosion of their telomeres. For example, populations of human mammary epithelial cells encounter their first growth barrier following 10–20 divisions in culture. This stage of senescence can be avoided if these cells are grown on fibroblast feeder layers or in a different type of medium (26, 27). Similarly, mouse embryo fibroblasts (MEFs) senesce after approximately ten divisions. It was recently demonstrated that when MEFs are propagated in 3% oxygen, rather than the commonly used 20% oxygen conditions, they can avoid senescence (28). This study also demonstrated that when grown in 20% oxygen, MEFs suffer from the rapid accumulation of DNA damage. Consequently, the cumulative oxidative damage induced by growth in conditions that are hyperoxic (by the standards of living tissues) leads to the onset of senescence in these cells. These findings provide a direct demonstration of how extrinsic physiologic stress experienced by cells can lead to replicative senescence.
In many cases, both types of mechanisms for the induction of replicative senescence — a telomere-based one and a stress-based one — seem to function together in the same cell population. Human fibroblasts are the best-studied example of a cell type in which the cause of senescence is attributed to critical telomere attrition, as their senescence can usually be prevented by ectopic expression of hTERT. However, these cells are hardly indifferent to their growth conditions in vitro: the timing of their entry into senescence is affected by various parameters of culturing, such as plating density, media composition, and others. Thus, when propagated in 1–3% oxygen instead of 20% oxygen, human fibroblasts are able to undergo more divisions prior to senescence; conversely, oxygen levels higher than 20% will shorten their lifespan (29–31). Clearly, a stress-based clock contributes to the effect of the telomere-based clock in these cells, and the combined effects of the two dictate the onset time of senescence.
Physiologic stress could hasten the onset of senescence in these cells in a manner independent of the process of telomere shortening. Alternatively, it could act by increasing the rates of telomere shortening. However, if the latter were true, then telomeric DNA could no longer be viewed as a counting device that advances autonomously at its own rate, but should rather be viewed as a cellular structure that responds to stress. In fact, it has been reported in the past that the telomeres of some cells shorten more quickly in high oxygen conditions (31). This observation received further substantiation in a recent study, demonstrating that in 2–5% oxygen conditions the rates of telomere shortening of some commonly studied human fibroblast lines — WI38 and IMR90 — are slower than the rates observed in 21% oxygen (32). Moreover, when the hTERT gene is ectopically introduced into various human fibroblast lines, resulting in the elongation of their telomeres, the rate of telomere elongation is much slower in 21% oxygen than in 2–5% oxygen growth conditions. This study clearly demonstrates that telomere shortening is not an intrinsic clock-like mechanism that operates independently of extrinsic physiologic stresses. This recent study also underscores a little-regarded observation, namely that many normal human fibroblast lines cannot be immortalized by ectopic expression of hTERT. Telomerase activity enables these cells to proliferate longer, but eventually they do undergo senescence, even though their telomeres have been elongated well beyond the lengths observed in early passage cells (32). Thus, the immortalizing capabilities of telomerase are only limited to a subset of cell types.
Different types of intrinsic and extrinsic stress signals are likely to converge on the activation of the p53 protein, the Rb protein, or both. In this manner, these two key tumor suppressor proteins might act as integrators of stress signals, and their combined level of activation would determine the onset of senescence (Figure 1). Recent studies have demonstrated that some cultured human fibroblast lines indeed express higher levels of the p16/INK4a gene than do others, presumably reflecting a higher degree of stress that these cells experience in vitro (33, 34). This stress-induced expression of p16, an activator of Rb, apparently acts to hasten the replicative senescence of cells, doing so in a manner independent of telomere length (34, 35). These and other findings suggest that telomere attrition leads mainly to the activation of the p53 protein, while culture stresses mainly activate Rb through p16.
Senescence as a general stress-response program. A variety of physiologic stresses, intrinsic and extrinsic, lead to the onset of senescence. These stresses stimulate various cellular signaling pathways, which are funneled down to activate either the p53 protein, the Rb protein, or both. p53 can be activated by the DNA damage signaling pathways, the ATM and ATR proteins, or by the p14/ARF protein, which responds to oncogene overexpression and other stresses. p21, a target of p53, can cause the activation of Rb. Most cellular stresses will activate the p16/INK4a gene, also leading to Rb activation. Different stress signals will have different relative effects on the p53 and Rb arms, and their combined level of activation dictates the onset of senescence. Once this program is activated, a series of changes in cell function and morphology take place. ATM, ataxia telangiectasia mutated; ATR, ATM-related; p14/ARF, alternative reading frame product of INK4a gene locus.
The emerging picture is that various types of intrinsic and extrinsic stress stimuli can activate the senescence program, and whether this occurs rapidly, or gradually following a period of proliferation, is mainly determined by the combined levels of these stresses. Moreover, extrinsic signals may affect cell-intrinsic processes such as telomere shortening. It seems, then, that there is no necessity in a functional distinction between replicative senescence and stress-induced senescence, as these titles merely reflect the fact that a spectrum of different stimuli feed into one response program.