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Cellular Senescence and Tissue Aging In Vivo

^a Department of Physiology and the Sam and Ann Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio

Peter J. Hornsby, Sam and Ann Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center, 15355 Lambda Drive STCBM 2.200, San Antonio, TX 78245 E-mail: hornsby{at}uthscsa.edu.

Decision Editor: James R. Smith, PhD

	Abstract

Top Abstract The Cell Culture Phenomenon Telomere Shortening in Tissues... Does Telomere Shortening... Human Versus Mouse Aging Do Senescent Cells Accumulate... Conclusions and Speculation References

 
A long-standing controversy concerns the relevance of cellular senescence, defined and observed as a cell culture phenomenon, to tissue aging in vivo. Here the evidence on this topic is reviewed. The main conclusions are as follows. First, telomere shortening, the principal known mediator of cellular senescence, occurs in many human tissues in aging. Second, it is not clear whether this results in cellular senescence or in some other cell fate (e.g., crisis). Third, rodents probably are not appropriate experimental models for these questions, because of important differences in telomere biology between rodent cells and cells from long-lived mammals (e.g., human or bovine cells). Fourth, better and more comprehensive observations on aging human tissues are needed to answer the question of the occurrence of senescent cells in tissues, and new experimental approaches are needed to elucidate the consequences of telomere shortening in tissues in aging.

IN the past, the debate over the relationship between cellular senescence and aging has been phrased in terms of whether this cell culture phenomenon is a model "for" aging. However, phrasing the question this way is misleading—an analogy is that it might well be correct to state that a toy car is a good model for a real car, but it would not make sense to suggest that a wheel is a model for a car. Either the wheel is part of the car or it is not, but it is not a model for it. Similarly, cellular senescence is either a part of aging or it is not. Recently, the viewpoint has been expressed that cellular senescence might be purely a cell culture phenomenon; for example, Hanahan and Weinberg ⁽¹⁾ state:

The above-cited observations [on cellular senescence] might argue that senescence, much like apoptosis, reflects a protective mechanism that can be activated by shortened telomeres or conflicting growth signals that forces aberrant cells irreversibly into a G0-like state, thereby rendering them incapable of further proliferation. If so, circumvention of senescence in vivo may indeed represent an essential step in tumor progression that is required for the subsequent approach to and breaching of the crisis barrier. But we consider an alternative model equally plausible: senescence could be an artifact of cell culture that does not reflect a phenotype of cells within living tissues and does not represent an impediment to tumor progression in vivo. Resolution of this quandary will be critical to completely understand the acquisition of limitless replicative potential. (p. 63)

Clearly, therefore, it is important to understand the current state of the evidence for the occurrence of cellular senescence in tissues in vivo and what evidence exists for its playing a role in the age-related changes in tissues. It is even more important to appreciate the extent of our ignorance in this field and what still has to be resolved.

	The Cell Culture Phenomenon

Top Abstract The Cell Culture Phenomenon Telomere Shortening in Tissues... Does Telomere Shortening... Human Versus Mouse Aging Do Senescent Cells Accumulate... Conclusions and Speculation References

 
As the mechanisms of cellular senescence have been increasingly well understood, it has become clear that the process and the end point are separable biological phenomena. The well-known counting process that human cells undergo as they are grown in culture, which has been termed a "mitotic clock" or "mitotic counter," is generally thought to result from the progressive and inexorable erosion of telomeres, until telomere length reaches a critical value. Telomeres shorten in most dividing human somatic cells because of the lack of telomerase activity that is required for telomere maintenance ⁽²⁾⁽³⁾. The lack of telomerase activity results from the absence of expression of the reverse transcriptase subunit (TERT) of the telomerase ribonucleoprotein complex ⁽⁴⁾⁽⁵⁾. When cells divide in the absence of telomerase activity, approximately 40–100 bp of the terminal telomeric repeat DNA is not replicated ⁽²⁾⁽³⁾. After a normal human cell has divided a certain number of times, that number varying with the specific cell type and culture conditions, the telomeres become so short that they trigger a cell cycle checkpoint that puts the cell into a terminally nondividing state. These events have usually been termed "cellular senescence," but it is now understood that cultured cells can be made to enter a biochemically similar senescent state by means that do not involve telomere shortening. As a result, the term cellular senescence has now come to mean the process whereby cells are forced into this senescent state, whether or not the process involves telomere shortening. Therefore the term replicative senescence will be used here to mean cellular senescence resulting from telomere shortening; the term M1 is also used. As cells undergo replicative senescence, further cell division is blocked by inhibitors of cell proliferation such as p21^{SDI1/WAF1/CIP1} and p16^{INK4A (6)(7)}. When replicative senescence is abrogated by oncoproteins such as SV40 T antigen, this first checkpoint is bypassed and cells eventually enter a second state, termed crisis or M2 ⁽⁸⁾. In this state the much shorter telomeres undergo end-to-end fusions and chromosomal breakage-fusion cycles that cause the cells to undergo apoptosis. There is massive loss of cells from the culture, whereas in replicative senescence (M1) cells do not die ⁽⁷⁾.

Replicative senescence involves widespread changes in gene expression. In fibroblasts the pattern resembles that of fibroblasts in inflammation ⁽⁹⁾. Of particular significance is the production of proteases that may erode the surrounding extracellular matrix and the production of cytokines that could have effects on neighboring cells ^{(10)(11)(12)(13)}. Interestingly, other cell types (retinal pigmented epithelial cells and endothelial cells) show different patterns of alteration of gene expression when they reach replicative senescence ⁽⁹⁾. A useful biochemical marker for senescent cells, although one of unknown biological significance, is the high level of ß-galactosidase enzymatic activity with a pH optimum of 6.0, which is called senescence-associated ß-galactosidase (SA-ßgal) ⁽¹⁴⁾. The currently available data are consistent with the hypothesis that the triggering of the block to DNA synthesis that is characteristic of replicative senescence is accompanied by dysregulation of expression of various other genes, and that the pattern of dysregulation will be cell-type specific.

Cultured cells can be made to undergo cellular senescence by various means that do not involve cell division at all, such as oxidative stress, radiation, and the ectopic expression of some signal transduction molecules and cyclin-dependent kinase inhibitors ^(15)(16)(17). Because cells can be forced into cellular senescence by manipulations that do not necessarily involve telomere shortening, the process of mitotic counting must be distinguished from its consequence, replicative senescence. Therefore the question relating cellular senescence to in vivo aging must be divided into several separate questions: whether telomere shortening occurs in tissues in vivo; whether this results in replicative senescence; if senescent cells are formed, whether they persist in tissues in vivo; and if senescent cells are found in tissues, whether they were formed by replicative senescence or by another process. For the first of those two questions, whether telomere shortening occurs in vivo, there is a variety of evidence suggesting that it does, although to varying extents in different tissues. However, there is much less evidence on the occurrence in tissues in vivo of cells that have reached replicative senescence.

	Telomere Shortening in Tissues During Aging

Top Abstract The Cell Culture Phenomenon Telomere Shortening in Tissues... Does Telomere Shortening... Human Versus Mouse Aging Do Senescent Cells Accumulate... Conclusions and Speculation References

 
Human cell proliferation in aging has often been investigated by making primary cultures of cells from donors of different ages and by studying their proliferation in culture. In some cases the complete replicative potential of such cells has been studied; that is, the cells have been grown until they reached replicative senescence. In others, colony-forming efficiency has been studied in the primary culture, as a surrogate measure for total replicative capacity ⁽¹⁸⁾.

Much attention has been paid to the observation originally made in the 1970s that the replicative capacity of human fibroblasts in culture decreases as a function of donor age ⁽¹⁹⁾. It was well known even at the time of these initial observations that there was much variation within each decade of age in the maximal and minimal proliferative capacity of the different cell samples. Subsequently the generality of the observation was challenged by suggesting that the decrease as a function of donor age applied only to fibroblasts isolated from diabetic and "prediabetic" patients ⁽²⁰⁾, and that it was not evident in fibroblasts obtained from non–sun-exposed skin ^(21)(22). However, other sets of data have upheld the original observations ⁽²³⁾. Fibroblasts from older donors also show a higher level of expression of collagenase, characteristic of senescent cells ⁽²⁴⁾. Additionally, cells from patients with Werner syndrome, a segmental progeroid syndrome, have a decreased replicative potential and accelerated telomere shortening ^(25)(26).

Nevertheless, skin fibroblasts may not be the ideal cell type to study because they probably undergo little proliferation over a life span ⁽²⁷⁾. Studies that have been done on nonfibroblast cell populations have shown much larger decreases in proliferative capacity than was observed as a function of donor age in fibroblasts. In some nonfibroblast cell types, many cells in the population isolated from older donors have very limited or no proliferative capacity. Some examples are age-related decrements in proliferative potential in lens epithelial cells ^(28)(29), retinal pigmented epithelial cells ⁽³⁰⁾, smooth muscle cells ^(31)(32)(33), osteoblasts ^(34)(35)(36), and chondrocytes ⁽³⁷⁾. In the author's laboratory, studies on the effects of age on the proliferation of adrenocortical cells from donors as a function of age show a great decrease in proliferative capacity associated with short telomeres in cells from older donors ⁽³⁸⁾.

Other examples are provided by endothelial cells and myoblasts. Proliferative capacity is closely related to telomere length in endothelial cells. Telomere length in endothelial cells decrease as a function of donor age, with a greater decline being observed in cells isolated from the iliac artery in comparison with cells from the thoracic artery ⁽³⁹⁾. The greater decline in telomere length was observed in the cells that had likely undergone more proliferation in vivo, because they resided in a part of the vascular system where blood flow might cause most chronic damage to the endothelium. Unfortunately, because the data are from human specimens, it is difficult to test this hypothesis directly. Skeletal muscle satellite cells can be isolated from human muscle samples and exhibit a limited replicative potential in culture. They show decreasing proliferative potential as a function of donor age and decreased telomere length, but muscle fiber nuclei show stable telomere length ⁽⁴⁰⁾.

These examples are consistent with the hypothesis that cell proliferation occurring over the life span of the donor causes telomere shortening, and that cell cultures are then initiated with cells that have a lowered remaining proliferative potential because continued cell division in culture shortens telomeres to a point where replicative senescence occurs.

	Does Telomere Shortening Interfere With Cell Proliferation in Tissues In Vivo?

Top Abstract The Cell Culture Phenomenon Telomere Shortening in Tissues... Does Telomere Shortening... Human Versus Mouse Aging Do Senescent Cells Accumulate... Conclusions and Speculation References

 
No data show a complete loss of cell proliferative potential in any human organ as a function of age, and it is unlikely that the day-to-day functions of tissues are compromised by changes in replicative potential. However, tissues may suffer a loss of ability to repair damage efficiently or to restore sudden losses of cells during aging. Under conditions where there is a chronic stimulus to divide, there is evidence, albeit limited, that cells can reach a stage of impaired replication under in vivo conditions. In patients with chronic ulcers, fibroblasts were observed to have decreased proliferative capacity and increased senescence markers ^(41)(42). These effects were more pronounced in ulcers that had been present for >3 years ⁽⁴¹⁾. In conditions of muscle fiber death and excessive cell turnover in the muscle, such as Duchenne muscular dystrophy, satellite cells show a short replicative potential even when isolated from young donors, and this becomes worse over time ⁽⁴³⁾. More examples are found in the hematopoietic system and the liver ⁽²⁷⁾.

Whether these cells have truly reached a terminally nonproliferating state in vivo before isolation is difficult to assess. An unanswered question is whether the short-telomere signal for replicative senescence can be modulated by factors in the environment of the cell. Because the senescent state can be triggered by many manipulations other than telomere shortening, there is the possibility that some factor in the cell culture environment (e.g., some degree of DNA damage by exposure to oxygen and light) that by itself does not cause replicative senescence can synergize with the short-telomere signal. Thus although donor-age effects clearly indicate the potential for exhaustion of replicative capacity, the imperfect cell culture environment might amplify the effect. However, telomere shortening can be accelerated in cell culture by DNA damage ⁽⁴⁴⁾, indicating the possibility that cells isolated from tissues may not always have acquired their short-telomere state by repeated cell division.

There is a large body of data, too extensive to be reviewed here, on changes in telomere length in the hematopoietic system and other stem cell systems in human aging ⁽²⁷⁾. Stem cells often have some low level of telomerase activity, whereas their descendants usually do not. The data are consistent with the hypothesis that stem cell proliferative potential is adequate for the supply of the system over a normal human life span, but has a limited excess capacity that is significant in very old age or under pathological conditions. The limitation of replicative capacity, by suppression of TERT in most of the system, can be viewed as an evolutionary trade-off. In a system that is so dependent on cell proliferation for normal function, the opportunity for neoplastic transformation over the life span is very large. Suppression of TERT may prevent many abnormal preneoplastic clones of cells from evolving into lethal cancers (see the paragraphs that follow), but this evolutionary strategy may leave the system vulnerable to exhaustion in old age.

	Human Versus Mouse Aging

Top Abstract The Cell Culture Phenomenon Telomere Shortening in Tissues... Does Telomere Shortening... Human Versus Mouse Aging Do Senescent Cells Accumulate... Conclusions and Speculation References

 
Laboratory strains of mice and some other rodents have very long telomeres, have telomerase activity in many tissues, and do not show telomere shortening in aging, except in telomerase RNA knockout mice ⁽⁴⁵⁾. An argument heard quite often is that if telomere shortening is irrelevant to mouse aging then it must be irrelevant to human aging. That argument makes an unwarranted assumption—that all aging processes in all mammals are the same. The point that is missed is that although some aging processes may be shared among mammals (an assertion that almost all gerontologists would agree with), some may not. Some may be confined to large, long-lived mammals. In such mammals cancer suppression is an important factor in longevity.

Because TERT appears to be reexpressed in the majority of human cancers, it has been hypothesized that the process by which TERT is repressed in most somatic cells is an anticancer mechanism. It may contribute to the large difference in susceptibility to cancer (calculated on a per cell basis) between mice and humans. Suppose that mice and humans have the same risk of dying of cancer over their life spans, which is approximately true at least for some strains of mice ⁽⁴⁶⁾. However, a human being is approximately 3000 times heavier than a 25-g mouse and lives approximately 30 times as long. Consider also that cells are approximately the same size in mice as in humans and that cell turnover occurs at approximately the same rate. All these assumptions may not be entirely correct, but this does not substantially affect the basic validity of this argument. Then it is evident that human cells are approximately 90,000 times more resistant to tumorigenic conversion per unit of time than are mouse cells. Presumably, as part of the evolution of the life history of the human species, anticancer mechanisms evolved that were not present in short-lived ancestors. In this case the anticancer process may provide an example of antagonistic pleiotropy, the genetic event (repression of TERT) having beneficial effects in early life span and possibly negative effects in late life span ^(47)(48). The best evidence that TERT repression is indeed an anticancer mechanism in human cells comes from data showing that the well-known oncoproteins Ras and SV40 T antigen cannot transform a normal human cell into a tumor cell unless they are also expressed together with TERT ⁽⁴⁹⁾.

Another human–mouse contrast is provided by the fate of telomerase-deficient mouse cells in culture. Cultured embryonic stem cells from telomerase-deficient mice show progressive telomere shortening in culture that eventually results in growth arrest accompanied by frequent chromosomal abnormalities ⁽⁵⁰⁾. However, chromosomal abnormalities are not thought to be a cause of replicative senescence in human cells. Telomere shortening in human fibroblasts and other human cell types in culture results in a replicative arrest that is not associated with chromosomal abnormalities ⁽⁵¹⁾. Before human cells senesce, there is an increase in the number of chromosomes with short telomeres. This is thought to cause replicative arrest, although the correlation between the number of short-telomere chromosomes and senescence is not yet clear ⁽⁵¹⁾. Moreover, it is noteworthy that viable animals have been cloned by nuclear transfer from bovine fibroblasts that were close to replicative senescence, and there was no decrease in the success rate when such presenescent cells were used ⁽⁵²⁾. This implies that such cells have at most only mild chromosomal defects.

To state the situation in an alternate form, it appears that telomerase-deficient mouse cells have no easily recognizable M1 form of replicative senescence, but continue to M2 or crisis, whereas cultured human cells stop dividing when their telomeres reach the M1 length and only proceed to the M2 length when forced to bypass replicative senescence. However, some human cell types, such as mammary epithelial cells, may also lack an M1 form of replicative senescence ^(53)(54).

	Do Senescent Cells Accumulate in Tissues?

Top Abstract The Cell Culture Phenomenon Telomere Shortening in Tissues... Does Telomere Shortening... Human Versus Mouse Aging Do Senescent Cells Accumulate... Conclusions and Speculation References

 
If cells that reach the M1 telomere length truly "senesce" in vivo, and then undergo the same kinds of changes in gene expression as they do in culture, this process could certainly have adverse effects on tissue function. In this regard one can pose a series of related questions. First, do cells undergo telomere shortening to the extent of reaching the M1 telomere length? Second, if so, is the consequence of this the same as it is in culture, that is, the generation of senescent cells, or do they suffer some other fate (crisis)? Third, if they become senescent, do such cells accumulate in tissues, or are they eliminated by some part of either the acquired or innate immune system? Fourth, if they do accumulate in tissues, do they exert a procarcinogenic effect because they secrete proteases and cytokines?

As reviewed herein, and more extensively elsewhere ⁽²⁷⁾, there is considerable evidence that telomere shortening occurs in tissues in vivo and one might expect therefore that short-telomere cells would stop dividing at the M1 telomere length. However, observations made in the human genetic disease dyskeratosis congenita (DKC) suggest that perhaps this does not occur in vivo. DKC is a disease of impaired telomerase activity and shortened telomeres ^(55)(56)(57). In one form of the disease (X-linked) the DKC1 gene is defective; its protein product, dyskerin, is required for proper RNA processing, including the RNA of the telomerase ribonucleoprotein complex. In an autosomal dominant form of DKC, telomerase RNA is mutated. In these syndromes there are proliferative defects in tissues known to have telomerase-positive stem cells (hematopoietic system and skin). DKC patients have very short telomeres in fibroblasts and white blood cells. They usually die of bone marrow failure at a young age. However, the disease is also associated with chromosomal abnormalities and early death from some malignancies. Whether replicative senescence accounts for some of the pathology in DKC is unknown, but the chromosomal instability and increased cancer suggest that shortening telomeres in human tissues in vivo might lead to crisis rather than replicative senescence. If so, to some extent this resembles the situation in telomerase-deficient mice ⁽⁴⁵⁾, in which chromosomal aberrations cause defects in proliferation.

The most significant evidence for the occurrence of senescent cells in aging tissues is the occurrence of cells that stain for SA-ßgal in tissues as a function of age. The presence of SA-ßgal⁺ cells was first reported for human skin ⁽¹⁴⁾ and was subsequently shown in the rhesus monkey in retinal pigmented epithelium ⁽⁵⁸⁾ and in the epidermis ⁽⁵⁹⁾. In these studies the number of SA-ßgal⁺ cells increased as a function of donor age. These intriguing observations raise several questions. First, we do not know the mechanism by which such cells are formed; if their existence has consequences for tissue function, the mode by which they become senescent should be understood, so that appropriate interventions (both experimental and clinical) can be designed and tested. Second, whether such cells in vivo actually have the same range of changes in gene expression observed in replicative senescent cells in culture is also unknown. This is important, because it has been speculated that these changes may result in a procarcinogenic state in tissues that could aid the growth of premalignant cells and provide a permissive environment for tumor progression ^(48)(60)(61). It is conceivable that many properties of aging tissues might result from the presence of relatively small numbers of replicative senescent cells.

Many more studies are needed in this area. First, the variety of tissues and the range of donor ages that have been surveyed so far is very small, and it is not yet possible to determine whether the occurrence of SA-ßgal⁺ cells is an inevitable part of normal aging or is alternatively evidence of a pathological process. Some recent studies in the prostate, liver, and vascular endothelium are suggestive of an accumulation of SA-ßgal⁺ cells in disease states ^(62)(63)(64). Second, more studies are needed to show whether SA-ßgal⁺ cells are generated by telomere shortening or by some other process. The suspicion that in some cases telomere shortening is not involved exists for retinal pigmented epithelial cells, because these cells are mostly postmitotic in adult life ⁽⁶⁵⁾. In culture it is known that oxidative damage can cause cells to enter a senescent-like state; this could be a source of damage in tissues in vivo that might force cells into senescence. Conversely, more studies are needed to show the fate of cells with shortening telomeres in tissues. One consequence of our lack of knowledge in this area is that SA-ßgal⁺ staining cannot be used as an in situ assay for cells that have exhausted their replicative capacity in vivo by telomere shortening.

One final point is that there has not yet been enough consideration given to alternate fates of cells that have undergone telomere shortening in vivo and whose telomeres have reached the M1–senescent length. The possibility should be considered that their changes in gene expression might become so marked that they are no longer recognized as "self" and are eliminated by either acquired or innate immune functions, much as incipient cancer cells are eliminated by immune surveillance ⁽⁶⁶⁾.

	Conclusions and Speculation

Top Abstract The Cell Culture Phenomenon Telomere Shortening in Tissues... Does Telomere Shortening... Human Versus Mouse Aging Do Senescent Cells Accumulate... Conclusions and Speculation References

 
Independent of the question of whether replicative senescent cells affect tissue function, it is clear that telomere shortening does occur in human tissues in vivo, potentially putting cells ever closer to replicative senescence. It is important to distinguish the phenomenon of telomere shortening from its significance. The significance is still under debate, but the fact that it does indeed occur should not be considered controversial.

The senescent state appears to be a universal process that is a reaction of mammalian cells to certain kinds of damage, including telomere shortening. Cellular senescence presents a puzzle in terms of evolutionary biology. The kinds of damage that cause cells to enter the senescent state are very similar to those types of damage that cause other cells to enter apoptosis. From the point of view of the organism and the genome, making cells undergo apoptosis makes sense because the damaged cell and its progeny, carrying potentially damaged copies of the genome, are removed from the body. One may consider cells to be very cheap in terms of the overall economy of the body—millions of cells are born and die every day and there would seem to be no reason why cells should be preserved by means of the cellular senescence process, rather than killed off by means of apoptosis. In view of this, renewed efforts should be made to determine the prevalence and significance of senescent cells in tissues, both by a much more extensive survey of the incidence of SA-ßgal⁺ cells and by the development of new detection methods. Such studies may be aided by alternate approaches, such as the use of cell transplantation to create tissues comprising or containing senescent cells ⁽⁶⁷⁾.

	Acknowledgments

 
Work from my laboratory was supported by grants from the National Institute on Aging (Grants AG 12287, AG 13663, and AG 20752) and by a Senior Scholar Award from the Ellison Medical Foundation.

I am very grateful to Dr. Robert Marciniak and to an anonymous reviewer for their comments on an earlier version of this article.

Received February 25, 2002

Accepted March 19, 2002

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Top Abstract The Cell Culture Phenomenon Telomere Shortening in Tissues... Does Telomere Shortening... Human Versus Mouse Aging Do Senescent Cells Accumulate... Conclusions and Speculation References

 

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