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Replicative Senescence: An Old Lives' Tale?

1996; Cell Press; Volume: 84; Issue: 4 Linguagem: Inglês

10.1016/s0092-8674(00)81023-5

1097-4172

Judith Campisi,

Genetics, Aging, and Longevity in Model Organisms

Normal animal cells, with few exceptions, do not divide indefinitely. This property, termed the finite replicative life span of cells, leads to an eventual arrest of cell division by a process termed cellular or replicative senescence. Although predicted and observed earlier, replicative senescence was first formally described over 30 years ago when Hayflick and his colleagues reported that human fibroblasts gradually and inevitably lost their ability to proliferate upon continual subculture (10Hayflick L. Exp. Cell Res. 1965; 37: 614-636Crossref PubMed Scopus (4038) Google Scholar). Since then, many cell types from many animal species have been shown to have a finite replicative life span (see21Stanulis-Praeger B. Mech. Aging Dev. 1987; 38: 1-48Crossref PubMed Scopus (155) Google Scholar). Most of these studies have used cells in culture. However, a limited number of in vivo experiments, as well as the evidence discussed here and elsewhere (21Stanulis-Praeger B. Mech. Aging Dev. 1987; 38: 1-48Crossref PubMed Scopus (155) Google Scholar, 2Campisi, J., Dimri, G., and Hara, E. (1996). In Handbook of the Biology of Aging, Fourth Edition, E. Schneider and J. Rowe, eds. (New York: Academic Press), pp. 121–149.Google Scholar), strongly suggest that cellular senescence is not an artifact of culture. In higher organisms, particularly mammals, two views suggest that replicative senescence may have important physiologic consequences. One view holds that cellular senescence is a tumor suppressive mechanism. There is substantial molecular, cellular, and in vivo evidence to support this idea (2Campisi, J., Dimri, G., and Hara, E. (1996). In Handbook of the Biology of Aging, Fourth Edition, E. Schneider and J. Rowe, eds. (New York: Academic Press), pp. 121–149.Google Scholar). This evidence will not be reviewed here, but is briefly summarized. First, senescence prevents cells from acquiring the multiple mutations that are needed for malignant transformation. Indeed, many, if not most, malignant tumors contain cells that have an extended or indefinite division potential. Thus, tumorigenesis selects for cells that can wholly or partly bypass senescence. Second, certain oncogenes—both cellular and viral—act at least in part by extending replicative life span. Thus, oncogenic mutations and the strategies of oncogenic viruses may and do entail mechanisms to overcome senescence. Third, among the genes needed to establish and maintain senescence are the p53 and retinoblastoma susceptibility genes. These are well-recognized tumor suppressors that, together, are the most commonly lost functions in human cancer. A related idea suggests that tumor suppression is the adaptive value of senescence, but senescence may have evolved to fine-tune tissue modeling during development (15Martin G.M. J. Gerontol. 1993; 48: 171-172Crossref Scopus (19) Google Scholar). Of course, there is also intuitive and teleologic appeal to the idea that a growth-limiting process may suppress tumorigenesis. A second view regarding the physiologic significance of cell senescence holds that it reflects processes that occur during organismic aging and may constitute an underlying cause of organismic aging. This idea is much more controversial and less intuitively obvious. How did it come about, and what is the evidence for it? The observation that cell division is inherently limited contradicted an earlier belief, championed by Carrel, that vertebrate cells can proliferate indefinitely once removed from the organism. Carrel's belief stemmed in part from his apparent (but since irreproducible) ability continually to subculture chick cells. This study and others (see10Hayflick L. Exp. Cell Res. 1965; 37: 614-636Crossref PubMed Scopus (4038) Google Scholar) spawned the idea that cells may be intrinsically “immortal.” Implicit in some of these studies was the idea that understanding cell immortality might uncover the basis for organismic mortality (3Carrel A. J. Exp. Med. 1912; 15: 516-528Crossref PubMed Scopus (213) Google Scholar). With this idea as a backdrop, then, Hayflick asserted that “normal human diploid cell strains in vitro are in fact 'mortal'” and suggested that replicative life span in culture reflects processes that occur during the chronological life span, or aging, of the organism (10Hayflick L. Exp. Cell Res. 1965; 37: 614-636Crossref PubMed Scopus (4038) Google Scholar). In retrospect, there was little basis for equating cell replicative life span with organismic life span. Certainly, replicatively immortal cells can die just as readily as replicatively mortal cells. Moreover, mammals are replicatively immortal (through the germline), even though individuals age and die. Why, then, was the parallel between the replicative life span of cells and organismic life span, or aging, accepted? In fact, it was not. There were 30 years ago, and remain today, many skeptics. Nonetheless, Hayflick and other biologists, perhaps intuitively, pursued the idea that cellular senescence and aging are related. As a result, the idea has garnered increasing experimental support and increasing interest among biologists. When examined carefully, cells that can divide—with a few notable exceptions—undergo replicative senescence. However, whether cell senescence is a limited or universal phenomenon has not been adequately explored and, for some cells, may not be easy to determine. Cells from some species—for example, many rodents—spontaneously escape senescence (immortalize) at a measurable frequency (1 in 104–106 cells). Because immortal cells rapidly overgrow senescing cultures, it can be difficult to assess the replicative life span of some cells in mass culture. Nonetheless, several cell types from a wide (but hardly exhaustive) variety of species have been shown to have a finite replicative life span (20Rohme D. Proc. Natl. Acad. Sci. USA. 1981; 78: 5009-5013Crossref PubMed Scopus (307) Google Scholar; see21Stanulis-Praeger B. Mech. Aging Dev. 1987; 38: 1-48Crossref PubMed Scopus (155) Google Scholar). Replicative senescence is especially stringent in human cells, which almost never spontaneously immortalize (17McCormick J.J. Maher V.M. Mutat. Res. 1988; 199: 273-291Crossref PubMed Scopus (117) Google Scholar). Even some single-celled organisms, such as Saccharomyces cerevisiae, clearly senesce when individual cells are monitored (12Jazwinski S.M. Genetica. 1993; 91: 35-51Crossref PubMed Scopus (81) Google Scholar). This suggests that a finite replicative life span may be a very primitive phenotype. There is, however, a major difference in this regard between higher eukaryotes and S. cerevisiae. In higher organisms, daughter cells inherit the replicative “age” of their mother, minus one division. Thus, mammalian cultures accumulate senescent cells more or less exponentially, until they contain only senescent cells. By contrast, S. cerevisiae daughters do not strictly inherit their mother's replicative age, and cultures appear immortal. S. cerevisiae daughter cells are easily identified and separated from mothers, but this is not true for many other cells. We do not know whether immortal cultures from other species also contain cells that senesce but do not transmit their replicative age to daughters. Only two, perhaps three, higher eukaryotic cell types may have an unlimited division potential. Certainly the germline is capable of continuous replication (although mature sperm and ova are not). In addition, as noted above, tumor cells are often immortal. Finally, some stem cells may be immortal (for example, inner cell mass cells, spermatogonia, hematopoietic stem cells), but this has yet to be critically demonstrated. Replicative senescence entails an irreversible arrest of cell proliferation and altered cell function. It is controlled by multiple dominant-acting genes and depends on the number of cell divisions, not time. It also depends on the cell type and on the species and age of the donor (see21Stanulis-Praeger B. Mech. Aging Dev. 1987; 38: 1-48Crossref PubMed Scopus (155) Google Scholar, 5Cristofalo V.J. Pignolo R.J. Physiol. Rev. 1993; 73: 617-638Crossref PubMed Scopus (238) Google Scholar, 2Campisi, J., Dimri, G., and Hara, E. (1996). In Handbook of the Biology of Aging, Fourth Edition, E. Schneider and J. Rowe, eds. (New York: Academic Press), pp. 121–149.Google Scholar). Cells acquire three characteristics upon senescence. First, they stably arrest growth with a G1 DNA content, irreversibly losing the ability to enter S phase in response to physiologic mitogens. The cells remain metabolically active, and many genes remain mitogen inducible, but there are changes in a few key growth regulators. These include the repression of three positive-acting transcriptional regulators and overexpression of a cyclin-dependent protein kinase inhibitor (see2Campisi, J., Dimri, G., and Hara, E. (1996). In Handbook of the Biology of Aging, Fourth Edition, E. Schneider and J. Rowe, eds. (New York: Academic Press), pp. 121–149.Google Scholar). Second, senescent cells acquire altered functions. In fact, by the criteria of a stable irreversible growth arrest and change in function, senescent cells resemble terminally differentiated cells (8Goldstein S. Science. 1990; 249: 1129-1133Crossref PubMed Scopus (583) Google Scholar, 2Campisi, J., Dimri, G., and Hara, E. (1996). In Handbook of the Biology of Aging, Fourth Edition, E. Schneider and J. Rowe, eds. (New York: Academic Press), pp. 121–149.Google Scholar). Third, senescent cells acquire resistance to apoptotic (programmed) cell death (22Wang E. Cancer Res. 1995; 55: 2284-2292PubMed Google Scholar) and thus are quite stable. The perception that cellular senescence leads to cell death is incorrect. It may stem from the early finding that senescent cells can be lost when subcultured (10Hayflick L. Exp. Cell Res. 1965; 37: 614-636Crossref PubMed Scopus (4038) Google Scholar; cf.14Linskens M.H. Harley C.B. West M.D. Campisi J. Hayflick L. Science. 1995; 267: 17Crossref PubMed Scopus (71) Google Scholar) or from confusion over the distinction between replicative mortality and cell viability. What is the evidence that cells senesce in vivo, and how might this cause or contribute to organismic aging? Cells cultured from old donors tend to senesce after fewer population doublings (PDs) than cells from young donors. There is considerable scatter in the data, particularly in humans, which may in part be due to genetic or life history differences (or both). Nonetheless, several independent studies show a significant inverse relationship between donor age and replicative capacity of cultured cells (16Martin G.M. Sprague C.A. Epstein C.A. Lab. Invest. 1970; 23: 86-92PubMed Google Scholar; see21Stanulis-Praeger B. Mech. Aging Dev. 1987; 38: 1-48Crossref PubMed Scopus (155) Google Scholar, 5Cristofalo V.J. Pignolo R.J. Physiol. Rev. 1993; 73: 617-638Crossref PubMed Scopus (238) Google Scholar, 2Campisi, J., Dimri, G., and Hara, E. (1996). In Handbook of the Biology of Aging, Fourth Edition, E. Schneider and J. Rowe, eds. (New York: Academic Press), pp. 121–149.Google Scholar). For example, human fetal fibroblasts typically senesce after 60–80 PDs, fibroblasts from young to middle-aged adults may do so after 20–40 PDs, and cells from old adults may senesce after 10–20 PDs. These studies suggest that cells in renewable tissues may progressively exhaust their replicative life span in vivo during aging. If true, tissues and individuals may vary considerably in the rate at which replicative potential declines because cell turn over varies widely among tissues and is very likely influenced by disturbances such as infection, inflammation, or injury. Interspecies comparisons suggest that cell replicative life span and organismic life span are genetically related. Although limited in scope, these studies show that cells from short-lived species tend to senesce after fewer PDs than cells from long-lived species (20Rohme D. Proc. Natl. Acad. Sci. USA. 1981; 78: 5009-5013Crossref PubMed Scopus (307) Google Scholar; see21Stanulis-Praeger B. Mech. Aging Dev. 1987; 38: 1-48Crossref PubMed Scopus (155) Google Scholar, 2Campisi, J., Dimri, G., and Hara, E. (1996). In Handbook of the Biology of Aging, Fourth Edition, E. Schneider and J. Rowe, eds. (New York: Academic Press), pp. 121–149.Google Scholar). For example, mouse fibroblasts typically senesce after 10–15 PDs, whereas Galapagos tortoise fibroblasts proliferate for >100 PDs. These studies suggest that the replicative life span of cells and chronological life span of organisms may be controlled by overlapping or interacting genes. A genetic link between aging and replicative life span is also supported by studies of hereditary premature aging syndromes in man (16Martin G.M. Sprague C.A. Epstein C.A. Lab. Invest. 1970; 23: 86-92PubMed Google Scholar; see21Stanulis-Praeger B. Mech. Aging Dev. 1987; 38: 1-48Crossref PubMed Scopus (155) Google Scholar). The best studied of these is the Werner syndrome (WS), caused by a recessive mutation on chromosome 8p11-p12 (18Oshima J. Yu C.E. Boehnke M. Weber J.L. Edelhoff S. Wagner M.J. Wells D.E. Wood S. Disteche C.M. Martin G.M. et al.Genomics. 1994; 23: 100-113Crossref PubMed Scopus (30) Google Scholar). WS patients are fairly asymptomatic early in life. Thereafter, they prematurely develop many, but not all, phenotypic correlates of age. These include hair and dermal thinning, atherosclerosis, osteoporosis, and cancer. WS is a segmental progeroid syndrome because only some aging phenotypes are premature. WS cells senesce well ahead of age-matched controls. Senescence may also be segmental in WS cells. Senescent WS fibroblasts are similar to normal counterparts in many ways. However, c-fos repression, a hallmark of normal senescence, does not occur (19Oshima J. Campisi J. Tannock T.C. Martin G.M. J. Cell. Physiol. 1995; 162: 277-283Crossref PubMed Scopus (73) Google Scholar). Thus, a single locus mutation accelerates age-related processes in vivo and cellular senescence in culture. The WS gene is not yet cloned but, once identified, may yield extraordinary insights into cell senescence, aging, and many age- related diseases. Cell senescence and organismic aging also share physiologic and molecular features. For example, senescent cells and aged tissues are more sensitive to a variety of stresses. At a molecular level, the stress inducibility of heat shock protein 70 is markedly attenuated in senescent human fibroblasts (4Choi H.S. Lin Z. Li B.S. Liu A.Y. J. Biol. Chem. 1990; 265: 18005-18011Abstract Full Text PDF PubMed Google Scholar) and several tissues from aged rodents (11Heydari A.R. Wu B. Takahashi R. Strong R. Richardson A. Mol. Cell. Biol. 1993; 13: 2909-2918Crossref PubMed Scopus (260) Google Scholar, 7Fawcett T.W. Sylvester S.L. Sarge K.D. Morimoto R.I. Holbrook N.J. J. Biol. Chem. 1994; 269: 32272-32278Abstract Full Text PDF PubMed Google Scholar). In both cases, the attenuation is due to reduced binding of a heat shock transcription factor. Similar culture/in vivo parallels exist for the regulation of c-fos, certain protease inhibitors, and collagenase (see5Cristofalo V.J. Pignolo R.J. Physiol. Rev. 1993; 73: 617-638Crossref PubMed Scopus (238) Google Scholar, 2Campisi, J., Dimri, G., and Hara, E. (1996). In Handbook of the Biology of Aging, Fourth Edition, E. Schneider and J. Rowe, eds. (New York: Academic Press), pp. 121–149.Google Scholar). Another feature shared by senescence in culture and aging in vivo is telomere shortening. Telomere shortening is perhaps the best candidate for a cell division “counting” mechanism in normal somatic cells of higher organisms (see9Harley C.B. Villeponteau B. Curr. Opin. Genet. Dev. 1995; 5: 249-255Crossref PubMed Scopus (291) Google Scholar, 24Wright W.E. Shay J.W. Trends Cell Biol. 1995; 5: 293-297Abstract Full Text PDF PubMed Scopus (126) Google Scholar). In such cells, telomerase is absent or present at low levels, and telomere length shortens with PDs in culture and organismic age. In human fibroblasts, for example, mean telomere length decreases about 50 bp per doubling in culture and 15 bp per year of donor age (1Allsopp R.C. Vaziri H. Patterson C. Goldstein S. Younglai E.V. Futcher A.B. Greider C.W. Harley C.B. Proc. Natl. Acad. Sci. USA. 1992; 89: 10114-10118Crossref PubMed Scopus (1885) Google Scholar). By contrast, telomerase is relatively abundant in replicating germ cells, tumor cells, and lower eukaryotes, where telomere length is stable. Finally, a neutral β-galactosidase activity is expressed by several types of human cells upon senescence in culture and increases with age in human skin cells (6Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley C. Medrano E. Linskens M. Rubelj I. Pereira-Smith O. Peacocke M. Campisi J. Proc. Natl. Acad. Sci. USA. 1995; 92: 9363-9367Crossref PubMed Scopus (5284) Google Scholar). Because this activity is not associated with quiescence, terminal differentiation, or immortality, it has provided in situ evidence that senescent cells do in fact accumulate in aged tissue in vivo. Organismic aging entails changes in both proliferative and postmitotic (for example, fat, nerve, and muscle) cells. Age-related changes in postmitotic cells cannot, of course, be due to replicative senescence. Thus, renewable and postmitotic tissues may age by different (albeit interacting) mechanisms. If so, model organisms such as Drosophila melanogaster or Caenorhabditis elegans, in which adults are composed entirely of postmitotic cells, may provide insights into the aging of postmitotic tissue, but not the aging of renewable tissues. Genes that extend the life span of the postmitotic organism C. elegans have been identified and are discussed in by Kenyon (1996 [this issue of Cell]). We do not yet know whether these genes also extend the replicative life span in C. elegans embryonic cells, nor whether they or their homologs alter the rate of aging in mammals. In summary, there is substantial evidence, mostly but not wholly correlative, that senescent cells exist and accumulate with age in vivo. It has not yet been demonstrated that senescent cells cause or contribute to aging. However, the molecular biology of senescence is just developing, as is the prospect of detecting and controlling senescent cells in vivo. This said, how might senescence cells contribute to aging in higher organisms? One might imagine that cellular senescence could inhibit tissue repair. However, substantial numbers of proliferative cells can often be recovered from very old tissues, and wounds do heal even in very old mammals (although sometimes more slowly). Of course, senescent cells may comprise only a small fraction of aged tissue. Indeed, in situ staining for the senescence-linked β-galactosidase suggests that senescent cells are sparsely distributed in aged skin (6Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley C. Medrano E. Linskens M. Rubelj I. Pereira-Smith O. Peacocke M. Campisi J. Proc. Natl. Acad. Sci. USA. 1995; 92: 9363-9367Crossref PubMed Scopus (5284) Google Scholar). Thus, the growth arrest of senescent cells may not be a major problem in aged tissues, although it could certainly compromise the organism by delaying repair. By contrast, the altered function of senescent cells may have a substantial impact in aged tissue. For example (see2Campisi, J., Dimri, G., and Hara, E. (1996). In Handbook of the Biology of Aging, Fourth Edition, E. Schneider and J. Rowe, eds. (New York: Academic Press), pp. 121–149.Google Scholar), senescent human skin fibroblasts overexpress collagenase and underexpress collagenase inhibitors. This may well cause or contribute to the collagen breakdown and thin dermis typical of aged skin. Senescent fibroblasts also underexpress interleukin-6 and overexpress interleukin-1, cytokines with pleiotropic inflammatory and immune effects. Thus, senescence-linked changes in differentiation could, at least in principle, have rather profound and far-ranging consequences for tissue function. Moreover, relatively few senescent cells would be needed for some of these effects. Finally, resistance to apoptotic death may explain why senescent cells are not cleared and thus accumulate with age. Indeed, it was recently proposed that caloric restriction, which delays many age-related changes, may do so by increasing the incidence of apoptosis (23Warner H.R. Fernandes G. Wang E. J. Gerontol. 1995; 50A: B107-B109Crossref Scopus (56) Google Scholar). Thus, age-related decrements in tissue function may, at least in part, derive from an accumulation of senescent cells—which cannot proliferate, which resist apoptotic death, and which have an altered phenotype. Evolutionary theories of aging have suggested that traits selected to optimize health during the period of reproductive fitness can have unselected deleterious effects later in life. Replicative senescence may have been selected, at least in mammals, to help ensure the relative freedom from cancer that characterizes early adulthood. However, this trait may be deleterious late in life because dysfunctional senescent cells accumulate. Moreover, because cancer incidence rises with age, it also appears that cellular senescence is an imperfect tumor suppressive mechanism that fails increasingly with age. In fact, it would seem that late in life replicative senescence wreaks havoc—whether it succeeds or fails.