The definition of cellular senescence has undergone some revision since the phenomenon was described by Leonard Hayflick as cessation of replication of cultured human cells after a finite number of population doublings (Hayflick et al., Exp. Cell Res. 37:585-621, 1961). Senescent cells remain metabolically active but do not divide and resist apoptosis for long periods of time (Goldstein, Science 249:1129-1133, 1990). Cellular senescence is characterized by growth cycle arrest in the G1 phase, absence of S phase, and lifespan control by multiple dominant genes (Stanulis-Praeger, Mech. Ageing Dev. 38:1-48, 1987).
Cellular senescence differs from quiescence and terminal differentiation in several important aspects. Senescent cells have characteristic morphological changes such as enlargement, flattening, and increased granularity (Dimri et al., Proc. Nat. Acad. Sci. USA 92:9363-9367, 1995). Senescent cells do not divide even if stimulated by mitogens (Campisi, Trends Cell Biol. 11:S27-S31, 2001). Senescence involves activation of p53 and/or Rb and their regulators such as p16INK4a, p21, and ARF. Except when p53 or Rb is inactivated, senescence is irreversible. Senescent cells express increased levels of plasminogen activator inhibitor (PAI) and exhibit staining for β-galactosidase activity at pH 6 (Sharpless et al., J. Clin. Invest. 113:160-168, 2004). Irreversible G1 arrest is mediated by inactivation of cyclin dependent kinase (CdK) complexes which phosphorylate Rb. P21 accumulates in aging cells and inhibits CdK4-CdK6. P16 inhibits CdK4-CdK6 and accumulates proportionally with β-galactosidase activity and cell volume (Stein et al., Mol. Cell. Biol. 19:2109-2117, 1999). P21 is expressed during initiation of senescence but need not persist; p16 expression helps maintain senescence once initiated.
Replicative senescence, the type of senescence originally observed by Hayflick, is related to the progressive shortening of telomeres with each cell division. Senescence is induced when certain chromosomal telomeres reach a critical length (Mathon and Lloyd, Nat. Rev. Cancer 3:203-213, 2001; Martins, U. M. Exp Cell Res. 256:291-299, 2000). Senescence can be abrogated by the expression of telomerase which lengthens telomeres; human fibroblasts undergo replication indefinitely when transfected to express telomerase. Most cancers express telomerase in order to maintain telomere length and replicate indefinitely. The minority of cancers that do not express telomerase have alternative lengthening of telomere (ALT) mechanisms.
Indirect evidence suggests some relationship between replicative senescence and aging. Cultured cells from old donors exhibit senescence after fewer population doublings than cells from young donors (Martin et al., Lab. Invest. 23:86-92, 1970; Schneider et al., Proc. Nat. Acad. Sci. USA 73:3584-3588, 1976). Cells from short-lived species senesce after fewer population doublings than cells from long-lived species (Rohme, D., Proc. Nat. Acad. Sci. USA 78:5009-3320, 1981). Cultured cells from donors with hereditary premature aging syndromes such as Werner's syndrome show senescence after fewer replications than cells from age-matched controls (Goldstein, Genetics of Aging, 171-224, 1978; Martin, Genetic Effects on Aging, 5-39, 1990). Whether replicative senescence actually contributes to aging or age-related symptoms in vivo is questionable on the basis of theoretical estimates of the number of cell divisions that occur in vivo and the absence of strong empirical evidence.
There are, however, other pathways to senescence than replication. Collectively, these are often referred to as stress-induced premature senescence (SIPS). Oxidative stress can shorten telomeres (von Zglinicki, Trends Biochem. Sci. 27:339-344, 2002) and hyperoxia has been shown to induce senescence. Gamma irradiation of human fibroblasts in early to mid G1 phase causes senescence in a p53-dependent manner (Di Leonardo et al., Genes Dev. 8:2540-2551, 1994). Ultraviolet radiation also induces cellular senescence. Other agents that can induce cellular senescence include hydrogen peroxide (Krtolica et al., Proc. Nat. Acad. Sci. USA 98:12072-12077, 2001), sodium butyrate, 5-azacytadine, and transfection with the Ras oncogene (Tominaga, Mech. Ageing Dev. 123(8):927-936, 2002). Chemotherapeutic agents including doxorubicin, cisplatin, and a host of others have been shown to induce senescence in cancer cells (Roninson, Cancer Res. 63:2705-2715, 2003). 5-bromodeoxyuridine treatment results in cellular senescence in both normal and malignant cells (Michishita et al., J. Biochem. 126:1052-1059, 1999). Generally speaking, agents that damage DNA can cause cellular senescence. The existence of cellular senescence in vivo has been demonstrated. In a study by Dimri et al., published in 1995, senescent fibroblasts were shown to exhibit staining for β-galactosidase activity at pH 6. These cells failed to incorporate tritiated thymidine and retained β-galactosidase activity after replating but did not divide. Quiescent fibroblasts did not show staining. Keratinocytes, umbilical vein endothelial cells, and mammary epithelial cells all showed increased staining with increased population doublings. Immortalized cells and terminally differentiated keratinocytes did not show staining. Staining was performed on skin biopsies to test whether senescence is observed in vivo. An age-dependent pattern in which an increased number of cells showed staining with increased donor age was observed in the dermis and epidermis (Dimri et al., Proc. Nat. Acad. Sci. USA 92:9363-9367, 1995). The existence of an increase in the number of senescent fibroblasts has been shown in the lungs of subjects with emphysema relative to subjects without emphysema (Müller et al., Resp. Res. 7:32-41, 2006).
Cellular senescence confers a number of functional changes on the cell that likely have clinical relevance. Senescent endothelial cells secrete elevated levels of plasminogen activator inhibitor 1 (PAI-1; Kletsas et al., Ann. N.Y. Acad. Sci. 908:11-25, 2000). Senescent fibroblasts over express collagenase and under express collagenase inhibitors (West et al., Exp Cell Res 184:138-147, 1989). Serial passages of human fibroblasts from a 25 year old donor showed increased elastase endopeptidase type activity (Homsy et al., Journal of Investigative Dermatology 91:472-477, 1988). Endothelial cells obtained from tissue overlying atherosclerotic plaques were observed to have a senescent morphology and express increased levels of PAI-1 and intracellular adhesion molecule 1 and decreased levels of nitric oxide (Davis et al., British Heart Journal 60:459-464, 1988; Arterioscler. Thromb. 11:1678-1689, 1991; Finn et al., Circulation 105:1541-1544, 1976; Comi et al., Exp. Cell Res. 219:304-308, 1995; Chang et al., Proc. Nat. Acad. Sci. USA 92:11190-11194, 1995). Indirect evidence that cellular senescence may play a role in cardiovascular disease also is provided by the observation that shorter leukocyte telomere length is associated with an increased risk of premature myocardial infarction (Brouilette et al., Arteriosclerosis, Thrombosis, and Vascular Biology 23:842-846, 2003).
Cancer cells are immortal, meaning that they can replicate indefinitely without exhibiting senescence. A preponderance of opinion in the scientific community says that the teleological purpose of senescence is to prevent cancer by limiting the number of cell divisions that can occur. This view is supported by experiments in mice showing that p53 knockout results in increased cancer incidence and severity. Indirect evidence that senescence suppresses cancer occurrence includes the observations that oncogenes immortalize or extend cellular lifespan and tumor suppressors Rb and p53, which are critical for senescence, suffer a loss of function mutation in cancer (Shay et al., Biochem. Biophys. Acta. 1071:1-7, 1991).
Senescent cells can also promote tumorigenesis. Senescent stromal cells express tumor promoting as well as tumor suppressing factors that exert a paracrine effect on neighboring epithelial cells; such effects include mitogenicity and antiapoptosis (Chang et al., Proc. Nat. Acad. Sci. USA 97(8):4291-4296, 2000). Senescent fibroblasts have been shown to stimulate premalignant and malignant epithelial cells but not normal epithelial cells to form tumors in mice; this occurred when as few as 10% of the fibroblasts were senescent (Krtolica et al., Proc. Nat. Acad. Sci. USA 98:12072-12077, 2001). Tumor promoting factors secreted by senescent cells are partly mediated by p21waf1/cip1/sdi1 (Roninson, Cancer Res. 63:2705-2715, 2003). A threshold of senescent stromal cells appears to provide a milieu allowing adjacent premalignant epithelial cells to survive, migrate, and divide (Campisi, Nat. Rev. Cancer 3:339-349, 2003).
In summary, cellular senescence does occur in vivo and is a likely sequel to environmental insults. Its prevalence increases with age at least in some tissue compartments. Senescence confers functional changes on the cell which have been associated to some degree with various age-related diseases (Chang et al., Proc. Nat. Acad. Sci. USA 97(8):4291-4296, 2000). Senescent cells also contribute to tumor formation. There exists a need for agents that are capable of detecting senescent cells in vivo and for treating or preventing diseases and disorders related to or caused by cellular senescence.