Biogerontology 5: 1–10, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
1
Review article
Mechanisms of cellular senescence in human and mouse cells Koji Itahana1 , Judith Campisi2 & Goberdhan P. Dimri3,∗ 1 Department
of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA; 2 Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA; 3 Division of Cancer Biology, Department of Medicine, Evanston Northwestern Healthcare Research Institute, 1001 University Place, Evanston, IL 60201, USA; ∗ Author for correspondence (e-mail:
[email protected]; fax: +1-847-570-8011)
Received 10 June 2003; accepted in revised form 15 August 2003
Key words: p16, p53, pRb, senescence, telomere
Abstract Telomere erosion is considered to be the main cause of the onset of replicative senescence. However, recent findings suggest that a senescent phenotype can be induced by a variety of other stimuli that act independently of telomeres. Moreover, telomere-dependent replicative senescence depends on the species of cell origin, in particular whether cells are of human or rodent origin. In addition, the tissue of origin may also dictate the pathway by which cells undergo replicative senescence. In this Review article, we categorize cellular senescence into two types, which for simplicity we term intrinsic or extrinsic senescence, focus on the differences between human and mouse cells, and discuss the roles of the p53 and pRb tumor suppressor pathways in cellular senescence.
Introduction More than 40 years ago, Hayflick and colleagues reported that normal human fibroblasts have finite replicative lifespan (Hayflick and Moorhead 1961). Human fibroblasts irreversibly arrested growth and exhibited large and flat cell morphology after a limited number of cell divisions. These irreversibly growth-arrested cells survived for long periods of time in tissue culture without any obvious signs of cell death. This stable growth-arrested state has since been termed as replicative senescence, and is now believed to be a specific example of a more widely induced process termed cellular senescence. It is generally believed that cellular senescence reflects some of the changes that occur during the aging of organisms. Senescent cells differ from presenescent cells in multiple aspects of cellular physiology (Campisi 2000; Itahana et al. 2001). For example, senescent cells exhibit a different repertoire of gene expression, including changes in the expression of various transcription factors (Campisi et al.
1996; Shelton et al. 1999). Although the relationships between cellular senescence and aging in vivo is not very clear yet, senescent cells were reported to accumulate in aging human skin (Dimri et al. 1995), primate retina (Mishima et al. 1999) and human liver (Paradis et al. 2001). Senescent cells have also been detected at sites of age-related pathology, such as benign hyperplastic prostate (Choi et al. 2000) and atherosclerotic lesions (Vasile et al. 2001). Finally, senescent cells have been detected near preneoplastic lesions in the liver (Paradis et al. 2001), and were recently shown capable of stimulating the malignant progression of premalignant keratinocytes and breast epithelial cells (Krtolica et al. 2001). Because senescent cells frequently secrete degradative enzymes and cytokines that can disrupt tissue architecture, these findings suggest that senescent cells may contribute to the age-related decline in tissue structure and/or the genesis of certain age-related pathologies. Recent reports suggest that cellular senescence may be one of the mechanisms by which cancer chemotherapy drugs work in vivo (Schmitt et al. 2002; te Poele et al. 2002).
2 At the molecular level, cellular senescence is controlled by the tumor suppressor proteins pRb and p53 (reviewed in Dimri and Campisi 1995; Campisi 2001). Inactivation of these tumor suppressors results in bypass of senescence. Due to its essentially irreversible growth arrest and the requirement for p53 and pRb function, cellular senescence is considered a potent tumor suppressor mechanism. Numerous studies, primarily in human fibroblasts, suggest that telomere shortening is the primary cause of replicative senescence (Harley et al.,1990; reviewed in Kim et al. 2002). It is thought that telomere shortening beyond a certain limit triggers a DNA damage response, thereby activating a checkpoint mediated by p53 (reviewed in Itahana et al. 2001). It is not clear how pRb mediates the response to telomere dysfunction. While the inactivation of p53 and pRb is sufficient to bypass the senescence of human fibroblasts, their inactivation does not lead to cell immortalization (Wright and Shay 1996).
Intrinsic (telomere-dependent) senescence Since eukaryotic cells have linear chromosomes, each chromosome shortens from the ends, or telomeres, during every round of cell division due to the biochemistry of DNA replication. Telomeres are the repetitive DNA sequence (TTAGGG in vertebrates) and specialized proteins that caps the ends of linear chromosomes. Telomeres are essential structures that prevent chromosome end fusions and genomic instability (de Lange 2002; McClintock 1941). Human germ cells express the enzyme telomerase, which synthesizes telomeres to keep the length of chromosomes constant. However, most human somatic cells do not express telomerase, and therefore lose telomeric DNA during each round of DNA replication. Several lines of evidence suggest that human fibroblasts undergo senescence before they acquire critically short telomeres, thereby avoiding genomic instability leading to cancer (reviewed in Kim et al. 2002). This telomere-dependent senescence growth arrest is intrinsic, and inevitable for most somatic cells, which do not express telomerase. Human cells undergo intrinsic senescence in response to repeated cell divisions (Figure 1). Telomere shortening is so far the only known intrinsic mechanism, which acts to “count” cell divisions. Cultured cells may have mechanisms to “count” other events, such as those proposed to be controlled by a circadian pacemaker
and recently described in fibroblasts (Yagita et al. 2001). Nevertheless, for the purposes of this Review article, intrinsic senescence is used synonymously with telomere-dependent senescence.
Intrinsic senescence does not exist in laboratory mouse fibroblasts Mouse embryonic fibroblasts (MEFs), like human fibroblasts, have a finite replicative lifespan and show senescent phenotype at the end of that lifespan. However, it is believed that the replicative senescence of mouse fibroblasts is not induced intrinsically, but rather is induced by extrinsic factors, specifically the culture environment (Sherr and DePinho 2000; Wright and Shay 2000). Since laboratory mice have very long telomeres (40–60 kb) compared to humans (5–15 kb) and, in some cases, express telomerase, it is unlikely that the replicative senescence of mouse cells is due to telomere shortening (Sherr and DePinho 2000; Wright and Shay 2000). To study the effect of telomere erosion in mouse cells, the telomerase RNA component (mTR) was deleted from the mouse germ line, and telomerase negative transgenic mice were generated (Blasco et al. 1997) The first generation of mTR−/− mice did not show any distinguishing phenotypes compared to their wild type littermates. However, cells from fourth generation mTR−/− mice began to exhibit significant loss of telomere repeats, aneuploidy, and end-to-end chromosome fusions. Cells from fifth or sixth generation mTR−/− mice showed p53 dependent growth arrest and apoptosis due to genomic instability. These mice were also sterile due to inability of germ cells to divide and maintain proliferation. To further understand the role of p53 in the senescence of mTR−/− mice, they were crossed with p53+/− mice. It was found that p53 dependent apoptosis was attenuated in mTR−/− p53+/− mice and these mice survive up to eight generations (Chin et al. 1999). These data suggest that mouse cells can be growth arrested by telomere erosion, similar to human cells. This conclusion was supported by studies of mTR−/− embryonic stem cells, which stop proliferating after approximately 450 doubling (Niida et al. 1998). The fifth generation mTR−/− mice are resistant to carcinogen induced skin tumors in a p53 dependent manner (Gonzalez-Suarez et al. 2000). In addition, sixth generation mTR−/− mice show several signs of premature aging symptoms, including reduced
3
Figure 1. Two types of senescence in mammalian cells. Senescence induced by telomere attrition ordysfunction is termed intrinsic senescence. Senescence induced by signals that originate outside cells or by telomere independent signals is termed extrinsic senescence. These signals include cell culture conditions, DNA damage caused by radiation or chemicals, oncogenic or mitogenic stimuli and exogenously overexpressed tumor suppressors such as p16, p14ARF and p21.
capacity to respond to stresses such as wound healing and hematopoietic ablation (Rudolph et al. 1999). Telomere loss in fifth generation mTR−/− mice also leads to cardiac failure associated with p53 upregulation (Leri et al. 2003). Together, these results suggest that telomere shortening is not only a tumor suppressor mechanism but also a mechanism that may contribute to aging and age-related pathologies in vivo. Why, then, do wild type MEFs, with their long telomeres and relative high expression of telomerase, senesce under standard culture conditions? Recent findings indicate that the replicative senescence of MEFs in culture is in fact a response to oxidative stress. Standard culture conditions include atmospheric O2 , roughly 20%, which is substantially above the O2 tension experienced by most cells in vivo (Vaupel 1989). MEFs that undergo replicative senescence in 20% O2 show several signs of severe oxidative stress, including substantial oxidative damage to DNA; moreover, MEFs do not replicatively senesce when cultured under lower (3%), more physiological O2 tensions (Parrinello et al. 2003). Strikingly, human fibroblasts accumulated much less oxidatively damaged DNA in 20% O2 than MEFs. This finding suggests that murine cells are much more sensitive to oxidative stress than human cells, which may at
least partly explain the vastly different rates at which humans and mice age and develop cancer. Interestingly, MEFs from mice that carry germline deletions in the p53 pathway do not senesce in 20% O2 (Harvey et al. 1993), whereas MEFs from mice with germline deletions in the pRb pathway do (Sharpless et al. 2001). These results also suggest that the activity of p53 may drive cellular responses, such as cellular senescence, that can contribute to aging. Recently, mice were generated in which one allele of the p53 gene had a deletion of the first six exons (Tyner et al. 2002). These mice had hyperactive p53, as assessed by enhanced apoptosis in response to genotoxic stress. Interestingly, these mice also displayed premature aging phenotypes (Tyner et al. 2002). Similar results were obtained in a BRCA1-p53 transgenic study, where it was reported that premature activation of p53 due to BRCA1 deficiency causes accelerated aging in vivo (Cao et al. 2003). On the other hand, mice carrying one extra copy of the normal p53 gene did not show any indication of premature aging (Garcia-Cao et al. 2002). However, although these mice were cancer resistant, they did not have an increased lifespan, despite cancer being a major cause of death in mice (Garcia-Cao et al. 2002). These observations suggest that while normally regulated
4 p53 can provide enhanced tumor suppression, uncontrolled p53 activity generates phenotypes that mimic in vivo aging. In mTR−/− mice, if the p53 checkpoint is not functional, telomere shortening can cause genomic instability and the generation of telomerase independent cancers. Indeed, ectopic expression of mTR prevents transformation mediated by overexpresison of Myc and RAS in MEFs derived from sixth generation mTR−/− p53+/− mice (Chin et al. 1999). In addition, the combined expression of MDM2, which inhibit p53 functions, Ha-RasV12 and adenovirus E1A is sufficient to transform normal human fibroblasts to tumorigenicity in nude mouse assays (Seger et al. 2002). Although the expression of telomerase has long been thought a facilitating, if not necessary, step in order to covert a normal cell into a cancer cell, this clearly is not the case if p53 function is abrogated.
Intrinsic senescence in human cells Normal human fibroblasts senesce after 50–100 doublings, most commonly due to the telomere shortening that results from cell division in the absence of telomerase activity. Moreover, studies using mTR−/− mice suggest that intrinsic senescence, triggered by telomere shortening, could be p53 dependent. One possibility is that p53 detects a disrupted telomeric end structure (the T-loop) or the end to end fusion of chromosomes that result from telomere dysfunction (de Lange 2002; Griffith et al. 1999). p53 is able to bind telomeric single strand overhangs and T-loop junctions in vitro (Stansel et al. 2002). Moreover, p53 activity is enhanced by a variety of stresses such as DNA damage (Di Leonardo et al. 1994), oncogenic insults (Serrano et al. 1997), and quiescence by growth factor deprivation (Itahana et al. 2001; Itahana et al. 2002). p53 activation in response to stressful conditions causes cell cycle arrest, apoptosis, or senescence. Inactivation of p53 extends the lifespan of human fibroblasts (reviewed in Itahana et al. 2001). Indeed p53 activity is high in senescent cells (Atadja et al. 1995; Itahana et al. 2002) and expression of p21, which is a p53 target gene, increases in senescent cells (Noda et al. 1994). Since targeted deletion of p21 gene is sufficient to bypass senescence in human fibroblasts (Brown et al. 1997), p21 may have a major role in the induction of cellular senescence in human fibroblasts by inhibiting the activity of cyclin dependent kinases (CDKs).
What transduces the signal of telomere erosion to p53? p53 shows a distinct phosphorylation pattern in senescent fibroblasts compared to early passaged cells (Webley et al. 2000). One candidates p53 kinase is ATM, which is known to transduce the signals of DNA damage (Pandita 2002). Telomere shortening is accelerated in fibroblasts patients with ataxia telangiectasia (AT), a hereditary syndrome resulting from inactivating mutations in ATM (Metcalfe et al. 1996; Vaziri et al. 1997). Apoptosis of cancer cells, induced by expressing a dominant negative form of TRF2, the wild type of which is important for maintaining the telomeric T-loop, is p53- and ATMdependent (Karlseder et al. 1999). These data suggest that ATM may have an important role in sensing a disrupted telomere structure and contributing to p53 signaling. The other known post-translational modification that activates p53 is acetylation. p53 is acetylated in senescent MEFs (Pearson et al. 2000). p300/CBP, PML, and Sir2 are known to regulate p53 acetylation (Bischof et al. 2002; Langley et al. 2002; Pearson et al. 2000). However, at present the connection between p53 acetylating agents and intrinsic senescence is unknown. p53 can be stabilized by p14/ARF (p19/ARF in mice) via MDM2 (Pomerantz et al. 1998; Zhang et al. 1998). Since many human cancer cells have a deletion of ARF, ARF is widely recognized as a tumor suppressor in human as well as mouse cells, although there are some functional differences between mouse and human ARF (Sugihara et al. 2001, Wadhwa et al. 2002). While ARF mRNA levels are very low in human fibroblasts, its expression rises when some, but not all, human fibroblasts undergo replicative senescence (Dimri et al. 2000; Wei et al. 2001). Nonetheless, p53 protein levels rise little, if at all, in senescent human fibroblasts (Afshari et al. 1993; Atadja et al. 1995; Itahana et al. 2001; Vaziri et al. 1997), suggesting that ARF may contribute to senescence and tumor suppression through p53 independent mechanisms. Indeed ARF has been also reported to inhibit cell growth independent of p53, MDM2 and p21 (Korgaonkar et al. 2002).
Extrinsic senescence (telomere independent senescence) Cellular senescence can be induced by a variety of extrinsic factors, such as X- or UV-irradiation, H2 O2 , and ectopic expression of certain oncogenes and tumor
5 suppressors (Figure 1; Campisi 2001). Here, we define extrinsic senescence as the senescence that occurs even when cells still have functional telomeres and the potential to proliferate under suitable culture conditions (Figure 1). In this case, signals to senesce originate from sources independent of telomeres.
Extrinsic senescence in mouse cells As discussed above, MEFs senesce after 10–20 doublings in culture (20% oxygen), despite retaining long telomeres (extrinsic senescence). What are the proteins responsible for this type of senescence? There are two widely recognized major tumor suppressor pathways, p19/ARF-p53 and p16/pRb, that regulate cellular senescence (Campisi 2001). It has been suggested that the p16/pRb pathway does not contribute to senescence in MEFs because MEFs derived from pRb or p16 null mice senesce similar to wild type MEFs in 20% O2 (Sherr and DePinho 2000; Krimpenfort et al. 2001; Sharpless et al. 2001). On the other hand, MEFs from mice carrying a p16 binding deficient mutant of CDK4 fail to senesce in culture (Rane et al. 2002). Furthermore, MEFs from transgenic mice generated by the targeted disruption of all pRb family proteins (pRb, p107, and p130) do not senesce (Dannenberg et al. 2000; Sage et al. 2000). Therefore, the importance of p16/pRb pathway in senescence of mouse cells is still debatable. MEFs from p19/ARF or p53 null mice do not senesce in standard culture conditions, suggesting p19/ARF-p53 pathway is primarily responsible for the replicative senescence of mouse cells in 20% O2 (Harvey et al. 1993; Kamijo et al. 1997). However, MEFs from p21 null mice senesce normally (Pantoja and Serrano 1999). These data suggest that p21, which is required for the replicative senescence of human fibroblasts, is not a major player in senescence of mouse cells and that other factors downstream of the p19/ARF-p53 pathway may limit the growth of MEFs under the oxidative stress of standard culture (Figure 2). Conclusions based on cells from knock out mice need to be interpreted with caution. It is known that disruption of genes in the germ line can sometimes be compensated for by other genes during development. Thus, it is possible that the senescence of p16, pRb, and p21 null MEFs is caused by compensatory mechanisms. Indeed, conditional disruption of the pRb gene in normally developed MEFs was recently
shown to reverse the arrest of a fraction of MEFs that senesced under standard culture conditions (Sage et al. 2003).
Extrinsic senescence in human cells The p53/p21 and p16/pRb pathways have been long known to play a role in the replicative senescence of human fibroblasts. However, the extent to which p16 and p21 contributes to senescence remains to be elucidated. Using two commonly used human fibroblast strains, WI-38 and BJ, we reported that the replicative senescence in human fibroblasts can also entail a component of intrinsic senescence (Figure 3; Itahana et al. 2003). While cells that express high levels of p16 continuously arise even in early passaged cultures of WI-38 cells, cultures of BJ cells were essentially devoid of p16 even after replicative senescence. p21 expression increased as cells approached replicative senescence in both WI-38 and BJ cells. These data indicated that BJ-type fibroblasts senesce only by an intrinsic (telomere-dependent) senescence mechanism, independent of the p16/pRb pathway, whereas WI-38-type fibroblasts senesce by both intrinsic and extrinsic (p16/culture stress) senescence mechanisms (Figure 3).
Contribution of the polycomb protein Bmi-1 to senescence of human fibroblasts What are the factors that induce p16 in WI-38-type fibroblasts? Recently, it was reported that MEFs from Bmi-1 null mice showed accelerated senescence due to premature accumulation of p16 and p19/ARF (Jacobs et al. 1999). In addition, overexpression of Bmi-1 abrogated the senescence of MEFs and extended the replicative lifespan of human fibroblasts (Jacobs et al. 1999; Itahana et al. 2003). Bmi-1 is a transcriptional repressor belonging to the Polycomb group gene family (van der Lugt et al. 1994). Polycomb group proteins, and the counter-acting Trithorax group proteins, are crucial for maintaining proper gene expression patterns during development (Pirrotta 1998). We investigated the mechanism of lifespan extension mediated by Bmi-1 (Itahana et al. 2003). Overexpression of Bmi-1 in WI-38 cells resulted in the disappearance of high p16 expressing cells in early passage cultures. A deletion mutant of Bmi-1 lacking
6
Figure 2. Pathways of senescence in mouse and human cells. In mouse embryo fibroblasts (MEFs), the p19/ARF-p53 pathway (solid lines) is dominant. In MEFs, the p16 pathway (dashed lines) may not play a significant role in cellular senescence, although recent data question this assumption. In human fibroblasts, the telomere-p53/p21 pathway is a major pathway of senescence, but in some cells, depending on extrinsic factors, the p16 pathway may play a role in senescence. Signals from telomere dysfunction may be mediated by ATM and other factors. Various regulators of p16, such as Bmi-1, Id1, Ets1, are likely to contribute to extrinsic senescence.
the RING finger domain exhibited dominant negative activity, inducing p16 and accelerating senescence. Bmi-1-overexpressing cells eventually senesced with shorter telomeres compared to control cells. Senescent cells maintained for a month in culture showed reduced levels of Bmi-1, concomitant with a rise in p16 expression. Based on these observations, we propose two means by which human fibroblasts senesce in culture (Figure 3). In cultures of cells such as WI-38, extrinsic senescent cells, which still have long telomeres, continuously arise because the cells are sensitive to environmentally induced stress. Overexpression of Bmi-1 rescues these cells from extrinsic senescence by repressing p16 and thus extends the replicative capacity of the culture (WI-38 + Bmi-1, Figure 3). However, telomere erosion causes Bmi-1-overexpressing cells to eventually senesce intrinsically. On the other hand, in cultures of cells such as BJ, cells may be resistant to stress induced by extrinsic factors, incapable of inducing p16, and undergo extrinsic senescence. Thus, these cells undergo senescence only
by the intrinsic telomere dependent pathway (Figure 3). Consistent with this idea, Bmi-1 overexpression could not extend the replicative lifespan of BJ cells, and BJ cells senesce with shorter telomeres (4.7 kb) than those of senescent IMR-90 or WI-38 cells (6 to 7 kb). Moreover, Bmi-1-overexpressing WI-38 cells senesced with telomere lengths similar to those of senescent BJ cells. In addition, WI-38 cells are more difficult to immortalize by ectopic expression of telomerase compared to BJ cells, and telomeraseexpressing WI-38 cells continually produce senescent cells (unpublished results). Thus, Bmi-1 has a role in senescence by repressing p16 in WI-38 and similar types of fibroblasts.
Extrinsic factors inducing p16 What are the extrinsic factors that induce p16? Reactive O2 has been reported to induce senescence. As discussed above, atmospheric O2 (20%), in which most cells are cultured, is much higher than the O2
7
Figure 3. Model of senescence mechanisms in human fibroblasts. WI-38 type fibroblast cultures constantly produce senescent cells by the extrinsic (p16-dependent) mechanism (p16 STOP), as well as the p53/p21 based intrinsic senescence (telomere-dependent) mechanism (p53/p21 STOP). BJ type fibroblasts on the other hand produce no senescent cells by the p16 based mechanism (p16 STOP), but produce senescent cells only by the p53/p21-telomere based mechanism (p53/p21 STOP). Solid bar (black bar – subtelomeric region; gray bar – telomeric repeat region) below each cell represents hypothetical telomere(s) status. Telomere(s) shorten in both cases, however, but the senescence telomere length is greater in cells that undergo senescence via the p16 dependent pathway. Bmi-1 overexpression in WI-38 cells (WI-38 + Bmi-1) abrogates the p16 pathway, and cells senesce solely by the p53/p21-telomere based mechanism in this case.
concentrations found in vivo (Vaupel et al. 1989). Reduced O2 has long been known to promote the growth and extend the lifespan of cultured human cells (Packer and Fuehr 1977; Saito et al. 1995), and, as discussed earlier, completely abrogates the replicative senescence of MEFs (Parrinello et al. 2003). We hypothesized that some human fibroblasts might be hypersensitive to oxygen, and that the effects of high O2 might induce p16 in such cells. However, p16 expression was unaffected when human fibroblasts were cultured in 3% O2 (Itahana et al. 2003). On the other hand, p21 was clearly reduced in 3% O2 . Moreover, Bmi-1 extended the lifespan of WI-38 cells to the same extent in3% O2 as it did in 20% O2 . These data suggest that oxygen may preferentially affect the p53/p21 pathway but not the p16/pRb pathway. Low O2 may prevent the telomere damage and contribute to the extension of lifespan. Consistent with this view, it was recently reported that p16 can be repressed by manipulating culture conditions (Ramirez et al. 2001). Human mammary epithelial cells (HMECs) are known to undergo senescence (M0, similar to extrinsic
senescence) mediated by the induction of p16 (Wong et al. 1998; Brenner et al. 1997). However, when HMECs were cultured on feeder layers, p16 induction was significantly delayed and replicative lifespan was simultaneously extended (Ramirez et al. 2001). Our results predict that transcription factors that induce p16 in response to stress should exist in WI-38 cells. One of such candidate is Ets1. Ets1 can directly induce p16 and senescence, while Id-1 is an inhibitor of Ets1 (Ohtani et al. 2001). Different strains of fibroblasts, such as WI-38 or BJ, may have different sensitivity to stress and ability to regulate p16, by virtue of different levels of Ets1, Id-1, Bmi-1 and possibly other regulators of p16. We also note that WI-38 fibroblasts are lung-derived fibroblast, while BJ cells are skin-derived fibroblasts. Most lung-derived fibroblasts appear to have a shorter replicative lifespan compared to skin-derived fibroblasts. We speculate that this difference in lifespan depending on the tissue origin reflects the ability to undergo p16 mediated extrinsic senescence. This hypothesis is currently being tested in one of our laboratories (GPD).
8 Conclusions To understand the mechanism of senescence in human cells and its role as a tumor suppressor mechanism, a variety of studies have been done using knockout mice. However, senescence mechanisms clearly differ in mouse and human cells, and thus the regulation of senescence in MEFs can only be applied to human cells once critical human–mouse differences are elucidated. Many human senescence studies have been done using WI-38 fibroblasts, which do not represent all human fibroblast strains with respect to p16 regulation. As discussed here and elsewhere, senescence mechanisms may differ in different cell types, cell strains and depends on the species. The mechanism of inducing senescence may also depend on how cells respond to environmental stresses. Study of senescence in multiple cell types and cell strains derived from different tissue origins will help us understand the role of senescence in aging and cancer in different tissues, and help devise effective treatments for age-related pathologies and cancer in different tissues.
Acknowledgements Authors gratefully acknowledge the financial support from National Institute on Aging (J.C., G.P.D), NEMC Cancer Center Start Up Funds (G.P.D), The Charlotte Geyer Foundation (G.P.D) and USAMRMC Award Number DAMD17-02-1-0509 (G.P.D).
References Afshari CA, Vojta PJ, Annab LA, Futreal PA, Willard TB and Barrett JC (1993) Investigation of the role of G1/S cell cycle mediators in cellular senescence. Exp Cell Res 209: 231– 237 Atadja P, Wong H, Garkavstev I, Veillette C and Riabowol K (1995) Increased activity of p53 in senescing fibroblasts. Proc Natl Acad Sci USA 92: 8348–8352 Bischof O, Kirsh O, Pearson M, Itahana K, Pelicci PG and Dejean A (2002) Deconstructing PML-induced premature senescence. EMBO J 21: 3358–3369 Blasco MA, Lee HW, Hande MP, Samper E, Lansdorp PM, DePinho RA and Greider CW (1997) Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91: 25–34 Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S and Wright WE (1998) Extension of lifespan by introduction of telomerase into normal human cells. Science 279: 349–352
Brenner AJ, Stampfer MR and Aldaz CM (1998) Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation. Oncogene 17: 199–205 Brown JP, Wei W and Sedivy JM (1997) Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science 277: 831–834 Campisi J (2000) Cancer, aging and cellular senescence. In vivo 14: 183–188 Campisi J (2001) Cellular senescence as a tumor-suppressor mechanism. Trends Cell Biol 11: 27–31 Campisi J (2003) Cellular senescence and apoptosis: how cellular responses might influence aging phenotypes. Exp Gerontol 38: 5–11 Campisi J, Dimri GP and Hara E (1996) Control of replicative senescence. In: Schneider EL and Rowe JW (eds) Handbook of the Biology of Aging, 4th ed, pp 121–149. Academic Press, San Diego, California Cao L, Li W, Kim S, Brodie SG and Deng CX (2003) Senescence, aging, and malignant transformation mediated by p53 in mice lacking the Brca1 full-length isoform. Genes Dev 17: 201–213 Chin L, Artandi SE, Shen Q, Tam A, Lee SL, Gottlieb GJ, Greider CW and DePinho RA (1999) p53 deficiency rescues the adverse effects of telomere loss and cooperates with telomere dysfunction to accelerate carcinogenesis. Cell 97: 527–538 Choi J, Shendrik I, Peacocke M, Peehl D, Buttyan R, Ikeguchi EF, Katz AE and Benson MC (2000) Expression of senescenceassociated beta-galactosidase in enlarged prostates from men with benign prostatic hyperplasia. Urology 56: 160–166 Dannenberg JH, van Rossum A, Schuijff L and te Riele H (2000) Ablation of the retinoblastoma gene family deregulates G(1) control causing immortalization and increased cell turnover under growth-restricting conditions. Genes Dev 14: 3051–3064 de Lange T (2002) Protection of mammalian telomeres. Oncogene 21: 532–540 DiLeonardo A, Linke SP, Clarkin K and Wahl GM (1994) DNA damage triggers a prolonged p53-dependent G1 arrest and longterm induction of Cip1 in normal human fibroblasts. Genes Dev 8: 2540–2551 Dimri GP and Campisi J (1995) Molecular and cell biology of replicative senescence. Cold Spring Harbor Lab Symp Quant Biol Mol Genet Cancer 54: 67–73 Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith OM et al. (1995) A novel biomarker identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 92: 9363–9367 Dimri GP, Itahana K, Acosta M and Campisi J (2000) Regulation of a senescence checkpoint response by the E2F1 transcription factor and p14(ARF) tumor suppressor. Mol Cell Biol 20: 273– 285 Garcia-Cao I, Garcia-Cao M, Martin-Caballero J, Criado LM, Klatt P, Flores JM, Weill JC, Blasco MA and Serrano M (2002) “Super p53” mice exhibit enhanced DNA damage response, are tumor resistant and age normally. EMBO J 21: 6225–6235 Gonzalez-Suarez E, Samper E, Flores JM and Blasco MA (2000) Telomerase-deficient mice with short telomeres are resistant to skin tumorigenesis. Nat Genet 26: 114–117 Griffith JD, Comeau L, Rosenfield S, Stansel RM, Bianchi A, Moss H and de Lange T (1999) Mammalian telomeres end in a large duplex loop. Cell 97: 503–514 Harley CB, Futcher AB and Greider CW (1990) Telomeres shorten during ageing of human fibroblasts. Nature 345: 458–460
9 Harvey M, Sands AT, Weiss RS, Hegi ME, Wiseman RW, Pantazis P, Giovanella BC, Tainsky MA, Bradley A and Donehower LA (1993) In vitro growth characteristics of embryo fibroblasts isolated from p53-deficient mice. Oncogene 8: 2457–2467 Hayflick L and Moorhead PS (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25: 585–621. Itahana K, Dimri G and Campisi J (2001) Regulation of cellular senescence by p53. Eur J Biochem 268: 2784–2791 Itahana K, Dimri GP, Hara E, Itahana Y, Zou Y, Desprez PY and Campisi J (2002) A role for p53 in maintaining and establishing the quiescence growth arrest in human cells. J Biol Chem 277: 18206–18214 Itahana K, Zou Y, Itahana Y, Martinez JL, Beausejour C, Jacobs JJ, Van Lohuizen M, Band V, Campisi J and Dimri GP (2003) Control of the replicative lifespan of human fibroblasts by p16 and the polycomb protein bmi-1. Mol Cell Biol 23: 389–401 Jacobs JJ, Kieboom K, Marino S, DePinho RA and van Lohuizen M (1999) The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature 397: 164–168 Kamijo T, Zindy F, Roussel MF, Quelle DE, Downing RA, Ashmun G, Grosveld G and Sherr CJ (1997) Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell 91: 649–659 Karlseder J, Broccoli D, Dai Y, Hardy S and de Lange T (1999) p53and ATM-dependent apoptosis induced by telomeres lacking TRF2. Science 283: 1321–1325 Kim SH, Kaminker P and Campisi J (2002) Telomeres, aging and cancer: in search of a happy ending. Oncogene 21: 503–511 Korgaonkar C, Zhao L, Modestou M and Quelle DE (2002) ARF function does not require p53 stabilization or Mdm2 relocalization. Mol Cell Biol 22: 196–206 Krimpenfort P, Quon KC, Mooi WJ, Loonstra A and Berns A (2001) Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice. Nature 413: 83–86 Krtolica A, Parrinello S, Lockett S, Desprez P Y and Campisi J (2001) Senescent fibroblats promote epithelial cell growth and tumorigenesis: a link between cancer and aging. Proc Natl Acad Sci USA 98: 12072–12077 Langley E, Pearson M, Faretta M, Bauer UM, Frye RA, Minucci S, Pelicci PG and Kouzarides T (2002) Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence. EMBO J 21: 2383–2396 Leri A, Franco S, Zacheo A, Barlucchi L, Chimenti S, Limana F, Nadal-Ginard B, Kajstura J, Anversa P and Blasco MA (2003) Ablation of telomerase and telomere loss leads to cardiac dilatation and heart failure associated with p53 upregulation. EMBO J 22: 131–139 McClintock B (1941) The stability of broken ends of chromosomes in Zea mays. Genetics 26: 234–282 Metcalfe JA, Parkhill J, Campbell L, Stacey M, Biggs P, Byrd PJ and Taylor AM (1996) Accelerated telomere shortening in ataxia telangiectasia. Nat Genet 13: 350–353 Mishima K, Handa JT, Aotaki-Keen A, Lutty GA, Morse LS and Hjelmeland LM (1999). Senescence-associated beta-galactosidase histochemistry for the primate eye. Invest Ophthalmol Vis Sci 40: 1590–1593 Morales CP, Holt SE, Ouellette M, Kaur KJ, Yan Y, Wilson KS, White MA, Wright WE and Shay JW (1999) Absence of cancer-associated changes in human fibroblasts immortalized with telomerase. Nat Genet 21: 115–118 Niida H, Matsumoto T, Satoh H, Shiwa M, Tokutake Y, Furuichi Y and Shinkai Y (1998) Severe growth defect in mouse cells lacking the telomerase RNA component. Nat Genet 19: 203–206
Noda A, Ning Y, Venable SF, Pereira-Smith OM and Smith JR (1994) Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res 21: 90–98 Ohtani N, Zebedee Z, Huot TJ, Stinson JA, Sugimoto M, Ohashi Y, Sharrocks AD, Peters G and Hara E (2001) Opposing effects of Ets and Id proteins on p16INK4a expression during cellular senescence. Nature 409: 1067–1070 Packer L and Fuehr K (1977) Low oxygen concentration extends the lifespan of cultured human diploid cells. Nature 267: 423–425 Pandita TK (2002) ATM function and telomere stability. Oncogene 21: 611–618 Pantoja C and Serrano M (1999) Murine fibroblasts lacking p21 undergo senescence and are resistant to transformation by oncogenic Ras. Oncogene 18: 4974–4982 Paradis V, Youssef N, Dargere D, Ba N, Bonvoust F and Bedossa P (2001) Replicative senescence in normal liver, chronic hepatitis C, and hepatocellular carcinomas. Hum Pathol 32: 327–332 Parrinello S, Samper E, Krtolica A, Goldstein J, Melov S and Campisi J (2003) Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nature Cell Biol 5: 741–747 Pearson M, Carbone R, Sebastiani C, Cioce M, Fagioli M, Saito S, Higashimoto Y, Appella E, Minucci S, Pandolfi PP and Pelicci PG (2000) PML regulates p53 acetylation and premature senescence induced by oncogenic Ras. Nature 406: 207–210 Pirrotta V (1998) Polycombing the genome: PcG, trxG, and chromatin silencing. Cell 93: 333–336 Pomerantz J, Schreiber-Agus N, Liegeois NJ, Silverman A, Alland L, Chin L, Potes J, Chen K, Orlow I, Lee HW et al. (1998) The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2’s inhibition of p53. Cell 92: 713–723 Ramirez RD, Morales CP, Herbert BS, Rohde JM, Passons C, Shay JW and Wright WE (2001) Putative telomere-independent mechanisms of replicative aging reflect inadequate growth conditions. Genes Dev 15: 398–403 Rane SG, Cosenza SC, Mettus RV and Reddy EP (2002) Germ line transmission of the Cdk4(R24C) mutation facilitates tumorigenesis and escape from cellular senescence. Mol Cell Biol 22: 644–656 Rudolph KL, Chang S, Lee HW, Blasco M, Gottlieb GJ, Greider C and DePinho RA (1999) Longevity, stress response, and cancer in aging telomerase-deficient mice. Cell 96: 701–712 Schmitt CA, Fridman JS, Yang M, Lee S, Baranov E, Hoffman RM and Lowe SW (2002) A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy. Cell 109: 335–346 Sage J, Mulligan GJ, Attardi LD, Miller A, Chen S, Williams B, Theodorou E and Jacks T (2000) Targeted disruption of the three Rb-related genes leads to loss of G(1) control and immortalization. Genes Dev 14: 3037–3050 Sage J, Miller AL, Pérez-Mancera PA, Wysocki JM and Jacks T (2003) Acute mutation of Rb is sufficient for cell cycle re-entry in quiescent and senescent cells. Nature 424: 223–228 Saito H, Hammond AT and Moses RE (1995) The effect of low oxygen tension on the in vitro-replicative lifespan of human diploid fibroblast cells and their transformed derivatives. Exp Cell Res 217: 272–279 Seger YR, Garcia-Cao M, Piccinin S, Cunsolo CL, Doglioni C, Blasco MA, Hannon GJ and Maestro R (2002) Transformation of normal human cells in the absence of telomerase activation. Cancer Cell 2: 401–413 Serrano M, Lin AW, McCurrach ME, Beach D and Lowe SW (1997) Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88: 593–602
10 Sharpless NE, Bardeesy N, Lee KH, Carrasco D, Castrillon DH, Aguirre AJ, Wu EA, Horner JW and DePinho RA (2001) Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis. Nature 413: 86–91 Shelton DN, Chang E, Whittier PS, Choi D and Funk WD (1999) Microarray analysis of replicative senescence. Curr Biol 9: 939– 945 Sherr CJ and DePinho RA (2000) Cellular senescence: Mitotic clock or culture shock? Cell 102: 407–410 Stansel RM, Subramanian D and Griffith JD (2002) p53 binds telomeric single strand overhangs and t-loop junctions in vitro. J Biol Chem 277: 11625–11628 Sugihara T, Kaul SC, Kato J, Reddel RR, Nomura H and Wadhwa R (2001) Pex19p dampens the p19ARF-p53-p21WAF1 tumor suppressor pathway. J Biol Chem 276: 18649–18652 te Poele RH, Okorokov AL, Jardine L, Cummings J and Joel SP (2002) DNA damage is able to induce senescence in tumor cells in vitro and in vivo. Cancer Res 62: 1876–1883 Tyner SD, Venkatachalam S, Choi J, Jones S, Ghebranious N, Igelmann H, Lu X, Soron G, Cooper B, Brayton C et al. (2002) p53 mutant mice that display early ageing-associated phenotypes. Nature 415: 45–53 van der Lugt NM, Domen J, Linders K, van Roon M, RobanusMaandag E, te Riele H, van der Valk M, Deschamps J, Sofroniew M, van Lohuizen M et al. (1994) Posterior transformation, neurological abnormalities, and severe hematopoietic defects in mice with a targeted deletion of the bmi-1 proto-oncogene. Genes Dev 8: 757–769 Vasile E, Tomita Y, Brown LF, Kocher O and Dvorak HF (2001) Differential expression of thymosin beta-10 by early passage and senescent vascular endothelium is modulated by VPF/VEGF: evidence for senescent endothelial cells in vivo at sites of atherosclerosis. FASEB J 15: 458–466 Vaupel P, Kallinowski F and Okunieff P (1989) Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res 49: 6449–6465 Vaziri H, West MD, Allsopp RC, Davison TS, Wu YS, Arrowsmith CH, Poirier GG and Benchimol S (1997) ATM-dependent telomere loss in aging human diploid fibroblasts and DNA damage lead to the post-translational activation of p53 protein involving poly(ADP-ribose) polymerase. EMBO J 16: 6018– 6033
Vaziri H, Squire JA, Pandita TK, Bradley G, Kuba RM, Zhang H, Gulyas S, Hill RP, Nolan GP and Benchimol S (1999) Analysis of genomic integrity and p53-dependent G1 checkpoint in telomerase-induced extended-lifespan human fibroblasts. Mol Cell Biol 19: 2373–2379 Wadhwa R, Sugihara T, Hasan MK, Taira K, Reddel RR and Kaul SC (2002) A major functional difference between the mouse and human ARF tumor suppressor proteins. J Biol Chem 277: 36665–36670 Webley K, Bond JA, Jones CJ, Blaydes JP, Craig A, Hupp T and Wynford-Thomas D (2000) Post-translational modifications of p53 in replicative senescence overlapping but distinct from those induced by DNA damage. Mol Cell Biol 20: 2803–2808 Wei W, Hemmer RM and Sedivy JM (2001) Role of p14(ARF) in replicative and induced senescence of human fibroblasts. Mol Cell Biol 21: 6748–6757 Williams G (1957) Pleiotropy, natural selection, and the evolution of senescence. Evolution 11: 398–411 Wong DJ, Foster SA, Galloway DA and Reid BJ (1999) Progressive region-specific de novo methylation of the p16 CpG island in primary human mammary epithelial cell strains during escape from M(0) growth arrest. Mol Cell Biol 8: 5642–5651 Wright WE and Shay JW (1996) Mechanism of escaping senescence in human diploid cells. In: Holbrook NJ, Martin GR and Lockshin R (eds) Modern Cell Biology Series – Cellular Aging and Cell Death, pp 153–167. Wiley & Sons, New York Wright WE and Shay JW (2000) Telomere dynamics in cancer progression and prevention: fundamental differences in human and mouse telomere biology. Nat Med 6: 849–851 Yagita K, Tamanini F, van Der Horst GT and Okamura H (2001) Molecular mechanisms of the biological clock in cultured fibroblasts. Science 292: 278–281 Zhang Y, Xiong Y and Yarbrough WG (1998) ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell 92: 725–734
