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Replicative Senescence Revisited

^a Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, and Department of Medicine, McGill University, Montréal, Québec, Canada.
^b Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Kentucky

Eugenia Wang, Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, KY 40292 E-mail: eugenia.wang{at}louisville.edu.

Decision Editor: James R. Smith, PhD

	Abstract

Top Abstract Tumor Suppression Mechanism Stress-Induced versus Telomere... References

 
Forty years after its discovery, replicative senescence remains a rich source of information about cell-cycle regulation and the progression from a normal to a transformed phenotype. Effectors of this growth-arrested state are being discovered at a great pace. This review discusses the latest findings on the players responsible for establishing replicative senescence, as well as the associated telomere shortening.

IN the early 1960s, the remarkable observation by Hayflick that normal cells have a finite ability to replicate in vitro took a conservative scientific community by surprise ⁽¹⁾. This limited replicative potential and its accompanying phenotype—irreversible growth arrest, increased cell size and distinct flat enlarged morphology, senescence associated ß-galactosidase (SA-ß-gal) activity, accumulation of lipofuscin granules, and a wide change in gene expression—is termed "replicative senescence," sometimes referred to as Hayflick's limit. Now, 40 years later, the scientific community still has no clear idea of its importance in vivo; emerging data suggest that replicative senescence may be a tumor suppressor mechanism, a theory that emerged following cell fusion experiments in which the senescent is dominant over the transformed phenotype ⁽²⁾⁽³⁾. This observation, along with complementation group experiments, also suggested that this phenotype is part of a genetic program controlled by a small number of dominant genes. It was proposed early on that replicative senescence in culture mimics organismal aging, particularly with regard to some phenotypic aspects of aging, such as delayed wound healing, declining immune response, and thinning of the skin. Presently, the identification of the dominant genes involved is well underway, and a general understanding of how this phenotype functions to shut down cell replication has emerged. This review discusses the latest findings associated with replicative senescence.

	Tumor Suppression Mechanism

Top Abstract Tumor Suppression Mechanism Stress-Induced versus Telomere... References

 
The Rb Checkpoint
The cell cycle is divided into four highly regulated stages: G₁ (G stands for "gap"), which allows the cells to prepare for successful DNA synthesis; S, the stage in which DNA synthesis occurs; G₂, in which cells prepare themselves to complete the cell cycle by undergoing mitosis; and M, for mitosis itself. Once the cell cycle is completed, cells either undergo another round of cell replication, if exposed to the proper mitogenic signals, or exit the cell cycle by entering a nondividing state referred to as G₀. Progression from G₁ to the S phase is a highly regulated transition, in which the retinoblastoma protein (Rb) plays an essential role. In normal cells, growth arrested either by contact inhibition or lack of growth-factor stimulation, Rb is in an active unphosphorylated form, sequestering E2F protein and preventing it from activating the transcription of a variety of genes involved in the S phase of the cell cycle. Following growth-factor stimulation or escape from contact inhibition, along with proper nutrient availability, cyclin D is synthesized, and heterodimerizes with cyclin-dependent kinase-4 or kinase-6 (cdk4 or cdk6), well-characterized serine/threonine kinases, which then phosphorylate Rb, leading to the release of E2F. Then, in association with DP, E2F activates cyclin E–cdk2 complexes, which sequentially phosphorylate Rb, leading to a full commitment to cell-cycle progression. Two families of cyclin-dependent kinase inhibitors (CKIs) restrain the phosphorylation of Rb. The first family, INK4, includes p15^INK4b, p16^INK4a, p18^INK4c, and p19^INK4d, binding specifically to cdk4 and cdk6 to prevent the formation of cyclin D–cdk complexes; p21^{CIP1/WAF1/Sdi1}, p27^KIP1, and p57^KIP2 form the CIP/KIP family, with a broader specificity, binding to cdk4–cyclin D, cdk6–cyclin D, cdk2–cyclin E, and cdk2–cyclin A complexes. The association with cdk4– and cdk6–cyclin D complexes is necessary for cell-cycle progression, whereas the association with cdk2 complexes is inhibitory (Fig. 1) ⁽⁴⁾.

View larger version (18K): [in this window] [in a new window]   Figure 1. The Rb cell-cycle checkpoint.

View larger version (25K): [in this window] [in a new window]   Figure 2. Relationship between cell numbers and telomere length in human fibroblasts during serial passaging in culture. As cells are passaged, telomere length decreases to 4 kb, where cells enter M1 or replicative senescence. This stage can be bypassed by using viral oncoproteins; in that case, cells replicate further for 10–20 population doublings and telomeres reach a critical length around 1.5 kb. Cells enter a crisis state referred to as M2, from which very rare transformed clones emerge (1 x 10-7). These two stages can be bypassed by introducing hTERT (the protein component of telomerase).

p16^INK4a
p16^INK4a was discovered as a cdk4-interacting protein that inhibits the catalytic activity of cdk4–cyclin D complexes ⁽¹⁷⁾, and then it emerged as a major tumor suppressor when it was found to be mutated in a large variety of human cancers, such as lung and brain cancer; melanoma, glioma, kidney, bladder, and ovary cancer, and leukemia ^(18)(19). Consequently, p16^INK4a expression is low in young replicating or quiescent cells, but high in senescent cells in different cell types ^{(20)(21)(22)(23)(24)}: in keratinocytes, increased p16^INK4a expression is seen only in senescent, but not differentiated, keratinocytes. Moreover, epithelial cells that display increased proliferative potential following an initial onset of senescent phenotype after 20–25 population doublings lack p16^INK4a expression ⁽²⁵⁾. Furthermore, a genetic analysis of p16^INK4a alleles reveals that, in human tumor cells, while deletions are frequent in one allele, the second allele is often methylated, leading to complete abrogation of p16^INK4a expression. Accordingly, treatment of these particular tumors with a demethylating agent such as 5-aza-2'-deoxycytidine (5-aza-dC) restores a high p16^INK4a protein level, inhibits cell growth, and induces a senescent phenotype ^{(23)(25)(26)(27)(28)}. Moreover, overexpression of p16^INK4a in some tumor cells induces a senescent phenotype ^(29)(30), whereas antisense p16^INK4a delays replicative senescence in human diploid fibroblasts ⁽³¹⁾. This effect is likely to depend on Rb status, as human bladder carcinomas show an inverse relationship between p16^INK4a and Rb; when p16^INK4a is deleted in carcinomas, Rb is functional, and when Rb is deleted, p16^INK4a is expressed ⁽³²⁾.

Similarly, when the papillomavirus protein E7, which inhibits Rb, is expressed in epithelial cells, they sustain p16^INK4a expression, whereas the papillomavirus protein E6 induces the loss of p16^INK4a in immortal bypassers ⁽³³⁾. In fact, in Rb-negative cells, p16^INK4a levels are higher than in Rb-positive cells, suggesting that Rb negatively regulates p16^INK4a expression ⁽²²⁾. Furthermore, reintroduction of functional Rb in Rb/p53 negative tumor cell lines induces cell senescence. Although the p16^INK4a status was not verified in this study, it is likely, on the basis of the previously mentioned results, that it must have been wild type ⁽³⁴⁾. Although overexpression of p16^INK4a likely causes replicative senescence by sequestering cdk4 and cdk6 and preventing Rb phosphorylation ⁽²⁰⁾, it may accelerate the loss of other factors involved in cell proliferation ⁽²⁹⁾. Consistent with p16^INK4a overexpression experiments, mouse embryonic fibroblasts (MEFs) null for either cyclin D1 or cyclin D2 undergo premature senescence, suggesting that the cdk4–cdk6 phosphorylation of Rb is the committing step in replicative senescence regulation ⁽³⁵⁾.

The way in which Rb promotes replicative senescence probably has to do with its ability to associate with chromatin remodeling complexes. Introduction of Bgr1, which is a component of the SNF–SWN chromatin-remodeling complex, into SW13 cells, which do not express any endogenous Brg1, induces features of replicative senescence ^(36)(37)(38). The induction of the flat morphology is dependent on the ability of Brg1 to bind Rb, since a non-Rb-binding mutant will not induce replicative senescence. Moreover, Rb's ability to stop cell division is dependent on Brg1, as a nonphosphorylable Rb mutant is unable to induce features of replicative senescence in cells deficient for Brg1 ⁽³⁹⁾. LKB1, a serine/threonine kinase defective in Peutz-Jeghers syndrome, associates with Bgr1 in vitro; this association is not dependent on the kinase domain of LKB1, and LKB1 does not phosphorylate Bgr1, but LKB1 kinase activity is needed to allow Bgr1 to induce replicative senescence ⁽⁴⁰⁾. Therefore, it seems likely that chromatin-remodeling complexes play an essential role in replicative senescence, a conclusion inferred by results from Ogryzko and colleagues ⁽⁴¹⁾.

MEFs have a greater rate of spontaneous immortalization than human cells. After 15–20 passage doublings, the cell population reaches a plateau from which several independent clones emerge to take over the senescent population. With the use of this cell system it is easy to assay the potential effect of different proteins on the senescent checkpoint; when MEFs are cultured, there is a rapid induction of both p16^INK4a and p15^{INK4b (42)}. Interestingly, p16^INK4a expression is absent from nearly all tissues examined, which has prompted some to suggest that p16^INK4a expression is stress regulated and a cell culture artifact (see later text); a hypothesis supported by the mild tumor profile of mice deficient for p16^INK4a, unless subjected to carcinogens ⁽⁴³⁾. Moreover, using MEFs from p16^INK4a knock-out mice, Sharpless and colleagues ⁽⁴³⁾ recently showed that p16^INK4a is not required for senescence, as these MEFs have the same growth kinetic as normal wild type MEFs, although p16^INK4a-negative MEFs have a slightly higher immortalization rate, suggesting that in murine cells p16^INK4a is not a major contributor to senescent growth arrest.

As in human cells, antisense targeted toward p16^INK4a extends MEF life span, and p16^INK4a-mediated growth arrest is Rb dependent ⁽⁴⁴⁾. Furthermore, MEFs bearing a mutated cdk4 (cdk4^R24C) unable to bind p16^INK4a escape cellular senescence ⁽⁴⁵⁾; these cells, however, are not transformed but rather immortalized, because they are unable to form colonies on soft agar, or to develop into tumors in nude mice. Mice bearing a germ line mutation in cdk4 are highly susceptible to tumor formation. These results are hard to reconcile with the p16^INK4a knock-out mice, but they suggest that in the former a compensatory mechanism other than p16^INK4a inhibits cdk4 activity; the mutation in the latter does not initiate this mechanism, because cdk4^R24C abrogates binding of other INK4 proteins as well ⁽⁴⁶⁾. In contrast, Rb-deficient MEFs still undergo replicative senescence, suggesting either that the Rb pathway in murine cells is not an important mediator of replicative senescence, or that other Rb proteins, p107 and p130, may compensate for the loss of Rb. This explanation is likely, because triple knock-out MEFs are readily immortalized ^(47)(48), and Rb is spontaneously mutated in a large variety of different cancers ⁽⁴⁹⁾.

Culprits for the increased expression of p16^INK4a in replicative senescence are slowly starting to surface. Id1-deficient MEFs undergo premature senescence accompanied by an increase in p16^INK4a. In wild type MEFs, Id1 represses the p16^INK4a promoter and keeps p16 expression in check ⁽⁵⁰⁾. Id1 expression is usually reduced in normal replicative senescence, which is consistent with the results reported in MEFs deficient for Id1 ⁽⁵¹⁾. Id1 sequesters Ets1 and Ets2 to prevent them from binding the p16^INK4a promoter, where they activate p16^INK4a transcription; this effect is potentiated by Mek expression. These enforced expressions are consistent with the pattern of expression seen in normal replicative senescence, where Ets1 is upregulated and Id1 is downregulated ⁽⁵²⁾. Moreover, overexpression of Id1 in keratinocytes is able to delay senescence, as a result of decreased p16^INK4a expression ⁽⁵³⁾. The polycomb protein Bmi-1 is also a negative regulator of p16^INK4a, and MEFs deficient for Bmi-1 undergo rapid premature senescence ⁽⁵⁴⁾. The premature senescence phenotype induced in Bmi-1-deficient MEFs is more severe and rapid than in Id1-deficient MEFs; this result is easily explained by the other transcriptional functions of Bmi-1 (see the paragraphs that follow). Positive regulators of p16^INK4a include JunB, with three AP1 binding sites in the p16^INK4a promoter; JunB overexpression induces premature senescence in primary cells, an effect lost in INK4a^-/- MEFs ⁽⁵⁵⁾.

p19^ARF–p53–p21 Connection
A role for p53 in replicative senescence emerged from the observation that both antisense p53 and papillomavirus protein E6, which mediates p53 degradation, lead to extension of life span in human diploid fibroblasts ^(5)(6)(56). Furthermore, introduction of mutant p53 into human diploid fibroblasts and p53-deficient MEFs results in an increased life span and a greater rate of immortalization ^(57)(58)(59). In the case of normal replicative senescence, although one study observes no increase in p53 mRNA and protein but a clear increase in its transcriptional activity, which likely explains the high p21 expression in these senescent cells, other reports found an increase in protein level ^{(60)(61)(62)(63)}. Moreover, introducing wild type p53 into a bladder carcinoma cell line lacking any functional p53 induces a senescence program, accompanied by p21 and mdm-2 upregulation and cyclin A, cyclin B, and cdc2 downregulation, consistent with previous observations ^(12)(13). The same senescence program is induced in these cells, albeit with slower kinetics, by overexpression of p21, suggesting that the program induced by p53 is mainly caused by upregulating p21; other CKIs known to induce replicative senescence, such as p15, p16, and p27, are not increased, ruling out a nonspecific effect ⁽⁶⁴⁾.

Accordingly, microinjection of anti-p53 antibodies into senescent fibroblasts abrogates the senescence program; these cells are now able to reenter the cell cycle, an effect mediated by a decrease in p21 expression, leading to a reversion of morphology back to a "younger" phenotype ⁽⁶⁵⁾. Moreover, cells defective in p53 but overexpressing p21 undergo senescence, which suggests that the main downstream effector of p53 is p21 ⁽⁶⁶⁾. Furthermore, p21^-/- human diploid fibroblasts bypass replicative senescence ⁽⁶⁷⁾. Cells deficient for ATM, a known activator of p53, undergo premature senescence, caused by a greater rate of telomere loss at each replication step; they also exhibit increased p21 expression, which is completely abolished in ATM^-/- p53^-/- double-null fibroblasts, suggesting that the increase in p21 is due to residual p53 activity in ATM^-/- cells. Moreover, the premature senescence seen in ATM^-/- MEFs is completely abolished in ATM^-/- p21^-/- double-null MEFs ⁽⁶⁸⁾. These results all point toward p21 as the major effector of p53 in inducing replicative senescence in human fibroblasts.

The picture is far from simple, however. These results are in sharp contrast with previous results obtained by Medcalf and colleagues ⁽⁶⁹⁾, demonstrating that p21 is not required for senescence in Li–Fraumeni human fibroblasts. This strain is defective for p53 and accordingly does not express any p21, but surprisingly still undergoes senescence, albeit with a higher immortalization frequency. This suggests that both p53 and p21 are dispensable for replicative senescence. Surprisingly, a second study using Li–Fraumeni immortalized fibroblasts demonstrates replicative senescence after p21 introduction ⁽⁷⁰⁾. However, introducing mutant p53 into human diploid cells completely abolishes p53 transcriptional activity and delays replicative senescence, without inhibiting p21 ⁽⁷¹⁾. Moreover, cells deficient for p53 and p21, induced to undergo a senescentlike phenotype following DNA damage, still show phenotypic markers of senescence, albeit with a decreased efficiency than wild type cells, suggesting the presence of p53- and p21-independent signals toward induced replicative senescence ⁽⁷²⁾. In this study, the p53-mediated effect is p21 dependent, which again makes the argument that p53's senescence-inducing activity is totally dependent on p21. This is also supported by the fact that overexpression of mdm-2 or microinjection of p21 antibodies into senescent cells similarly induce DNA synthesis ^(73)(74). Arguing against this result, however, is the response seen after microinjection of p21 or p53 antibodies; whereas antibodies against p53 restore a younger phenotype as well as the capacity to divide, p21 antibodies only induce DNA synthesis, but not cell division.

In a study by Pantoja and Serrano ⁽⁷⁵⁾, murine fibroblasts lacking p21 undergo senescence at the same rate as wild type fibroblasts. In fact, a clone-to-clone study reveals that cells that escape senescence have acquired a mutation in p53 or in the INK4a locus. This observation has serious implications for p21 being the only effector of p53-induced senescence in MEFs, since MEFs deficient for p53 do not undergo replicative senescence ^(58)(59). This is further supported by results showing that whereas p53^-/- Ras-overexpressing MEFs are readily transformed, Rb^-/- p107^-/- are immortalized but not transformed, suggesting that a downstream target of p53 other than p21 confers both immortalization and transformation potential ⁽⁷⁶⁾. Nevertheless, p53 and likely p21 play a major role in replicative senescence in a cell-type-dependent manner; clearly in MEFs p53 may require other factors than p21 to mediate this effect. Moreover, conclusions from experiments with p53-mutated cells should take into consideration the fact that a lack of p53 increases genomic instability, and therefore may increase the likelihood of cellular defects other than mutated p53. Interestingly, several genes found to be upregulated in replicative senescence are also upregulated following p21 ectopic expression ^(77)(78). Therefore, p21 clearly induces genes implicated in replicative senescence.

Recently, a new tumor suppressor gene was cloned out by subtractive hybridization; this protein, p33^ING1, is upregulated severalfold in senescent cells. Infection of presenescent fibroblasts with an antisense p33^ING1 fragment leads to a 10% extension in life span ⁽⁷⁹⁾, an increase similar to that seen with dominant-negative p53 ⁽⁵⁷⁾. In fact, p33^ING1-mediated growth arrest is dependent on wild type p53 and accompanied by an increase in p21 ⁽⁸⁰⁾. Intriguingly, p33 enhances DNA repair following UV damage, an effect mediated by p53 ⁽⁸¹⁾. Moreover, p33 associates with deacetylase complexes ⁽⁸²⁾, again suggesting an important role for chromatin-modifying enzymes in the control of replicative senescence.

The INK4a locus is a very peculiar region, encoding two tumor suppressors that differently regulate cell-cycle progression on different reading frames ⁽⁸³⁾. The first transcript, p16^INK4a, binds to cdk4 and cdk6 and prevents cyclin D–cdk complexes from phosphorylating Rb, keeping Rb functional by sequestrating E2F, a transcription factor responsible for S phase progression. The second transcript, p19^ARF, which bears no homology to any CKIs, encodes a protein that can dominantly inhibit cell-cycle progression; however, this effect is mediated at the level of p53 activity, rather than the Rb checkpoint. p19^ARF binds mdm-2, a negative regulator that binds p53 and mediates its degradation, sequestering it in the cytoplasm and preventing its association with p53 ^{(84)(85)(86)(87)}; the outcome is a net increase in p53 activity. Both proteins are upregulated after serial passaging of explanted MEFs in culture ^(43)(88); accordingly, MEFs deficient for p16^INK4a and p19^ARF or p19^ARF alone are easily immortalized in culture and bypass the senescence checkpoint. Interestingly, p16^INK4a-/- cells in which p19^ARF is overexpressed undergo senescence, whereas p19^ARF-/- cells overexpressing p16^INK4a fail to stop dividing and remain immortalized, suggesting that p19^ARF regulates not only the p53 but also the Rb checkpoint. Accordingly, dominant-negative p53 fails to overcome p19^ARF-mediated growth arrest. The effect of p19^ARF on the Rb checkpoint is mediated through mdm-2, as previously described ⁽⁸⁹⁾. These results are consistent with the recent generation of p16^INK4a-/- mice, in which MEFs have the same growth characteristics as wild type cells, suggesting that from the INK4a locus, p19^ARF is responsible for passage-induced senescence ⁽⁴³⁾.

In senescent human fibroblasts, p19^ARF expression has been reported to be both upregulated ⁽⁹⁰⁾ and downregulated ⁽⁹¹⁾. However, the downstream targets of p19^ARF, mdm-2 and p53, are not affected in senescent cells ^(92)(93), although p53 activity is increased ⁽⁶⁰⁾. Ectopic expression of p19^ARF induces replicative senescence in human fibroblasts, dependent on both p21 and p53, but not p16^{INK4a (91)}. Accordingly, overexpression of proteins leading to overexpression of p19^ARF causes replicative senescence in human fibroblasts; this propriety is ascribed to E2F and ß-catenin ^(90)(94). In contrast, Pex19p, a protein essential for peroxisomal biogenesis, sequesters p19^ARF in the cytoplasm on overexpression, leading to an increase in p53–mdm-2-mediated degradation. An antisense strategy targeted toward Pex19p leads to an increase in p53 and p21, and to a phenotype resembling replicative senescence ⁽⁹⁵⁾. Moreover, factors leading to downregulation of CKIs enjoy a considerable selective advantage in immortalizing normal cells. Indeed, transcription factors such as Bmi-1, which promotes downregulation of both p16^INK4a and p19^ARF and to a lesser extent p15^INK4b, cooperates with activated Ras to promote transformation ⁽⁵⁴⁾. In a screen to identify proteins that can bypass Bmi-1^-/--induced premature senescence, TBX2 immortalizes MEFs by repressing the expression of p19^ARF but not p16^{INK4a (96)}. TBX3, related to TBX2, also represses p19^ARF and replicative senescence; interestingly, a mutation that hampers its ability to repress senescence and p19^ARF expression is found in ulnar-mammary syndrome, a disease characterized by hypoproliferation ⁽⁹⁷⁾.

An intriguing emerging story is the role of promyelocytic leukemia-associated (PML) protein in replicative senescence, reviewed by Pearson and Pellicci ⁽⁹⁸⁾. PML protein and promyelocytic oncogenic domains (PODs) are increased in Ras-induced as well as normal senescence in human cells. Ectopic expression of PML protein induces features of replicative senescence, such as ß-galactosidase, PAI-1, p16^INK4a, p53, p21, and mdm-2. Moreover, p53 and Rb are relocalized to PODs following Ras-induced senescence ⁽⁹⁹⁾. PML^-/- MEFs bypass replicative senescence and are immortalized but not fully transformed, as they are unable to grow on semisolid substrates ⁽¹⁰⁰⁾; there is no Ras-induced senescence in PML^-/- MEFs. PML overexpression also induces premature senescence in MEFs, characterized by increased p53 and p21; no premature senescence is seen in p53^-/- or p21^-/- MEFs, nor p16^INK4a increase, contrary to human cells. PML or Ras overexpression causes shifting of p53 and CBP/p300 in PODs, and formation of a ternary complex of p53–CBP–PML; this association seems to be required for p53 acetylation and activation ⁽¹⁰¹⁾. Therefore, the role of PML in replicative senescence may be a consequence of its regulation of p53 activity.

Other CKIs
Whereas a role for p16^INK4a and p21 in replicative senescence is undisputed, data supporting a role for other CKIs are scarce and seem to be highly system dependent. p15^INK4b, p16^INK4a, p21, and p27 all induce features of replicative senescence when overexpressed in human diploid fibroblasts, albeit with different efficiencies; p15^INK4b and p16^INK4a are more efficient than p21 and p27, respectively. Moreover, the extent of growth arrest correlates well with ß-galactosidase expression ⁽¹⁰²⁾. Induced replicative senescence is not a hallmark of human diploid fibroblasts alone, as adenoviral transfer of p15^INK4b5 and p16^INK4a into human glioma or osteogenic sarcoma cells induces replicative senescence ^(29)(103), although both require intact Rb. Some results suggest that p15^INK4b and p18^INK4c play no role in inducing replicative senescence in mouse cells ⁽¹⁰⁴⁾. These disagree with a result obtained by Malumbres and colleagues ⁽¹⁰⁵⁾, who demonstrate a role for p15^INK4b in inducing a replicatively senescent phenotype when overexpressed in MEFs; they also found that it is upregulated after Ras-induced premature senescence, mediated via the Raf–Mek–Erk pathway ⁽¹⁰⁵⁾. In fact, p15^INK4b induction kinetics follow p16^INK4a expression kinetics. Moreover, MEFs deficient in p15^INK4b are more prone to transformation by oncogenes than wild type cells. TGF-ß induces replicative senescence in cancer cells ⁽¹⁰⁶⁾; this may reflect an increase in p15^INK4b, because TGF-ß is known to activate this particular CKI by bringing Smad and Miz-1 to its promoter. Furthermore, Miz-1 overexpression leads to replicative senescence characterized by upregulation of p15^{INK4b (107)(108)}. p15^INK4b is upregulated during replicative senescence in T lymphocytes, reflected in mice deficient in p15^INK4b with lymphoproliferative disorders ⁽²¹⁾. Lastly, p57 induces features of replicative senescence in epithelial cells, when overexpressed with the same kinetics as p16^{INK4a (109)}.

Tumor cells deficient in both p53 and Rb, into which a wild type Rb is introduced, undergo replicative senescence characterized by posttranscriptional upregulation of p27, independently from E2F ⁽¹¹⁰⁾. LY294002, a specific PI-3 kinase inhibitor, causes a senescencelike growth arrest in human fibroblasts ⁽¹¹¹⁾; the same effect is mediated in MEFs, accompanied by an increase in p27 but no other CKIs, p53, or p19^ARF. The Forkhead protein, AFX, is likely the downstream mediator of PI-3 kinase inhibition, because ectopic expression of AFX mimics p27 overexpression ⁽¹¹²⁾. This senescencelike growth arrest is also partly dependent on p130, a Rb-related family member, because p27^-/- MEFs still undergo growth arrest. Recently, PTEN was found to sequester mdm2 in the cytoplasm, leading to a net increase in p53 transcriptional activity ⁽¹¹³⁾; it is therefore surprising that PI-3 kinase inhibitors, which likely mimic the PTEN-mediated effect, are unable to upregulate p53 and p21. Although a different cell system, as well as other potential physiological targets for PI-3 kinase inhibitors, may be involved, further studies are needed to clarify how p27 and the PI-3K–Akt–PTEN pathway may regulate replicative senescence.

	Stress-Induced versus Telomere-Induced Senescence

Top Abstract Tumor Suppression Mechanism Stress-Induced versus Telomere... References

 
Although the proteins described herein are responsible for establishing a replicatively senescent phenotype, they are not causative of that state; they are merely a consequence of an upstream signal. Telomere shortening is a likely candidate to signal replicative senescence in human cells, whereas a different signal seems to dictate this phenomenon in mouse cells. The section that follows describes recent results explaining the intrinsic differences between mouse and human cells, and the consequences of these differences on the establishment and the bypass of the replicative senescence checkpoint; these were recently reviewed by Serrano and Blasco ⁽¹¹⁴⁾ and by Sherr and DePinho ⁽¹¹⁵⁾.

Telomeres
Chromosome ends are capped by a specialized structure referred to as telomeres, made up of highly specific tandem nucleotide repeats. As human cells are passaged in vitro, telomere repeats are gradually lost until cells reach a critical limit and enter a crisis state, referred to as replicative senescence or the M1 barrier. Human cells entering this crisis usually have a mean telomeric length of less than 4 kb. By the use of viral oncoproteins, this first crisis state can be bypassed and cells can undergo 10–20 more population doublings until they reach a second crisis state, M2, characterized by extensive cell death. At this point, telomere length is around 1.5 kb (Fig. 2) ⁽¹¹⁶⁾. From the early work of McClintock, these highly specialized structures were recognized as being different from regular DNA breaks, and as being responsible for preventing chromosome end-to-end fusion ^{(117)(118)(119)}. Telomeres are basically made up of tandem repeats of TTAGGG double-stranded DNA, with a small 3' single-stranded overhang of approximately 200 nucleotides, with an overall length of 10–15 kb in human cells. Proteins involved in forming a higher-order structure bind to both the double- and single-stranded DNA regions. The 3' single-stranded overhang is recognized by telomerase, a nucleoprotein reverse transcriptase that specifically recognizes telomeric repeats and elongates the double-stranded DNA region.

A strong argument for telomere shortening as an initiator of the replicative senescence phenotype is the fact that introducing the catalytic subunit telomerase into presenescent human cells leads to stabilization of telomere length, unlimited replicative capacity, and bypass of M1 and M2 ^(120)(121). These fibroblasts show no sign of oncogenic transformation, as they remain dependent on cell contact, serum, and a solid support for cell proliferation ⁽¹²²⁾; moreover, the G₁ checkpoint is intact. Following ionizing radiation, p53 and p21 proteins are efficiently expressed and DNA damage response is normal ⁽¹²³⁾. However, these results do not argue against telomerase itself being able to inhibit a senescence-inducing signal. In human keratinocytes and epithelial cells, ectopic expression of telomerase is not sufficient to promote immortalization; a complementing deletion of p16^INK4a expression is required to bypass the senescent-mediated growth arrest ^(124)(125). Cells expressing an active in vitro telomerase, but unable to elongate telomeres in vivo, fail to escape crisis and senescence; thus it would appear that it is the elongation of telomeres per se that signals inhibition of replicative senescence, unless the elongating activity of telomerase signals to something else ⁽¹²⁶⁾. However, results from immortalized fibroblasts with an active hTERT, the protein component of telomerase, suggest that telomerase is unlikely to inhibit a senescence-inducing signal. These cells, with a subsenescent telomeric length, fail to undergo replicative senescence before 20 extra population doublings when transfected with a dominant-negative mutant ⁽¹²⁷⁾. Interestingly, in conditions in which only low levels of telomerase activity are available, telomerase preferentially elongates the shortest telomeres, indicating that a cis-acting signal recruits telomerase to these short telomeres; accordingly, the shortest telomere, not the mean telomere length, limits cell survival and chromosome stability ⁽¹²⁸⁾. Moreover, despite high telomerase activity in transformed cells, mean telomere length reaches a maximum and is thereafter kept constant, arguing for another cis-acting mechanism that signals to telomerase when and where to elongate telomeres in a cell-specific manner ⁽¹²⁹⁾.

Telomeres give rise to a highly specialized structure referred to as a T-loop, in which the single-stranded TTAGGG overhang loops back and inserts itself into the double-stranded telomeric region, supposedly "hiding" from being recognized as a DNA break ⁽¹³⁰⁾. Forming this T-loop requires several proteins, among which TRF1 and TRF2 play an essential role. Overexpression of TRF1 in telomerase-positive cells induces progressive loss of telomeric repeats, whereas dominant-negative TRF1 increases telomere length ⁽¹³¹⁾; thus it would appear that TRF1 is part of the cis-acting signal that limits telomere lengthening in the presence of telomerase. Two mutants of TRF2 induce features of replicative senescence when overexpressed in telomerase-positive immortalized cells ⁽¹³²⁾, likely caused by loss of the single-stranded 3' overhang. This senescent phenotype is dependent on either the p53 or Rb checkpoint, as both E6 and E7 are required to prevent replicative senescence by TRF2 mutants ⁽¹³³⁾. Overexpression of TRF2 leads to the same effect as TRF1, a progressive loss of telomeric repeats ⁽¹³⁴⁾. Telomerase itself may play a self-protective role other than telomere elongation, because telomerase prevents otherwise subsenescent telomere lengths from inducing senescence without net telomere lengthening ^(135)(136). These results demonstrate that uncapping telomeres induces replicative senescence, suggesting that not only telomere length, but also the structure itself, produces a signal-inducing replicative senescence. In fact, the structure of telomeres may be the sole signal-promoting replicative senescence, because short telomeres likely impede the capping status of telomeres. This has led to a probabilistic model in which capping status determines whether or not cells exit the cell cycle ⁽¹³⁷⁾. Recent results suggest that proteins such as TRF2 are probably implicated in the counting mechanism and the signal to induce replicative senescence; this is a reflection of telomere structure, not telomere length ⁽¹³⁸⁾.

When MEFs are cultured in vitro, they undergo 15–20 population doublings before entering "replicative senescence." This phenotype is likely not mediated by telomere shortening, because telomeres in mice are much longer than those in human cells. Moreover, keratinocytes and epithelial cells are not immortalized by telomerase, but instead require mutation in the Rb–p16 checkpoint pathway; this raises the possibility that some signal other than telomere shortening induces replicative senescence. Recently, this phenotype was challenged as being true replicative senescence, because rodent cells can be passaged indefinitely using the "proper" cell culture conditions, raising the possibility that the phenotype observed in MEFs is in fact a stress response, similar to Ras-induced senescence (see the paragraphs that follow) ^(139)(140). Moreover, in human cells, recent evidence demonstrates that telomere-independent mechanisms inducing replicative senescence are likely caused by inadequate cell culture conditions. All cell culture conditions tested led to an increase in p16^INK4a, a protein usually not expressed in vivo ⁽⁴²⁾; therefore, if in vitro conditions reproduced in vivo conditions, mutation in that particular pathway would not be required ⁽¹⁴¹⁾. A caveat behind these results, however, is that the conditions used in these experiments may not reflect the actual conditions in vivo. These results also suggest that replicative senescence is induced by telomere shortening only. Nevertheless, even though inadequate cell culture conditions may induce a phenotype similar to replicative senescence, it remains a great tool for fishing out tumor suppressor genes as well as oncogenes.

How does telomere shortening signal to cell-cycle checkpoints to induce replicative senescence? Senescent cells are characterized by chromosomal rearrangements and aneuploidy. Moreover, when cells exhibit extended life span following viral oncoprotein expression, they undergo a crisis referred to as M2, characterized by genomic instability and extensive chromosome end-to-end fusions. This suggests that short telomeres may mimic DNA double-strand breaks; in fact, the T-loop structure is proposed to "hide" the G' overhang strand from the DNA repair machinery. As mentioned earlier, depletion of TRF2 from telomeres leads to replicative senescence in some cell types, whereas in others, which likely lack the Rb checkpoint, apoptosis is induced dependent on p53 and ATM, two proteins involved in DNA damage response ⁽¹⁴²⁾. When both pathways are mutated, genomic instability leads to the selection of clones that upregulate telomerase activity and become transformed. The story is far from being as simplistic and linear as ATM's recognizing short telomeres and signaling these "breaks" to p53, leading to replicative senescence. Ataxia telangiectasia (AT) fibroblasts, defective in ATM, have a reduced life span in culture; MEFs from the ATM knock-out mice share this property ^{(143)(144)(145)}. ATM^-/- fibroblasts show a rapid loss of telomeric DNA sequences, which suggests that ATM may actually be involved in telomere stability. This possibility is reviewed by Pandita ⁽¹⁴⁶⁾. This telomere loss is a likely cause of premature replicative senescence, because ectopic expression of hTERT in AT fibroblasts restores normal life span ⁽¹⁴⁷⁾. Moreover, p53 DNA binding activity is higher in AT fibroblasts than in matched controls ⁽⁹²⁾; therefore, signaling from the telomere to p53 is not totally dependent on ATM. Interestingly, inhibition of Pin2/TRF1 expression in AT cells rescues the telomere-shortening phenotype ⁽¹⁴⁸⁾, suggesting that in normal cells Pin2/TRF1 is kept in check by ATM, because overexpression of TRF1 in normal cells induces telomere lengthening ⁽¹³¹⁾. Accordingly, ATM associates with and phosphorylates Pin2/TRF1 in vivo ⁽¹⁴⁹⁾. Another target of ATM, c-abl, also seems to play a negative role in telomere elongation, because MEFs deficient for c-abl have elongated telomeres ⁽¹⁵⁰⁾.

ATM is not the only DNA-damage-sensing enzyme implicated in telomere biology; recently poly(ADP-ribose) polymerase (PARP), involved in single-stranded DNA strand breaks, was found to function in telomere length regulation. In fact, PARP-1 deficient cells show decreased telomere length, a phenotype reminiscent of AT cells ⁽¹⁵¹⁾, and increased chromosomal end fusions. The shorter telomere phenotype is rescued in the presence of p53, which suggests a functional interaction between PARP and p53 in telomere maintenance ⁽¹⁵²⁾; p53-null cells show no change in telomere length. These results are controversial, because a more recent study found no difference in telomere length between wild type and PARP-deficient cells ⁽¹⁵³⁾. At this point, there are no clear explanations for this major discrepancy. Interestingly, tankyrase, an enzyme with PARP activity, localizes to human telomeres, where it ADP-ribosylates TRF1 and reduces its ability to bind telomeric DNA ⁽¹⁵⁴⁾. Consistent with the dominant-negative TRF1 study, overexpression of tankyrase leads to telomere elongation, likely by inhibiting TRF1 binding to telomeres in telomerase-positive cells ⁽¹⁵⁵⁾. Several other proteins interact with telomeric sequences and affect the formation and the stability of the T-loop, of which some are involved in the nonhomologous end joining (NHEJ) repair pathway. DNA-dependent protein kinase (DNA-PK) is made of three subunits: Ku70 and Ku80, the DNA binding moiety of the enzyme, and the DNA-PK catalytic subunit (DNA-PKcs) possessing the enzymatic activity; all three components are localized to telomeres ^(156)(157). Ku80 protein is tethered to telomeric DNA by binding to TRF1 ⁽¹⁵⁸⁾, where it plays an essential role in preventing chromosome end-to-end fusion, as suggested in MEFs deficient for Ku80, which exhibit a large increase in chromosome fusion compared with wild type cells; these fusion events do not show any overall decrease in telomere length. This increase in chromosomal end fusion is seen in other repair-deficient MEFs, such as Ku70 and DNA-PKcs knock-outs ^{(156)(159)(160)}. Interestingly, Ku80^-/- MEFs undergo premature replicative senescence, dependent on a functional p53 response, because they are abrogated in a p53-null environment ⁽¹⁶¹⁾. Finally, TRF2 associates with the RAD50–MRE11–NBS1 complex, implicated in homologous recombination DNA repair ⁽¹⁶²⁾; a defect in NBS1, the gene mutated in the Nijmegen breakage syndrome, also leads to rapid telomere shortening, and NBS1 is a substrate of ATM following DNA damage (Fig. 3). The effects of these DNA-repair and -damage signaling proteins on telomere length and maintenance were recently reviewed by Goytisolo and Blasco ⁽¹⁶³⁾.

View larger version (55K): [in this window] [in a new window]   Figure 3. Schematic of a T-loop at the end of a telomere. The 3' overhang telomeric region folds back and inserts itself into the double-stranded telomeric region. This T-loop requires several proteins to fold properly, which are depicted in the figure. Upon telomere shortening or T-loop destabilization, telomeres are recognized supposedly as DNA breaks and, depending on p53 and Rb status, elicit a senescence or apoptosis signal.

Do telomere-independent signals inducing replicative senescence exist? Introducing constitutively active Ras into primary fibroblasts induces a phenotype similar to replicative senescence, accompanied by increased expression of p16, p21, p53, PAI-1 and SA-ß-gal activity, as well as a decrease in cyclin A and cdk2-associated kinase activity; moreover, these cells fail to induce c-fos after serum stimulation ⁽¹⁶⁶⁾. However, MEFs defective for p53 or lacking the INK4a/ARF locus are readily transformed by Ras overexpression, suggesting that the Ras-induced senescence is dependent on these key players, regulating two different key pathways. However, MEFs defective for only p16^INK4a still undergo premature senescence following Ras overexpression ⁽⁴³⁾, suggesting that p19^ARF is the key player from the INK4a locus, mediating replicative senescence in MEFs. Moreover, p15^INK4b-deficient MEFs are transformed rather than growth arrested following Ras overexpression, which suggests that Ras participates in establishing this phenotype ⁽¹⁰⁵⁾. The premature senescence phenotype is dependent on the mitogen-activated protein kinase pathway, because Raf overexpression induces the same replicative senescent phenotype as Ras ⁽¹⁶⁷⁾; Raf is also able to induce a senescentlike phenotype in prostate cancer cells ⁽¹⁶⁸⁾. However, murine fibroblasts lacking p21 and transfected with Ras still undergo premature senescence; thus it would appear that p21 is not an important downstream effector, whereas in human cells p21 is in a linear pathway with p53 ⁽⁶⁷⁾. In contrast, Zhu and colleagues reported that Raf fails to induce premature senescence in p21-deficient mouse fibroblasts ⁽¹⁶⁷⁾. Moreover, a pathway independent of the conventional Ras–Raf–Mekk pathway is responsible for the characteristic morphological changes in senescent cells, because Raf-induced premature senescence does not yield the typical flattened morphology of senescent cells seen with Ras. Ras induces a replicative senescent phenotype in telomerase-expressing cells ^(122)(169), despite continuously high expression of telomerase. Moreover, ectopic expression of p19^ARF in normal human fibroblasts modified to express telomerase activity ⁽⁹¹⁾, or of p15^INK4B or p16^INK4A in human glioma cells ⁽¹⁰³⁾, induces replicative senescence; in the glioma cells, this replicative senescence is accompanied by an inhibition of telomerase activity.

Interestingly, introducing Rb into Rb-defective tumor cell lines induces replicative senescence as well as telomerase activity inhibition ⁽³⁴⁾, which suggests that the p16^INK4a–Rb pathway may regulate telomerase activity; the cell lines used are also p53 defective, which suggests that neither senescence nor telomerase inhibition require a functional p53 pathway. In fact, overexpression of the bovine papillomavirus E2 protein, which binds to the promoter region of the E6 and E7 proteins and represses their expression, induces replicative senescence in HeLa cells, despite high telomerase activity and long telomeres ^(170)(171). These results have tremendous potential implication for therapy, because telomerase is activated in more than 85% of all tumors ⁽¹⁷²⁾.

The Ras-induced premature senescence phenotype may be mediated by increasing levels of reactive oxygen species ⁽¹⁷³⁾. This is an intriguing result, because oxygen radicals are known to cause DNA damage, suggesting that Ras overexpression may actually mimic the hyperoxic cell culture conditions normally used to grow cells in culture. However, these observations fit with the increasing evidence that oxidative stress may play a role in both cellular and organismic aging ⁽¹⁷⁴⁾.

Conclusions
Forty years later, what have we learned? Certainly, compelling evidence suggests that replicative senescence is a tumor suppressor checkpoint ... in vitro. Although ß-galactosidase ⁽¹⁷⁵⁾ stains replicatively senescent cells, this is not a true specific marker because it also stains cells exposed to different stresses, such as DNA damaging drugs, oxygen species, or Ras overexpression (Fig. 4). Although this stress-induced senescence uses the same downstream effector, there is no clear evidence demonstrating how well this state mimics the replicative senescence phenotype caused by telomere shortening; therefore it should be referred to as stress-induced senescence, until these concepts are proven identical. In fact, microarray analysis suggests that they may indeed be different ^(78)(176). In vivo cellular senescence, which seems to increase with aging, may result from stresses that cells sustain throughout their lifetime, and not because they are replicatively exhausted. Accordingly, telomere length in fibroblasts does not correlate with donor age, as fibroblasts from centenarians still undergo many population doublings in vitro ⁽¹⁷⁷⁾.

View larger version (18K): [in this window] [in a new window]   Figure 4. Several signals lead to terminal growth arrest resembling replicative senescence, referred to as stress-induced senescence. Both growth arrest states share similar phenotypes, such as ß-galactosidase expression, enlarged and flattened morphology, and a characteristic G1 growth arrest. Whether both states are identical, and the extent of their contribution to aging, is not known.

Ample evidence now demonstrates the role of telomere shortening as a cause of replicative senescence. However, even this model is likely oversimplified, in light of recent results dealing with the structure of the telomeric complex, recently reviewed by Chan and Blackburn ⁽¹⁸⁰⁾. Recent results using mouse models suggest that telomere dysfunction in these models induces a tumor spectrum similar to that seen in humans (reviewed in ⁽¹⁶³⁾), suggesting that telomere shortening may indeed contribute to the increased incidence of cancer in elderly patients, as well as some of the aging-associated phenotype. The observation that most tumor cells can be induced to undergo replicative senescence following the introduction of negative cell-cycle regulators, anti-telomerase peptides, or drug treatment, suggesting that the machinery to establish this phenotype is still functional, is of great interest for potential anticancer treatment (even more so in light of the recent results by Karselder and colleagues) ⁽¹³⁸⁾ demonstrating that replicative senescence is not induced by short telomeres but by the disruption of telomeric structures when telomeres get shorter. Therefore, a better understanding of the telomeric structure could lead to ways to disrupt that structure and lead to replicative senescence even in tumor cells. Therefore, one would hope, replicative senescence has reached the end of the in vitro era, and can now progress to the in vivo era.

	Acknowledgments

 
This work was supported by Grant RO1 AG07444 to E. Wang from the National Institute on Aging of the National Institutes of Health. The authors are indebted to Chantal Cossette for preparation of Fig. 1, and to Alan N. Bloch for proofreading this manuscript.

Received March 29, 2002

Accepted April 18, 2002

	References

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