Living on a break: cellular senescence as a DNA ...

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senescence

Living on a break: cellular senescence as a DNA-damage response Fabrizio d’Adda di Fagagna

Abstract | Cellular senescence is associated with ageing and cancer in vivo and has a proven tumour-suppressive function. Common to both ageing and cancer is the generation of DNA damage and the engagement of the DNA-damage response pathways. In this Review, the diverse mechanisms that lead to DNA-damage generation and the activation of DNAdamage-response signalling pathways are discussed, together with the evidence for their contribution to the establishment and maintenance of cellular senescence in the context of organismal ageing and cancer development.

IFOM Foundation — FIRC Institute of Molecular Oncology Foundation, via Adamello 16, 20139 Milan, Italy. e‑mail: fabrizio.dadda@ ifom-ieo-campus.it doi:10.1038/nrc2440

Nuclear DNA is undoubtedly the most precious component of a cell. It is not surprising therefore that any kind of damage that introduces a discontinuity in the DNA double helix elicits a prompt cellular reaction. An evolutionary conserved pathway senses DNA damage and relays this information throughout the cell by engaging a signal amplification cascade that is known as the DNA-damage response (DDR)1. This response has two distinct, but coordinated, functions: it prevents or arrests the duplication and partitioning of damaged DNA into daughter cells to impede the propagation of corrupted genetic information and it coordinates cellular efforts to repair DNA damage and maintain genome integrity. The actions that are devoted to the alteration of cell-cycle progression are known as checkpoint functions and, together with those that are devoted to DNA repair, are collectively known as the DDR. If the DNA damage that is generated in proliferating cells is promptly and properly fixed, cells will quickly resume normal proliferation. By contrast, when DNA damage is particularly severe, cells may undergo programmed cell death (apoptosis), a cellular form of suicide that removes damaged cells from a cell population2. However, an additional outcome is also possible: cells may initiate cellular senescence, a naturally irreversible cell-cycle arrest that is induced by DDR signalling. It is still unclear what determines the choice between apoptosis and senescence, but determinants may include cell type and the intensity, duration and nature of the damage. In contrast to quiescence, senescence cannot be reversed by altering the cellular environment, by removing cell contact inhibition or providing abundant nutriments in vitro. Furthermore, senescence is distinct from terminal differentiation, as it is not the end of a programmed differentiation process.

Senescent cells have been observed in vivo in mammals in association with ageing and with the early phases of tumorigenesis. In this Review, I discuss the current evidence for the involvement of the DDR in the control of cellular senescence.

The DNA-damage response Ruptures of the sugar-phosphate DNA backbone that can lead to the exposure of single-stranded DNA and/or the generation of DNA double-strand breaks (DSBs) are powerful activators of the DDR. These are dangerous events because they compromise the structural stability of chromosomes. Single-stranded DNA and DSBs are sensed by specialized complexes that recruit and activate two large protein kinases, ataxia telangiectasia and Rad3-related (ATR) or ataxia-telangiectasia mutated (ATM), respectively, at the site of the DNA lesion1,3 (FIG. 1). The recruitment of either of these apical kinases to the lesion causes the local phosphorylation in cis of the histone H2AX, which is a key step in the nucleation of the DDR4. At DSBs, γH2AX (the phosphorylated form of H2AX) recruits additional ATM complexes in a positive feedback loop, thereby increasing local ATM activity and consequently fuelling the spread of γH2AX along the chromatin. Crucial to the establishment of this positive feedback loop are mediator of DNA-damage checkpoint 1 (MDC1)5–7 and p53-binding protein 1 (53BP1)8, so-called DDR mediators that facilitate the recruitment of ATM complexes to γH2AX9–12. When single-stranded DNA is exposed instead, the single-stranded DNA-binding protein replication protein A (RPA) binds to it, thereby generating a signal for ATR recruitment13. ATR kinase activity is additionally boosted by the heterotrimeric 9–1–1 complex (composed

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REVIEWS At a glance • Cells respond to the perception of DNA damage by arresting cell-cycle progression and attempting repair: collectively these actions are known as the DNA-damage response (DDR). In mammals, proliferation is not resumed until DNA damage is fixed. • Cellular senescence is a condition in which cells, despite being alive, are unable to proliferate further. This is a stress response, and therefore is different from quiescence or terminal differentiation. • Replicative senescence limits the proliferation of normal human cells. Proliferation that is associated with progressive telomere shortening leads to senescence establishment when critically short telomeres are recognized as DNA damage and trigger a DDR. • Mammalian ageing is associated with the progressive accumulation of senescent cells and DDR accumulation in stem or progenitor cells and more differentiated cells. In the skin of primates, DDR markers associate with the telomeres. • Oncogene activation also causes DDR activation and cellular senescence. Oncogene-induced DNA damage is caused by altered DNA replication, and oncogene-induced senescence is a barrier to cancer. Senescent cells can be observed in vivo in preneoplastic lesions. • Collectively, mounting evidence indicates that senescence, triggered by different stimuli, is the outcome of a protracted DDR.

of RAD9, RAD1 and HUS1)14, a trimer that is structurally similar to proliferating cell nuclear antigen (PCNA; an accessory DNA-replication factor), and by topoisomeraseII-binding protein 1 (TOPBP1), an amplifier of ATR kinase activity15,16. Therefore, DSBs, such as those generated by ionizing radiation, primarily activate ATM, whereas RPA-coated single-stranded DNA that is next to a single-strand to double-strand DNA transition, as generated during perturbed DNA replication, triggers ATR activity. However, resection of one DNA strand at DSBs in cells in the S and G2 phases of the cell cycle provides a suitable substrate for ATR activation and therefore an opportunity for the engagement of both kinases at the same lesion17. An increase of local ATM and ATR activity above a certain threshold is necessary to engage DDR factors that function far from the site of DNA damage. When DNA damage causes this threshold to be exceeded, the checkpoint kinase CHK2 is activated by ATM phosphorylation18; CHK2 then freely diffuses in the nucleoplasm, spreading DDR signalling by phosphorylating its substrates throughout the nuclear space19. Similarly, once CHK1 is activated by phosphorylation, mainly by ATR but also by ATM, it is not retained at DNA lesions and diffuses throughout the nucleus20. Ultimately, checkpoint enforcement results from multiple, often redundant, signalling pathways that converge on key decision-making factors, such as p53 and the cell-division cycle 25 (CDC25) phosphatases. DNA-damage-induced CDC25 inactivation causes a rapid cell-cycle arrest, as these phosphatases are essential for proliferation21. By contrast, slower p53 induction following phosphorylation by DDR kinases22 leads to its stabilization and enhancement of its ability to induce the transcription of p21, a cyclin-dependent kinase inhibitor23, which results in a stable cell-cycle arrest. γH2AX spreads hundreds of kilobases away from the DNA-damage site, which generates a molecular ‘velcro’ that attracts and retains a high number of DDR factors24

and results in the formation of cytologically detectable nuclear foci that contain multiple copies of the same protein (BOX 1). The dimensions of DDR foci range from 0.6 to 1.6 µm, growing in size over time25. They are therefore similar in dimension to some prokaryotic organisms, such as Escherichia coli. The substantial size of these subnuclear domains resembles so-called DNA replication factories, to which they might be similar in structure and function. DNA replication factories are specific regions of the nucleus where different DNA replication forks polymerize nascent DNA. They are thought to promote the optimal use of nuclear space and available DNA replication enzymes and to reconcile different DNA replication events with topological constraints26. Previous studies suggested that DDR foci that were induced by individual DNA breaks could merge27. However, more recent live imaging indicates that DSBs and DDR foci are positionally stable28,29. As the juxtaposition of multiple DSBs in a shared focus can cause mistaken DNA ligation reactions and consequent chromosomal fusions, it would seem wise to separate multiple DSBs rather than bring them together. Therefore, DDR foci represent single DNA lesions where high concentrations of factors are achieved, probably to favour protein–protein interactions and enzymatic reactions and establish feedback loops. Indeed, it has recently been shown that the juxtaposition of DDR sensors alone is sufficient to trigger cell-cycle arrest despite the absence of physical DNA damage30. Once a DNA lesion is repaired, DDR foci are disassembled. This is probably due to the action of both chromatin remodelling machines and the dephosphorylation of γH2AX by dedicated phosphatases24. Therefore, promptly repaired lesions are expected to lead to transient and relatively small foci, whereas harder to repair and more persistent DNA breaks will stimulate more protracted DDR signalling and increased γH2AX spreading and consequently produce visibly bigger foci. Checkpoint enforcement is a direct consequence of the signalling events that occur in DDR foci. This is particularly relevant for the study of the mechanisms that lead to the establishment and maintenance of cellular senescence, as it seems that senescence is associated with large DDR foci and protracted DDR signalling.

Cellular senescence The popular use of transformed cell lines in the laboratory has generated the widespread fallacious perception that checkpoint activation is a transient phenomenon and, unless apoptosis occurs, cell proliferation will inevitably ensue. However, the use of normal and primary cultures shows that the generation of DNA damage can cause a permanent cell-cycle arrest31. The irreversible condition in which damaged cells remain alive but are unable to proliferate is known as cellular senescence32. This cellular phenotype was first observed as the result of in vitro replicative exhaustion of human fibroblasts (replicative senescence)33,34 (BOX 2). However, we now know that senescence can also result from the aberrant activation of proliferative pathways, such as following the expression of activated oncogenes. The robust activation of DDR

nature reviews | cancer

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REVIEWS

DNA-damage sensors

P H2AX

P H2AX

NBS1 RAD50 MRE11 Apical local kinases

DNA-damage mediators (signal boosters)

Downstream diffusible kinases Effectors

RPA

P H2AX P RAD17–RFC H2AX HUS1 RAD9 RAD1 RPA ATRIP

ATM

ATR

MDC1

TOPBP1

53BP1

Claspin

CHK2

CHK1 p53

CDC25

Transient checkpoint

Cellular senescence

Apoptosis

Figure 1 | The DNA-damage response. The DDR is robustly activated by DSBs and/or the exposure of RPA-coated single-stranded DNA. DSBs are sensed by the MRE11– Nature Reviews | Cancer RAD50–NBS1 (MRN) complex130 and the carboxyl (C) terminus of NBS1 recruits the apical protein kinase ATM131. ATM undergoes autophosphorylation, and activated ATM phosphorylates the histone H2A variant H2AX at the site of DNA damage. γH2AX (phosphorylated H2AX) is an epitope that is recognized by a phospho-specific domain of MDC1. MDC1 recruitment to γH2AX fuels the additional accumulation of MRN (to which MDC1 binds), which leads to amplified local ATM activity and the spreading of γH2AX along the chromatin from the DSB. This in turn causes an increase in the local concentration of several DDR factors at the site of DNA damage, thereby generating a positive feedback loop that amplifies ATM activity. It has also been suggested that exposure of modified histone residues further boosts the accumulation of the DNAdamage mediator 53BP1 at the site of DNA damage, in addition to the ability of 53BP1 to bind to MDC1 directly132. Once RPA-coated single-stranded DNA forms, the recruitment of the heterodimeric complex that comprises ATR (a paralogue of ATM) and its DNA-binding subunit ATRIP is favoured. Although a defined feedback loop has not been identified for ATR, its activity is boosted by additional ATR targets, such as the RAD9–HUS1–RAD1 (9–1–1) and RAD17–RFC complexes. In addition, ATR activity is stimulated by TOPBP1, an ATR phosphorylation target that can also activate ATR in the absence of DNA, and claspin, which is necessary for CHK1 phosphorylation. Not all DDR factors accumulate at sites of DNA damage. The protein kinase CHK2 is thought to transiently localize at sites of DNA damage only for enough time to be phosphorylated and activated by ATM, an event that is dependent on the so-called DDR mediators MDC1 and 53BP1. Similarly, although a fraction of CHK1 is chromatin bound in undamaged cells, ATR-dependent phosphorylation of CHK1 allows it to freely diffuse in the nucleus133. Therefore, both CHK1 and CHK2 are responsible for DDR signalling in distant nuclear regions from the DNA-damage site. Finally, p53 and the CDC25 phosphatases are the bottom elements of the DDR signalling cascade that interface this pathway with the core of the cell-cycle progression machinery. p53 induces cell-cycle arrest by activating the transcription of p21, a CDK inhibitor that blocks cell-cycle progression. CDC25 phosphatases are important for normal cell proliferation, as they activate CDKs and cause their DDR-mediated inactivation, by either proteolytic degradation or exclusion from the nucleus. DDR-mediated arrest can be transient, and if DNA damage is effectively removed cells resume normal proliferation. However, if DNA damage is particularly severe, cells may undergo apoptosis or enter a protracted DDR-induced cell-cycle arrest that is termed cellular senescence. 53BP1, p53-binding protein 1; ATM, ataxia-telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related; ATRIP, ATR-interacting protein; DDR, DNAdamage response; CDK, cyclin-dependent kinase; DSB, DNA double-strand break; MDC1, mediator of DNA-damage checkpoint 1; MRE11, meiotic recombination 11; NBS1, Nijmegen breakage syndrome 1; RPA, replication protein A; RFC, replication factor C; TOPBP1, DNA topoisomerase-II-binding protein 1. Figure modified, with permission, from Nature Reviews Molecular Cell Biology (REF. 32)  (2007) Macmillan Publishers Ltd. All rights reserved.

pathways and the generation of DDR foci in senescent cells (also known as senescence-associated DNA-damage foci; SDFs) were recently shown to have a causative role in the establishment and maintenance of both replicative and oncogene-induced senescence (OIS). Replicative cellular senescence. Telomeres, the tips of linear chromosomes composed of repetitive 5′-TTAGGG-3′ DNA sequences, do not activate a DDR and a consequent cell-cycle arrest, despite being physical DNA ends35. This may be due to numerous reasons, such as their chromatin structure and resistance to resection and/or their DNA configuration36, but most importantly because telomere-associated factors inhibit DDR activity in cis. In vitro and in vivo evidence has shown that telomeric repeat-binding factor 2 (TRF2; also known as TERF2), a double-stranded telomeric DNA-binding protein, and protection of telomeres 1 (POT1), a singlestranded telomeric-binding protein, inhibit the checkpoint activity of ATM and ATR, respectively37,38 (FIG. 2). The crucial role of telomere-associated factors was proven by the observation that telomeres trigger a powerful DDR if experimentally ‘uncapped’ by stripping TRF2 or POT1 off telomeric DNA. Owing to an intrinsic peculiarity in the DNA replication apparatus, chromosome DNA ends are incompletely replicated. In the absence of specialized telomere maintenance mechanisms, each DNA replication cycle therefore reduces the number of telomere repeats. Telomere attrition limits the proliferative lifespan of many human cells and causes cells to undergo replicative senescence with short telomeres39. In molecular terms, telomere shortening leads progressively to the loss of telomere-bound inhibitors of ATM and ATR. When telomeres shorten below a threshold length, an unrestrained DDR is activated that is manifested by the appearance of DDR foci in proximity to telomeric DNA and proteins and by the tight association of DDR factors with telomeric DNA in chromatin immunoprecipitation experiments40–42. Importantly, senescence is not determined by the average telomere length within a cell, but by the presence of a few telomeres that are sufficiently short to trigger the DDR42,43. The DDR plays an essential part in both senescence initiation and maintenance. Functional inactivation of CHK2, or TP53 (which encodes p53) and CDKN1A (which encodes p21) gene deletion, extends the proliferation of human fibroblasts in culture beyond their senescence limit44–46. Transient inactivation of ATM, alone or together with ATR, and combined CHK1 and CHK2 inactivation leads to an escape from senescence and re-entry into S phase of the cell cycle41,42. In summary, progressive telomere shortening eventually causes chromosome ends to be recognized as DNA breaks, which consequently activates a DDR and enforces senescence. This attractively simple model of replicative cellular senescence is probably substantially correct, but there are still some gaps in our knowledge. As telomeric attrition is expected to become apparent at the end of the DNA replication process, cells should accumulate and arrest with a nearly 4N DNA content. However, despite

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REVIEWS Box 1 | Origin, structure and importance of DDR foci DNA-damage response (DDR) foci are the sites in cells where DDR signalling originates, and understanding their formation and function is therefore crucial to understanding how DDR activities are exerted. Factors that lie upstream in the DDR signalling cascade and function close to the DNA-damage site are constituents of DDR foci. Downstream factors, such as CHK1, CHK2, p53 and CDC25, do not take part. Importantly, in the absence of H2AX, the initial recruitment of ATM (ataxiatelangiectasia mutated) to DNA discontinuities is unaffected. However, the formation of visible foci through immunofluorescence microscopy of activated ATM and other DDR factors is disrupted, which highlights the difference between their initial recruitment to the DNA lesion and their subsequent accumulation in foci that depends mainly on phospho-specific protein–protein interactions123. Photobleaching experiments suggest that foci are stable sites of dynamic accumulation of DDR factors19. Importantly, continuous activity of upstream kinases is essential for their stability because, at least in the context of cellular senescence, transient inhibition of ATM kinase activity disrupts their detection51. This suggests that the maintenance of upstream kinases is the result of continuous kinase activity and therefore their detection reveals the presence of as-yet-unrepaired DNA damage that fuels ATM kinase activation. The high local concentration of DDR factors at damage sites also provides a conveniently robust and specific set of markers for the detection of an activated DDR. Staining DNA-damaged cells with antibodies against γH2AX or other DDR factors that accumulate at DNA-damage sites generates a distinctive nuclear pattern of discrete, bright foci. As foci detection reveals the number and position of the DNA lesions within a cell, immunocytological staining is generally considered a highly sensitive and informative approach. Furthermore, because immunocytological staining can detect even a single focus, it can provide us with information about the activation of a cellular response at the single-cell level. This is particularly useful if DDR activation occurs in a small fraction of cells and in histologically complex tissues. However, although DDR-foci detection is generally accepted as proof of DNA breaks, it should be noted that DDR foci are a read out of DDR, not of physical DNA damage. Therefore, a cell can experience DNA damage, even in the absence of any detectable DDR focus, if upstream DDR signalling pathways are impaired. The contraposition also holds true: a few DDR factors accumulate in the absence of DNA damage in discrete subnuclear structures that resemble foci. However, the investigator must take care not to be misled; for example, by components of the MRE11–RAD50–NBS1 complex that reside in promyelocytic leukaemia protein bodies124 and γH2AX that associates with silenced XY bodies125.

some variability, most senescent cells tend to display a prominent 2N peak when analysed by fluorescenceactivated cell sorting47. This suggests the enforcement of a G1/S checkpoint, rather than the expected late‑S or G2/M checkpoint. A number of possible explanations can be postulated. For example, telomere erosion may take place at the next cell cycle in G1 by one or more DNA exonucleases that function following the completion of DNA replication. Indeed, evidence for active telomere remodelling has been reported48. Alternatively, the G1/S checkpoint may be more sensitive to exposed DNA ends than the G2/M checkpoint. It was recently suggested that the G2/M checkpoint has a higher activating threshold and that nearly 20 DSBs are needed for its enforcement49. Conversely, it has been proposed that a single unrepaired break leads to the enforcement of a G1/S checkpoint50. When analysed cytologically, senescence induced by telomere attrition is usually characterized by less than 20 SDFs and therefore a less stringent G2/M checkpoint might explain the accumulation in G1. The presence of multiple SDFs in most senescent cells — less than 20 but more than 1 (Ref. 51) (F.d’A.d.F, unpublished observations) — raises other questions.

Given the heterogeneity in telomere length among chromosomes and telomere alleles52,53, it is likely that one single telomere becomes critically short and triggers a DDR before the others. If so, why do senescent cells tend to show more than one SDF? This observation could be explained by either the simultaneous fall below a threshold length of more than one telomere at the end of the same round of DNA replication (which is unlikely) or by the appearance of telomeres in separate successive cell cycles. This would imply that a single exposed telomere is not enough to trigger an effective checkpoint and cells can proliferate despite the presence of an exposed DNA end. It should be noted, however, that when bona fide senescent cells in the skin of ageing primates were analysed in vivo, they displayed a single SDF at a single telomere54,55, suggesting that a tighter checkpoint response might be enforced in vivo than in vitro. Thus, the question of how many telomeres are required to trigger senescence remains open for debate. Related to the issues raised above is the ability of cells to repair exposed telomere ends. DNA ends can be repaired by two main repair pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). Although HR has been reported to elongate telomeres in some transformed cell lines, known as ALT (alternative lengthening of telomere) cells 56, this pathway is suppressed by telomere-associated factors in normal cells57. Widespread telomeric fusions have been observed in mice that lack TRF2, which indicates that NHEJ is able to repair uncapped telomeres58. However, the poor efficacy of this mechanism in cells that are undergoing telomere attrition could be explained by the observation that DSBs in mammals occupy a relatively fixed position in the nucleus and therefore cannot roam the nuclear space in search of a DNA fusion partner. It should also be considered that if only one telomere is critically short in a cell, it would not be able to undergo NHEJ, because of the lack of a fusion partner. In addition, recent in vitro evidence has shown that telomeric DNA is intrinsically less amenable to in vitro DNA ligation by NHEJ in a manner that is dependent on the binding of TRF2 and its associated factors59. The model of cellular senescence induced by telomere attrition is based on the assumption that a telomere is recognized as a DSB when it shortens below a threshold length. Indeed, SDFs tend to co-localize with the shortest telomeres 60. However, the number of telomeric repeats below which a DDR is triggered remains unclear. DNA sequence analysis of telomere fusion points that result from the ligation of two chromosome ends with critically short telomeres suggests that telomeric DNA tracts are shorter than 12.8 repeats61. However, in senescent cells, DDR foci co-localize with telomeric DNA, as detected by peptide nucleic acid (PNA) probes42. This is usually regarded as proof of telomere involvement in DDR signalling in senescent cells. However, the observation that DDR markers associate with detectable telomeric DNA tracts suggests that these telomeres are unlikely to be only a few repeats long, because the detection limit of PNA probes is 150–200 base pairs on metaphase chromosome spreads (M.P. Hande, personal



REVIEWS Box 2 | Are my cells really arresting because of telomere shortening? The field of cellular senescence began with the seminal observations of Leonard Hayflick in 1961, who discovered that human foetal lung fibroblast cell lines, such as WI38, did not exhibit unrestrained proliferative capacity as many thought at the time, but instead were capable of only a limited and reproducible number of population doublings (cell divisions applied to bulk populations of cells) before entering a prolonged state of proliferative arrest that he termed replicative senescence33,34. Subsequent work by other researchers who used a different set of human fibroblast cell lines indicated that telomere attrition could be the cause of replicative senescence39. This was finally demonstrated when forced expression of telomerase, an enzyme that adds telomere repeats at the end of chromosomes and therefore counteracts natural telomere shortening, provided the cells with unlimited proliferative capacity126. However, we now know that the expression of telomerase is not sufficient to immortalize all human fibroblast cell lines127. WI38 and other cell lines undergo premature senescence as the result of a so-called ‘culture shock’: a combination of oxidative stress, artificial growth conditions and excessive mitotic stimulation that leads to cellular stress and senescence. Culture-shockinduced senescence has also been observed when keratinocytes are grown in the absence of a layer of feeder cells128 and mouse embryo fibroblasts are cultivated at atmospheric oxygen tension129. Ironically, it could therefore be proposed that the entire cellular senescence field stemmed from an artefactual observation that was caused by suboptimal in vitro cell-growth conditions. In conclusion, the question of whether cells are arresting because of telomere shortening or because of stressful culture conditions can be easily addressed experimentally: if the expression of TERT (telomerase reverse transcriptase) immortalizes a particular cell line, telomere attrition is the cause of the observed limited proliferation and senescence. By contrast, if cells senesce despite the expression of telomerase, other causes that are unrelated to telomere attrition are forcing cells to stop proliferating.

communication) and probably lower in interphase cells. Therefore, the threshold length below which a DDR is triggered at a telomere is unclear. Oncogene-induced cellular senescence. Oncogene activation is a hallmark of cell transformation and cancer. However, oncogene activation in a normal cell does not lead to cell transformation, but instead induces cellular senescence62. OIS is therefore a tumour suppressive mechanism that impedes the proliferation of a cell that expresses high levels of an aggressive oncogene. Importantly, this type of senescence is established independently of any telomere attrition or dysfunction63. It has recently been observed that cellular senescence, which is mediated by many different oncogenes and signalling factors (see Table 1 for a comprehensive list of senescence-inducing genes), is associated with DDR activation64–71. Indeed, aberrant activation of signal transduction pathways and positive cell-cycle regulators can lead to the accumulation of DNA damage, full engagement of the DDR cascade and appearance of SDFs. Time-course studies have revealed that expression of the oncogenic form of HRAS leads to a biphasic response72 (FIG. 3). Ras drives an initial hyperproliferation phase in which cells proliferate faster than control cells. However, this burst of proliferation is transient and is quickly followed by a proliferation slow down and the eventual establishment of cellular senescence72. This biphasic response has also been observed following the expression of other oncogenes, such as BRAF, E2F1 and MYC71,73–76. DDR activation coincides in time with the end of hyperproliferation and entry into senescence. Thus, DDR activation blunts oncogene-driven hyperproliferation and enforces senescence. Consistent with this model, DDR activation has a causative role both in senescence establishment and maintenance. Inactivation of key DDR gene products leads to senescence avoidance and cell proliferation and allows cell transformation64,65,70. When OIS is established, transient inactivation of individual DDR genes allows

the restart of DNA replication and cell proliferation64. These observations provide a mechanistic explanation for the known tumour-suppressive role of DDR checkpoint genes77. Interestingly, the engagement of the DDR pathways is subtly different in telomere attrition-induced and OIS. Telomere attrition mainly triggers ATM activation, with ATR engagement being detectable only in the absence of ATM42. Conversely, ATR, its DNA-binding subunit ATRIP (ATR interacting protein) and RPA, together with RPA phosphorylation and increased chromatin association, have been detected in response to OIS64,65. Although much remains to be learned about the mechanisms that lead to DNA-damage generation and DDR activation following oncogene activation, oncogeneinduced DDR activation and senescence have been proposed to result from altered DNA replication. The best evidence that links an oncogene to DNA replication regulation is the recent discovery that MYC binds to DNA replication origins and activates the replication machinery. However, this is associated with the accumulation of DNA damage and activation of a DDR76. Furthermore, oncogenic HRAS expression impacts on DNA replication and increases the number of simultaneously active DNA replication origins64. Increased DNA-replication-origin usage caused by oncogenic HRAS is associated with increased rates of fork stalling and reduced symmetry between DNA replication forks that depart from the same origin64. This suggests that increased origin activity comes at the expense of reduced processivity and smooth fork progression. Similar observations have been made following cyclin E overexpression65. Although still unproven, it is conceivable that excessive origin firing triggers a DDR by depleting limiting DNA replication factors or by generating regions of single-stranded DNA, which activate the ATR-dependent checkpoint. This straightforward interpretation has recently been challenged by the demonstration that DNA-damage-induced stalling of

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REVIEWS ATM

TRF2 ATRIP ATR

5′

POT1 3′

Figure 2 | DDR inhibition at telomeres. Telomeric Reviews | Cancer repeat-binding factor 2 (TRF2) is aNature double-stranded telomeric DNA-binding protein that is necessary to maintain the capping functions of telomeres. Protection of telomeres 1 (POT1) is a single-stranded DNA-binding protein that is necessary for efficient telomere elongation by telomerase and inhibition of telomeric DNA recombination134,135. Both proteins contribute to the prevention of DDR activation at the telomeres. It has been shown that TRF2 can directly bind to the kinase domain of ATM (ataxia-telangiectasia mutated) and inhibit its activity37, whereas POT1 can inhibit ATR (ataxia telangiectasia and Rad3-related) activation and POT1 inactivation allows ATR activation136. Although the precise mechanism (or mechanisms) of action of POT1 on ATR activity is unclear, it is likely that POT1 inhibits ATR activation by preventing it from binding to telomeric single-stranded DNA. ATRIP, ATR-interacting protein.

replication forks induces the firing of otherwise dormant origins78. Therefore, the observed higher number of active origins might follow DNA damage, rather than cause DNA damage. More research is needed to clarify this point. Re-firing of the same origin before chromosome segregation64 — that is, DNA re-replication — is an additional alteration of the DNA replication pattern that can be induced by oncogene activation (although so far it has only been reported for HRAS-expressing cells). It has previously been shown that experimentally induced DNA re-replication is sufficient to generate DSBs and trigger a DDR79. How activated Ras alters DNA replication is currently unknown, although Ras expression increases CDC6 levels72. CDC6 is a positive regulator of DNA-replication-origin activity and its experimental overexpression is sufficient to generate DNA damage and trigger a robust DDR and senescence65,79. Therefore, although still unproven, it is possible that aberrantly active signal transduction pathways can induce genome instability in a manner that is dependent on core DNA replication regulators. Importantly, Ras activation or MYC expression in cells that are unable to enter S phase does not trigger DDR activation64,76. These results, together with the observation that activated DDR markers localize at sites of ongoing DNA replication in oncogene-expressing cells65, indicate that oncogene-induced DDR activation is a strictly DNA-replication-dependent event.

So far, the exact structure and genomic locations of DNA lesions that are induced by oncogene activation and lead to DDR activation remain unknown. However, when studied in senescent cells, DNA damage that is generated by oncogenes does not seem to be randomly distributed. Both in in vitro cell-culture systems and in human tumours, chromosomal fragile sites are distinctly unstable compared with the rest of the genome64,80,81. Fragile sites are notoriously hard-to-replicate regions of the chromosomes and their observed instability is consistent with the altered DNA replication patterns that are observed in oncogene-expressing cells, the involvement of ATR in fragile-site stability and ATR activation in these cells64.

The role of heterochromatin A distinguishing feature of cellular senescence, which is most apparent following oncogene activation, is the development of global genome heterochromatinization72,82. Senescent cells display a characteristic pattern of nuclear DAPI (4′,6-diamidino-2-phenylindole) staining in which chromosomes appear to be individually compacted in structures known as SAHFs (senescence-associated heterochromatin foci). This mechanism has been proposed to be exploited by the cell to prevent transcription of E2F target genes, which are associated with cell-cycle entry and proliferation83. It remains unclear how the DDR and global heterochromatinization relate to each other. It has recently been observed that activated CHK1 is a chromatin modifier that mediates transcriptional repression of some genes84 and therefore the potential direct contribution of DDR signalling in transcriptional silencing in senescent cells cannot currently be excluded. In principle, it is conceivable that the DDR can impact SAHF formation. Indeed, at sites of DNA damage, a number of chromatin modification events occur85. However, most seem to lead to chromatin relaxation86,87. In vivo imaging shows that DNA breaks lead to a local chromatin expansion29, an effect that is dissimilar to the chromatin compaction that is observed in senescent cells. Therefore, our present knowledge of the impact of DNA-damage generation and DDR activation on chromatin suggests that SAHF formation is an event that occurs in senescent cells independently from the generation of DNA damage. It is still possible, however, that low but protracted levels of endogenous DNA damage have long-range and belated consequences that are different from the local and immediate, possibly transient, outcomes of the generation of exogenous DNA damage. It could also be speculated that SAHF formation can result from a cellular attempt to counteract DNA-damage-induced chromatin relaxation and DDR activation. Cellular senescence in vivo in ageing and cancer Although much of our knowledge on the mechanisms that lead to the establishment of cellular senescence has been gained from in vitro experiments, evidence for the presence of senescent cells in vivo in the context of organismal ageing or tumorigenesis is mounting.



REVIEWS Table 1 | Cellular senescence-inducing genes Gene

Refs

Mitogenic signalling EGFR

M. Schartl, personal communication

PTEN

102

NF1

137

RAS

64,138

RAC1 RAF

66 139

MOS

65

MEK

140

β-catenin

Cytokine signalling

67

TGF‑β

141

IFN‑β

69

SMURF2 STAT5 PML

Cell-cycle regulators

142 70 143

E2F

75

Cyclin E

65

MYC

71

CDC6

65

CDT1

144

CDC6, cell-division cycle 6; CDT1, chromatin licensing and DNA replication factor 1; EGFR, epidermal growth factor receptor; IFN-β, interferon-β; MEK, MAPK kinase; NF1, neurofibromin 1; PML, promyelocytic leukaemia; PTEN, phosphatase and tensin homologue; RAC1, Ras-related C3 botulinum toxin substrate 1; SMURF2, SMAD-specific E3 ubiquitin protein ligase 2; STAT5, signal transducer and activator of transcription 5; TGF-β, transforming growth factor-β.

The discovery of increased levels of lysosomal β‑galactosidase in senescent cells88,89 provided the first marker for their detection in vivo. The fraction of senescence-associated β‑galactosidase-positive cells increases with age in the skin of human healthy individuals88. The progressive increase of cells that have entered senescence, presumably following replicative exhaustion, is thought to make an important contribution to the ageing phenotype by impairing tissue homeostasis90. More recently, molecular evidence of the accumulation of senescent cells in vivo during ageing was provided by the observed increase in the relative number of DDR-positive cells in the skin of baboons with age54,55. These cells exhibit DDR foci that contain γH2AX and 53BP1 and co-localize with telomeric DNA, which is consistent with telomere attrition and replicative exhaustion. In old baboons, nearly one of every three skin fibrocytes contained DDR foci, which highlights the wide impact that DDR activation has in this tissue. Senescence-associated β‑galactosidasepositive detection was not reported for the same samples.

However, the presence of DDR foci at telomeres and the increased detection of heterochromatin markers, which are typical of cells that are undergoing senescence, suggest they are indeed senescent. Therefore, it seems that DDR activation and senescence in tissues become widespread during ageing, although it will be important to extend these observations to more tissues and other species. More recently, stem cells have been shown to accumulate DNA damage with age. It was observed that DDR foci accumulate in haematopoietic stem cells from ageing wild-type mice91 and that NHEJ impairment dramatically increases their appearance92. Little is known about the way in which stem cells respond to DNA damage and, at least in mouse embryonic stem cells, p53 and some crucial checkpoint kinases, such as CHK2, may not be functional93,94. Clearly, if confirmed and extended to somatic stem cells, this could have important consequences for the potential impact that oncogene activation has on this cell compartment: if the DDR is partially impaired in these cells, the tumour suppressive functions of cellular senescence might not be effective. Whether stem cells can undergo cellular senescence has not been established. However, p16 (encoded by CDKN2A), a crucial mediator of the senescence process under many, but not all, settings95, is increased in the stem-cell compartments of different mouse organs during ageing96–98 and p16 inactivation rescues the proliferative impairment that is associated with ageing. Although polycomb factors that regulate CDKN2A transcription respond to DNA damage99, a clearly established connection between the DDR and p16 induction is missing. Overall, the available evidence indicates that DDR activation, and probably senescence, becomes progressively detectable in stem and differentiated cells of ageing organisms. In the context of tumorigenesis, the presence of senescent cells in the early phases of the neoplastic process has been observed by several groups in different tumours of different histotypes. Reports that were either based on transgenic mice67,73–75,100–102 or on the analysis of human tumour samples65 have shown that cells that express senescence markers accumulate in the initial stages of tumour formation. DDR markers in early cancer lesions associate with senescence-associated heterochromatin markers and senescence-associated β‑galactosidase staining, which indicates that DDR-associated senescence occurs in vivo in the early pre-neoplastic or dysplastic phase of tumorigenesis65,103 before rampant genomic instability is detected104. When DDR activation was studied in different tumour types throughout different disease stages — lung squamous-cell carcinoma, non-small cell lung cancer, colon adenomas and bladder cancer — DDR markers peaked in the early dysplastic precancerous stage and were progressively lost during cancer progression80,103,104. These observations are consistent with a model in which DDR activation and senescence represent an initial barrier to oncogene-induced proliferation, and cancer progression depends on the bypass of this barrier. Mounting evidence points to DDR inactivation by genetic mutation of DDR genes in advanced cancers, and ATM is one of the protein kinase genes that most frequently carries driver mutations in human somatic cancers105. The observation of a

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Total number of cells

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∞ Oncogenic Ras Empty vector Hyperproliferation

Time following oncogene activation

Slow down

Senescence DDR activation

Figure 3 | Ras expression leads to a biphasic response. Expression of the activated, Nature Reviews | Cancer and therefore oncogenic, form of HRAS (HRAS-V12) in normal fibroblasts leads to an initial burst of proliferation that causes Ras-expressing cells to proliferate faster than normal cells. This is in keeping with the mitogenic functions of this signal transducer molecule. However, hyperproliferation leads to cell-cycle slow down and, eventually, to senescence establishment. DNA-damage-response (DDR) activation coincides with cell-cycle slow down and accompanies senescence throughout its maintenance.

selective pressure in somatic cells for the inactivation of DDR genes during cancer progression is consistent with known cancer predisposition in patients and mouse models that carry germline mutations which impair DDR gene functions77,106. However, it should be noted that most DDR genes can control not only the induction of senescence but also apoptosis, another powerful tumour-suppressor mechanism. It should also be noted that tumours do not always engage the DDR machinery to the same extent. Although it is not completely clear what determines the impact of DDR activation, it has recently been observed that colorectal adenomas which overexpress the proto-oncogene cyclin D1 (CCND1) are less prone to the display of markers of DDR activation compared with those that express cyclin E107. Finally, the fate of senescent cells in vivo remains unclear. In specific experimental settings, it was observed that re-expression of p53 in sarcoma and liver carcinomas108,109 leads to senescence establishment, which is followed, in the liver, by clearance of senescent cells in a manner that is dependent on the innate immune system, a biological system that is devoted to the removal of infectious and potentially hazardous cells. However, skin naevi (moles) — clones of cells that have undergone senescence following oncogene activation73 — are apparently stable for many years and do not seem to be cleared. As DDR activation stimulates the activation of the innate immune system110, it would be interesting to learn whether DDR activities contribute to the clearance of senescent cells in vivo. The link between cellular senescence and molecules of the immune system seems tighter and more complex than expected. OIS is associated with a secretory phenotype that comprises a number of cytokines32, some of which seem to positively regulate senescence by controlling both DDR activities and SAHF formation111,112 (M. Bonafé, personal communication).

Open questions In summary, an element that is common to both replicative and oncogene-induced cellular senescence is the induction of the DDR. Senescence is a permanent condition, and DDR signalling is required for both its initiation and its maintenance. Despite the recent progress in our understanding of the molecular mechanisms that trigger the DDR and senescence establishment, and the identification of senescent cells in vivo and appreciation of their roles, a number of crucial questions remain open. The distinguishing element (or elements) between the transient activation of a DDR that allows recovery of cellcycle progression and the activation of a DDR that leads to senescence remains elusive. The generation of DNA damage above a threshold amount cannot be a determining element, as senescent cells display relatively few SDFs. However, it is worth noting that SDFs are usually larger than DDR foci, which are formed immediately after the generation of exogenous DNA damage. As DDR foci grow in size with time, and because SDFs can be observed years after the establishment of replicative senescence in some cell types of human primary fibroblasts, despite some earlier reports113 (F.d’A.d.F., unpublished observations), it is likely that SDFs arise from lesions that are not promptly repaired. Therefore, it seems that persistent DDR signalling is a distinguishing feature of cellular senescence and, possibly, an important contributor to the decision of a cell to undergo senescence. This leads to the question of why the DDR remains sustained and is not inactivated. DDR could remain activated because of either a deficiency of DDR inactivation despite efficient DNA-damage repair, or because of an inability to repair DNA damage. Indeed, not all DSBs are repairable with similar kinetics and some specialized enzymes, such as Artemis (also known as DCLRE1C), facilitate the repair of hard-to-repair ‘dirty’ DNA ends114. In addition, there might also be regions within the nucleus where DNA damage is less easily repaired, possibly in a manner that is also affected by chromatin structure. More work is needed to discriminate between these possibilities. As senescence can be a stable condition and activated DDR signalling does not seem to be reduced with time, it seems that, in contrast to the yeast Saccharomyces cerevisiae, human cells do not adapt to DNA damage. In S. cerevisiae, adaptation to DNA damage involves the inactivation of DDR kinases and resumption of proliferation despite DNA damage115. The tighter checkpoint activation in human cells makes sense if we consider the potentially devastating consequences of a ‘loose’ checkpoint in multicellular organisms that are under the constant threat of developing cancer. However, as discussed in the previous sections, the presence of multiple SDFs raises some important questions about the complete lack of adaptation in cultured mammalian cells. SDFs and transient DDR foci share many components, but it is unclear whether there are unique factors that belong exclusively to only one component. If such factors exist, their identification would shed light on the molecular mechanisms that control foci formation and



REVIEWS provide researchers with a set of powerful markers to study their presence, especially in in vivo settings. In addition, it is currently hard to distinguish SDFs of different origins, particularly in vivo. Telomere attrition, oncogene activation and additional DNA-damage-generation events that are associated with inflammation 103 or exogenous DNA damage can all induce DDR foci. Identification of proteins, or their modified forms, that are specifically associated with only one stimulus would provide researchers with a powerful tool to dissect the individual contribution of distinct events to DDR activation. This is especially relevant in cancer, for which different genotoxic events might contribute to DDR activation within the same tumour mass. The recent demonstration that telomere shortening is a tumoursuppressive barrier in vivo that functions by limiting the proliferation of oncogene-expressing cells116,117 indicates that both telomere shortening and oncogene-induced altered DNA replication can induce DDR activation and senescence in the same tumour setting. In the context of animal ageing, cellular senescence is generally considered to be a pro-ageing event, and p53 and the INK4 locus are recognized as positive modulators of senescence32. However, it has recently been reported that mice that express an extra endogenous copy of Trp53 and the INK4 locus (which expresses the tumour suppressors INK4a, INK4b and ARF) exhibit an extended median lifespan118. This unexpected result contrasts with the notion that these tumour-suppressor genes enforce senescence. In these transgenic mice, DDR-positive cells are reduced. At least two interpretations can be proposed: DNA-damage generation is reduced in these mice or damaged cells are removed more efficiently. It is therefore possible that augmented p53 signalling removes senescent cells and that this has a positive impact on lifespan. ARF is a tumour suppressor that is thought to function by positively regulating TP53 induction following Shiloh, Y. The ATM-mediated DNA-damage response: taking shape. Trends Biochem. Sci. 37, 402–410 (2006). Taylor, R. C., Cullen, S. P. & Martin, S. J. Apoptosis: controlled demolition at the cellular level. Nature Rev. Mol. Cell Biol. 9, 231–241 (2008). 3. Zou, L. Single- and double-stranded DNA: building a trigger of ATR-mediated DNA damage response. Genes Dev. 21, 879–885 (2007). 4. Celeste, A. et al. Genomic instability in mice lacking histone H2AX. Science 296, 922–927 (2002). 5. Lou, Z., Minter-Dykhouse, K., Wu, X. & Chen, J. MDC1 is coupled to activated CHK2 in mammalian DNA damage response pathways. Nature 421, 957–961 (2003). 6. Goldberg, M. et al. MDC1 is required for the intra‑S‑phase DNA damage checkpoint. Nature 421, 952–956 (2003). 7. Stewart, G. S., Wang, B., Bignell, C. R., Taylor, A. M. R. & Elledge, S. J. MDC1 is a mediator of the mammalian DNA damage checkpoint. Nature 421, 961–966 (2003). 8. Abraham, R. T. Checkpoint signalling: focusing on 53BP1. Nature Cell Biol. 4, E277–E279 (2002). 9. Lukas, C. et al. Mdc1 couples DNA double-strand break recognition by Nbs1 with its H2AX-dependent chromatin retention. EMBO J. 23, 2674–2683 (2004). 10. Stucki, M. et al. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell 123, 1213–1226 (2005). 11. Lou, Z. et al. MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals. Mol. Cell 21, 187–200 (2006). 1.

2.

oncogenic stress119. ARF has an important role in the establishment of senescence in mice, and oncogenic Ras-induced senescence is abolished in ARF-inactivated MEFs120. However, which primary stimulus triggers ARF accumulation upon oncogenic expression is currently unclear. Although ARF is not generally considered a canonical DDR effector, it has not been excluded that altered DNA replication and consequent DDR signalling of the type that is induced by activated oncogenes have an impact on ARF accumulation in mouse cells. Finally, escape from senescence in vivo has yet to be proven. This is particularly relevant in cancer, as it is unknown whether increased cell proliferation of more advanced tumours is the result of the expansion of a small fraction of cells that never entered senescence because they carry a mutation in senescence-enforcing genes or whether it is the result of an escape from senescence that is achieved through mutations incurred during the senescence state. In this regard, accumulation of DNA mutations in non-proliferating cells in vivo has been observed121. In conclusion, DNA-damage generation and activation of the DDR is a proven cause of cellular senescence. However, caution should be taken when considering cellular senescence as being uniquely caused by DNAdamage and activation of a DDR. Recently, it was shown that triggering ATR activation in the absence of physical DNA damage is sufficient to induce cellular senescence in cultured cells122. Therefore, senescence might be caused by the activation of upstream DDR kinases, regardless of the initial trigger. Extending this concept speculatively, it is also conceivable that other pathways, which are distinct from canonical DDR signalling and are not triggered by DNA damage yet engage some elements of the DDR cascade, could also generate a senescence phenotype. In the words of the American journalist H.L. Mencken: “For every complex problem, there is a simple solution…and it is wrong.”

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Acknowledgements

F.d’A.d.F. is supported by the Associazione Italiana per la Ricerca sul Cancro, the Association for International Cancer Research, the Human Frontier Science Program, the FP6 and FP7 program of the European Union and the EMBO Young Investigator Program.

DATABASES Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene 53BP1 | ATM | ATR | ATRIP | BRAF | CCND1 | CDC6 | CDKN1A | CDKN2A | CHK1 | CHK2 | DCLRE1C | E2F1 | H2AX | HRAS | MDC1 | MYC | PCNA | POT1 | TERF2 | TOPBP1 | TP53 National Cancer Institute: http://www.cancer.gov/ Bladder cancer | lung squamous-cell carcinoma | non-small cell lung cancer | sarcoma

FURTHER INFORMATION Fabrizio d’Adda di Fagagna’s homepage: http://www.ifomieo-campus.it/research/dadda.php All links are active in the online pdf

522 | july 2008 | volume 8
