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Human cell senescence as a DNA damage response Article in Mechanisms of Ageing and Development · February 2005 DOI: 10.1016/j.mad.2004.09.034 · Source: PubMed
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Mechanisms of Ageing and Development 126 (2005) 111–117 www.elsevier.com/locate/mechagedev
Human cell senescence as a DNA damage response T. von Zglinickia,*, G. Saretzkia, J. Ladhoffa, F. d’Adda di Fagagnab, S.P. Jacksonc a
Henry Wellcome Biogerontology Laboratory, Newcastle University, Newcastle upon Tyne NE4 6BE, UK b IFOM Foundation – The FIRC Institute of Molecular Oncology Foundation, Milan, Italy c Wellcome Trust and Cancer Research UK Gurdon Institute, Department of Zoology, Cambridge University, Tennis Court Road, Cambridge CB2 1QR, UK Available online 20 October 2004
Abstract It has been established that telomere-dependent replicative senescence of human fibroblasts is stress-dependent. First, it was shown that telomere shortening, which is a major contributor to telomere uncapping, is stress-dependent to a significant degree. Second, the signalling pathway connecting telomere uncapping and replicative senescence appears to be the same as the one that is activated by DNA damage: uncapped telomeres activate signalling cascades involving the protein kinases ATM, ATR and, possibly, DNA-PK. Furthermore, phosphorylation of histone H2A.X facilitates the formation of DNA damage foci around uncapped telomeres, and this in turn activates downstream kinases Chk1 and Chk2 and, eventually, p53. It appears that this signalling pathway has to be maintained in order to keep cells in a senescent state. Thus, cellular senescence can be regarded as a permanently maintained DNA damage response state. This suggests that antibodies against DNA damage foci components might be useful markers for senescent cells in vivo. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Telomeres; Senescence; DNA damage; Histone; H2A.X; Aging
1. Telomeres and replicative senescence Cellular senescence has been defined by Hayflick (Hayflick and Moorehead, 1961) as the ultimate and irreversible loss of replicative capacity occurring in primary somatic cell culture. These authors found that cell growth was reproducibly blocked after a fairly well-defined number (under constant culture conditions) of population doublings (PD), and this suggested the idea of a ‘biological clock’ or, more specifically, a replication counter (for review, see (Hayflick, 2000)) that counts biological time in numbers of cell divisions, and after a reproducible number of divisions triggers signalling pathways that block cellular division. Several different processes have been suggested as possible clocking mechanisms (Boukamp, 2003), but telomere uncapping (Blackburn, 2000) is by far the best established and most extensively investigated of these. In 1990 it was * Corresponding author. Tel.: +44 191 256 3310; fax: +44 191 256 3445. E–mail address:
[email protected] (T.v. Zglinicki).
shown that as human primary fibroblasts are cultured towards the end of their replicative lifespan, telomere length gradually decreases (Harley et al., 1990). When it was demonstrated that the activity of telomerase, the enzyme that counteracts telomere shortening by re-elongating telomeres, is strong in immortal cells but essentially absent from human somatic cells and that human primary fibroblasts can be immortalised by transfection of hTERT, the catalytic subunit of human telomerase (Bodnar et al., 1998), telomere attrition was firmly established as a trigger for cellular senescence (Hayflick, 2000). It is believed that telomere shortening destabilizes telomeric loops (Griffith et al., 1999) and thus increases the probability of telomere uncapping.
2. Telomere-dependent senescence is stress-dependent The telomere-driven checkpoint is not the only one that has been shown to be capable of inducing senescence-like growth arrest. Human epithelial cells, for instance, encounter
0047-6374/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2004.09.034
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a telomere-independent, p16-dependent growth arrest that might be triggered in response to suboptimal culture conditions (Stampfer and Yaswen, 2003). Furthermore, over-expression of oncogenes, such as activated RAS or RAF, can induce a senescence-like arrest in primary human or mouse cells (Lin et al., 1998; Dimri et al., 2002; Ferbeyre et al., 2002) in a p16-dependent manner (Benanti and Galloway, 2004). Modification of chromatin towards a more decondensed structure, for instance by inhibitors of histone deacetylases, also induces a senescent phenotype (Ogryzko et al., 1996). Last but not least, DNA-damaging stresses have been shown to induce cellular growth arrest. Such stresses include various types of radiation (Herskind and Rodemann, 2000), drugs generating DNA double-strand breaks (Robles and Adami, 1998) and different ways of generating oxidative stress, for example by increasing the ambient oxygen tension (Balin et al., 1977; von Zglinicki et al., 1995) and treatment with hydrogen peroxide (Chen and Ames, 1994; Frippiat et al., 2001) or butylhydroperoxide (Dumont et al., 2000). Notably, such growth arrest can be induced essentially immediately by acute intense stress (Chen et al., 2001; Gorbunova et al., 2002) or more slowly under chronic or semi-chronic treatment regimens (Balin et al., 1977; von Zglinicki et al., 1995; Dumont et al., 2000), thus suggesting stress-induced growth arrest being a premature form of senescence (Toussaint et al., 2000). There is now a broad consensus to use the term premature senescence (or stress-induced premature senescence SIPS) for those senescence-like growth arrests that are stress-dependent and telomere-independent. Phenotypic markers of senescence, from cellular morphology, and expression of senescence-associated b-galactosidase activity (Dimri et al., 1995) to expression of senescence-associated genes at the mRNA (Saretzki et al., 1998; Frippiat et al., 2001) or protein level (Dierick et al., 2002), have shown to be significantly overlapping if not identical between telomere-dependent replicative senescence and stress-induced premature senescence. Importantly, telomere shortening is in itself strongly influenced by cellular stress. Thus, the average telomere length decreases faster under elevated oxidative stress (von Zglinicki et al., 1995; Adelfalk et al., 2001) and is better maintained under low-stress conditions (von Zglinicki et al., 2000; Forsyth et al., 2003; Saretzki et al., 2003; Serra et al., 2003) due to a telomere-specific deficiency in DNA single-strand break repair (Petersen et al., 1998; Sitte et al., 1998). Telomere shortening rates vary in a stochastic manner between cells in single human fibroblast clones, and this has been related to the well-known sub-clonal heterogeneity in replicative lifespan (Smith and Whitney, 1980; Thomas et al., 1997), because those cells among the progeny of a single clone that develop a senescent phenotype early have telomeres that are significantly shorter than those in their still cycling counterparts (Martin-Ruiz et al., 2004). Thus, increased stress accelerates replicative, telomere-dependent senescence. In addition, even under the most favourable conditions one might expect that cellular stress will make a contribution towards the rate of progress towards senescence.
The results described above have some interesting consequences: first, they help to clarify the distinction between SIPS and (accelerated) replicative senescence insofar as the first is telomere-independent and the latter is telomere-dependent. Second, they suggest that telomeredriven replicative senescence is a stress response in essentially all-practical cases (von Zglinicki, 2002). Third, they cast serious doubts on the concept of replicative senescence as a stepwise and ’clocked’ process, and instead suggest that the observed reproducibility of both replicative lifespans and average telomere shortening rates might be much more the result of stochastic then programmed events (von Zglinicki, 2003). Finally, they allow the possibility of viewing cellular senescence in all its forms including telomere-dependent and -independent senescence, as one common type of cellular stress response, in a way comparable to apoptosis (von Zglinicki, 2001, 2002, 2003; Campisi, 2003; Ben-Porath and Weinberg, 2004; Itahana et al., 2004). Accepting the above points, we wanted to know whether telomere-dependent senescence and growth arrest induced by DNA-damaging stress share not only the final phenotype but also the major steps in the signalling pathway(s) involved.
3. The DNA damage response pathway triggers replicative senescence In recent years, the early events in DNA damage-induced signalling have been very well characterised (for review, see (Shilo, 2003)). Signalling kinases, notably ATM, ATR and, possibly, DNA-PK, are recruited to the site of damage and are activated, leading to phosphorylation of Ser-139 of histone H2A.X molecules (g-H2A.X) adjacent to the site of DNA damage. It is thought that this phosphorylation of histone H2AX facilitates the focal assembly of checkpoint and DNA repair factors including 53BP1, MDC1/NFBD1 and NBS1, and also promotes the activation by phosphorylation of the transducer kinases Chk1 and Chk2, which converge the signal on p53. It was known already that telomere dysfunction induces growth arrest via activation of ATM/ATR (Karlseder et al., 1999; Rouse and Jackson, 2002), and, eventually, p53 and (in human but not mouse fibroblasts) p16 (Smogorzewska and De Lange, 2002). Thus, we hypothesized that uncapped telomeres might trigger senescence via formation of (senescence-associated) DNA damage foci (SDFs). To test this hypothesis, we first measured the fractions of cells containing at least one DNA damage focus (by staining with antibodies against both gH2A.X and 53BP1) as cultures of human MRC5 and BJ fibroblasts were grown to senescence. We found that these fractions increased gradually from 10 to 20% in young, proliferating cultures towards about 80% or more in senescent cultures, but remained at background levels of 10–20% in hTERT-immortalised fibroblasts at postsenescent PDL (d’Adda di Fagagna et al., 2003). Some of the foci
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found in young cultures are most probably responses to ongoing DNA replication, as we found consistently less staining in quiescent cells than in cycling cells. The remaining foci are regarded as indicative for the existence of senescent cells in young cultures, as predicted by the heterogeneity of replicative lifespan (Smith and Whitney, 1980; Martin-Ruiz et al., 2004). In addition to g-H2A.X and 53BP1, the foci observed in the senescent cells contained the DNA damage checkpoint protein MDC1, the DNA doublestrand break repair and checkpoint factor NBS1 as well as the phosphorylated form of SMC1 (phospho-Ser966) as shown by colocalisation studies. We also detected phosphorylation of Rad17 (pSer-645), Chk1 (pSer-345), Chk2 (pThr-68) and p53 (pSer-15) as well as stabilisation of p53 and p21 in senescent cells, thus indicating that fully functional foci indistinguishable from those induced by DNA damage are present in senescent fibroblasts (d’Adda di Fagagna et al., 2003).
4. Senescence-associated DNA damage foci form at uncapped telomeres Next, we wanted to know whether SDFs were localised directly at telomeres in order to distinguish between three possible scenarios. First, senescence might be triggered by DNA damage (i.e. double-strand breaks) at sites distinct from telomeres. This has been suggested in a recent study (Sedelnikova et al., 2004) because the authors did not find colocalisation between g-H2A.X antibody staining and fluorescence in situ hybridisation using a telomere (TTAGGG) PNA probe. Second, senescence might be triggered by uncapped telomeres but in an indirect fashion, i.e. via telomere-dependent chromosomal end-to-end fusions followed by chromosome breakage. Increased fractions of end-to-end fusions have been found both in senescent cells and following experimental uncapping of telomeres by over-expressing dominant negative TRF2 (van Steensel et al., 1998). Third, uncapped telomeres might directly trigger a DNA damage response, and only in this case one would expect a significant association of g-H2A.X foci with telomeres. To find out which of these possibilities was correct, we first used a system that allows the conditional overexpression of a dominant negative TRF2 mutant in fibrosarcoma cells (van Steensel et al., 1998). This results in the induction of a senescence-like arrest with uncapped, but long telomeres. Chromatin immunprecipitation revealed preferential association of SDF components g-H2A.X, 53BP1, Rad1 and NBS1, but not of Chk1 and Chk2, with telomeric DNA following TRF2 over-expression (d’Adda di Fagagna et al., 2003), consistent with the colocalisation of DNA damage foci and TRF1 found in human fibroblasts transfected with the dominant negative TRF2 or with TRF2 siRNA (Takai et al., 2003). Activation of ATM was also found following transfection of dominant-negative TRF2 in
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quiescent G0 fibroblasts (using lentiviral transduction) without any cell cycling (Bakkenist et al., 2004). Together, these data show that chromosome breakage is not necessary for the induction of a DNA damage response if telomeres are uncapped by interference with TRF2. In senescent human fibroblasts, we could not find colocalisation of SDFs to TRF2 (Fig. 1) in accordance with (Sedelnikova et al., 2004), although some colocalisation between foci components and telomeric DNA has recently been shown in near-senescent fibroblasts by high-resolution confocal imaging (Herbig et al., 2004). Similarly, chromatin immunprecipitation was not unambiguous: even by using highly sensitive real-time PCR to detect telomeric TTAGGG sequences (Martin-Ruiz et al., 2004), we could not find a significant enrichment of telomeres in DNA precipitated with the monoclonal g-H2A.X antibody from senescent cells, although we found it using the polyclonal g-H2A.X and the anti-53BP1 antibody (data not shown). However, by analysing the immunprecipitated DNA on genomic DNA chips (‘CHIP-on-Chip’), we demonstrated significantly increased binding of g-H2A.X to sub-telomeric DNA in senescent fibroblasts at nearly half of all chromosome ends (d’Adda di Fagagna et al., 2003). Very recently, it has been shown that some chromosome ends lose all recognizable telomeric signal in near-senescent fibroblasts with rather high probability (Zou et al., 2004). More than half of the chromosome ends that were without telomeric signal in at least 10% of nearsenescent metaphases were among those that were enriched for SDFs in the CHIP-on-Chip analysis. That some chromosomes in near-senescent cells can lose all telomeric repeat sequence has also been shown by measurements of the lengths of individual Xp/Yp telomeres (Baird et al., 2003). For the three chromosome ends with the highest probability of complete telomere loss, colocalisation of sub-telomeric BAC probes with SDFs has been confirmed (Zou et al., 2004). Taken together, these data strongly suggest that it is these very short telomeres that are devoid of most of their repeat sequences, which trigger DNA-damage foci formation. So far, the data do not exclude the formation of non-telomeric foci in senescent cells, which would not be visible if they were randomly distributed throughout the genome. Thus, DNA damage responses originating from broken chromosomes or elsewhere unrepaired double-strand breaks might be part of a composite signal as cells progress towards senescence. However, progression through mitosis is not necessary for foci formation in fibroblasts transfected in G0 with dominant negative TRF2, and many of these cells appear to arrest before reaching M-phase, thus indicating that chromosome fusion-breakage cycles are not necessary for senescent growth arrest (Takai et al., 2003). Together, the data show clearly that direct induction of SDFs at short uncapped telomeres is a very important part of the signal transduction pathway in replicative senescence.
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Fig. 1. g-H2AX and TRF2 staining patterns do not colocalise in senescent cells. Senescent MRC5 fibroblasts were grown on coverslips, fixed and sequentially stained for g-H2A.X (red) and TRF2 (green) as described (d’Adda di Fagagna et al., 2003).
5. Telomeric single-stranded overhangs can induce foci formation How do short telomeres induce foci formation? It is widely accepted that ATM and, to a lesser degree, DNA-PK phosphorylate H2A.X in response to DNA double-strand breaks generation by ionizing radiation (Bakkenist and Kastan, 2003, Stiff et al., 2004), while ATR responds to single-stranded DNA tracts coated with multiple RPA molecules (Zou and Elledge, 2003). Activated ATM was found in telomeric foci in both senescent fibroblasts (Herbig et al., 2004) and in cells over-expressing dominant negative TRF2 (Takai et al., 2003). However, telomere uncapping induces senescence efficiently in ATM/ fibroblasts (Takai et al., 2003), and colocalisation of ATR and ATRIP to telomeres was observed in ATM/ cells (Herbig et al., 2004), suggesting possible ‘back-up’ roles for DNA-PK and/ or ATR. If one accepts that unscheduled opening of the tloop is the physical substrate of an uncapped telomere, a signalling telomere would represent a double-strand break ending in a rather long (some ten to some hundred nucleotides) single-stranded stretch of G-rich DNA. It is possible that this structure is processed towards a bluntended double-strand break before signalling. Loss of overhangs has been reported in cells over-expressing dominant negative TRF2 (van Steensel et al., 1998) and in two human fibroblast strains as they progress towards senescence (Stewart et al., 2003, Keys et al., 2004), but not in a number of other fibroblast strains (Keys et al., 2004). It is, however, not at all clear whether overhang processing is
necessary for signalling, as recently suggested (Stewart et al., 2003), or whether it simply occurs as a consequence of senescence. In fact, there are data to suggest that the overhang itself might be essential for senescence signalling. Thus, in budding yeast, uncapped telomeres are singlestranded and activate DNA damage checkpoint pathways mainly via the ATR homolog Mec1 (Gardner et al., 1999; Maringele and Lydall, 2002). Short single-stranded telomeric G-rich DNA oligonucleotides, but not C-rich ones, activate a senescence-like, p53-dependent growth arrest in human cells (Saretzki et al., 1999) and might thus be regarded as a model for telomere uncapping (von Zglinicki, 2001). Phosphorylation of p53 was largely ATM-dependent and was accompanied by phosphorylation of Nbs1 (Eller et al., 2003). Moreover, single-stranded G-rich oligonucleotides induce massive phosphorylation of H2A.X in human fibroblasts (Fig. 2). Together, these data suggest that the unscheduled exposure of telomeric overhangs is necessary and sufficient to trigger the formation of telomeric SDFs and, thus, senescence as suggested earlier (von Zglinicki, 2000, 2001). Evidently, complete processing of telomeric overhangs and production of a blunt end is not necessary to generate telomeric substrates that are able to activate ATM.
6. Senescence is a permanently maintained DNA damage response state According to a number of studies, the fractions of SDFcontaining cells increase with culture time until all cells
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Fig. 2. G-rich telomeric single-stranded DNA induces phosphorylation of histone H2A.X. Proliferating MRC5 fibroblasts (PD 34) were treated for 3 days with 60 mM of either (TTAGGG)2 or (CCCTAA)2 telomeric single-stranded oligonucleotides and stained 2 days later for g-H2A.X as described (d’Adda di Fagagna et al., 2003). Treatment with the G-rich oligonucleotide, but not with the C-rich one, significantly decreased the fraction of growing cells as measured by Ki67staining (data not shown).
senesce (d’Adda di Fagagna et al., 2003; Herbig et al., 2004; Sedelnikova et al., 2004) and DNA damage foci do not disappear from senescent cells for many months after the cells became senescent (data not shown and Sedelnikova et al., 2004). In contrast, one recent study reported loss of ATM activation, g-H2A.X-containing foci and p53 accumulation in human fibroblasts during their last three population doublings, concluding that the telomere dysfunctioninduced stress response disappears in fully senescent cells (Bakkenist et al., 2004). So far, it is not understood what causes these controversial results. However, the constant presence of SDFs in senescent cells in our hands suggested to us the possibility that senescence is a state that is maintained by the constant activity of signalling kinases. In fact, a concerted knock-down of ATM/ATR and/or Chk1/ Chk2 in senescent BJ fibroblasts by either siRNA or microinjection of kinase-dead mutants resulted in reentrance into S-phase of a significant percentage of cells as shown by BrdU incorporation (d’Adda di Fagagna et al., 2003). This treatment also raised the expression of the Sphase marker MCM7 and reduced the number of g-H2A.X foci. This is in accord with a recent study showing that treatment of senescent cells with the rather unspecific protein kinase inhibitor 2-aminopurine, or by transfection with ATM and ATR siRNAs resulted in a large number of cells loosing SDFs and entering S-phase (Herbig et al., 2004). In this latter study, a fibroblast strain was used that shows a mosaic expression of p16 at senescence, and it was shown that some cells in this strain senesce via a pathway that leads from telomere attrition via SDF formation to p53 and p21 upregulation, while other senesce in a telomereindependent, p16-dependent manner. Accordingly, inhibition of ATM/ATR reversed senescence exclusively in p16negative cells (Herbig et al., 2004). However, BJ fibroblasts as used in the senescence reversal experiments by d’Adda di Fagagna et al. (2003) do not upregulate p16 (Itahana et al., 2003), and there must be other reasons for the incomplete reversal of senescence in our experiments. We do not know
whether we did not achieve a complete repression of the DNA damage responses, or whether downstream processes like those that regulate the induction of secondary phenotypes of senescence including cell morphology, senescence-associated b-galactosidase activity or expression of matrix-modifying gene products (Frippiat et al., 2001) might have prevented complete reversal of senescence in some cells. However this might be, the data clearly demonstrate that cellular senescence is accompanied and actively maintained by a persistent activation of DNA damage signalling kinases and SDFs. This response might be triggered by either widespread, non-telomeric DNA damage, resulting in SIPS, or be telomere uncapping leading to replicative senescence more or less accelerated by stress acting on telomeres. Both cases can be characterised as cellular senescence (von Zglinicki, 2001, 2002, 2003; Campisi, 2003; Ben-Porath and Weinberg, 2004; Itahana et al., 2004), and we can thus define cellular senescence as a permanently maintained DNA damage response state. It still remains to be seen whether the above definition is valid for such forms of senescence that are not induced by either DNA damage or telomere uncapping. However, we can conclude that persistence of SDFs is a marker for cellular senescence. This immediately suggests the potential to use immunstaining for foci components like g-H2A.X or 53BP1 as markers to detect senescent cells in vivo. ATRdependent foci will also be formed in cells undergoing active proliferation and DNA replication, especially in situations that lead to frequently stalled replication forks as, for instance, chromosomal rearrangements during meiotic cell division. However, such cells will generally be confined to specific organs, for instance the seminiferous tubuli in testes, or developmental stages and will hardly interfere with the observation of cellular senescence. DNA damage foci will also be found in cells undergoing DNA repair or apoptosis following acute DNA damage, however, this response is short-lived and should thus be easily distinguishable from the permanent pattern of senescent cells. So far, most of what
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we know about the existence of senescent cells in vivo and their relationship to aging of organisms stems from the use of a single marker, senescence-associated b-galactosidase activity, which is only indirectly related to the senescent phenotype (Dimri et al., 1995). It will be interesting to see whether the new markers identified by recent studies will confirm what we have learnt so far about the involvement of replicative senescence in organismic aging, and will lead to a further extension of our understanding of cellular senescence, both within in vitro and in vivo settings.
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