Replicative stress, stem cells and aging

Abstract

DNA synthesis is a remarkably vulnerable phase in the cell cycle. In addition to introduction of errors during semi-conservative replication, the inherently labile structure of the replication fork, as well as numerous pitfalls encountered in the course of fork progression, make the normally stable double stranded molecule susceptible to collapse and recombination. As described in this issue, maintenance of genome integrity in the face of such events is essential to prevent the premature onset of age-related diseases. At the organismal level, the roles for such maintenance are numerous; however, the preservation of stem and progenitor cell pools may be particularly important as indicated by several genetically engineered mouse models. Stresses on stem and progenitor cell pools, in the form of telomere shortening (Terc^−/−) or other genome maintenance failures (ATR^mKO, Ku86^−/−, LIG4^Y288C, XPD^R722W/R722W, etc.), have been shown to degrade tissue renewal capacity and accelerate the appearance of age-related phenotypes. In the case of telomere shortening, exhaustion of replicative potential appears to be at least partially dependent on the cell cycle regulatory component of the DNA damage response. Therefore, both the genome maintenance mechanisms that counter DNA damage and the cell cycle checkpoint responses to damage strongly influence the onset of age-related diseases and do so, at least in part, by affecting long-term stem and progenitor cell potential.

Introduction

Deficiencies in genome maintenance are associated with various aspects of premature aging in both humans and genetically modified mouse strains. In humans, for example, Werner syndrome, Cockayne syndrome, trichothiodystrophy, dyskeratosis congenita, and ataxia-telangiectasia are characterized by specific age-related diseases, and all are caused by mutation of genes involved in the efficient repair of DNA damage or the cell cycle regulatory response to such damage (Kipling et al., 2004). Age-associated changes, such as graying hair, alopecia, kyphosis, osteoporosis, and impaired tissue regeneration, have also been observed in a number of mouse models in which targeted mutations have been generated in DNA damage response (DDR) regulators. These mouse models include Ku86^−/− (Vogel et al., 1999), XPD^R722W/R722W (de Boer et al., 2002), BRCA1^Δ11/Δ11p53^+/− (Cao et al., 2003), XRCC4^−/−p53^S18A/S23A (Chao et al., 2006), LIG4^Y288C (Nijnik et al., 2007), Terc^−/− (Rudolph et al., 1999), Wrn^−/−Terc^−/− (Chang et al., 2004; Du et al., 2004; Pignolo et al., 2007), and those expressing putatively hypermorphic variants of p53 (Maier et al., 2004; Tyner et al., 2002).

The loss of tissue homeostasis through stem and progenitor cell attrition has previously been proposed as a model to explain the general organismal decline associated with aging (Chen, 2004; Pelicci, 2004; Sharpless and DePinho, 2004; Van Zant and Liang, 2003). Consistent with this model, hematopoietic deficiencies in Ku86^−/−, LIG4^Y288C, p53^+/m, Terc^−/−, XPD^R722W/R722W mice have recently been shown to be accompanied by the premature decline of stem and progenitor cell numbers and/or an inability to generate downstream progenitors efficiently (Choudhury et al., 2007; Dumble et al., 2007; Nijnik et al., 2007; Rossi et al., 2007). In addition, the functional decline in certain stem cell compartments has been shown to be associated with the down-regulation of several DNA repair genes in the course of wild-type mouse aging (Chambers et al., 2007). While it is unlikely that loss of regenerative capacity consequential to failures in genome maintenance causes all aspects of aging, the evidence described above argues that many characteristics of aging are strongly influenced by the maintenance of genome integrity in adult stem cells.

In response to what genetic insults are DDR genes required? Certainly, the sources of DNA damage in the course of normal cellular metabolism are plentiful. They include oxidative DNA damage resulting from normal mitochondrial respiration, programmed damage occurring in the course of immune cell maturation (VDJ recombination) and exogenous sources of DNA damage, such as UV irradiation. However, potent forms of DNA damage are generated as cells transit into DNA synthesis, leading to types of damage for which a broad array of DDR genes are required to counter. Effectively, DNA synthesis magnifies the effects of common forms of damage. For example, the slow replacement of an excised base during base excision repair would result in a transient single strand nick if occurring in the G1 phase; however, during DNA synthesis, such nicks lead to the collapse of active replication forks into DNA double strand breaks. Collapsed forks require homologous recombination to restart replication and prevent large deletion events that can span megabases of DNA or whole chromosome arms (Paulsen and Cimprich, 2007). Furthermore, as discussed in detail in this review, deficiencies in cell cycle checkpoint mechanisms, such as those mediated by the ATR protein kinase, can lead to compound failures not only in maintaining replication fork stability (RFS), but also in preventing cell cycle progression. Thus, while the causes of many initial forms of DNA damage may not be particularly increased during DNA replication, the potential effects of this damage are accentuated, making cells more reliant on the DNA damage response.

In this review, we describe evidence indicating that events occurring during normal DNA replication create sufficient DNA damage over time to cause stem and progenitor cell attrition and deplete tissue renewal capacity (Fig. 1). Since decreased regenerative capacity is associated with several aspects of normal aging, the efficiency of the DNA damage response in stem cells may be particularly important to prevent the premature degenerative decline of many tissues. In addition, we explore how stem and progenitor cell fates, e.g. senescence versus apoptosis, may substantially impact tissue renewal and the onset of age-related diseases.

Figure 1.

Unrepaired DNA damage in the course of DNA replication initiates checkpoint responses that lead to cell senescence or death. Stem and progenitor cell proliferation and differentiation acts as a means to reconstitute affected tissues following the death and clearance of these mutant cells. However, replicative DNA damage, both in stem cells and niche forming cells, has recently been shown to alter the multipotent properties of these cells or cause their loss from the proliferative pool. These cyclic deteriorating events provide one potential cause of declining regenerative potential and aging over time.

DNA replication: an Achilles' heel in genome maintenance

Simply put, the second law of thermodynamics (the tendency for ordered systems to decay into general disorder) applies to all molecules, including DNA. The most common forms of DNA damage, depurination and depyrimidation, occur at normal pH and temperature via nucleophilic attack by water. Depurination/pyrimidation occurs at an estimated 10,000 bases per day in each cell (Friedberg et al., 2005) and is repaired by AP endonuclease-and Pol β-catalyzed ribose ring excision and Pol β base replacement (base excision repair, BER). Other common forms of DNA damage include cytosine deamination (100 bases/cell/day) and hydroxyl radical-mediated oxidative damage, a consequence of normal metabolic respiration. Cytosine deamination and oxidative DNA damage leads to an assortment of modified bases repaired predominantly by BER and mismatch repair. Repair of each of these forms of DNA damage are dramatically facilitated by the fact that they occur on one strand, leaving the opposing strand as a template for efficient repair.

However, at the DNA replication fork, the double-stranded nature of DNA is temporarily suspended. This process allows normally innocuous single strand nicks to be converted into potentially more destructive double strand breaks upon collision with actively progressing replication forks. At unprocessed damaged bases, the failure of replication fork stabilizing mechanisms, such as those mediated by the ATR protein kinase pathway, can also lead to collapse, presumably as a result of prolonged replication stalling and subsequent recombinatorial intervention (Paulsen and Cimprich, 2007). Thus, a large complement of genes is required to prevent replication fork collapse, be it through efficient repair of DNA ahead of fork progression or through stabilization of stalled forks at the site of damage (Table1).

Table I.

Genes that purportedly help prevent replication fork collapse or initiate restart

Genes that reverse common damage that causes stalling and/or collapse

Single strand break repair: XRCC1, Lig3, Lig1, PARP-1, PARP-2

Base excision repair: OGG1, MYH, MPG, NEI1, NEIL2, NEIL3, Apex1, Apex2, Pol β, HMGB1

Nucleotide excision repair: XPA, Rad23B, XPC, XPG, XPF, ERCC1, CSA, CSB, DDB1, DDB2, XPB, XPD, GTF2H1-5, RPA1-3

Genes involved in preventing collapse

Translesion synthesis-mediated bypass of damage: Rad6, Rad18, PCNA, Polη, Polι, Polκ (DINB), Rev1, Rev3, Rev7

ATR fork-stabilizing pathway: ATR, ATRIP, RPA1-3, TOPBP1, Rad17, RFC2-5, Rad9, Rad1, Hus1, Clspn, Tim, Tipin, Chk1

Genes involved in fork recovery

Break recognition: H2AX and MDC1 (phos. by ATM, DNA-PK & ATR), chromatin ubiquitylation by Ubc13 and RNF8 to recruit BRCA1, Bard1, Rap80 and others

Strand resection: Mre11, Rad50, Exo1, CtIP

Strand invasion: BRCA1, BRCA2, Rad51, Rad51B, Rad51D, Xrcc2, Xrcc3, Rad52, Rad54, Rad55, Rad57, RPA1-3, SMC5 & 6, and sister chromatid cohesion mediators (SMC1, SMC3, Scc1, Scc3)

Resolution: Blm and Wrn helicases and HJ resolvases (Mus81, Eme1, Rad51C)

An equally complex process (homologous recombination) is utilized to restart replication forks should collapse occur. Homologous recombination takes place in several stages (double strand break recognition, strand invasion and holiday junction resolution) and requires over 50 genes (Table1). Failure of this response pathway can lead to reliance on more mutagenic types of repair and the persistence of DNA damage, either in the original form of the DSB or in bridge-breakage fusion cycles occurring as a consequence of chromosome translocations. Fork collapse and recombinatorial restart has also been proposed to occur at difficult-to-replicate regions of the genome, such as triplet repeats and common fragile sites, and higher order chromatin structures, such as replication fork barriers (Lambert et al., 2007). Finally, in addition to replication fork collapse, another common form of DNA damage that occurs unavoidably in the course of DNA replication is the misincorporation of nucleotides, forming base mismatches (reviewed in this issue). In aggregate, the process of DNA replication requires several groupings of repair and checkpoint genes that are essential to safeguard the genome at multiple levels during this precarious stage.

The replication fork instability appears to be a relatively common occurrence in the course of normal DNA replication. For example, phosphorylation of H2AX, a histone variant phosphorylated in response to double strand breaks, is stimulated upon entry into DNA replication in cultured cells (Mirzoeva and Petrini, 2003). Other double strand break response proteins, such as Rad51 and BRCA1, are also recruited to chromatin specifically during S phase, indicative of DNA damage processing (Scully et al., 1997). In addition, it has long been known that sister chromatid exchange (SCE), a marker of homologous recombination between sister chromatids, occurs at a rate of approximately 10 exchanges per S phase in cultured cells. Because of the multivariable nature of recombination intermediate resolution, 10 exchanges per S phase likely provides only a lower limit to the actual recombination rate during normal DNA synthesis. These exchange events are indicative of the replication fork collapse and reinitiation scenarios such as those described above (Wilson and Thompson, 2007). Finally, it is important to note that greater than half the genes listed in Table I are essential for embryonic development and long-term proliferation in culture, again consistent with their importance in the absence of exogenous DNA damage. As discussed herein, loss of genome integrity during DNA synthesis can affect tissue regenerative capacity by initiating prolonged checkpoint responses that cause the functional loss of stem and progenitor cells. In this view, each of the broad array of genes listed in Table I would be expected to play some role in impeding tissue degeneration over time.

The ATR protein kinase pathway regulates replication fork stability

Replication fork stability is maintained by a variety of mechanisms, including sensory complexes that preserve the ability to reinitiate replication fork progression once the source of fork stalling is overcome. These sensory complexes were first described as checkpoint genes based on their role in regulating cell cycle progression; however, in the last several years it has come to light that such complexes are also required to prevent replication fork collapse into double strand breaks when replication stalls. This function in DNA synthesis was first discovered in studies of Mec1, an S. cerevisae homolog of mammalian ATR. In these studies, mutation of either Mec1 or the downstream checkpoint kinase, Rad53, prevented reinitiation of replication fork progression following DNA damage or deoxyribonucleotide depletion (Lopes et al., 2001; Tercero and Diffley, 2001; Tercero et al., 2003). It was later shown that these checkpoint kinases are generally required to prevent replication fork collapse and double strand break formation at sites of stalled replication, including regions of the genome that are inherently difficult to replicate (Cha and Kleckner, 2002). Most recently, it has been proposed that the main role for these checkpoint kinases in replication fork stability is to maintain the replicative polymerases (α, δ and ε) at stalled DNA replication forks; thus, providing the means to reinitiate replication fork progression once such impediments have been removed (Cobb et al., 2003).

Subsequent to these ground-breaking studies, similar functions have been demonstrated for the ATR and Chk1 protein kinases in vertebrate cells. ATR is essential for early stages of murine embryonic development and cell proliferation in culture, and proliferative failure of ATR knockout cells correlates with a dramatic increase in chromosome breakage (Brown and Baltimore, 2000). In particular, ATR is required to prevent double strand breaks upon replication fork stalling and, similar to Mec1 mutation, ATR's absence is associated with an inability to maintain replicative polymerases at forks (Brown and Baltimore, 2003; Trenz et al., 2006). Chk1 loss has also been shown to be associated with replication fork collapse (Durkin et al., 2006; Nghiem et al., 2001; Zachos et al., 2003), indicating that the entire ATR pathway complex regulating Chk1 phosphorylation and activation is likely to be involved in preventing fork collapse. Finally, common fragile sites, which are favored regions of instability in oncogene-transformed cells and preneoplastic lesions (Bartkova et al., 2005; Gorgoulis et al., 2005), are significantly more unstable (5-10 fold) in the absence of ATR or Chk1 (Casper et al., 2002). Given that ATR heterozygous mice have an increased rate of late onset tumor incidence (Brown and Baltimore, 2000), it is possible that many ATR pathway components play important tumor suppressing roles. However, as an essential genome maintenance regulator in S phase, one would also expect that complete loss of ATR in adults could dramatically affect tissue regenerative capacity. This expectation has recently been fulfilled through the use of a Cre/LoxP-conditional allele of ATR.

Deletion of ATR in adult mice: a model system that correlates replicative stress, stem cell loss and aging

As described above, DNA damage occurs constitutively in all cell types as a byproduct of normal metabolic respiration and from unavoidable failures during DNA replication. Thus, one model of aging predicts that replicative and oxidative stress in the course of stem cell proliferation causes the slow attrition of these cells throughout adulthood and that this attrition ultimately degrades tissue homeostasis (Rossi et al., 2008; Sharpless and DePinho, 2004; Van Zant and Liang, 2003). One way to examine such a model would be to ablate the proliferative potential of a large fraction of cells in young adult animals while leaving the remaining cells relatively untouched. Taking a large fraction of cells out of the proliferative pool early in life at one stroke should shorten the length of time in which a particular stem cell compartment will be able to continue to contribute to tissue renewal and, thus, would accelerate aging.

As described above, ATR is essential for embryonic development (Brown and Baltimore, 2000). To determine the effect of ATR loss in adult mice, a Cre/LoxPconditional allele of ATR and a ubiquitously expressed form of Cre recombinase (Cre-ERT2) that is drug inducible were used to produce mosaic deletion of ATR (ATR^mKO). Mosaic ATR deletion was shown to lead to the rapid loss of proliferating ATR knockout cells, followed by the reconstitution of most tissues with cells that failed to undergo Lox-Pmediated recombination and continued to express ATR. Despite ample reconstitution with ATR-expressing cells, ATR^mKO mice ultimately developed a broad range of age-related phenotypes including hair graying, alopecia, kyphosis, osteoporosis, premature thymic involution, loss of spermatogenesis, and increased tissue fibrosis (Ruzankina et al., 2007). Consistent with the importance of stem cells in preventing age-related disease, some phenotypes were shown to be associated with reductions in tissue-specific stem and progenitor cell pools. For example, the frequencies of both Lin⁻Sca1⁺c-Kit⁺ (LSK) stem and multi-potent progenitor cells in the bone marrow and early T lineage progenitor (ETP) cells in the thymus were suppressed one year following ATR deletion, and these declines were associated with premature thymic involution. In addition, reduced numbers of CD34⁺ stem cell and progenitor cells in the hair follicle bulge were observed in ATR knockout skin, and this decline correlated both with increased hair follicle loss and delayed regeneration. Importantly, stem cell attrition in ATR^mKO mice occurred despite the previous reconstitution of these tissues with ATR-expressing populations. Thus, the elimination of ATR knockout cells in these mice appeared to lead to the exhaustion of the residual ATR-expressing stem and progenitor cell pools, which in turn caused the degeneration of tissue renewal capacity (Krishnamurthy and Sharpless, 2007; Ruzankina et al., 2007).

While many of the regenerative failures observed in ATR^mKO mice are likely due to an over-reliance on residual ATR-expressing cells, the mechanisms leading to decline of specific stem and progenitor cell pools are not clear at the present time. For example, it is possible that residual ATR-expressing stem cells are lost over time in the course of normal organismal function or by the additional replicative stress acquired during pool regeneration. This possibility is supported by the consistent delays in CD34⁺ stem and progenitor cell expansion and hair follicle regeneration observed in the course of consecutive follicle cycles in ATR_mKO skin (Ruzankina et al., 2007). However, it is also possible that ATR deletion affects the niches that maintain stem cells. Such a mechanism may contribute to reductions in LSK and ETP pools in ATR_mKO mice (Krishnamurthy and Sharpless, 2007; Rossi et al., 2008; Ruzankina et al., 2007). This interpretation suggests that replicative stresses on the mesenchymal stem and progenitor cells that maintain the niche may have a particularly potent impact on age-related decline by directly and indirectly inhibiting the regeneration of multiple tissue compartments. Consistent with the plausibility of this model, functional decline of the hematopoietic stem cell niche and the mesenchymal progenitor cells that presumably maintain this niche have been strongly implicated as the cause of decreased B cell generation in Terc^−/− mice (Ju et al., 2007). Together, these studies imply that replicative stresses on stem and progenitor cell pools may affect regenerative potential by multiple mechanisms, including combinations of niche deterioration and direct effects on stem cell maintenance (Fig. 1).

DNA damage checkpoints in response to telomere attrition: the effectors of tissue degeneration

The means by which DNA replication-associated damage leads to stem and progenitor cell loss have been well studied in telomerase defective mice (Terc^−/−). Telomere dysfunction in humans has been proposed to contribute to accumulation of DNA damage and aging. Without maintenance by telomerase, telomeres shorten during DNA replication to a critical point in which telomere capping protein complexes can no longer bind and protect the ends of chromosomes from recombinatorial processes and DNA damage checkpoint activation. Terc^−/− mice exhibit significantly shortened telomeres after three generations in the absence of telomerase (G3). This shortening leads to chromosome end fusions and persistent genomic instability through the bridge-breakage-fusion cycle (Rudolph et al., 1999). Consequentially, at G3 and beyond, Terc^−/− mice exhibit impaired hematopoiesis, atrophy of the intestinal epithelium, decreased spermatogenesis, and reduced regenerative capacity in response to wounds and hematopoietic ablation (Rudolph et al., 1999). Accelerated telomere shortening has been observed when genes that regulate alternative telomere lengthening (ALT), such as the gene mutated in Werner (WRN) and Bloom (BLM) syndromes, are combined with telomerase deficiency. These compound mutants have accentuated degenerative phenotypes, which appear at earlier generation times, and suffer additional pathologies, such as bone loss and kyphosis (Chang et al., 2004; Du et al., 2004; Pignolo et al., 2007). Importantly, progenitor cells, which normally express telomerase to maintain regenerative potential, are particularly sensitive to telomerase loss (Flores et al., 2006; Hiyama and Hiyama, 2007). Thus, increased genomic instability from telomerase absence in HSCs, spermatogonia and progenitor cells in the intestinal crypts is likely to be the cause of homeostatic failure in the bone marrow, testes and intestines, respectively (Choudhury et al., 2007; Rudolph et al., 1999). Importantly, B cell deficiencies in G4 Terc^−/− mice are strongly influenced by deterioration of the HSC niche and decreased replicative potential and function of the mesenchymal progenitor cells that are thought to maintain this niche (Ju et al., 2007). It is important to note that, in addition to the niche, the systemic environment of the organism can play an important role in maintaining proliferation and the regenerative capabilities of progenitor cells (Brack et al., 2007; Conboy et al., 2005). Therefore, these data indicate that decreased regenerative capacity can occur by multiple mechanisms, including repopulation failures in stem and progenitor cells that directly maintain the tissue and in those that maintain the niche.

Recent studies indicate that stem cell attrition in Terc^−/− mice is not directly caused by DNA damage itself, but rather by the response to DNA damage. As described above, DNA damage leads to the activation of checkpoint signaling pathways mediated by the ATR and ATM protein kinases, which, among other things, phosphorylate and stabilize the p53 transcription factor (Kastan and Bartek, 2004). Upon activation, p53 stimulates the expression of cell cycle regulators, such as p21, and in some cell types, initiates cell death programs via upregulation of pro-apoptotic genes. Importantly, elimination of p21 largely rescues hematopoiesis and intestinal villi generation in late generation Terc^−/− mice (Choudhury et al., 2007). HSC (CD34^lo/−Lin⁻Sca1⁺c-Kit^hi) potential for generating downstream progenitors is enhanced in G4 Terc^−/−p21^−/− mice and their numbers are maintained at near normal levels. The number and proliferative potential of intestinal progenitors in Terc^−/− mice were also dramatically enhanced by elimination of p21. These degenerative phenotypes were lessened by p21 absence without rescuing either telomere attrition or DNA damage, as measured by increased H2AX phosphorylation (Choudhury et al., 2007). These data strongly argue that the response to DNA damage is a proximal effector in causing decreased tissue renewal capacity.

Hyperactivation of the p53 pathway and aging

Accumulation of senescent cells has long been viewed as potential cause of aging (Campisi, 2005), and it has been shown that abnormally high levels of apoptosis achieved by genetically altering mitochondrial function (PolgA^D257A/D257A) leads to the premature appearance of age-related phenotypes (Kujoth et al., 2005). p16^INK4A and p53 can affect continued cellular proliferation through either senescence (p16^INK4A or p53) or apoptosis (p53), and each is induced by DNA damage, albeit with significantly differing kinetics. Therefore, it is conceivable that the stochastic induction and activation of these genes in the course of redundant stem and progenitor cell replication might influence long-term tissue homeostasis. According to this model, one would predict that hyperactivation of these tumor suppressing pathways over and above that caused by normal replicative stresses would accelerate aging. Each of these expectations has been at least partly fulfilled by the generation and analysis of mouse models that alter the activity and/or expression of p16^INK4A and p53.

The p16^INK4A and p53 tumor suppressor genes are upregulated in response either to oncogenic stress or to DNA damage. Following DNA damage, the p53-p21 transcriptional axis provides an immediate cell cycle regulatory response; however, once these factors wane, p16^INK4A levels are elevated to levels that cause irreversible cell cycle exit via senescence (Campisi, 2005; Kim and Sharpless, 2006). Consistent with 16^INK4A-mediated senescence being involved in natural aging, 16^INK4A has been observed to be upregulated in various tissues in aged humans and rodents, and overexpression of p16^INK4A in mice causes decreased β-islet regeneration (Krishnamurthy et al., 2006; Krishnamurthy et al., 2004; Ressler et al., 2006). If p16^INK4A elevation in the course of natural aging causes the loss of regenerative capacity in some tissues, then one would expect that deletion of p16^INK4A would suppress age-associated stem and progenitor cell attrition and tissue degeneration. At least for some tissues, this effect has been observed. For example, tissue regeneration and proliferation of forebrain progenitors, pancreatic β-islet cell and HSC is preserved in p16^INK4A knockout mice in comparison to age-matched control mice (Janzen et al., 2006; Krishnamurthy et al., 2006; Molofsky et al., 2006). These studies indicate that the naturally occurring elevation of p16^INK4A with age leads to a rate-limiting impediment to regeneration of some tissues. However, in other tissues, stem and progenitor cells were not protected from age-related decline in p16^INK4A knockout mice, indicating that the reduced proliferative potential of some cell types with age does not rely on p16^INK4A (Molofsky et al., 2006) and that other rate-limiting factors may be involved.

As exemplified by several mouse models, the p53 tumor suppressor gene also appears to play a role in the age-associated decline of tissue homeostasis. In one mouse model, a putative 24kD N-terminally truncated form of p53 (p53^m) was generated in the course of conventional gene targeting (Tyner et al., 2002). In the background of a p53 null allele (p53^m/−), p53^m appears to be non-functional; however, in the presence of a wild-type copy of p53 (p53^+/m), the p53^m allele causes a small but detectable hypermorphic effect on the transcription of p53 target genes and suppresses oncogene-mediated transformation in culture. Presumably, the effect of the 24kD protein is mediated through higher order complex formation with wild-type p53 in p53^+/m mice. p53 hyperactivation in these mice is accompanied by a ~20% shorter median lifespan and the premature appearance of several age-related phenotypes including osteoporosis, kyphosis, generalized organ atrophy, impaired wound healing, hair regrowth defects, and slowed recovery following myeloablation. More recently, HSC number and proliferative potential have been shown to be decreased in p53^+/m mice and increased in late age haploinsufficient p53^+/− mice (Dumble et al., 2007). Unfortunately, the deletion leading to this truncation also causes haploinsufficiency in 23 nearby genes (Gentry and Venkatachalam, 2005). Although the potential synergistic effects of such a large deletion significantly complicates interpretation of these findings, the phenotypes observed in p53^+/m, p53^m/−, p53^+/−, and p53^−/− mice suggest that the premature aging phenotypes in p53^+/m mice are most likely related to the p53 truncation (Vijg and Hasty, 2005). Similar phenotypes have been observed in transgenic mice expressing a naturally occurring N-terminally truncated isoform of p53 called p44 (Maier et al., 2004). Transgenic p44 mice manifest several aspects of premature aging as well as a significantly reduced lifespan (32-week median). Finally, consistent with the importance of p53 in suppressing regeneration, Lamin A deficient mice (Zmpste24^−/−) exhibit age-related phenotypes that are partially reversed by p53 absence (Varela et al., 2005). These studies indicate that aberrant hyperactivation of the p53 pathway can cause the premature appearance of age-related phenotypes.

In apparent contrast to the observations above, it has more recently been shown that an additional wild-type copy of p53 under normal regulatory control (super-p53 mice) or reduced expression of the p53 inhibitor Mdm2 does not accelerate aging (Garcia-Cao et al., 2002; Matheu et al., 2007; Mendrysa et al., 2006). Furthermore, the super-p53 transgene in combination with an extra-copy of the Arf locus (super-p53/super-Arf), extends lifespan and protects against oxidative DNA damage and cancer (Matheu et al., 2007). Therefore, increased quantities of p53 under normal regulatory control lead to the opposite outcome from that of constitutive activation (p53^+/m). The fact that super-p53/super-Arf mice live longer than wild-type counterparts indicates either that DNA damage is suppressed, as suggested by increased expression of genes that possess anti-oxidant activities (Matheu et al., 2007), and/or that the cellular clearance mechanism utilized in these transgenic mice has been altered in a way that favors regeneration. In other words, super-p53/super-Arf may enhance clearance and suppress the accumulation of mutant cells without causing the wholesale loss of all cell types, such as has been proposed to occur from aberrant regulation of p53 in p53^+/m mice. Therefore, while indiscriminant cellular clearance forces constitutive tissue reconstitution and undue replicative stress that accelerates aging (ATR_mKO, PolgA^D257A/D257A, p53^+/m, Zmpste24^−/−, etc.), enhanced checkpoint responses and cellular clearance in ways justified by DNA damage and proto-oncogenic stress (super-p53/super-Arf) may suppress aging.

Stem and progenitor cells fates: contrasting the potential impacts of apoptosis, senescence and defective immune surveillance

As described above, replicative stress leads to DNA damage, which in turn can speed stem and progenitor cell attrition and the appearance of age-related phenotypes. This model raises important questions about how such cells are lost from the proliferative pool and how this fate will affect regenerative potential. For example, apoptosis may deplete regenerative potential if these cells are not ultimately replaced; however, if such cells can be replaced, then their efficient clearance would facilitate regeneration. On the other hand, stem and progenitor cells that survive damage but are rendered replication-incompetent (senescent) might be expected to have more severe effects on regenerative potential as these cells could continue to occupy the niche, preventing more able-bodied stem cells from filling the compartment.

While according to this viewpoint the persistence of senescent cells may accelerate aging, an increasing body of evidence suggests that, in some circumstances, senescent cells may be actively cleared by the immune system. For example, senescent cells have been noted to upregulate the expression of a number of inflammatory cytokines and other immune regulators (Krtolica and Campisi, 2002). In a recent study, p53-reactivation in Ras^G12V-transformed liver progenitor cells led to induction of senescence in vivo as determined by senescence-associated β-gal staining (Xue et al., 2007). Senescence was associated with tumor regression, even after expression was turned off, and a progressive inflammatory response. This inflammatory response was marked by infiltration of neutrophils, macrophages and natural killer (NK) cells near the vasculature followed by spreading throughout the tumor. In addition, inflammatory cytokines both in cultured cells (Csf1, Mcp1, Cxcl1 and IL-15) and in vivo(Ncf2, Ncf4, Mgl2, MSR2, CD68, Klrb1 and Klrd1) were shown to be elevated soon after p53 reactivation. These cytokines are known to attract and activate the infiltrating leukocytes observed. Importantly, specific inhibition of neutrophils, macrophage and NK cells in tumor-bearing animals inhibited clearance of predominantly senescent tumor cells. These studies indicate that transformed liver progenitor cells require the immune system for efficient clearance following p53-induced senescence. In addition, other studies have shown that suppression of clearance mechanisms through depletion or impaired recruitment of macrophages can cause the accumulation of senescent cells and the appearance of select age-related changes in rodents (Ambati et al., 2003; Giuliani et al., 2005). Together, this evidence is consistent with the hypothesis that lack of efficient immune-mediated clearance of senescent or otherwise defective cells can influence the progression of age-related diseases (Fig. 1).

DNA damage itself may be an important trigger for immune system-mediated cellular clearance. The NK cell ligand, NKG2D, has recently been shown to be upregulated in response to stalled replication and double strand breaks. This response is dependent on ATR and ATM and independent of p53 (Gasser et al., 2005). Antibody blocking studies indicate that NKG2D engagement is a major mechanism by which tumor cells are eliminated in vivo; although, NKG2D activation is clearly not the only means for clearance (Jamieson et al., 2002), consistent with an important role for p53-mediated apoptotic and senescence pathways. Such immune-mediated mechanisms might provide alternative, albeit slower and less efficient, means for clearance of defective cells. If clearance of defective stem and progenitor cells is beneficial for tissue regeneration, then one would predict that inhibition of each of these clearance pathways (apoptosis, immune cell-mediated) should lead to devastating effects on tissue homeostasis and cause the rapid onset of aging phenotypes. However, such a model has yet to be tested.

Summary

DNA replication represents a precarious phase in cell proliferation and, therefore, tissue renewal. Evidence provided herein suggests that inevitable replication-associated DNA damage leads to replicative exhaustion and degeneration of tissue homeostasis with age. Declining regenerative capacity depends, in part, on apoptotic and cell cycle regulatory effectors of the DNA damage response, such as p16^INK4A, p53 and p21. Current evidence suggests that the organismal effects of replicative stress are most potent in those cells that carry the largest burden in tissue renewal, that is, adult stem and progenitor cells. Adult stem cell pools are generated and positioned during development, giving each tissue and tissue sub-compartment the means for some self-renewal. However, in the course of long-term renewal, replication-associated mutations in these cells and those that maintain the niche cause stochastic failures in tissue regeneration (Fig. 1). It is important to note that in some cases, stem cell pools are mobile and can expand to occupy available niches, e.g. in bone marrow; however, in other cases, this may not be as easily achievable, e.g. in hair follicles. A key question in this area will be to determine which degenerative disorders may be amenable to stem cell-based therapies, and which may not be. Finally, emerging evidence suggests that clearance of damaged cells may also play an important role in preserving tissue homeostasis. Therefore, just as excision and replacement of damaged bases in the repair of oxidative DNA damage is essential for maintaining genome integrity, so may be the removal of damaged and senescent cells for maintaining tissue homeostasis and organismal preservation.