dna damage response in adult stem cells
TRANSCRIPT
Blood Cells, Molecules and Diseases 52 (2014) 147–151
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Blood Cells, Molecules and Diseases
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Review
DNA damage response in adult stem cells
Alessandra Insinga a, Angelo Cicalese a, Pier Giuseppe Pelicci a,b,⁎a Department of Experimental Oncology, European Institute of Oncology, IEO, 20141 Milan, Italyb Dipartimento di Medicina, Chirurgia e Odontoiatria, Università degli Studi di Milano, 20122 Milan, Italy
⁎ Corresponding author at: Department of Experimentof Oncology, IEO, 20141 Milan, Italy.
E-mail address: [email protected] (P.G. Pelic
1079-9796/$ – see front matter © 2013 Published by Elsehttp://dx.doi.org/10.1016/j.bcmd.2013.12.005
a b s t r a c t
a r t i c l e i n f oArticle history:Submitted 8 November 2013Available online 28 January 2014
(Communicated by Grover C. Bagby, M.D.,5 December 2013)
Keywords:Stem cellsDNA damageSelf-renewalCancer aging
This review discusses the processes of DNA-damage-response and DNA-damage repair in stem and progenitorcells of several tissues. The long life-span of stem cells suggests that theymay respond differently to DNA damagethan their downstream progeny and, indeed, studies have begun to elucidate the unique stem cell responsemechanisms to DNA damage. Because the DNA damage responses in stem cells and progenitor cells are distinctlydifferent, stem and progenitor cells should be considered as two different entities from this point of view. Hema-topoietic and mammary stem cells display a unique DNA-damage response, which involves active inhibition ofapoptosis, entry into the cell-cycle, symmetric division, partial DNA repair and maintenance of self-renewal.Each of these biological events depends on the up-regulation of the cell-cycle inhibitor p21. Moreover, inhibitionof apoptosis and symmetric stem cell division are the consequence of the down-regulation of the tumor suppres-sor p53, as a direct result of p21 up-regulation. A deeper understanding of these processes is required beforethese findings can be translated into human anti-aging and anti-cancer therapies. One needs to clarify and dissectthe pathways that control p21 regulation in normal and cancer stem cells and define (a) how p21 blocks p53functions in stem cells and (b) how p21 promotes DNA repair in stem cells. Is this effect dependent on p21s abil-ity to inhibit p53? Such molecular knowledge may pave the way to methods for maintaining short-term tissuereconstitution while retaining long-term cellular and genomic integrity.
© 2013 Published by Elsevier Inc.
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147DNA damage processing in SCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148Impact on aging and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150Conflict of Interest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Introduction
DNA integrity is fundamental for cell survival and maintenance oftissue homeostasis. Any kind of DNA damage (DD) is rapidly sensed bythe cell and activates evolutionary conserved and well-characterizedsignaling pathways, collectively known as the DNA damage response(DDR) [1,2]. This response has two important outcomes: it preventscell-cycle progression and coordinates efforts devoted at repairingDNA. Briefly, single-stranded DNA and double-strand breaks are detect-ed by specialized sensor complexes which then recruit and activate
al Oncology, European Institute
ci).
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apical protein kinases, respectively ATR (ataxia telangiectasia and Rad-3 related) or ATM (ataxia telangiectasia mutated), to the site of damage[1,3,4]. The recruitment of these signal transducers to the DNA lesioncauses phosphorylation of the histone H2A histone-variant (H2AX),which is essential in the nucleation of the DDR [5]. At sites of DD, ATMand ATR activities are amplified by DD mediators [2,6–10]. When localATM or ATR activity exceeds a certain threshold, DDR factors that func-tion far from the site of damage are engaged [11]. ATM phosphorylatesand activates the checkpoint kinase CHK2, while CHK1 is principallyphosphorylated by ATR (but also by ATM). These downstream kinasesdiffuse throughout the nucleus and spread the DDR signal by phosphor-ylating their substrates [12,13]. Ultimately, activation of the DDR signal-ing cascade converges upon the key decision-making factor p53 [2,14].p53 is phosphorylated and stabilized; it accumulates in the nucleusand activates its target genes. Depending on the target genes that are
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activated, p53 regulates different cellular outcomes: cell-cycle arrest(through induction of the cyclin-dependent kinase inhibitor p21), or ap-optosis (through transcriptional activation of the bcl2 family membersbax, noxa, and puma). The DDR signaling cascade has been extensivelystudied and characterized in cell lines and primary cells like murine em-bryo fibroblasts (MEFs). In contrast, little is known about the DDRin vivo. Interestingly, in vivo evidence of DD and DDR cascade activationcomes from several recent reports on human and mouse pre-malignantlesions of different origins, namely lung, prostate, bladder, melanocyticnevi and lymphoid derivation [15–21]. DD in these cells accumulatesdue to oncogene-driven cell-division cycles and is associated with DNAreplication. These benign lesions consist of senescent cells that expressactivated oncogenes, show H2AX phosphorylation, display active formsof ATM/ATR, Chk1 and,most importantly, showp53 stabilization. Indeed,genetically, oncogene induced senescence is dependent on p53, as ele-gantly shown in the prostate and confirmed in the lung and pancreasand in lymphoid cells. Notably, active DDR markers and a senescencestatus were detected in bulk pre-cancerous lesions, mostly consistingof progenitor cells, while no information was provided on stem cells(SCs). Equally, the mechanism through which normal SCs respond todouble strand breaks was (until recently) an unexplored territory, bothin vitro and in vivo. However, we recently discovered that expressionof leukemia-associated oncogenes in hematopoietic SCs (HSCs) inducesDNA damage, p53-independent p21-upregulation, DNA repair andextended self-renewal [22], suggesting that a specific cellular responseto DD takes effect in normal SCs and is de-regulated (or constitutivelyactivated) by oncogenes. In this chapter, we review recent progressesin our understanding of the DDR in stem and progenitor cells, in vivo,as well as our group's contribution to this field.
DNA damage processing in SCs
Adult SCs self-renew and maintain tissue homeostasis throughoutthe life of an individual. Therefore, it has long been speculated thatthey possess evolutionary features allowing them to survive and repop-ulate the original tissue shortly after acute insults [23]. Importantly, DDprocessing in SCs must ensure genome integrity for daughter SCs anddownstream lineages. Indeed, the impact of damage accrued in individ-ual SCs is potentiated through the self-renewal and differentiation pro-cesses, with possible ramifications to all levels of the developmentalhierarchy. This is particularly true for highly regenerative tissues suchas the skin, the gut and the hematopoietic system.
The blood systemhierarchy is verywell characterized and comprisesa small number of long-term SCs that are predominantly quiescent andwhich ensure the lifelong production of all the diverse hematopoieticcell types, and progenitors that proliferate and progressively differenti-ate. Hematopoietic stem cells (HSCs) are probably the best character-ized among somatic SCs, owing to the availability of well-establishedcell surface markers that facilitate their purification; they can be quan-titatively and qualitatively assayed with high resolution [24,25].Mouse long-termHSCmarkers are particularly refined and allow purifi-cation of a population – termed LT-HSC (expressing the lin− Sca1+
cKit+ Flk2− CD34− markers) – that generates long-term reconstitutionin about 30% of single cell repopulation transplants [26]. HSCs, alongwithother tissue-specific SCs, are equipped with a variety of cytoprotectivemechanisms to ensure protection of their genomes beyond that ofother cell types. For example, they possess a high ABC transporter activ-ity that pumps genotoxic compounds out of the cell. Moreover, they ex-perience exogenous protection because of the hypoxic niche where theyreside. Additionally, they display a quiescent, metabolically inactive statethat generates low levels of endogenous free radicals and reactiveoxygen species [27–29]. Nevertheless, DNA lesions accumulate in SCs.So what happens once they have occurred?
Mohrin and colleagues addressed this issue by using hematopoieticstem/progenitor cells (HSPCs) – defined as lin−/c-kit+/Sca-1+/Flk2−
bone marrow cells – and myeloid progenitors, isolated from young
mice [30]. They found that, in agreement with earlier studies [31],these cells were more resistant than the progenitor cells to apoptosisinduced by low doses of ionizing radiation (2 Gy). This unique cellintrinsic mechanism, which ensures survival of HSPCs following DNAdamage, included enhanced expression of pro-survival genes (likeBcl2 andBcl-xl) and robust induction of p53, resulting in a strong upreg-ulation of both proapoptotic target-genes (like Bax and Puma) and p21expression. A strong induction of p53 and of its target genes was alsoobserved in myeloid progenitors that, in contrast, undergo apoptosis.The researchers thus hypothesized that high levels of pro-survival fac-tors in HSPCs might limit apoptosis while supporting p21-mediatedgrowth arrest, DNA repair and survival. Importantly, however, thereare two observations by the same group that question the role of p53in the unique response of HSCs to DNA damage. First, when HSPCsare induced to cycle through prolonged culture or treatment with thecytokineG-CSF, they are protected from ionizing radiation (IR)-mediatedapoptosis in the absence of a p53-mediated response. Second, p53−/−HSPCs show increased radioresistance.
Strikingly, in contrast to this study, in a companion study Milyavskyand colleagues reported that human umbilical cord blood (CB)-derivedHSPCs (a fraction containing few HSCs and many MPPs, defined as lin-age− CD34+ CD38− CD45RA−) undergo p53-dependent apoptosisafter irradiation [32]. The DDR differences seen in CB from human ver-sus bone-marrow frommice may have a different basis. In our opinion,they most likely reflect cell-intrinsic differences between SCs at differ-ent stages of ontogeny. Indeed, human umbilical CB-derived HSCs arehighly proliferative cells that are considered to be of fetal origin; theirgenomic integrity is expected to be highly protected in order to obtaina pool of cells able to sustain lifelong hematopoiesis in the organismand for reproduction. In contrast, bone-marrow HSCs are largely quies-cent adult SCs; the main functions of these cells are to quickly respondto hematopoietic needs and provide immediate blood homeostasis[27,33]. However, a direct comparison between the studied cells is notpossible because the antibodies used to purify SC and progenitor popu-lations are different between human andmouse (beingmore refined inthe mouse).
In an attempt to elucidate themechanisms throughwhichHSCs pro-cess DNA damage and resolve these discrepancies, our group studiedthe responses elicited in vivo by X-ray treatment of wild-type andp53−/− mice [34]. We characterized the response to DNA damage inhighly purified HSCs (LT-HSCs: long-term reconstituting HSCs, Lin−/c-Kit+/Sca-1+/Flk2−/CD34−) and in different progenitor subpopulations(ST-HSCs: short-term reconstituting HSCs, Lin−/c-Kit+/Sca-1+/Flk2−/CD34+; MPPs: multipotent progenitors, Lin−/c-Kit+/Sca-1+/Flk2+/CD34+; CMPs: common myeloid progenitors, Lin−/c-Kit+/Sca-1−).Strikingly, we found that, upon irradiation, LT-HSCs do not activatep53, but activate p21 independently of p53. Importantly, p21 up-regulation inhibits p53 induction and prevents apoptosis. The apparentdiscrepancy between our data and those from Passegué and colleaguesfully depends on the SC populations analyzed. HSPCs mainly includeST-HSCs (about 75%) and thus the rare LT-HSC population (about25%) is likely overlooked. Indeed, when we analyzed purified ST-HSCs,we found p53 activation, as in other progenitor populations, namelyMPPs and CMPs. Strikingly, however, while MPPs and CMPs underwentp53-dependent apoptosis, ST-HSCs were radio-resistant, as reported byPassegué and colleagues, and their enhanced resistance to X-ray-induced apoptosis was p21-dependent. Our data thus show that stem/progenitor populations that differ for self-renewal and differentiationpotential display different DDRs: LT-HSCs respond to X-irradiationwith a p21-dependent inhibition of p53-activation and apoptosis; ST-HSCs show p53 activation and p21-dependent inhibition of apoptosis;and MPPs and CMPs undergo p53-dependent and p21-independentapoptosis. In other words, progressive loss of self-renewal correlateswith a switch from a p21-dependent response that inhibits p53functions and apoptosis to a p53-dependent response that activatesp21-dependent apoptosis. This “checkpoint maturation” that
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accompanies hematopoietic cell differentiation most likely underliesthe inconsistencies between our data and those reported byPassegué and colleagues.
Different adult tissue-specific SCs share the same task of preservingorgan functionality. We therefore studied p53 and p21 regulatory sys-tems after X-rays in another SC compartment, namely mammary SCs(MaSCs). Also in this system, we analyzed highly purified SCs, definedby the PKH-26 label-retaining assay [35]. The purified (PKH+) MaSCsare able to reconstitute a mammary gland in about 30% of single cellrepopulation transplants. Remarkably, irradiated MaSCs displayed a re-sponse to DNA damage identical to that observed in LT-HSCs, implyingthat the LT-HSC DDR is not unique to the hematopoietic system butconserved in SCs of different origins. Accordingly, Sotiropoulou and col-leagues reported a similar response (short duration of p53 activationand resistance to apoptosis) for a population enriched in hair folliclebulge SCs (BSCs) [36]. Interestingly however, hair follicle melanocyteSCs (MSCs), that are located in the same hair follicle niche as BSCs,have a different cell autonomous response to DD: p53 is transientlyactivated but cells are eliminated by premature differentiation (inde-pendently of p53) [37]. Another well-known exception of SCs beingresistant to DD is the intestine. Intestinal SCs (ISCs) are particularlysensitive to DD: they show enhanced p53 activation and undergo mas-sive apoptosis upon low doses of X-rays [38,39]. Remarkably, colon SCs(CoSCs), despite sharing a similar localization at the crypt bottom, areconsiderably more radioresistant than ISCs [40,41]. Importantly, theirradioresistance has been linked to a lower expression of p53 (and a
Fig. 1. DNA damage processing in stem cells. Adult SCs are resistant to DD-induced apoptosis oand enhanced expression of pro-survival genes. Additionally, p21-dependent inhibition ofrenewing divisions of SCs. p21 also activates DNA repair in SCs, thereby limiting accrual of Dterm organ functionality. However, continuousDD andDD repairmight favor accumulation of S(aging) or favor transformation (cancer).
higher expression of Bcl2) [42–46], strongly supporting the notionthat a unique and p53-independent DDR is conserved in most adultSCs. The main outcomes of this pathway are cell survival and repair ofdamaged DNA. Notably, DNA repair in HSPCs and BSCs has beenshown to occur by non-homologous end joining, an error-pronemechanism that is typical of quiescent cells [30,36]. As a result, chro-mosomal aberrations were identified in irradiated HSPCs and in theirprogeny, and engraftment defects were observed in secondary recip-ients [30]. Accordingly, using highly purified HSCs and MaSCs wefound that repair of damaged DNA is incomplete, since we detectedmoderate levels of persistent DD several months after irradiationof wild-type LT-HSCs, and reduced self-renewal. Importantly, botheffects (persistent DD after irradiation and reduced self-renewal)were exacerbated in the absence of p21, indicating that p21 regu-lates the cellular response to DD in SCs (HSCs and MaSCs), limitingDD accumulation and preventing exhaustion of their self-renewalcapability. To our surprise, when we investigated how p21 preventsDD accrual, we discovered that p21 up-regulation after X-ray in-duced cell-cycle entry and expansion of the absolute numbers ofLT-HSCs and MaSCs. Increased numbers of SCs after X-rays werecaused by induction of symmetric self-renewing divisions in MaSCsthat, mechanistically, relies on p21-dependent downregulation ofp53 activity [34].
Collectively our data, along with other published studies, reveal that,following DD, SCs inactivate apoptotic responses, limit DD accumulation,maintain self-renewal and enter symmetric self-renewing divisions.
r senescence. This event has been linked to up-regulation of p21, reduced p53 activationp53 activation and of p53 basal activity leads to cell cycle entry and symmetric self-D and exhaustion of their self-renewal potential. This unique response preserves short-Cs that harbor DNAmutations, which, over a lifespan, can reduce their functional efficiency
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Importantly, the outcome of this uniqueDDR is the expansion of a pool offunctional SCs able to fulfill their role of preserving short-term tissue re-constitution (Fig. 1).
Impact on aging and cancer
The ability of SCs to handle DD without committing apoptosis orundergoing senescence, but rather activating a DNA repair response,might have important physiological and pathological consequences.
Mammalian aging results, at least in some key aspects, from an age-associated decline in SC functionality. A number of recent studies impli-cate DD repair and maintenance of genomic integrity as importantmechanisms in the preservation of SC functions. These studies analyzedpatients with hereditable mutations, or mouse models with engineeredmutations in specific DNA repair pathways (that decrease DNA repairactivities). In humans, patients with deficiencies in genomic mainte-nance genes display bone marrow failure syndromes (with varyingdegrees of cytopenia) [47–49]. Significant functional defects weredetected in HSCs from mice deficient in DNA repair proteins involvedin homologous recombination, mismatch repair, nucleotide excisionrepair or interstrand crosslink repair, such as FANCD1, MSH2, orERCC1 [50–52]. A mouse strain with a hypomorphic mutation in DNAligase IV, which is involved in double-strand break repair by non-homologous end joining, showed a progressive loss in HSC numbersand functions during aging [53]. Furthermore, mice bearing a mutatedRad50 allele displayed profound bone marrow hypoplasia resultingfrom constitutive activation of the ATMpathway, suggesting that excessDD signaling reduces HSC numbers and/or functions [54,55]. Finally,Rossi and colleagues showed that mice with germline deficiencies innucleotide excision repair, non-homologous end joining or telomeremaintenance (XPDTTD, Ku80−/− and late-generation mTR−/− mice,respectively) displayed a premature decline in HSC functions. Most im-portantly, this study noted DD accumulation (in terms of γ-H2AX foci)in the LT-HSC compartment, with physiological aging even in wild-type mice [56]. Similarly, increased incidence of endogenous γ-H2AXfociwas observed during human aging in peripheral blood lymphocytesand in hematopoietic stem and progenitor cells [57,58]. Together, theseresults are consistentwith the view that endogenous DD is accumulatedduring physiological aging and results in decreased self-renewal of theaged SCs. Notably, as mentioned, we recently demonstrated that SCspossess specific mechanisms for the processing of the DNA damagegenerated by exogenous irradiation or by endogenous cellular activities,but that some damaged DNAs evade repair and accumulate over timelimiting their SC self-renewal potential. Therefore, adult SCs surviveDNA-damaging insults, but their imperfect DD response limits theirlife span. Genetically, this cellular response is dependent on p21 andindependent of p53. Indeed, in the absence of p21, DD accumulationin LT-HSCs during physiological aging was significantly more pro-nounced than in the WT, leading to their premature exhaustion [34].We propose that this flawed DNA repair response in SCs physiologicallyevolved as a mechanism of tumor suppression in these cells, as p53-mediated apoptosis or senescence is suppressed.
On the other hand, this response in SCs can work as a double-edgedsword during the lifetime of an individual. Continuous waves of DDand DD-repair in SCs might lead to accumulation of DNA-mutations(mutator phenotype), thus favoring tumor initiation. Furthermore,expression of activated oncogenes in SCs induces DD and DD-repairactivities ([22]), further increasing their propensity to accumulateDNA-mutations and progress toward full transformation. Indeed, wefound that hyper-activation of p21-dependent DNA repair mechanismcontributes to leukemia progression [22]. It is interesting to note thattissues in which SCs do not repair DD do not give rise to commonhuman tumors. For instance, hair follicle melanocyte SCs that undergopremature differentiation following DD do not give rise to melanomathat, in contrast, originates from skin melanocytes. Furthermore, apo-ptosis of intestinal SCs upon DD might explain the rarity of intestinal
neoplasias as compared to the frequency of colonic cancers, despitethe higher cellular turnover in the intestine.
Therefore, cancer and aging can be regarded as two different mani-festations of the same underlying mechanism, namely a specific wayof handling DD in SCs without undergoing apoptosis or senescence:both processes are the cost of a specific tumor suppression mechanismthat limits SC lifespan but results in accumulation of cellular damage(Fig. 1).
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgments
We would like to kindly thank Paola Dalton for critical reading andediting of the manuscript.
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