mechanisms of dealing with dna damage in terminally differentiated cells
TRANSCRIPT
Mutation Research 685 (2010) 38–44
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Review
Mechanisms of dealing with DNA damage in terminally differentiated cells
P. Fortini, E. Dogliotti ∗
Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
a r t i c l e i n f o
Article history:Received 17 November 2009Accepted 17 November 2009Available online 24 November 2009
Keywords:DNA repairCell differentiationDNA damage signallingApoptosisAutophagy
a b s t r a c t
To protect genomic integrity living cells that are continuously exposed to DNA-damaging insults areequipped with an efficient defence mechanism termed the DNA damage response. Its function is toeliminate DNA damage through DNA repair and to remove damaged cells by apoptosis. The DNA dam-age response has been investigated mainly in proliferating cells, in which the cell cycle machinery isintegrated with the DNA damage signalling. The current knowledge of the mechanisms of DNA repair,DNA damage signalling and cell death of post-mitotic cells that have undergone irreversible cell cyclewithdrawal will be reviewed. Evidence will be provided that the protection of the genome integrity interminally differentiated cells is achieved by different strategies than in proliferating cells.
© 2009 Elsevier B.V. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392. DNA damage repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.1. Nucleotide excision repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392.2. Base excision repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392.3. Mismatch repair and homologous recombination/non-homologous end joining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3. DNA damage signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414. Cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
4.1. Restricted apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424.2. Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
5. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Abbreviations: AAG, alkyladenine DNA glycosylase; Apaf1, apoptotic peptidase activating factor 1; ATG, autophagy-related protein 4; ATM, ataxia telangiectasia mutated;ATR, ataxia telangiectasia and Rad3 related; DNA-PK, DNA-phosphokinase; DRAM, damage-regulated autophagy modulator; E2F1, E2F transcription factor 1; FEN1, flapendonuclease 1; FoxM1, forkhead box M1; H2AX, histone H2A; LC3, light chain 3; MRE11, meiotic recombination 11; mTOR, mammalian target of rapamicin; MYH, mutYhomolog; NBS1, Nijmegen Breakage Syndrome 1; NEIL1, nei-like DNA glycosylase 1; NTH1, DNA glycosylase and apyrimidinic (AP) lyase (endonuclease III); OGG1, 8-oxoguanine-DNA glycosylase 1; PCNA, proliferating cell nuclear antigen; PI3K, phosphoinositide-3-kinase; pRb, phosphoretinoblastoma; PTEN, phosphatase and tensinhomolog; Puma, p53 up-regulated modulator of apoptosis; SMUG1, single-strand selective monofunctional uracil DNA glycosylase; TDG, thymine DNA glycosylase; TSC1,tuberous sclerosis 1; UNG2, uracil DNA glycosylase; XIAP, X-linked inhibitor of apoptosis protein; XRCC1, X-ray repair complementing defective repair in Chinese hamstercells 1; XRCC2, X-ray repair complementing defective repair in Chinese hamster cells 2.
∗ Corresponding author at: Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Section of Molecular Epidemiology, Viale Regina Elena299, 00161 Rome, Italy. Tel.: +39 6 49902580; fax: +39 6 49903650.
E-mail address: [email protected] (E. Dogliotti).
0027-5107/$ – see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.mrfmmm.2009.11.003
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. Introduction
A growing body of evidence indicates that based on their des-iny cells present different mechanisms to protect their genomentegrity from endogenous and exogenous stress. The DNA damageesponse (DDR) of cells that proliferate to generate proliferatingells is different from that of progenitor cells (multipotent or dif-erentiation committed) that in return differs from the response ofheir functional progeny (differentiated cells). Under stress stemells seem to favour apoptosis over DNA repair (reviewed in [1])hereas differentiated cells restrict apoptosis and limit DNA repair
o transcriptional domains (reviewed in [2]). Depending on theell type differentiated cells present significantly different turnoverhat might impact on the need of a strict control of their genomentegrity. For example, blood or skin cells have relatively short lifes-an and might deal with accumulated DNA damage better thanifferentiated cells like neurons or adipocytes that if damaged can-ot be replaced. Muscle satellite cells are an example of progenitorells that serve as a reservoir of undamaged cells that in responseo mechanical strain become activated and proliferate to reconsti-ute the intact myofibers. The general notion that DNA repair isown-regulated in terminally differentiated cells leaves open theuestion of how these cells deal with persisting damage. The para-oxical resistance of terminally differentiated cells to various typesf stress raises the question of how the cell death programme isegulated. Mechanistic insights into the regulation of DDR alonghe differentiation programme have been obtained by using dif-erentiation cell model systems. These studies will be specificallyighlighted in this mini-review.
. DNA damage repair
.1. Nucleotide excision repair
DNA repair capacity in terminally differentiated cells is expectedo be essential for both the preservation of the transcribed genomentegrity and the protection from cell death to guarantee tissueomeostasis. Pioneering studies on DNA repair capacity of non-ividing (i.e. post-mitotic) cells have been carried out by Hanawaltnd co-workers who extensively analysed the nucleotide excisionepair (NER) efficiency in several terminally differentiated humanissues, i.e. striated muscle, macrophages and neurons. Briefly, NERs a versatile DNA repair mechanism responsible for the removalf UV light induced photoproducts, bulky chemical adducts andntra-strand DNA cross-links. NER can be divided into two sub-athways: the global genome repair (GGR) and the transcriptionoupled repair (TCR). GGR and TCR recognize and remove DNAesions throughout either the entire genome or the transcribedtrands of the active genes, respectively. In terminally differen-iated cells, GGR is generally attenuated whereas, within activeenes, not only the transcribed but also the non-transcribed strandsre efficiently repaired (reviewed in [2,3]). It is conceivable that inerminally differentiated cells, that never divide, a severe surveil-ance of the entire genome is not necessary anymore, whereashe maintenance of efficient repair of the transcribed genome isequired for preservation of tissue specificity. By complementationtudies it was clarified that the reduction of NER efficiency in post-itotic macrophages was not due to the partial or total inactivity of
ny NER enzymes but could be restored by the ubiquitin-activatingnzyme E1. A model has been proposed which implies that a reduc-ion in phosphorylation of E1, as observed in differentiated cells,
ould lead to a reduced ubiquitination of TFIIH which, in turn,ould determine a decrease of its activity in GGR [4]. Initially thishenomenon was named differentiation-associated repair (DAR)5] but when a similar mechanism was found in quiescent mousembryo fibroblasts [6] as well as in actively growing cells [7,8] theesearch 685 (2010) 38–44 39
acronym was maintained but redefined as transcription domain-associated repair. Currently, the most accredited hypothesis is thatDAR is nothing more than a subset of GGR, which operates on bothDNA strands, and is restricted to the sub-nuclear compartmentswhere transcription occurs. This phenomenon, not discernablefrom GGR, becomes detectable only in cells that have little or noglobal NER.
DNA repair measurements after UV in terminally differentiatedkeratinocytes revealed that GGR is not reduced when comparedwith the undifferentiated counterpart [9] questioning whether DARis a general phenomenon that operates in all cell types. Moreover,basal and UV-induced levels of p53 were nearly undetectable dur-ing late differentiation stages when GGR was active [10] suggestingthat the mechanisms involved do not require p53.
2.2. Base excision repair
The information on the efficiency and mode of other DNA repairpathways along the cell differentiation program is still quite lim-ited. Base excision repair (BER) is the main repair mechanisminvolved in the processing of structurally non-distorting lesions,such as alkylated, oxidised bases and abasic sites. BER is a stepwisemulti-enzymatic pathway: the first step implies the removal of thedamaged base by specific DNA glycosylases which give rise to aba-sic sites which are rapidly converted into single-strand breaks (SSB)by an AP endonuclease. After this step, BER can proceed throughtwo different sub-pathways: the short patch BER (SP-BER) and thelong patch BER (LP-BER) that differ for the repair-patch length andthe specific players involved (reviewed in [11]). DNA polymerase �and DNA ligase III are responsible for the filling-in and the sealingstep of the SP-BER, respectively, whereas, in LP-BER, these reac-tions are catalysed by the PCNA-dependent DNA polymerases �/�and DNA ligase I. BER is the repair mechanism of election for oxida-tive DNA damage. Reactive oxygen species (ROS) are generatedas by-products of normal mitochondrial activity and, at moderatedoses, ROS can function as specific second messengers in signallingcascades involved in cell growth and differentiation. Cell differ-entiation can be promoted by ROS (reviewed in [12]). Changes inexpression levels of oxidative stress marker genes together with anincreased oxidation of DNA was detected in myotubes in an in vitroskeletal muscle differentiation cell system [13] and ROS have beenshown to prime Drosophila hematopoietic progenitors for differen-tiation [14]. However, if the ROS level is not properly controlled,their overproduction poses a serious threat to cell integrity sincedifferent cell constituents, such as lipids, proteins and nucleic acids,can be oxidised.
Various properties of skeletal muscle, first of all the redoxmodulation of muscle contraction, render this tissue particularlysusceptible to ROS injuries. We recently explored BER efficiencyin a murine skeletal muscle cell differentiation system [13].Muscle satellite cells, a physiological reservoir of differentiation-committed cells, can be readily isolated from mouse thigh, culturedand induced to fuse in multinucleated myotubes. These termi-nally differentiated cells faithfully recapitulate the process ofdifferentiation that occurs in vivo by showing repression of cellproliferation-associated genes, expression of muscle-specific genesand irreversible cell cycle exit. A clear impairment of BER effi-ciency in myotubes versus myoblasts was observed. Both BERsub-pathways resulted to be less efficient in post-mitotic cellsalthough the LP-BER, which shares several partners with DNA repli-cation, was more severely compromised. At molecular level the
BER impairment was ascribed to the nearly complete lack of DNAligase I and to the strong down-regulation of XRCC1, a scaffoldprotein known to be essential for DNA ligase III stabilization. Inline with this observation XRCC1 is a transcriptional target forFoxM1 [15] and E2F1 [16] that activate several cell cycle genes
40 P. Fortini, E. Dogliotti / Mutation R
Fig. 1. (A) In vitro muscle differentiation is associated with progressive down-regulation of XRCC1, E2F1 and FoxM1 gene expression. mRNA levels weredetermined by RT-PCR by using the comparative Ct method (ABI Prism 7000sequence detection system). Shadowed bars: 24 h; grey bars: 48 h and white bars:72 h, after differentiation induction. Values were normalized by setting the valuesof myoblasts (Mb) at 1. (B) Quantitative gene expression analysis of XRCC1, FoxM1aemi
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nd DNA ligase I in myotubes obtained by differentiation of mouse satellite cellsxpressing E2F1 (shadowed bars). Values were normalized by setting the values ofuscle cells infected with an empty vector at 1 (white bars). A typical experiment
s shown.
nd are both down-regulated during cell cycle exit in myotubes.ig. 1A shows the progressive down-regulation of XRCC1, E2F1nd FoxM1 expression during in vitro muscle cell differentiation.ctopic expression of E2F1 by infection of mouse satellite cells withn adenovirus expressing E2F1 stimulated the expression of FoxM1,RCC1 and DNA ligase I (Fig. 1B) supporting the hypothesis thaturing myogenesis XRCC1 is down-regulated as a consequence ofhe reprogramming of cell cycle related genes.
The attenuation of BER observed in myotubes was consistentlyirrored by the accumulation of SSB and phosphorylated H2AX
uclear foci upon cell exposure to hydrogen peroxide [13].Conversely, XRCC1 down-regulation was not observed in non-
ividing neural cells [17] which were protected by XRCC1 itselfgainst the cytotoxic effect induced by oxidising agents providingn example of tissue-type specificity in DNA damage repair.
A few studies have addressed the question of the regulation ofNA glycosylase activity during differentiation. Neurospheres haveeen used as a model for the study of the expression and activityf OGG1 in neural stem cells as compared to differentiated neu-ons [18]. OGG1 activity was high in neurospheres derived fromewborn mice and decreased in adults and upon induction of cellifferentiation. We have analysed gene expression levels of variousNA glycosylases in murine myotubes as compared to myoblasts.o significant difference in gene expression levels of a variety ofNA glycosylases, such as SMUG1, OGG1, TDG, NTH1, AAG andEIL1 was observed during differentiation with the exception ofNG2 and MYH (Fortini and Dogliotti, unpublished data). Inter-stingly, both UNG2 and MYH are involved in the processing ofost-replicative lesions and therefore their activity may be dispens-ble for cells undergoing irreversible cell cycle exit.
Since the mitochondrial genome is a susceptible target forxidative damage its repair should play an important role partic-larly in those tissues with high metabolism such as the skeletaluscle. It has been recently discovered that repair in mitochon-
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dria occurs not only by SP-BER but also by LP-BER. The LP-BER isinvolved in the processing of oxidative lesions [19] and requiresthe nuclease activity of FEN1 to repair damage both in nuclear andmitochondrial genome [20,21]. FEN1 is not only involved in LP-BER, but it is also essential for DNA replication. FEN1 is stronglydown-regulated in terminally differentiated cells such as myotubes(Fortini P., unpublished observation). How this might impact on theefficiency of mitochondrial BER in normal and pathological condi-tions should be investigated.
2.3. Mismatch repair and homologousrecombination/non-homologous end joining
Terminally differentiated cells are not replicating their DNA butmismatches can occur either spontaneously as in the case of deam-ination or during repair attempts by error-prone DNA polymerases.Hippocampal neurons of MSH2 heterozygous mice are more resis-tant to apoptosis after oxidative stress than wild-type neuronsindicating that MMR is indeed important for the genomic integrityof adult neurons [22]. The few studies addressing the question ofmismatch repair (MMR) functionality in the course of differenti-ation indicate that differentiated neurons possess MMR [23] andtheir repair activity seems to be similar to that of undifferentiatedcells [24,25].
In mammalian cells double strand breaks (DSB), which are con-sidered as the most lethal form of DNA damage, are repaired viatwo mechanistically distinct pathways: homologous recombina-tion (HR) and non-homologous end joining (NHEJ) (reviewed in[26]). HR is a high fidelity repair mechanism that involves a groupof RAD51-related proteins, including XRCC2. HR occurs preferen-tially during the late S and G2 phases of the cell cycle when asister chromatid is present and therefore it is precluded in non-cycling cells. However, HR can also use a homologous chromosomeas a template, and some HR is observed in GO/G1 cells, albeit at alevel lower than in S/G2/M (Saleh-Gohari and Helleday NAR 2004).NHEJ simply pieces together the broken DNA ends and specificallyrequires the DNA dependent protein kinase (DNA-PK) holoenzyme(Ku heterodimer and the DNA-PK catalytic subunit) which togetherwith XRCC4, Cernunnos and DNA ligase 4 (LIG4) reseals the DNAends previously trimmed by various nucleases. NHEJ is active dur-ing all cell cycle phases and it is the predominant pathway for DSBrepair in mammalian cells. An elegant study analysed the selectiverequirements for HR and NHEJ during nervous system development[27]. By using mice carrying a germ line disruption of XRCC2 (HRdefective) and LIG4 (NHEJ defective) the two pathways for recombi-nation were found to be spatiotemporally distinct: HR inactivationwas crucial from the early steps of embryogenesis leading to abun-dant apoptosis, whereas the disruption of NHEJ had deleteriousconsequences only at later developmental stages (not before E12).Since the late stages of the embryogenesis are characterized bymassive differentiation, these results imply that the HR pathwayhas an essential protective role against DSB-induced cytotoxicityin proliferating cells becoming dispensable in post-mitotic cellswhere NHEJ is the pathway of election. The key role of the NHEJin differentiated long-lived cells was also established in an in vitromurine adipogenesis cell system [28]. A faster DSB repair kineticsin adipocytes compared to their proliferating precursors was foundafter exposure to a radiomimetic chemical or ionizing radiation. Theincreased ability of adipocytes to repair DSB was mainly ascribedto the up-regulation of DNA-PK expression and activity.
If DNA-PK is involved in the repair of DSB in terminally differen-
H2AX following exposure to camptothecin (CPT) of primary lym-phocytes [29]. CPT induces Topoisomerase I (TopI)-linked SSB. Ithas been proposed that TopI cleavage complexes produce tran-scription arrest with R-loop formation and thus generate DSB [29]
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hat trigger the phosphorylation of the histone H2AX (see also Sec-ion 2). What are the pathways that are involved in the repairf transcription-associated DSB in post-mitotic cells awaits to belarified.
. DNA damage signalling
When cells enter a post-mitotic state genes involved in cellycle progression are permanently silenced. Among these therere genes belonging to the DDR that are down-regulated or theirroducts redistributed during terminal differentiation. An exam-le is p53 that in the absence of stress plays an important role
n the regulation of differentiation and development. P53 tran-cripts reach a maximum during differentiation of several tissuesut the levels decline strongly in cells undergoing terminal differ-ntiation. In some species (e.g. Xenopus) the absence of p53 duringevelopment leads to dramatic defects in differentiation [30]; inontrast approximately 80% of the p53-null mice develop normallyut are prone to increased incidence of spontaneous cancer [31].his might indicate an ancillary role of p53 in development andifferentiation but most probably indicate a compensatory role ofhe other p53 family members such as p63 and p73. In particu-ar, the truncated isoform of p73 blocks differentiation of murine
yoblasts and protects neurons and myotubes from cell death afterifferent apoptotic stimuli in part by inhibiting the pro-apoptoticole of p53 [32,33].
In the case of skeletal muscle differentiation early steps in geneeprogramming include the activation of p21 and the hypophos-horylation of pRb, which leads to the transcriptional repression of2F-activated genes. These steps, up to the expression of myogenin,an occur in the absence of the p53 family. After the cell cycle with-rawal p53, p63 and p73 contribute either directly or indirectlyo the activation of pRb, that cooperate with MyoD to transcribeater differentiation markers [34,35]. Cell-to-cell contacts can alsoncrease pRb expression thus overcoming the requirement of p5334] and likely accounting for the apparent normal muscle devel-pment of p53-null mice.
P53 can regulate proliferation and differentiation also of neu-al progenitor cells as well as axon outgrowth and regenerationhus challenging the idea that the role of p53 in neuronal biologys exclusively an apoptotic role [36]. Overall a concept is emerginghat p53 is also a “guardian of differentiation” although dependingn the specific cell type and differentiation program it may exertither a positive or a negative effect [36].
P53 is the major regulator of the cellular defense against DNAamage. Upon DNA damage p53 is stabilized and activated. Its level
s tightly controlled by its post-translational modification statusnd that of its E3 ubiquitinin ligases, in particular by site-specifichosphorylation, acetylation and ubiquitination. In a very simplis-ic model genotoxic stress activates one or more of the PI3K-likeinases like ATM, ATR and DNA-PK that in turn activate the his-one H2AX as well as downstream checkpoint kinases (i.e. Chk1nd Chk2) and p53. The processing of DNA lesions is associatedith specific DDR sub-pathways. The activation of the ATM-Chk2athway has been described after induction of DSB by IR whereasulky lesions as those induced by UV trigger the ATR-Chk1 branchia replication blocks [37]. ATR is suppressed in post-mitotic cells38,39] thus leaving ATM and DNA-PK as key candidate kinases inhese cells.
In neurons as well as in myotubes nuclear ATM seems to carryut the same role as in proliferating cells, thus mobilizing the DDR.
fter exposure of C2C12 myotubes to IR, ATM is activated by phos-horylation and then phosphorylates H2AX and recruits MRE11nd NBS1 at DSB sites [40]. Fig. 2 shows phosphorylation of H2AXollowing treatment of post-mitotic myotubes with hydrogen per-xide. The formation of �H2AX foci is reversible, although slowerFig. 2. DNA damage induction and repair kinetics as detected by �H2AX foci for-mation in proliferating (white bars) and terminally differentiated (shadowed bars)murine muscle cells after treatment with 500 �M H2O2 for 30 min. NT, untreatedcells. A typical experiment is shown.
than in proliferating cells, which is consistent with the repair ofDNA lesions. Hydrogen peroxide mostly induces SSB thus ques-tioning whether phosphorylation of this histone marks exclusivelyDSB. Phosphorylation of H2AX occurs also in post-mitotic neuronsfollowing genotoxic stress. Interestingly, �H2AX foci have beenrecently described in primary neurons following treatment withCPT that induces transcription-dependent DSB (see Section 2.3).Even if the activation of ATM is maintained in post-mitotic cellsthe overlapping in function with proliferating cells is questioned byemerging evidence. During neurogenesis ATM is required for DNAdamage-induced apoptosis but only when NHEJ is disrupted butnot HR [27]. Another example is provided by the lack of phospho-rylation of p53 and apoptosis after exposure of C2C12 myotubes toionizing radiation (IR) but not after exposure to doxorubicin. Fol-lowing IR exposure ATM is autophosphorylated but DDR is blockeddownstream of ATM [40]. These data reveal tissue- and cell-type aswell as DNA lesion specificity for DDR during differentiation.
Chk 1 has been shown to decline during differentiation ofC2C12 myoblasts thus confirming that the ATR-Ck1-p53 pathwayis suppressed in non-cycling cells [38]. Conversely, Chk2 levels aremaintained in terminally differentiated cells and the protein can beactivated upon damage [41].
The effects of DNA damage on the differentiation program isnot specifically addressed in this review but it is important toknow that precursor cells when damaged activate a transcriptionalcheckpoint that leads to inhibition of the differentiation program.The first observation of this phenomenon has been reported incultured myoblasts [42] whereas myotubes were unable to trig-ger this response. It has been speculated that the inhibition oftranscription of differentiation genes allows DNA repair to occurbefore cells enter into a post-mitotic state (for a review see[43]).
Unlike proliferating cells in which DNA damage typically trig-gers cell cycle checkpoints post-mitotic neurons under stress aswell as in pathological conditions activate their cell cycle machin-ery. The re-entry into the cell cycle leads to neuronal death.Attempts to re-enter the cell cycle in stressed neurons may be a partof the DDR as suggested by the attenuation of cell cycle re-entry anddeath of post-mitotic neurons upon suppression of ATM (reviewedin [44]). The function of this response might be the removal of DNAdamage (activation of cell cycle associated DNA repair pathways?)or the elimination of damaged cells via apoptosis. Whether thismechanism is confined to neurons or extends to other types ofpost-mitotic cells waits to be investigated.
4. Cell death
In response to irreparable DNA damage and depending on thecellular context, inactivation of the cells by apoptosis or senescencemay occur. A crucial event of apoptosis is the activation of caspaseproteases. In mammalian cells, including post-mitotic cells, DNA
4 tion Research 685 (2010) 38–44
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Fig. 3. DNA damage response changes during differentiation: the example of theskeletal muscle cell differentiation system. Microphotographs captured with a con-
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amage can activate the intrinsic apoptotic pathway leading to theelease of cytochrome c from mitochondria, its binding to Apaf-that in turn induces the formation of the apoptosome complex
45]. This complex results in the activation of caspase 9 and then ofownstream caspases that are responsible for cell death. P53 playsn important role in the regulation of apoptosis in response to DNAamage in proliferating as well as differentiated cells [46,47]. P53egulates cytochrome c release both directly and by transcriptionalnduction of Bcl-2 proteins such as Bax, Puma and Noxa. Terminallyifferentiated cells are characterized by resistance to apoptosisrestricted apoptosis) that is presumably important to ensure theong-term survival of post-mitotic cells. Another recently described53-regulated cellular process is autophagy, a lysosomal pathwayf cellular self-digestion. Autophagy plays a key role in the mainte-ance of normal cellular homeostasis and occurs at basal levels inost tissues. Its failure is associated with neurodegeneration andyopathies.
.1. Restricted apoptosis
In post-mitotic cells including neurons and cardiomyocyteseath by apoptosis requires an additional step that is to relieve the
nhibition of caspases by the X-linked inhibitor of apoptosis proteinXIAP). XIAP regulates caspases by directly binding and inhibitingheir function. In the case of neurons two distinct mechanisms haveeen identified to relieve XIAP between nerve growth factor (NGF)eprivation and DNA damage. In the first case XIAP is selectivelyegraded whereas in the case of DNA damage the relief of XIAPequires p53-mediated induction of Apaf-1 [48]. The XIAP “brake”as also been confirmed in skeletal muscle myotubes where theechanism involves the decrease in Apaf-1 that occurs during dif-
erentiation [49]. In order for myotubes to die they may degrader inactivate XIAP or up-regulate Apaf-1. C2C12 myotubes that areesistant to IR have been shown to die after doxorubicin treatmenthat activates p53 [40] likely by up-regulating Apaf-1 and relievinghe XIAP “brake”.
.2. Autophagy
Recently another type of cell death, macroautophagy (calledutophagy from now on), has been reported as novel responseo various stress stimuli including starvation, oxidative dam-ge by ROS and exposure to DNA-damaging agents such asR and temozolomide. During autophagy portions of the cyto-lasm are encapsulated in a double membrane structure calledutophagosome. Autophagosomes then fuse with lysosomes andheir contents are degraded by lysosomal hydrolases (reviewed in50]). Autophagy plays an important role as intracellular qualityontroller and is constitutively active in many tissues includingkeletal muscle and brain. Accordingly, loss of autophagy resultsn neurodegeneration as in Alzheimer’s disease [51] and disregu-ated autophagy contributes to muscle wasting disorders [52]. Anmportant player in autophagy is LC3 that is an ubiquitin-like pro-ein that is activated by a series of proteases like ATG4, ATG7, ATG3o form II that becomes membrane bound [53].
P53 is an important regulator of both basal and DNA damage-nduced autophagy by playing a bidirectional control [54]. It acts asnegative regulator in the absence of stress suggesting that basal
evels of p53 inhibit autophagy. It has been shown that inhibitionf p53 leads to enhanced autophagy thus improving the survivalf p53 defective cells by allowing them to maintain high levels of
TP [55]. This finding might be of relevance in the case of post-itotic tissues where the expression of p53 is strongly decreased56] and basal autophagy is activated. In response to genotoxictress, the signal transduction between the nucleus (where damageccurs) and the cytoplasm (where autophagy takes place) involves
focal fluorescence microscope in bright field of cycling and non-cycling muscle cells.The direction of the arrows indicates either a decrease or an increase of the efficiencyof the indicated pathway during differentiation.
p53 as positive regulator. P53 is believed to stimulate autophagythrough signalling events that may involve either activation of theAMP kinase (AMPK) and inhibition of the serine/threonine kinasemTOR (mammalian target of rapamycin) or up-regulation of mTORinhibitors (such as PTEN and TSC1) or the cell death gene DRAM(reviewed in [57]).
The potential role of autophagy in the long-life maintenanceof differentiated tissues and its regulation are an exciting area forfuture investigation.
5. Conclusion
Our increasing understanding of the mechanisms of DNA repairand damage signalling illuminates the role played by cell typeand status on the response to exogenous and endogenous stress.We began this review by asking how post-mitotic cells deal withaccumulation of DNA lesions (due to inefficient DNA repair) andhow the cell death programme is regulated. Based on the informa-tion available they might limit repair to transcriptional domainsand/or rely on restricted apoptosis to guarantee the maintenanceof long-lived tissues (Fig. 3). However, the accumulation of lesionsin non-transcribed genes might pose a threat to differentiated tis-sues by causing genetic instability, apoptosis or senescence. Manyissues are still unexplored and should be addressed by futureresearch. Finally, the comprehension of how DDR is differentiallyregulated between mitotic and post-mitotic cells is of clinical sig-nificance as it might guide the design of drugs that could modulatecell death in selective cells in the context of diseases that targetpost-mitotic cells (e.g. neurodegenerative or muscle diseases) orcancer.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
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P. Fortini, E. Dogliotti / Mut
cknowledgements
We would like to thank Prof. Ciro Isidoro and Dr. Roberta CastinoUniversità del Piemonte Orientale “A. Avogadro”, Novara, Italy)or sharing with us their knowledge and expertise in the field ofutophagy and Dr. Marco Crescenzi’s laboratory (Istituto Superiorei Sanità, Roma, Italy) for the long-standing and fruitful collabo-ation on the skeletal muscle cell differentiation system. We arerateful to Dr. Laura Narciso for her important contribution to theharacterization of the DNA damage response in terminally differ-ntiated muscle cells. Grant support: Associazione Italiana per laicerca sul Cancro (AIRC), MIUR/FIRB (RBNE01RNN7).
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