DNA Damage-Induced Apoptosis

DNA damage-induced apoptosis

Chris J Norbury1 and Boris Zhivotovsky*,2

1Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK; 2Institute of Environmental Medicine, Karolinska Institutet, Box 210, Nobels va¨g. 13, SE-171 77 Stockholm, Sweden

Unicellular organisms respond to the presence of DNA phila melanogaster.The combination of data from these lesions by activating cell cycle checkpoint and repair genetically amenable models with those from mamma- mechanisms, while multicellular animals have acquired the lian and other vertebrate species has revealed conserva- further option of eliminating damaged cells by triggering tion of several key molecular mechanisms, as well as apoptosis. Defects in DNA damage-induced apoptosis species-specific variations on these themes. contribute to tumorigenesis and to the resistance of cancer Most of the morphological criteria that were first used cells to a variety of therapeutic agents. The intranuclear to distinguish apoptosis from necrosis relate to the mechanisms that signal apoptosis after DNA damage nucleus (Kerr et al., 1972). Degradation of chromoso- overlap with those that initiate cell cycle arrest and DNA mal DNA into oligonucleosome length fragments in repair, and the early events in these pathways are highly irradiated lymphoid tissues was reported as early as conserved. In addition, multiple independent routes have 1976 (Skalka et al., 1976). This observation was first recently been traced by which nuclear DNA damage can linked to endonuclease activation in 1980 (Wyllie, 1980) be signalled to the mitochondria, tipping the balance in and has since been used as a biochemical marker of favour of cell death rather than repair and survival. Here, apoptosis.Transcriptional activation of gene expression we review current knowledge of nuclear DNA damage is also a common, though not universal, feature of signalling, giving particular attention to interactions apoptosis.Unsurprisingly, the interest of many re- between these nuclear events and apoptotic processes in searchers in the field was therefore initially focused on other intracellular compartments. understanding the nuclear involvement in apoptosis Oncogene (2004) 23, 2797–2808. doi:10.1038/sj.onc.1207532 signalling.Questions about the primacy of nuclear events were raised when NUC-1, a protein essential Keywords: DNA damage; DNA repair; apoptosis; for apoptotic DNA degradation in C. elegans, was mechanisms shown to act downstream of the key apoptotic regulators CED-3 and CED-4 (Ellis and Horvitz, 1986).In some cases, DNA degradation required prior engulfment of apoptotic cells by phagocytes, again indicating that nuclease activation is a late event. Introduction In addition, upon treatment with staurosporine or antibodies against Fas (Apo-1/CD95), enucleate The integrity of genomic DNA is constantly under cells (cytoplasts) underwent morphological changes threat, even in perfectly healthy cells.DNA damage can characteristic of apoptosis, which could be inhibited result from the action of endogenous reactive oxygen by overexpression of the antiapoptotic protein species, or from stochastic errors in replication or Bcl-2 (Jacobson et al., 1994; Schulze-Osthoff et al., recombination, as well as from environmental and 1994). therapeutic genotoxins.While unicellular organisms A variety of strands of evidence subsequently respond to the presence of DNA lesions by activating indicated that caspases (cysteine-aspartate proteases) cell cycle checkpoint and repair mechanisms, multi- play central roles in apoptotic signalling and execution cellular animals have the additional possibility of (Thornberry, 1999 and references therein).The shift of eliminating the damaged cells by triggering their death. focus from the nucleus to the cytoplasm became even Induction of apoptosis has been recognized as a possible more pronounced when, in the mid-1990s, Wang and outcome of DNA damage for more than 20 years co-workers demonstrated that the release of cytochrome (Wyllie et al., 1980). The earlier literature in this area is c from mitochondria into the cytosol resulted in caspase dominated by studies of mammalian systems, but it has activation and the execution of apoptosis (Liu et al., since become apparent that DNA damage can also 1996).This, along with related key observations, led to induce apoptosis in diverse experimental model organ- an explosion in research focusing on mitochondrial isms, most notably Caenorhabditis elegans and Droso- regulation of apoptosis (for a review, see Cory and Adams, 2002).It is clear that any coherent view of the *Correspondence: B Zhivotovsky; apoptotic response to DNA damage must take into E-mail: [email protected] account events in the nucleus and the cytoplasm, as well DNA damage and apoptosis CJ Norbury and B Zhivotovsky 2798 as the regulatory traffic within and between these compartments.

Sensitivity and resistance to DNA damage-induced apoptosis

Animal cells differ markedly in their propensity to commit to apoptotic death following DNA damage. Thymocytes, for example, are exquisitely primed to engage apoptosis in response to DNA damage as well as a variety of other triggers.By contrast, primary fibroblasts appear to undergo DNA damage-induced apoptosis (DDIA) rarely, if at all – a feature of these cells familiar to anyone who has irradiated them for use as ‘feeder’ layers in cell culture systems.These differing apoptotic thresholds presumably reflect differing basal patterns of expression of pro- and anti-apoptotic regulators, and generally relate to the importance of Figure 1 Life versus death decisions following DNA damage. apoptosis during the normal developmental programme Common damage detection and signal transduction mechanisms of the cells in question.The relative radiosensitivity of are shared between pathways governing DNA repair, cell cycle thymocytes, for instance, can be related to their checkpoint activation and apoptosis.The consequence of any given propensity for apoptosis during negative selection in level of DNA damage is cell type-specific.By allowing the survival and proliferation of cells with damaged DNA, failure of these vivo.Cells less prone to triggering apoptosis can use processes contributes to tumorigenesis and resistance to cancer alternative strategies to avoid the propagation of therapies mutations that might otherwise threaten the health of the whole organism. (Di Leonardo et al., 1994; Baus et al., 2003). In this way, the threat posed by proliferation of mutated cells is Consequences of cell survival after DNA damage effectively eliminated without recourse to apoptosis.On the other hand, it is important that the apoptotic Loss of apoptosis undoubtedly plays a major role in programme, once initiated, is completed (Figure 1). tumorigenesis (Igney and Krammer, 2002).Studies in Transient activation of the caspase-activated DNase this area have frequently addressed the significance of (CAD) followed by cell recovery might even contribute loss of p53-dependent apoptosis in response to oncogene to the initiation of site-specific chromosomal transloca- activation or hypoxia, but loss of DDIA probably also tions during tumorigenesis (Vaughan et al., 2002). contributes significantly to tumour development.The Perhaps for this reason amplification mechanisms, issue is complicated by the fact that many of the notably those involving release of mitochondrial pro- mediators of DDIA have additional functions in cell teins following permeabilization of the outer mitochon- cycle regulation and/or DNA repair, which in turn can drial membrane, drive the complete destruction of the have dramatic influences on genome stability.For cell following commitment to DDIA, as discussed example, loss of function of the XPB or XPD helicase, below. as seen in xeroderma pigmentosum patients, results in Loss of apoptotic potential probably also makes a defective DDIA as well as defective nucleotide excision significant contribution to the resistance of cancer cells repair and a hugely elevated frequency of UV-induced to therapies that induce DNA damage (Lowe et al., skin tumours (Wang et al., 1996). Similarly, the ATM 1993a).It would be a gross oversimplification to protein kinase is required for a variety of cell cycle consider cancer cells simply as apoptosis-defective checkpoint responses to DNA double-strand breaks versions of their normal counterparts.There is no (DSB), but roles for this protein in DNA repair and doubt, however, that loss of function of proapoptotic DDIA have also been identified, as discussed below.It is regulators such as p53 and/or overexpression of anti- therefore difficult to determine the extent to which the apoptotic proteins such as Bcl-2 can tilt the balance in excess tumour incidence seen in ataxia telangiectasia favour of tumour cell survival following DNA damage- (AT) (ATMÀ/À) patients is attributable specifically to based therapy. apoptotic defects. Even in the absence of faithful DNA repair, cell survival following DNA damage is not necessarily Signals linking DNA damage to cell cycle arrest, DNA associated with an increased risk of tumorigenesis.The repair or apoptosis lack of DDIA in primary fibroblasts, for example, is balanced by the capacity of these cells to maintain Any signalling pathway responding to DNA damage long-term p53-dependent cell cycle arrest in G1 or G2 must comprise one or more sensors, coupled via

Oncogene DNA damage and apoptosis CJ Norbury and B Zhivotovsky 2799 transducers to effector components (Figure 1).From an required for transduction of the DNA damage signal to evolutionary standpoint, it appears that the requirement downstream protein kinases essential for the execution for DNA damage sensors predated the emergence of of cell cycle checkpoint arrest (Norbury and Hickson, multicellularity by billions of years.DNA damage 2001).All of these components are conserved from responses such as homologous recombinational repair yeasts to human cells, and for the sake of simplicity here were clearly in place before the emergence of the we will use the human nomenclature for the RF-C eukaryotes.Cell cycle checkpoint controls that are related protein (RAD17), the sliding clamp (RAD1, found in all modern eukaryotes must also have arisen RAD9 and HUS1, otherwise known as the 9-1-1 early in eukaryotic evolution.It is debatable whether complex) and the downstream kinases (CHK1 and unicellular organisms could derive any selective advan- CHK2).Activation of the latter is brought about by tage from the acquisition of a mechanism for DDIA; their direct phosphorylation by the ATM/ATR protein indeed, these organisms lack apoptotic effectors such as kinases (Matsuoka et al., 1998; Ahn et al., 2000), but caspases that are found in multicellular animals.None- loading of the 9-1-1 complex is independent of ATM/ theless, molecular mechanisms that mediate DNA ATR function (Roos-Mattjus et al., 2002). In the yeast damage-induced cell cycle arrest and DNA repair in models, loss of function of any one of these proteins unicellular species have come to be involved additionally results in cell cycle checkpoint defects, and some in the activation of apoptosis in metazoans.The components of this checkpoint pathway have additional identification of additional mechanisms has helped to functions in, for example, stabilizing stalled replication explain how nuclear DNA damage signals can be forks.While these DNA damage signal transducers have coupled separately to the cytoplasmic events of apop- retained their cell cycle checkpoint roles in animal cells, tosis, in particular the release of apoptotic regulators like ATM/ATR they have also acquired important roles from mitochondria. in DDIA.CHK2-deficient mice, for example, exhibit All eukaryotic cells studied to date employ PI3- reduced radiation-induced apoptosis in neurons, thy- kinase-related protein kinases of the ATM/ATR family mocytes and splenocytes (Takai et al., 2002). to trigger a variety of DNA damage responses (Shiloh, Further targets of ATM and ATR have evolved more 2001).Members of this family were first identified recently and lack direct equivalents in the yeast models, through genetic screens in yeasts, where the protein yet are important for the efficient transduction of DNA kinase Mec1/Rad3 performs key roles in cell cycle damage signals to downstream effectors.These targets checkpoint responses to DNA damage.Subsequent include H2AX, a variant histone specifically and rapidly cloning of ATM, the gene defective in the human phosphorylated within its C-terminal tail by both ATM genetic disease AT, showed that it encodes a protein of and ATR in the nuclear microenvironment adjacent to the same family.Cells from AT patients ( ATMÀ/À) DNA damage (Fernandez-Capetillo et al., 2002). display a variety of cell cycle checkpoint defects after Phospho-H2AX is thought to be involved in the exposure to agents that induce DNA DSBs.ATR, concentration of repair and signalling proteins near identified through its similarity to other members of the DNA lesions.One such protein is BRCA1, which is also family, also performs important cell cycle checkpoint phosphorylated by ATM in response to DNA DSB functions.Current models place the ATM/ATR kinases (Cortez et al., 1999). Under these circumstances at or close to the sites of primary DNA damage, with BRCA1, best known as a tumour suppressor in familial ATM responding to DSB and ATR specific for partially breast cancer, forms nuclear foci that contain a variety single-stranded lesions coated with replication protein of proteins involved in mismatch repair, homologous A.AT patients suffer from extreme tissue sensitivity to recombination and checkpoint signalling (Wang et al., ionizing radiation.As well as defective cell cycle arrest 2000).BRCA1 is required for a specific subset of ATM/ on exposure to agents that induce DNA DSB, cells ATR-dependent phosphorylation events, including lacking ATM function exhibit a corresponding hyper- phosphorylation of p53 (Foray et al., 2003). 53BP1, sensitivity to such agents, as measured by cell survival yet another ATM target, interacts with phospho-H2AX, assays.It is therefore perhaps surprising that such cells CHK2, p53 and BRCA1, and is required for CHK2 have also been shown to be defective in DDIA, either in activation, BRCA1 phosphorylation, p53 stabilization culture or in vivo (Duchaud et al., 1996; Westphal et al., and BRCA1 focus formation after DNA damage 1997).Indeed the ataxia that is one of the hallmarks of (Fernandez-Capetillo et al., 2002; Wang et al., 2002). AT has been attributed to a failure to eliminate by While the details of these various interactions are apoptosis cells that have sustained DNA damage during complex and incompletely understood, many of these neural development (Lee et al., 2001). Clearly, in this components clearly have the capacity to influence the case at least, radiosensitivity in vivo cannot be correlated efficiency with which proapoptotic signals are trans- directly with the extent of DDIA. duced to p53, as well as governing the integrity of cell Extensive genetic and biochemical data have identi- cycle checkpoints and repair pathways.Indeed, mice fied further conserved components acting in concert deficient for H2AX exhibit defects in the efficiency and/ with ATM/ATR and the corresponding kinases in or fidelity of DNA DSB repair which, in the absence of unicellular eukaryotes.In yeasts, these include a p53 function, lead to massive genetic instability and a replication factor C (RF-C)-related protein, which is dramatically increased cancer incidence (Bassing et al., thought to load a PCNA-related heterotrimeric sliding 2003; Celeste et al., 2003). Furthermore, the kinetics of clamp onto damaged DNA.Each of these proteins is H2AX phosphorylation suggest that this chromatin

Oncogene DNA damage and apoptosis CJ Norbury and B Zhivotovsky 2800 modification could be induced by apoptotic DNA et al., 2001), but the identities of the key target genes fragmentation itself (Rogakou et al., 2000). activated in p53-dependent DDIA vary markedly Extension of the ATM/ATR paradigm might suggest among species, and even among different human cell the existence of a battery of damage sensors that could types.In Drosophila, these targets include the apoptotic recognize all possible DNA lesions, either directly or regulators reaper and sickle (Sogame et al., 2003), which indirectly after lesion processing.The recent discovery encode negative regulators of inhibitor of apoptosis of a chromatin-derived signal linking nuclear DNA (IAP) proteins.No homologous genes have been damage to mitochondria via the linker histone H1.2 identified among p53 targets in human cells, though suggests an alternative and more parsimonious model human p53 can effectively neutralize IAP proteins for damage signalling in DDIA (Konishi et al., 2003). indirectly through triggering release of apoptotic effec- This histone H1 isoform was identified through bio- tors from mitochondria.To this end, human p53 chemical fractionation of rat thymus cytosol as a factor directly activates transcription of several genes encoding mediating cytochrome c release from mitochondria members of the Bcl-2 family.This diverse family following X-irradiation, or exposure to other agents includes multidomain pro- and anti-apoptotic proteins that induce DNA DSB.H1.2,but not other H1 as well as a variety of ‘BH3-only’ proteins that together isoforms, in recombinant form was able to trigger this regulate mitochondrial permeability (Cory and Adams, apoptosis event in vitro, and endogenous H1.2 was 2002).The p53 targets considered most important in this released from the nucleus, along with all other H1 respect are the proapoptotic Bax and the BH3-only isoforms, following DNA damage in vivo.Downregula- proteins Noxa and PUMA (Miyashita and Reed, 1995; tion of H1.2 using small interfering (si) RNA or gene Oda et al., 2000; Nakano and Vousden, 2001). In deletion led to increased cell viability over a wide range mammalian cells, p53 also activated transcription of Fas of radiation doses.Although the mechanism of H1.2- following DNA damage, while Fas ligand expression induced cytochrome c release is unclear, this release is was induced independently of p53 (Owen-Schaub et al., correlated with activation of Bak and is dependent on 1995; Muller et al., 1998). These data raise the surprising multidomain proapoptotic Bcl-2 family members.This notion that nuclear DNA damage signals can be appealing model removes the requirement to postulate transduced via cell surface receptors.This mechanism multiple damage sensors, since a variety of primary renders cells with damaged DNA more susceptible to lesions could be sensed indirectly following the release of Fas ligand-induced apoptosis and cytotoxic T lympho- H1.2 into the cytoplasm. cyte-mediated killing (Chakraborty et al., 2003). None- theless, p53-mediated expression of FAS or any other transcriptional target of p53 is insufficient on its own to p53-mediated apoptosis in response to DNA damage induce DDIA (Reinke and Lozano, 1997). Aside from transcriptional activation, several other The key tumour suppressor p53 was the first determi- roles for p53 have been identified that could contribute nant of susceptibility to DDIA to be identified (Clarke to its ability to induce DDIA.The sequence-specific et al., 1993; Lowe et al., 1993b). In vertebrate cells p53 DNA binding activity of p53 can mediate transcrip- also has well-characterized roles in cell cycle check- tional repression, and p53-mediated downregulation of points, providing another example of the overlap transcription of genes including the IAP-family protein between the pathways controlling alternative DNA member survivin has been reported (Zhou et al., 2002). damage responses.p53 is functionally conserved in In addition p53 itself has been shown to bind DNA simpler multicellular animals, but is apparently absent strand breaks (Bakalkin et al., 1994), suggesting a from unicellular species.Although not required for possible role in DNA damage detection, or to translo- developmentally regulated apoptosis, and hence not cate to mitochondria specifically during p53-dependent identified in early screens for cell death genes, the p53 apoptosis (Marchenko et al., 2000; Mihara et al., 2003). homologue in C. elegans (cep-1) has since been shown to Once at the mitochondrial outer membrane, p53 appears be required for DDIA, as well as for chromosome to antagonize the antiapoptotic Bcl-2 and Bcl-XL segregation in the germ line (Derry et al., 2001; proteins directly by binding to them.Intriguingly, Schumacher et al., 2001). In Drosophila, p53 is required tumour-derived mutant forms of p53 that had pre- specifically for DDIA, but not for cell cycle checkpoint viously been shown to be defective in sequence-specific activation (Sogame et al., 2003). Together, these data DNA binding were also defective in binding to Bcl-2 suggest that the primordial role of p53 was probably and Bcl-XL.These findings are consistent with early more closely related to apoptosis than to cell cycle demonstrations that p53-mediated DDIA does not arrest.A detailed account of the role and regulation of necessarily involve new protein synthesis (Caelles et al., p53 in apoptosis is beyond the scope of this paper, but 1994), and suggest that the central importance of p53 excellent, more extensive reviews are available elsewhere transcriptional activation in DDIA should probably be (Shen and White, 2001; Vousden and Lu, 2002). re-evaluated.Functional p53 was also found to be There is a broad consensus that the primary required for the specific deamidation of two asparagine physiological role of p53 in DDIA is to act as a residues in Bcl-XL, neutralizing its ability to block the transcriptional activator of genes encoding apoptotic action of proapoptotic proteins such as Bax (Deverman effectors.The DNA-binding specificity of p53 is et al., 2002), but it is currently unclear whether this also conserved from C. elegans to human cells (Schumacher reflects a direct p53-Bcl-XL interaction.Interestingly, as

Oncogene DNA damage and apoptosis CJ Norbury and B Zhivotovsky 2801 was mentioned above, the release of histone H1.2 and other histone H1 isoforms from the nucleus after DNA damage was also p53-dependent (Konishi et al., 2003). It remains to be seen if the H1.2-releasing function of p53 requires its activity as a transcription factor, or one of the alternative activities outlined above, though H1.2 release was found to be upstream of caspase activation (Konishi et al., 2003).

Activation of p53 by ATM/ATR/CHK2

The first hint of a connection between the ATM/ATR- dependent pathways and p53 predated the identification of ATM itself, and derived from the observation that cells from AT patients showed delayed accumulation of Figure 2 An overview of DNA damage signalling in apoptosis. p53 protein after exposure to ionizing radiation (Lu and Early damage sensing in the nucleus involves the ATM and ATR Lane, 1993).The biochemical basis for this connection is protein kinases, the RAD1-RAD9-HUS1 (9-1-1) complex, and thought to involve ATM-dependent multisite phosphor- their downstream effector CHK2.Note that these components are also required for cell cycle checkpoint and DNA repair responses ylation of p53 (Caspari, 2000; Saito et al., 2002), and of (not shown).Once CHK2 is activated, the signalling processes can MDM2, preventing both MDM2-mediated inhibition of be grouped into p53-dependent (left) and p53-dependent events p53 DNA binding and its ubiquitin-dependent proteo- (right).The activities of p53 include transcriptional activation of lysis (Khosravi et al., 1999). Downstream from ATM/ the genes encoding Bax, Noxa, PUMA and Fas, as well as direct ATR, CHK2 can in turn phosphorylate p53 at Ser20, effects on mitochondrial permeabilization and mediating the release of histone H1.2 from the nucleus. The proapoptotic effects again potentially blocking the MDM2–p53 interaction of Bax are antagonized by the release of nuclear Ku70 into the and modulating the DNA-binding activity of p53 cytoplasm.The p53-independent pathways include CHK2- (Caspari, 2000).The relative contributions of these mediated signalling to PML, and redirection of E2F-1 towards various phosphorylation events to physiological DDIA proapoptotic transcriptional target genes, including those encoding have been difficult to assess, but valuable clues once p73 and procaspases.ATM also phosphorylates c-Abl, which promotes the neutralization of the antiapoptotic Bcl-2 and Bcl-XL again come from model systems.In Drosophila, expres- by RAD9.Caspase-2 and Nurr77 transduce p53-independent sion of a dominant-negative mutant form of Chk2 damage signals from the nucleus to mitochondria in less well- inhibited p53-mediated DDIA (Peters et al., 2002). defined ways.For further details, see text Evidence from Chk2 knockout mice further supports the idea that Chk2 is important for regulation of p53 Drosophila and human cells (Nahle et al., 2002; Zhou transcriptional activity, as well as stability (Takai et al., and Steller, 2003), and after DNA damage the specificity 2002). of E2F-1 was found to shift in favour of selected targets In addition to the negative regulation provided by including the gene encoding the p53-related protein p73 MDM2, the activity of p53 is limited by association with (Pediconi et al., 2003). The reduced DDIA seen in cells iASPP (inhibitory member of the ASPP family), which is lacking CHK2 therefore reflects defective p53-depen- conserved from C. elegans to human cells (Bergamaschi dent and p53-independent death pathways. et al., 2003). The steady-state level of this p53 inhibitor Components of the 9-1-1 complex also serve to couple may set the threshold level of damage required for DNA damage signalling to apoptotic effectors.In C. DDIA, since overexpression of iASPP conferred relative elegans the RAD1 homologue MRT-2 was found to be resistance to UV- and cisplatin-induced apoptosis. required for DDIA in addition to the core apoptotic regulators CED-4 and CED-3 (Gartner et al., 2000). In the same system, HUS1 was relocalized within the Further connections between checkpoint proteins and nucleus after DNA damage, and was required for the apoptosis CEP-1(p53)-induced transcriptional induction of egl-1, which encodes a proapoptotic BH3-only protein (Hof- The conserved DNA damage-signalling pathway down- mann et al., 2002). Damage signals relayed indepen- stream from ATM/ATR has been exploited in several dently by the ATM/ATR and 9-1-1 pathways therefore further ways during the evolution of apoptotic regula- appear to be integrated via CHK2 at the level of p53 tion (Figure 2).The induction of p53-independent activation. DDIA has been attributed to CHK2 acting through More direct involvement of 9-1-1 proteins in regula- the nuclear PML (promyelocytic leukaemia) protein tion of mitochondrial permeability may also be im- (Yang et al., 2002), although the downstream effectors portant in DDIA.Intriguingly, the RAD9 component of of this pathway have yet to be described.CHK2 has also this complex contains a BH3-like domain that is been found to phosphorylate and activate the E2F-1 conserved from yeast to human cells (Komatsu et al., transcription factor specifically after DNA damage 2000a), indicating that this structural motif is more (Stevens et al., 2003). E2F-1 activity determines the ancient than the Bcl-2 family itself.Human RAD9, level of expression of a variety of apoptotic effectors in which was found by coimmunoprecipitation to interact

Oncogene DNA damage and apoptosis CJ Norbury and B Zhivotovsky 2802 with the antiapoptotic Bcl-2 and Bcl-XL proteins, but apoptotic signal is sufficiently strong, Ku70 is released not with related proapoptotic proteins, induced apop- from the complex with Bax.As a result, the amino- tosis when overexpressed (Komatsu et al., 2000b). It is terminal region of Bax is exposed, allowing the protein to currently unclear whether RAD9 involved in such form a large, transient complex, the so-called ‘baxosome’ interactions would also participate in 9-1-1 complex that includes Bax itself, BH3-only proteins, membrane formation and, if so, how the nuclear 9-1-1 might lipid, cardiolipin, and possibly other unidentified pro- communicate with Bcl-2 family members, presumably at teins.Bax then undergoes conformational changes that the mitochondria.RAD9 was identified independently allow it to be inserted into the outer mitochondrial as a significant target of the c-ABL tyrosine kinase, with membrane and to oligomerize, resulting in the release of which it is also apparently stably associated (Yoshida mitochondrial apoptotic factors to the cytosol. et al., 2002a). The c-Abl-mediated phosphorylation of The concomitant upregulation of Ku70 and Bax after RAD9 occurred within the BH3 domain and promoted DNA breakage could favour the formation of Bax- the association between RAD9 and Bcl-XL, in response Ku70 complexes, which in turn could be central to the to DNA damage.Since c-Abl activation had previously decision of cells to repair the damage and survive, or to been shown to be dependent on ATM (Shafman et al., die.However, it is still unclear whether there are any 1997), these findings establish a further pathway linking links between upregulation of Ku70 and other p53- DNA damage sensing to regulation of Bcl-2 family activated proteins such as Noxa and PUMA.Interest- members, and hence mitochondrial permeability. ingly, downstream from the mitochondrial apoptotic events, activated caspase-3 cleaves another protein from DNA-PK complex, namely DNA-PKcs, thus destroying DNA-dependent protein kinase a key component of the cellular repair machinery.

A further member of the ATM/ATR family, DNA- dependent protein kinase (DNA-PK), has no obvious DAXX: a nuclear regulator of p53 and other apoptotic counterpart in unicellular eukaryotes but appears to effectors contribute to apoptotic regulation in mammals.DNA- PK is a heterotrimeric nuclear enzyme consisting of a DAXX, initially identified as a protein associated with 470 kDa catalytic subunit (DNA-PKcs) and the regula- the death domain of Fas (Fas Death Domain-Associated tory DNA binding subunits, Ku70 and Ku80 (Durocher protein XX), promotes Fas-induced cell death (Yang and Jackson, 2001).Cells deficient in DNA-PKcs, Ku70 et al., 1997). However, several lines of evidence suggest or Ku80 are hypersensitive to ionizing radiation and that this activity does not involve a direct association defective in V(D)J recombination, suggesting a primary between the two proteins.Most importantly, DAXX role for this kinase in DSB repair.Binding of the Ku was found to be exclusively nuclear and, upon induction heterodimer to DSB protects the DNA ends from of apoptosis by Fas, DAXX did not translocate out of degradation, and serves to recruit the catalytic subunit the nuclei (Torii et al., 1999). Moreover, DAXX was and additional factors essential for successful end joining. localized to subnuclear structures known as PODs A role for DNA-PK in p53-mediated death has been (PML-Oncogenic Domains), which are nuclear bodies suggested (Woo et al., 2002), although the nature of this primarily defined by their constituent proteins, most role is currently unclear.Ku70 was found to accumulate notably PML itself.PML-deficient mice display resis- following ionizing irradiation, and binding of nuclear tance to apoptosis induced by a variety of stimuli, clusterin, also known as TRPM-2, SGR-2 or XIP8, to including DNA damage, while PML overexpression can Ku70 correlated with dramatically reduced cell growth, induce cell death (Wang et al., 1998). In this light, it is colony-forming ability and increased cell death (Yang interesting that UV-induced apoptosis, which requires et al., 2000). On the other hand, recent studies have activation of the protein kinase JNK, was blocked by a identified associations between cell death and loss of dominant-negative form of DAXX (Wu et al., 2002). nuclear Ku70 (Song et al., 2003) and/or cytosolic DAXX directly binds to and activates the apoptosis accumulation of this protein (Um et al., 2003). In certain signal-regulating kinase 1 (ASK1), a mitogen-activated situations, Ku70 is even constitutively cytosolic (Fewell protein kinase kinase kinase that activates JNK (Chang and Kuff, 1996).Thus, it seems that Ku70 has a dual et al., 1998). It was suggested that ASK1 might function in DSB repair and apoptotic regulation. translocate DAXX to the cytoplasm, and that binding Cytosolic Ku70 can bind to Bax and neutralize its to DAXX could induce a conformational change within proapoptotic function (Sawada et al., 2003). Overexpres- ASK1 to activate its kinase activity.However, DAXX sion of Ku70-suppressed DDIA and Bax-induced was also reported to promote ASK1-mediated apoptosis apoptosis; conversely, Bax-mediated apoptosis was en- in a kinase- and caspase-independent manner (Charette hanced by downregulation of Ku70.The ability of Ku70 et al., 2001). The antiapoptotic protein HSP27 may to protect cells from apoptotic stimuli that do not induce inhibit cytosolic translocation of DAXX and block the DNA DSB probably reflects this cytosolic interaction DAXX-ASK1 interaction (Charette et al., 2000). In with Bax.Conformational changes of Bax and/or Bak addition, certain mutant forms of p53 may promote cell are essential for apoptotic permeabilization of mitochon- survival by inhibiting the DAXX-ASK1 pathway (Ohiro dria (Wei et al., 2001). However, Ku70 appears et al., 2003). Yeast two-hybrid studies indicated that specifically to control Bax-mediated killing.If the both p73a and its relative p53 can also associate with

Oncogene DNA damage and apoptosis CJ Norbury and B Zhivotovsky 2803 DAXX (Kim et al., 2003). This interaction involves the is rapidly induced by various stress stimuli, including oligomerization domain of p73a or p53 and the C- DNA damage.It was initially suggested that DNA terminal domain of DAXX, which is also the region that binding and transactivation by Nurr77 might be binds ASK1 and PML.Interestingly, transient coex- required for its proapoptotic effect.More recent data pression of DAXX resulted in strong inhibition of p73- showed that even a form of Nurr77 lacking DNA- and p53-mediated transcriptional activation of p53- binding ability could potently induce p53-independent responsive promoters.It is therefore possible that apoptosis in some tumour cells.Although normally DAXX modulates cell cycle arrest and apoptosis nuclear, in response to etoposide and 5-fluorouracil through regulation of the transcriptional activity of Nur77 was translocated to the cytosol where it was p53 and its family members. implicated in the release of mitochondrial cytochrome c Is DAXX normally pro- or anti-apoptotic? Targeted (Li et al., 2000; Wilson et al., 2003). It is unclear whether deletion of Daxx in the mouse results in severe Nur77 directly targets mitochondria or acts through developmental abnormalities in knockout embryos, as interaction with Bax and relocalization of Bax from the a result of unscheduled apoptosis during early embry- cytosol to mitochondria.Nuclear Nur77 was previously ogenesis (Michaelson et al., 1999). Similarly, DaxxÀ/À implicated in cell proliferation, so the cytoplasmic re- embryonic stem cells and siRNA-treated DAXX de- localization of Nur77 might play a key role in pleted cells are characterized by elevated levels of coordinating growth and apoptotic pathways through apoptosis (Michaelson et al., 1999; Michaelson and the cessation of the transcriptional program mediated by Leder, 2003).These and other data suggest that DAXX this protein after its translocation from the nucleus. acts as antiapoptotic regulator, probably via suppres- sion of proapoptotic gene expression.DAXX silencing might sensitize cells to multiple apoptotic pathways, Role of caspase-2 in DNA damage responses including DDIA (Chen and Chen, 2003). Following DNA damage, mitochondrial release of cytochrome c and the subsequent activation of procas- Nuclear FADD: a further regulator of the response to pase-9 are essential for activation of downstream DNA damage? apoptotic effectors.In addition to the contributions made by histone H1.2 release and other p53-mediated Fas-associated death domain protein (FADD) is an pathways reviewed above, a parallel line of investigation adaptor protein that bridges death receptors with has led to the notion that the link between nuclear DNA initiator caspases.FADD was at first considered to be damage responses and mitochondrial events also in- confined to the cytoplasm, since most studies focused on volves caspase activity.A low dose of etoposide, an its interaction with plasma membrane proteins.How- inhibitor of nuclear topoisomerase II that induces DNA ever, several groups recently demonstrated that FADD strand breaks, resulted in the release of a heat-labile is mainly nuclear (Gomez-Angelats and Cidlowski, factor(s) that, in turn, interacted with mitochondria to 2003; Screaton et al., 2003; Sheikh and Huang, 2003). elicit cytochrome c release (Robertson et al., 2000). This It was suggested that sequestration of FADD in the activity was inhibited by the general caspase inhibitor, z- nucleus might ensure that the death receptor pathway of VAD-fmk, and caspase-2 was the earliest caspase apoptosis is not constitutively activated.However, it activated in treated cells.This and subsequent studies seems that FADD may have a distinct role in the showed that, in response to DNA damage, activation of nucleus, where it interacts with the methyl-CpG binding caspase-2 is indeed required before mitochondrial domain protein 4 (MBD4), which excises thymine from permeabilization and apoptosis can take place (Guo GT mismatches in methylated regions of chromatin et al., 2002; Lassus et al., 2002; Paroni et al., 2002; (Screaton et al., 2003). The MBD4-interacting mismatch Robertson et al., 2002). Subcellular fractionation studies repair factor MLH1 was also found in a complex with revealed that, although procaspase-2 is present in several FADD.Consistent with the idea that MBD4 can signal intracellular compartments, including the Golgi, cytosol to an apoptotic effector, MBD4 regulated DDIA. and nucleus, it is the only procaspase present constitu- Interestingly, another death domain-containing protein tively in the nucleus (Zhivotovsky et al., 1999; Mancini LRDD, also known as PIDD, interacts with FADD et al., 2000). Nuclear localization of caspase-2 is strictly (Lin et al., 2000). LRDD is transcriptionally induced in dependent on the presence of the prodomain (Colussi a p53-dependent manner following exposure to DNA et al., 1998). Caspase-2 has been shown to associate via damaging agents.Together these data suggest that the its prodomain with RAIDD, a death adaptor molecule nuclear localization of FADD and its interaction with a that is thought to be involved in death receptor- genome surveillance/DNA repair protein might be mediated apoptosis through an association with the involved in regulation of DDIA. adaptor protein RIP and TRADD.Endogenous RAIDD is mostly localized to the cytoplasm, but in cells ectopically expressing caspase-2, a fraction of Nur77: a p53-independent response to DNA damage RAIDD is recruited to the nucleus (Shearwin-Whyatt et al., 2000). More recent observations revealed that The immediate early gene Nur77 (also known as NGF1- caspase-2 is spontaneously recruited to a large protein B and TR3), which encodes an orphan nuclear receptor, complex and that this recruitment is sufficient to

Oncogene DNA damage and apoptosis CJ Norbury and B Zhivotovsky 2804 mediate caspase-2 activation.No RAIDD was detected suggest that caspase-2 is an apical caspase in the in these high molecular weight fractions, ruling out the proteolytic cascade initiated by DNA damage and that involvement of RAIDD in caspase-2 complex formation it plays a key role in transduction of the DNA damage (Read et al., 2002). Using substrate-binding assays the signal to the mitochondria. authors provide clear evidence that caspase-2 activation It seems that the role of caspase-2 in apoptosis is cell might occur without processing of the precursor type-specific.Thus, neurons from caspase-2 knockout molecule.However, oligomerization is important step mice are more prone to apoptosis than neurons from for caspase-2 activation.Yeast two-hybrid system wild-type mice, whereas caspase-2-deficient oocytes or experiments with dominant-negative caspase-2 as bait lymphoblasts from the same animals are resistant to revealed the presence of a novel proapoptotic molecule apoptosis induced by chemotherapeutic drugs (Bergeron named proapoptotic caspase adaptor protein (PACAP) et al., 1998). Perhaps the upstream role assigned in some (Bonfoco et al., 2001; Lassus et al., 2002). In cell extracts cells to caspase-2 is performed by another caspase in and in cotransfection experiments, this protein bound to neurons.It is clear that many questions in this area have caspase-2 and -9 but not to caspase-3, -4, -7 or -8. yet to be resolved. Transient overexpression of PACAP-triggered apoptosis, which was prevented by inhibition of caspase activity.However, it is unclear whether this new adaptor Mitochondrial regulation of DNA damage-induced protein is important for caspase-2 complex formation.A apoptosis further study suggested that cyclin D3 is involved in activation of caspase-2 (Mendelsohn et al., 2002). Release of mitochondrial endonuclease G/CPS-6 and Expression of cyclin D3 and caspase-2 in human cells the oxidoreductase apoptosis-inducing factor (AIF/ potentiated apoptosis compared with expression of WAH-1) is important for chromatin fragmentation caspase-2 alone.Moreover, overexpression of cyclin during the execution phase of apoptosis, and is a feature D3 increased the amount of cleaved caspase-2.It was of the process conserved from C. elegans, where it occurs suggested that interaction with cyclin D3 may stabilize downstream from caspase activation, to human cells, caspase-2.This interaction may be just one way in which where its relation to caspase activation is less clearcut the baseline sensitivity of proliferating cells to apoptotic (Susin et al., 1999; Parrish et al., 2001; Arnoult et al., stimuli is increased, relative to that of the equivalent 2003).Release of mitochondrial components into the quiescent populations.A second such mechanism cytosol and nucleus has also assumed a more general involves the direct transcriptional activation of procas- role in the amplification of apoptotic signals, including pase genes by E2F-1 on cell cycle re-entry (Nahle et al., those activated by DNA damage, in vertebrates (Water- 2002). house et al., 2002). The best known of these components Following activation in response to DNA damage, is cytochrome c, which on permeabilization of the outer caspase-2 is involved in the release of apoptotic effectors mitochondrial membrane binds the apoptotic protease from mitochondria.Cells stably expressing antisense activating factor APAF-1 and promotes caspase activa- procaspase-2, or the corresponding transiently expressed tion.Smac/Diablo is also released on mitochondrial siRNA were refractory to cytochrome c release and as permeabilization and blocks the action of IAP proteins well as downstream events, such as procaspase-9 and -3 that would otherwise inhibit caspases.Cytochrome c activation, loss of phosphatidylserine asymmetry in the and Smac/Diablo release can both be caspase dependent plasma membrane, and DNA fragmentation, induced following DNA damage, indicating that these are by DNA damage (Lassus et al., 2002; Robertson et al., primarily amplification mechanisms, rather than routes 2002).Expression of a caspase-2 cDNA variant resistant by which caspase activation is initially brought about to siRNA restored the ability of cells to undergo (Adrain et al., 2001; Robertson et al., 2002). apoptosis.How does caspase-2 act in this sequence of events? During early apoptosis nuclear caspase-2 may trigger mitochondrial dysfunction without relocalizing Does mitochondrial DNA damage play a role in cell into the cytoplasm (Paroni et al., 2002). This view is death? based on the observation that release of cytochrome c occurred in the absence of obvious changes in nuclear/ Ionizing radiation and some anticancer drugs induce cytoplasmic transport, while caspase-2 was able to damage both in genomic and in mitochondrial DNA diffuse out of the nucleus only when the size limit for (mtDNA).Although the role of nuclear DNA damage passive diffusion through nuclear pores had increased in initiation of cell death has been discussed extensively, later in apoptosis.In any case, it is clear that caspase-2 the involvement of mtDNA is this process is contro- activity is required for translocation of Bax to the versial.Damage to mtDNA, if not repaired, could lead mitochondria, as well as for release of mitochondrial to disruption of the electron transport chain and the cytochrome c and Smac.Two groups demonstrated that production of reactive oxygen species (ROS).For some caspase-2 can induce release of these proteins from years, it has been known that mitochondrial and nuclear mitochondria directly (Guo et al., 2002; Robertson et al., DNA can be affected differently by DNA damaging 2002), or through cleavage of the proapoptotic protein drugs.Detailed investigation of DNA integrity in Bid, which moves to mitochondria and facilitates mitochondria suggested that DNA fragmentation dur- cytochrome c release (Guo et al., 2002). These data ing apoptosis is usually specifically nuclear (Murgia

Oncogene DNA damage and apoptosis CJ Norbury and B Zhivotovsky 2805 et al., 1992). Furthermore, when human leukaemia cell These data provide in vivo evidence that respiratory lines were exposed to conditions which resulted in chain deficiency predisposes cells to apoptosis.Interest- necrosis, mtDNA was damaged at approximately the ingly, Tfam is essential not only for mitochondrial gene same rate as nuclear DNA, but in apoptosis mtDNA expression, but also for maintenance and repair of was not degraded (Tepper and Studzinski, 1993).At this mtDNA.It was shown that Tfam preferentially time, it was believed that DNA degradation occurred recognizes cisplatin-damaged and oxidized DNA early after the delivery of the ‘lethal hit’ and before (Yoshida et al., 2002b). More detailed investigation intracellular organelles and the plasma membrane were revealed that binding of Tfam to cisplatin-modified damaged (Bursch et al., 1990). It is now clear that DNA DNA was significantly enhanced by p53, with which fragmentation is a relatively late event, so these data did Tfam was found to interact physically (Yoshida et al., not address the question of whether mtDNA and its 2003).Thus, although the role of mtDNA in cell death is damage might have a role to play in early apoptotic contentious, it is clear that its absence or impaired signalling. function can profoundly influence the rate of apoptosis. Not surprisingly, the human mtDNA depletion Mutations in the mtDNA leading to mitochondrial syndrome is lethal.However, cells without mtDNA dysfunction have been reported in a variety of cancers. can be maintained in long-term culture if they are Might these mutations influence cellular responses to provided with substrates for the glycolytic production of cancer therapy? In comparison with their r þ parents, r0 ATP.These so-called r0 cells undergo apoptosis in cells were extremely resistant to adriamycin, photody- response to staurosporine in the same way as the namic therapy or ionizing radiation, as judged by parental cells (Jacobson et al., 1993). Even though these clonogenic survival assays (Singh et al., 1999; Tang cells are defective for respiration, their apoptosis was et al., 1999). Importantly, resistance to adriamycin, characterized by cytochrome c release, caspase-3 activa- which induces ROS formation as well as acting as a tion and loss of mitochondrial membrane potential topoisomerase II inhibitor, was not due to the lack of (Jiang et al., 1999). Moreover, Bcl-2 overexpression was drug uptake, or to altered cell cycle responses.Thus, able to protect both parental and r0 cells from mitochondrial function is a determinant of cellular apoptosis.In contrast to staurosporine-induced apop- sensitivity to important cancer therapeutic agents. tosis, death of leukaemia cells in response to tumour necrosis factor (TNF) required mtDNA (Higuchi et al., 1997).However, in some cell lines TNF- a was able to Back to the nucleus: DNA damage-induced histone induce apoptosis despite the absence of mtDNA modifications in apoptosis (Marchetti et al., 1996), although with much slower kinetics than in the parental cells.A significant The data reviewed above concerning the involvement of difference in sensitivity of hepatocellular carcinoma histones H1.2 and H2AX in DNA damage signalling cells to TNF-related apoptosis-inducing ligand (TRAIL) were by no means the first suggestion of a connection was found on comparison of a r0 and parental cell line between histones and apoptosis.Compared with native (Kim et al., 2002). After TRAIL treatment, transloca- chromatin, apoptotic oligonucleosomal fragments con- tion of Bax, subsequent cleavage of Bid, release of tained less histone H1 (Borisova et al., 1984; Bell et al., cytochrome c and dissipation of mitochondrial trans- 1990).Interestingly, a rapid increase in poly(ADP- membrane potential were all seen in the parental line but ribose) polymerase (PARP) activity was observed upon not in r0 cells.However, parental and r0 cells did not induction of apoptosis by radiation, followed by the show differential susceptibility to agonistic anti-Fas poly(ADP-ribosyl)ation of histone H1 (Manome et al., antibody, TNF-a or staurosporine. 1993).Inhibition of PARP activity by nicotinamide or To what extent are the effects of mtDNA explicable in similar compounds inhibited DNA fragmentation.This terms of ROS production? Apoptotic r0 cells, unlike histone modification increases the accessibility of apoptotic parental cells, did not exhibit a change in the chromatin to nucleases and probably promotes DNA redox potential of glutathione (Jiang et al., 1999). This fragmentation during apoptosis.On the other hand, suggests that a change in redox potential or an increase histone H1.2 did not undergo any obvious post- in ROS generation does not play an essential role in translational modifications following DNA damage, apoptotic induction following treatment with stauros- and the nuclear and cytosolic forms of H1.2 were porine.Several observations revealed that oxidative indistinguishable (Konishi et al., 2003). Moreover, no stress induces damage in both nuclear and mtDNA and modifications in H1.2 were required for mitochondrial cells lacking mtDNA exhibit a marked resistance to cell cytochrome c release in vitro. death (Yoneda et al., 1995). Attempts to determine In certain experimental systems, induction of apop- whether loss of mtDNA and disturbance in respiratory tosis was accompanied by histone H1 phosphorylation chain function result in apoptosis in vivo were also (Lee et al., 1999) and activation of CDC2/CDK1 undertaken (Wang et al., 2001). Using embryos with (Meikrantz et al., 1994), events normally associated homozygous disruption of the gene encoding the with entry into mitosis.These data suggest that mitochondrial transcription factor Tfam, as well as apoptotic cells could be entering premature, and there- tissue-specific Tfam knockout animals with severe fore catastrophic, mitosis in response to DNA damage. respiratory chain deficiency in the heart, the authors However, CDC2/CDK1 activation was not required for showed massive apoptosis in both experimental systems. apoptosis in thymocytes (Norbury et al., 1994), and

Oncogene DNA damage and apoptosis CJ Norbury and B Zhivotovsky 2806 inactivation of CDC2/CDK1 even enhanced DDIA basal activities of these various components, in part by under some circumstances (Ongkeko et al., 1995). their upregulation during cell proliferation and in part Broadly speaking, similar data have been obtained for by the in-built amplification loops that operate via Fas histone H3, which is phosphorylated during mitosis and and the release of mitochondrial regulators of apoptosis. premature chromosome condensation, but not during Multiple independent routes have now been traced by apoptosis.Histone H2AX phosphorylation, as noted which nuclear DNA damage can be signalled to the above, plays a role in DNA damage signalling, but has mitochondria.These encompass p53-dependent tran- no known direct connection with the chromatin changes scriptional activation, less well-characterized direct that accompany apoptosis.Perhaps the most clearcut mitochondrial roles for p53, histone H1.2 release, case for direct involvement of histone phosphorylation Nurr77 and caspase-2 activation.A degree of coordina- in DDIA concerns mammalian histone H2B, which is tion between DNA repair and cellular commitment to phosphorylated at Ser14 in response to UV irradiation apoptosis may be achieved through the release of Ku70, or treatment with the topoisomerase II inhibitor etopo- conventionally considered to be a nuclear DNA repair side, which induces DNA strand breaks.This phosphor- protein, into the cytoplasm.Conversely, DAXX and ylation is mediated by mammalian sterile twenty (Mst1) FADD, initially identified through their association kinase (Cheung et al., 2003), activation of which is with the integral plasma membrane death receptor Fas, dependent upon its cleavage by caspase-3.Histone H2B now appear to play important nuclear roles in DDIA.It phosphorylation occurred immediately prior to DNA is also apparent that damage to the mitochondrial laddering and, like H1 poly(ADP-ribosyl)ation, may be genome, as well as nuclear DNA damage, has the involved in triggering this process. capacity to influence the extent of subsequent apoptosis. Each of these events is potentially of crucial importance both in tumour development and in the response of Conclusions cancer cells to therapies based on the induction of DNA damage. The wealth of recent literature has brought this field to a stage where it is now possible to begin to explain cellular Acknowledgements sensitivity to DDIA in terms of a cellular apoptotic We apologize to those authors whose work could not be cited ‘rheostat’.This comprises elements of the DNA damage directly due to space limitations.The work in our laboratories detection machinery, including proteins that also signal was supported by grants from Cancer Research UK, the cell cycle checkpoint arrest, signal transducers including Medical Research Council and the Association for Interna- CHK2, p53 and E2F-1, as well as the downstream tional Cancer Research (to CJN), and from the Swedish (3829- caspases and their regulators (Figure 2).The setting of B02-07XBC) and Stockholm (03:173) Cancer Societies, and the rheostat is determined in part by the abundance and EC grant (QLK3-CT-2002-01956) (to BZ).

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