(2013) 32, 3789–3797 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www..com/onc

REVIEW Various modes of death induced by DNA damage

O Surova and B Zhivotovsky

The consequences of DNA damage depend on the cell type and the severity of the damage. Mild DNA damage can be repaired with or without cell-cycle arrest. More severe and irreparable DNA injury leads to the appearance of cells that carry or causes a shift towards induction of the or cell death programs. Although for many years it was argued that DNA damage kills cells via or , technical and methodological progress during the last few years has helped to reveal that this injury might also activate death by or mitotic catastrophe, which may then be followed by apoptosis or necrosis. The molecular basis underlying the decision-making process is currently the subject of intense investigation. Here, we review current knowledge about the response to DNA damage and subsequent signaling, with particular attention to cell death induction and the molecular switches between different cell death modalities following damage.

Oncogene (2013) 32, 3789–3797; doi:10.1038/onc.2012.556; published online 3 December 2012 Keywords: DNA damage; cell death; ; senescence; response

INTRODUCTION cell-cycle arrest, whereas severe and irreparable injury shifts the Genomic integrity and stability is constantly threatened by DNA cellular response towards induction of the senescence or cell damage, which may arise from numerous environmental and death programs, such as apoptosis, mitotic catastrophe (MC), intrinsic sources. Failure to repair DNA damage can cause autophagy and necrosis. The molecular basis underlying the mutations, genomic instability, premature aging, mental multiplex decision-making process is currently the subject of intense retardation and other developmental disorders as well as . investigation. Here, we review current knowledge about the Consequently, eukaryotes have evolved a complex set of DDR with particular attention to cell death induction and the mechanisms to sense different types of DNA damage and molecular switches between different cell death modalities safeguard the genome, the so-called DNA damage response following DNA damage. (DDR). The DDR comprises a multiple networks of signal transduction pathways initiated by sensing damage, followed by signal transmission to effectors through activation of a cascade of CELL SENESCENCE IN RESPONSE TO DNA DAMAGE phosphorylation events. A group of proteins, including mediator Cellular senescence is one of the consequences of severe or of DNA damage checkpoint protein 1 (Chk1; MDC1/NFBD1), 53BP1 irreparable DNA damage, especially DNA DSBs, and it is (p53-binding protein 1), the Mre11-Rad50-NBS1 (MRN) and the characterized by an irreversible growth arrest of cells in the G1 Rad9-Rad1-Hus1 (9-1-1) complexes, have been implicated as DNA phase. Cellular senescence limits the proliferation of DNA- damage sensors and recognize both double- and single-strand damaged cells that are at risk of neoplastic transformation. breaks (DSBs and SSBs, respectively) as well as regions of However, these cells remain metabolically active, showing a replication stress.1–4 Binding of the MRN and 9-1-1 complexes to greatly altered pattern of . The outcomes of the sites of DNA damage in activates the signal cellular senescence following DNA damage are highly dependent transducing kinase ataxia-telangiectasia mutated (ATM), the ATM on the cell type and physiological context. Cells of epithelial and and Rad3-related (ATR) kinase and the DNA-dependent protein stromal origin undergo senescence following severe DNA damage, kinase (DNA-PK). Once activated, ATR and ATM amplify the while lymphoid cells undergo apoptosis. In most cell types, damage signal by phosphorylating various substrates, including activation of p53 is crucial for initiating the senescence response key DDR proteins H2AX, BRCA1, NBS1, SMC1 (structural following DNA damage, and in certain cells p53 is also important maintenance of protein 1), MDC1 and 53BP1, as for maintaining the senescent growth arrest. p21waf1/cip1,asa well as repair proteins Ku70/80, Artemis and Rad-like proteins.5,6 crucial target of p53 and mediator of senescence, inhibits a Additionally, in order to allow time for DNA repair, ATM spectrum of cyclin-dependent kinases that regulate the phos- temporarily arrests the by phosphorylation and phorylation of the (pRb). The hypopho- activation of cell-cycle checkpoints. p53, as one of the main sphorylated form of pRb binds to the E2F, targets of ATM/ATR, is a universal sensor of genotoxic stress and leading to transactivation of the genes essential for the G1-to-S has a key role in cellular responses to DNA damage, determining transition. On the other hand, can mediate transient DNA the fate of cells towards either survival, which involves cell-cycle damage-induced growth arrest. It is still unclear what determines delay accompanied by repair of DNA damage, or cell death.7 whether cells senesce or arrest transiently. It is likely that rapid Depending on the cell type and the severity and extent of DNA DNA repair will quickly shut down p53–p21 signaling, whereas damage, the activated DDR can elicit different cellular responses incomplete or incorrect repair results in prolonged signaling and (Figure 1). Mild DNA damage normally leads to the induction of senescence.8–10

Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. Correspondence: Professor B Zhivotovsky, Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Nobels vag. 13, Box 210, Stockholm SE-171 77, Sweden. E-mail: [email protected] Received 27 August 2012; revised 2 October 2012; accepted 3 October 2012; published online 3 December 2012 DNA damage and cell death O Surova and B Zhivotovsky 3790 pathways are intact, have a preference toward senescence compared with transformed cells and that senescence is usually induced by lower levels of damage than those leading to apoptosis.13 Overall, p53 stands at the center of a complex DNA damage signal transduction pathway following DNA damage. MRN 9-1-1 Phosphorylation of p53 at Ser15, Thr18 and Ser376 and dephosphorylation at Ser392 has been detected in fibroblasts undergoing senescence.14 By contrast, phosphorylation at Ser46 sensing damage was shown to be associated with the induction of p53AIP1 (p53- G1 MDC1 BRCA1 regulated apoptosis-inducing protein 1) and apoptosis. A study on 53BP1 NBS1 MCF-7 cells demonstrated the role of different phosphorylation M S Chk1/Chk2 Ku70/80 ATM/ATR sites (Ser15 and Ser46), induction of p21 and high or low (ROS) levels in determining the dosage of G2 adriamycin that induced either apoptosis or senescence. Cells in cell cycle signal DNA repair checkpoints transductions these two states exhibited quite distinct time course profiles of the proteins p53, p21 and . As mentioned above, DNA damage can also activate Rb through the pathway Cell Death to induce senescence. An in vivo study showed that, following TSC2/ PARP p53 cyclophosphamide treatment, a p53-dependent effect can also mTORC1 occur in an apoptosis-independent fashion by inducing cellular senescence in a p53- and p16-dependent manner.15 Thus, the genetic context, cell type or the specific activation of certain target genes upon DNA damage can favor either senescence or Necrosis Apoptosis Autophagy apoptosis. Figure 1. DNA damage response. DNA damage results from multiple environmental and intrinsic sources, activating a complex set of APOPTOSIS AS A FAST-ACTING RESPONSE TO DNA DAMAGE reactions called DNA damage response. This response is initiated by Like cellular senescence, apoptosis is activated in response to damage detection, followed by signal transmission to effectors and finally results in a number of cellular responses, such as induction of cellular stress and is executed when repair of DNA damage is slow cell-cycle arrest, repair of the lesions or, in case of non-repairable or incomplete. As mentioned above, it is still not fully understood damage, leads to activation of cell death program. A group of how cells decide whether to undergo senescence or apoptosis, proteins, including Mre11-Rad50-NBS1 (MRN complex) and the and despite the fact that most cells are capable of both, these Rad9-Rad1-Hus1 (9-1-1) complex, fulfilling a function of DNA processes seem to be mutually exclusive. Numerous studies have damage sensors, and recognized both DSBs and SSBs. Binding of clearly shown cross-talk between the processes of apoptosis and the MRN and 9-1-1 complexes to the sites of DNA damage in senescence, primarily at the level of p53, which is considered to be chromatin activates the signal transducing kinase ATM, preferen- a major player in the apoptotic response to genotoxins. The tially in response to DSBs and the disruption of chromatin structure. phosphorylation of p53 at Ser46 is the most prominent in priming By contrast, the ATR kinase is activating primarily in response to SSBs 16 and replication forks. The activation of DNA repair (orchestrated its apoptotic activity. Subsequently, homeodomain-interacting protein kinase 2 (HIPK2), a serine/threonine nuclear kinase, was by set of DNA damage response proteins, including BRCA1, NBS1, as 17,18 well as repair proteins Ku70/80 and others) and cell-cycle identified as the p53 Ser46 kinase. Following severe damage, checkpoints (regulated by MDC1, 53BP1 and checkpoint kinases HIPK2 binds p53 and is recruited to promyelocytic leukemia Chk1/Chk2) act as an immediate response to DNA damage to protein (PML) nuclear bodies, where PML acts as the main cofactor provide protection and recovery of injured cells, whereas activation for efficient HIPK2-mediated p53 phosphorylation. HIPK2- of cell death occurs much later and aims to eliminate the irreversibly mediated p53 Ser46 phosphorylation leads to enhanced damaged cell. Depending on type and the severity of stimulus and acetylation (Lys382) of p53 by the CREB-binding protein, which cellular context, DNA damage can induce different cell death also interacts with HIPK2 and colocalizes in PML nuclear bodies. programs, such as mitotic catastrophe, apoptosis, autophagy and necrosis. The latter results in complete transcriptional activation of p53 followed by potentiation of pro-apoptotic target gene expression.17,18 p53 can upregulate PML expression, thus Stimuli that produce DDR can also engage the p16–pRb potentiating the apoptotic signal. Depending on the extent of pathway. It was shown that in the terminal stages of growth DNA damage, p53 itself can either potentiate HIPK2 activity or arrest in the senescence of human diploid fibroblasts, the p16 negatively regulate HIPK2 stability.19,20 mRNA and cellular protein levels were upregulated almost 40-fold. Following activation, p53 initiates expression of its apoptotic p16 was also shown to be complexed to CDK4 (cyclin-dependent target genes, such as p53-upregulated modulator of apoptosis kinase 4) and CDK6 and is the major inhibitor of both kinases.11 (PUMA), p53AIP1, Bax and apoptotic protease activating factor 1 Although both p16 and p21 are CDK inhibitors, they are not (Apaf-1), in this way regulating the intrinsic apoptotic pathway. equivalent. Cells that undergo senescence solely due to activation Upon DNA damage, p53 also promotes the extrinsic apoptotic of p53–p21 can restart growth after inactivation of the p53 pathway through upregulation of the TRAIL (tumor necrosis pathway. However, cells that fully engage the p16–pRb pathway factor-related apoptosis-inducing ) receptors, death recep- cannot resume growth even after inactivation of p53, pRb or tor-4 and death receptor-5 (KILLER), as well as the CD95 receptor p16.12 Thus, both of these pathways can cause transient or (Fas/Apo-1) and the CD95 (Fas/Apo-1) ligand.21 Recently an reversible cell-cycle arrest, and it remains to be clarified exactly orphan nuclear receptor, RORa, was identified as a direct target how their activities are modified by senescence-inducing signals gene of p53. Upon DNA damage, RORa stabilizes p53 and activates and how cells make decisions in response to damage signals. p53 transcription, leading to the activation of a subset of p53 Both events, senescence and apoptosis, are under the control of target genes that are specifically involved in apoptosis.22 p53; therefore, it is difficult to understand the switch between It has also been discovered that DNA damage activates p53- these two modes of cellular response upon DNA damage-induced dependent expression of specific microRNAs (miRNAs), which activation of p53. It seems that normal cells, in which all signaling carry pro-apoptotic functions. The examination of miRNA

Oncogene (2013) 3789 – 3797 & 2013 Macmillan Publishers Limited DNA damage and cell death O Surova and B Zhivotovsky 3791 expression profiles in wild-type and p53-deficient mouse embryo- nic fibroblasts (MEFs) revealed that p53 affects the expression of 145 mouse miRNAs. These p53-induced miRNAs may amplify the Apoptosis p53 pro-apoptotic signals by targeting a network of existing transcripts + p53, Chk2, in response to DNA damage. As an example, p53 can induce the caspase-2 expression of miRNA-34a (miR-34a) in cultured H1299 lung cancer cells as well as in irradiated mice. Markedly, inactivation of miR- Mitotic 34a strongly attenuates p53-mediated apoptosis in cells exposed Autophagy DNA damage to genotoxic stress, whereas overexpression of miR-34a mildly catastrophe increases apoptosis.23,24 Although p53 primarily acts as a nuclear transcription factor, it p21waf/cip1 PARP - p53, Chk2, has also been shown to contribute to apoptosis induction in the caspase-2 cytoplasm by transcription-independent mechanisms. p53 was found to translocate to the cytoplasm in response to different Senescence Necrosis stress signals where it stimulates mitochondrial outer membrane Figure 2. Various modes of cell death induced by DNA damage. permeabilization and caspase activation, as well as antagonizes Severe and irreparable DNA damage evokes senescence or cell the anti-apoptotic proteins Bcl-2 and Bcl-XL by their direct death program such as apoptosis, mitotic catastrophe, autophagy 25 binding. In addition to p53, its homologs p73 and p63 are and necrosis. p53 has a central role in a complex signal transduction involved in apoptotic responses upon DNA damage. Following pathway following DNA damage, orchestrating primarily the etoposide-induced DNA damage, Chk1 and Chk2 are activated by apoptotic response, and regulates the switch between the apoptosis waf1/cip1 ATM and/or ATR, which leads to activation of the p73 transcription and cellular senescence. p21 is a crucial target of p53 and is factor E2F1, followed by increased levels of p73 protein. Although also a mediator of senescence. The DNA damage-induced necrosis the combined absence of p63 and p73 was shown to severely pathway is mediated mainly by ATP depletion followed by PARP activation. The mitotic catastrophe is initiated when the cell-cycle impair the induction of p53-dependent apoptosis in response to checkpoints are compromised and cells enter prematurely DNA damage, p73 can be pro-apoptotic itself even in the absence 26 before the completion of DNA repair; commonly, it serves as a pre- of p53. p73 elicits apoptosis via the mitochondrial pathway stage of apoptosis or necrosis. The final cell death modality in this using PUMA and Bax as mediators, followed by the release of case depends on the presence/absence of functional p53, caspase-2 cytochrome c. Moreover, p73 shares many pro-apoptotic target and Chk2-related proteins. Cellular context, nature and magnitude genes with p53, such as Noxa, Caspase-6 and CD95. Interestingly, of stress are essential for the type of autophagy response: p73 is also a substrate for caspases, and after processing its enhancing cell survival or inducing autophagic cell death, having truncated version, localizes to the mitochondria and amplifies tight cross-talk with the apoptotic pathway. apoptosis in response to TRAIL.27 The cytoplasmic functions of p73 remain to be investigated. Another p53-independent mechanism that can be engaged Caspase-2 deficiency is characterized by increased DNA damage upon DNA damage implicates the protein Nur77, also known as and genomic instability in proliferating cells. Casp2 À / À MEFs TR3 or NGFI-B (nerve IB). Similarly to p53, readily escape senescence in culture, exhibit increased micro- the pro-apoptotic action of Nur77 is believed to be exerted nuclei formation and sustained DNA damage during cell culture through its nuclear transactivation of gene expression. and following g-irradiation. Loss of caspase-2 leads to defective Accumulating data suggest that translocation of Nur77 from the p53-mediated signaling and decreased transactivation of p53 nucleus to the mitochondria represents an important mechanism target genes upon DNA damage.31 It seems that both p53- by which Nur77 initiates apoptosis. In response to differential dependent and -independent mechanisms can be activated upon apoptotic stimuli, Nur77 migrates from the nucleus to the DNA damage, leading to apoptosis induction; it might depend on cytoplasm where it targets mitochondria, which is associated the type of damage as well as the type of cells (Figure 2). with the release of cytochrome c and induction of apoptosis.28 However, recent studies showed the ability of Nur77 to inhibit apoptosis. When overexpressed in HEK293 cells, Nur77 promotes CELL DEATH VIA MC resistance to programmed cell death induced by death receptor To maintain genome integrity, cells respond to DNA damage by engagement, DNA-damaging agents and either a delay in cell-cycle progression, allowing time for proper stress. Nur77 overexpression leads to enhanced nuclear factor DNA repair, or by the elimination of cells that are irreparably (NF)-kB activity, which contributes to Nur77 anti-apoptotic injured. When checkpoints are compromised, cells can enter activity via induction of the anti-apoptotic gene cIAP1. These mitosis prematurely before the completion of DNA repair and results show that cross-talk between Nur77 and other initiate MC.32 MC occurs either during or shortly after a transcription factors contributes to cell-fate decisions in dysregulated/failed mitosis and can be accompanied by response to different apoptosis-inducing agents.29 Which factors morphological alterations, including micronucleation (which regulate the switch between the pro- or anti-apoptotic functions often results from and/or chromosome fragments of Nur77 remains unclear. that have not been distributed evenly between the daughter An additional mechanism that links DNA damage and the nuclei) and multinucleation (the presence of two or more nuclei of apoptotic pathway involves Caspase-2. Activation of this protein similar or heterogeneous sizes, derived from a deficient separation was shown to occur within PIDDosome, a protein complex that during cytokinesis). In addition to mitotic failure caused by contains PIDD (p53-induced protein with a death domain), an defective cell-cycle checkpoints, MC can be triggered by agents adaptor protein RAIDD (receptor-interacting protein-associated that influence the stability of microtubules and various anticancer ICH-1 homologous protein with a death domain) and Caspase-2. drugs. MC was also described as the main form of cell loss induced Increased PIDD expression results in spontaneous activation of by ionizing radiation. How does DNA damage induce MC and Caspase-2 and sensitization to apoptosis induced by genotoxic what is the link between MC and the cell death machinery? stimuli.30 The prodomain of Caspase-2 was shown to be required It is well known that tumors differ in their sensitivity to for its nuclear localization and activation, and further studies treatment with DNA-damaging agents. Sensitive cells die during demonstrated that nuclear Caspase-2 is rapidly cleaved in cells interphase, before entering mitosis. Cells that do not undergo upon DNA damage. death in interphase may become arrested in G1 and/or G2. If the

& 2013 Macmillan Publishers Limited Oncogene (2013) 3789 – 3797 DNA damage and cell death O Surova and B Zhivotovsky 3792 cellular machinery is not able to repair the damage during arrest, observed in a cisplatin-treated ovarian carcinoma cell line, SKOV-3. some cells can die by apoptosis after one or even several mitotic Necrosis following MC could be an effect of genetic instability cycles. In other circumstances, premature mitosis may lead to MC. caused by aneuploidy and/or polyploidy (Figure 2). However, depending on the severity of DNA damage, cells can Detailed analysis of the morphological markers associated with also exit mitosis to form 4N G1 cells, and after the second arrest at MC revealed that this process can also precede or occur in parallel G1, they die or survive. Dying cells are characterized by mitotic with cell senescence (a persistent arrest in the of the cell abnormalities, such as a multipolar meta- or anaphase, lagging cycle that manifests with cell flattening, increased b-galactosidase telophase, the random distribution of condensed chromosomes activity and decreased telomerase function).38 Taken together, it is throughout the cells, and so on. The biochemical mechanisms important to note that cells facing a mitotic-linked cell death can, responsible for these differences remain unclear. However, with- in fact, die by two separate mechanisms, either by apoptosis or out prematurely entering mitosis, a cell cannot undergo MC. It is necrosis, depending on the molecular profile of the cell, or important to note that cells that are sensitive to apoptosis and die undergo senescence. Accordingly, the disruption of MC blocks during interphase have very little or no likelihood of displaying cancer progression, and its induction might constitute a features of MC. There are several experimental models that have therapeutic endpoint.38 been utilized to clarify the biochemical changes associated with Mitotic catastrophe was long ago described to occur in MC. Thus, the inability of HCT116 colon carcinoma cells to response to DNA damage; however, it is still remains unclear sequester cyclin B and Cdc2 in the cytoplasm following how to modulate this process to favor different cell death doxorubicin-induced DNA damage is already a well-established outcomes. Induction of MC as a possible anti-cancer treatment MC model. However, more recent data indicate that Chk2, and not needs to be further evaluated, and more in vivo work is required in 14-3-3s, may be the main negative regulator of MC in this order to define the critical modulating factors. model.33 Abrogation of the ATR-initiated checkpoint cascade mediated through Chk1 also directs cells into MC and has been analyzed with respect to specific Chk1 phosphorylations. Other cell-cycle-regulating proteins whose normal function seems to DNA DAMAGE AND AUTOPHAGY inhibit entry into mitosis are the checkpoint mediators Brca1, p73 Autophagy is a well-known evolutionarily conserved catabolic and p53. In the absence of p53, cells have the ability to activate program for the degradation of proteins and other subcellular alternative routes to halt the cell cycle. One of the proteins that elements through lysosomal proteolysis. In most cases, autophagy may be involved in this process in addition to Chk1 is p38MAPK serves as a pro-survival mechanism, which adapts cells to stress (p38 mitogen-activated protein kinase).34 Depletion of p38MAPK/ conditions by providing metabolic precursors for cellular renewal MK2 in p53-deficient cells leads to checkpoint deregulation and and maintenance through the recycling of cellular components. MC irrespective of the type of DNA damage induced. This two-hit On the other hand, substantial activation of autophagy in model suggests that MC is the outcome of DNA damage only response to stress can lead to cell death. The cellular outcome when multiple central checkpoint proteins are suppressed and of inducing autophagy in response to stress is complex and other options for cell death and survival are minimized. depends on the cellular context, type and magnitude of stress. In One of the most prominent morphological characteristics of MC mild stress conditions, autophagy may enhance cell survival by is the formation of giant cells with abnormal nuclei.35 Several allowing the cell to engage repair mechanisms and checkpoint possible scenarios for the death of giant cells have been activation; higher levels of damage over a prolonged period of suggested. According to one of these, death is a direct cause of time may trigger autophagic cell death, also known as type II MC and is distinct from apoptosis. This conclusion was based on programmed cell death. the dissimilarities in morphology between MC and apoptotic cells. The typical DNA-damaging compounds 6-thioguanine and 5- Despite their distinct morphology, some authors have suggested fluorouracil were shown to induce autophagy. An important role that the two processes share several biochemical hallmarks. Thus, in this process was suggested for BNIP3 (Bcl-2/adenovirus E1B following inhibition of Chk2 in syncytia generated by the fusion of 19 kDa protein-interacting protein 3), which mediates autophagy asynchronous HeLa cells, metaphase-associated MC was in a p53- and mTOR (mammalian target of rapamycin)-dependent accompanied by sequential Caspase-2 activation, mitochondrial manner.39 Although the mechanisms of p53-dependent induction release of pro-apoptotic proteins, activation of Caspase-3, DNA of autophagy remain incompletely understood, there are fragmentation and chromatin condensation.36 However, it has also indications that both transcription-dependent (upregulation of been reported that dying multinucleated giant cells are mTOR inhibitors, PTEN (phosphatase and tensin homolog) and characterized by uncondensed chromatin and the absence of TSC1 (tuberous sclerosis protein 1) or DRAM (damage-regulated DNA ladder formation.37 It seems that the presence or absence of aautophagy modifier)) and -independent pathways (AMPK (50 chromatin condensation during MC depends on different events, adenosine monophosphate-activated protein kinase) activation) including the stage at which mitotic arrest takes place. There is no are involved.40,41 Interestingly, p53-induced autophagy may lead consensus concerning the requirement of caspases for MC, but it to cell death, and this can be blocked by DRAM small interfering is likely that the progression of MC is caspase-independent as RNA. However, under some circumstances, such as in c-Myc-driven neither inhibition nor down-regulation of caspases influences the lymphomas, p53-mediated autophagy can actually promote cell appearance of giant cells, although caspases are essential for the survival, as inhibition of autophagosomal maturation enhances termination of MC, suggesting that MC-related morphological p53-mediated tumor cell death. Thus, p53-mediated autophagy changes are followed by activation of the apoptotic machinery. can affect both life and death decisions depending on the cell Thus, MC is not an ultimate manifestation of cell death but rather type or type of stimulus, and it reflects the activation of different a process leading to apoptosis.37 sets of p53 signals (Figure 2). It is important to note that the role of Apoptosis, however, is not always required for MC lethality as p53 as a positive regulator of autophagy has been questioned by some giant cells can undergo slow death in a necrosis-like some authors. In particular, deletion, depletion or inhibition of p53 manner. This conclusion is based on similarities between was shown to induce autophagy in human, mouse and nematode morphological changes during MC and necrosis (the loss of cells. Inhibition of p53 led to autophagy in anucleated cells, and nuclear and plasma membrane integrities). Mitotic arrest in cytoplasmic, not nuclear, p53 is believed to repress the enhanced docetaxel-treated tumor cells was followed by massive cell autophagy of p53 À / À cells.42 Overall, the opposing control of destruction by means of cell lysis. Appearance of multinucleated autophagy by p53 raises many important questions for future giant cells that were terminated via a necrosis-like lysis was also research.

Oncogene (2013) 3789 – 3797 & 2013 Macmillan Publishers Limited DNA damage and cell death O Surova and B Zhivotovsky 3793 Recent data described a new cytoplasmic pathway linking ATM circumstances the selective targeting of mitochondria and autophagy, suggesting that ATM can engage the TSC2/ for autophagy can enhance apoptosis, as inhibition of mTORC1 signaling pathway in order to regulate autophagy PUMA or Bax-induced autophagy leads to a reduced apoptotic (Figure 1). Even though the molecular mechanisms regulating response.47 this phenomenon are not clear, it was indicated that a substantial There is an interesting link between the p53 and JNK signaling cytoplasmic pool of ATM is phosphorylated in the presence of pathways in terms of the regulation of autophagy. Ewing sarcoma leptomycin B and could initiate signaling via LKB1 (Serine/ cells treated with 2-methoxyestradiol (2-ME) are characterized by threonine kinase 11), AMPK and TSC2 in the cytoplasm to enhanced autophagy and apoptosis occurring by the activation of suppress mTORC1, resulting in an increased autophagic flux.43 both the p53 and JNK pathways. In this context, p53 regulates, at Poly ADP-ribose polymerase (PARP) has also been implicated in least partially, JNK activation, which in turn modulates autophagy the cell’s decision to commit to autophagy following DNA injury. via two distinct mechanisms: the promotion of Bcl-2 phosphoryla- Studies using PARP-1 wild-type and deficient cells revealed the tion, resulting in the dissociation of the Beclin 1-Bcl-2 complex, formation of autophagic vesicles and the increased expression and the upregulation of DRAM. The critical role of DRAM in of genes involved in autophagy (bnip-3, cathepsin b and beclin 1) 2-ME-mediated autophagy and apoptosis is underlined by the fact in wild-type cells treated with doxorubicin but not in PARP À / À that its silencing efficiently prevents the induction of both cells or cells treated with a PARP inhibitor.44 the processes.48 Overall, many important questions concerning New insights into how PARP-1 could have a role in the cell’s cross-talk between apoptosis and autophagy remain to be decision to undergo autophagy and how it determines the addressed, and the most significant involves identifying the cytotoxic effect to DNA damage have been described. The results exact biochemical switches that direct a cell towards apoptosis or indicate that doxorubicin induces PARP-1 hyperactivation in autophagy. immortalized MEFs, leading to a cell response with two compo- nents: cell death with characteristics of necrosis and autophagy, with a cytoprotective effect. Authors imply that cellular energy REGULATED FORM OF NECROTIC CELL DEATH INDUCED BY collapse (derived from PARP-1 overactivation) is a key event in the DNA DAMAGE cell’s decision to commit autophagy.44 Necrosis is an acute form of cell death usually initiated following Interestingly, recent data suggest that cells may use specific energy loss. Until recently, necrosis was considered to be an autophagy subpathways rather than recycle all the cellular unregulated form of cell death; however, accumulating experi- components, including those that are not damaged. It was mental data demonstrate that, except under extreme conditions, proposed that two yeast histone deacetylases Rpd3 and Hda1, and necrosis may be a genetically regulated process. Poly(ADP- the acetyltransferase Gcn5, control chromosome stability by ribosyl)ation was shown to have a central role in modulating the coordinating the ATR checkpoint and DSB processing with cellular response to severe genotoxic stress and induction of cell autophagy.45 Upon DSB formation, Rpd3 and Hda1 keep Sae2 death through necrotic mechanisms. Among the targets of PARP (human CtIP (C-terminal-binding protein interacting protein)) in are various proteins that are involved in DNA damage check- the deacetylated form, which influences Mre11 dynamics at the points, including p53, p21waf1, XPA ( DSB site. Sae2 is then released from the DSB site, perhaps in a group A), MSH6, DNA ligase III, XRCC1, DNA polymerase-e, DNA- multimeric form, and Gcn5-mediated acetylation shunts it into PKCS, Ku70, NF-kB, iNOS (inducible nitric oxide synthase), caspase- autophagy-mediated degradation. This last step might be needed activated DNase and telomerase.49 Because of the high negative to counteract extensive DSB resection and/or simply to eliminate charge on ADP-ribose polymers, poly-ADP-ribosylated proteins Sae2 once the first step of DSB processing has been accomplished. lose their affinity for DNA and, as a consequence, their biological Depending on the stimulus and cellular context, autophagy activities. ATP depletion is the central mechanism by which PARP may be a prerequisite for apoptosis by preceding and later turning activation is suggested to participate in DNA damage-induced it on. On the other hand, there are some circumstances in which necrotic death. PARP À / À fibroblasts resist necrotic death induced autophagy may antagonize or delay apoptosis, or in which these by MNNG (N-methyl-N’-nitro-N-nitrosoguanidine), and PARP À / À two processes may act exclusively and serve as reciprocal backup mice resist necrotic death during hemorrhagic shock. In mechanisms. Although it has become clear that cell fate in thymocytes, kidney and pulmonary artery, PARP inhibitors also response to stress, including DNA damage, can be determined by prevent necrotic, but not apoptotic, cell death induced by the functional status and the interaction between apoptosis and oxidants.50,51 Cleavage of PARP by caspases at a single site autophagy, the precise mechanisms underlying this switch occurs at an early stage of apoptosis and can be blocked by between these two stress-mitigating pathways remain obscure caspase inhibitors or Bcl-2 overexpression, suggesting that the loss (Figure 2). Resistance to apoptosis is believed to increase the of PARP function is required for efficient accomplishment of possibility that cells may undergo autophagic cell death in apoptosis.52 Caspase-mediated PARP cleavage promotes apop- response to chemotherapy and irradiation, promoting an alter- tosis due to the essential reduction of its repair function, thus native pathway for drugs and irradiation to facilitate tumor cell preventing unnecessary DNA repair and also facilitating the access killing. Studies of double-knockout MEFs indicate that after of endonucleases to chromatin. Caspase inhibition is known to treatment with etoposide, Bax À / À Bak À / À MEFs fail to undergo cause a switch from apoptosis to necrosis, suggesting that the apoptosis and exhibit massive autophagy followed by delayed cell induction of apoptosis is a protective mechanism against necrotic death. Elimination of the autophagy gene products Beclin-1 and damage (Figure 2). Atg5 was also shown to reduce the etoposide-induced cell death Some experimental data propose a role of poly(ADP-ribose) and in the same MEFs. In this cellular setting, no autophagy was PARP in p53-mediated pathways upon DNA damage, although induced when apoptosis was inhibited by other perturbations, opinions concerning the interaction of these two components are such as Apaf-1 À / À and caspase-9 À / À MEFs, or the addition of controversial. Studies of splenocytes, bone marrow cells and the caspase inhibitor zVAD-fmk to wild-type MEFs.46 This could embryonic fibroblasts from PARP À / À mice showed an increase in imply that Bcl-2-family proteins, such as Bax and Bak, may have p53 accumulation after treatment with an alkylating agent.53 direct or indirect roles in regulating autophagy that are X-ray-induced accumulation of p53 was found to increase upon independent of their role in modulating apoptosis. Furthermore, PARP inhibition. It has been established that the PARP inhibitor 3- PUMA, a key mediator of p53-dependent apoptosis, was aminobenzamide delays the repair of both SSBs and DSBs caused found to induce autophagy that leads to the selective removal by ionizing radiation and that cells treated with inhibitor of mitochondria. The study suggested that under some immediately after X-ray irradiation exhibit a prolonged p53

& 2013 Macmillan Publishers Limited Oncogene (2013) 3789 – 3797 DNA damage and cell death O Surova and B Zhivotovsky 3794 response. Later studies suggested that inactivation of PARP affects CROSS-TALK BETWEEN DIFFERENT CELL DEATH MODALITIES the duration, but not the magnitude, of p53 accumulation in UPON DNA DAMAGE AND ITS APPLICATION FOR THERAPY irradiated Burkett lymphoma cells. p53 accumulation is controlled As previously described, DNA damage elicits various cellular by PARP-dependent and PARP-independent pathways, but p53 responses and can lead to the execution of programmed cell activation is largely independent of PARP. It has also been death via different molecular mechanisms. However, there is still a reported that the activity of DNA-PK can be stimulated by PARP in gap in our knowledge about what defines these distinct death the presence of NAD þ in in vitro reactions and that DNA-PK ADP- 54 fates upon genotoxic stress. It is generally appreciated that the ribosylation stimulates p53 phosphorylation. Hence, the role of extent of DNA damage significantly influences the cell fate. Some PARP in p53 activation is complex, involving not only direct studies support the hypothesis that the nature of the DNA lesion activation, but also by acting through DNA-PK and possibly other also has a major impact on cell fate at comparable cytotoxic doses. proteins to transmit the DNA damage signal to p53. Overall, the For instance, the apoptotic cell death response triggered by current data suggest a passive rather than an active role of PARP different DNA lesions may differ qualitatively and quantitatively. in the apoptotic process. Two DNA-damaging drugs, adozelesin and bizelesin, are known to Energy collapse is not the sole mechanism by which PARP produce similar DNA lesions (alkylation) and their cytotoxic effect contributes to cell death. A ‘death link’ between PARP, mitochon- is mediated by both G2-M cell-cycle arrest and cell death.60 dria and AIF (apoptosis-inducing factor) has been established. AIF Despite these similarities, the drugs were shown to trigger is a bifunctional NADH (nicotinamide adenine dinucleotide qualitatively distinct cell death pathways in HCT116 colon phosphate-oxidase) oxidase that has both a pro-survival role carcinoma cells: adozelesin-induced cell death was associated through its yet unknown activity in mitochondria, and a lethal role with greater caspase activation than bizelesin-induced death. A upon its translocation to the nucleus. Once translocated to the low cytotoxic dose of adozelesin induced much more caspase nucleus, AIF is involved in chromatinolysis and cell death. In order activity than a higher dose of bizelesin. Another drug C-1027, to be released from mitochondria, AIF has to be cleaved into tAIF which is a radiomimetic compound that interacts with DNA and by calpain or cathepsins.55,56 The proteolytic process is Ca2 þ - induces SSBs and DSBs, or abasic sites by free radical attack of dependent, and in addition to a sustained intracellular Ca2 þ deoxyribose moieties, was shown to induce apoptotic cell death elevation, enhanced formation of ROS is a prerequisite step for AIF without triggering caspase activation. to be cleaved and released from mitochondria. The stimulated Another important determinant of the cell death mode ROS production leads to oxidative modification (carbonylation) of following DNA damage is the genetic status of the cell and the AIF, which markedly increases its rate of cleavage by calpain. profile of proteins involved in the regulation of the cell cycle. For Combined, these data provide evidence that ROS-mediated, post- example, the presence of p53 has a central role in DNA damage- translational modification of AIF is critical for its cleavage by 57 induced cell death, but in the absence of functional p53 delayed calpain and, thus, for AIF-mediated cell death. Recent data indicate that PARP activation is required for AIF translocation death referred to as MC can occur. We have previously shown that during cell death initiated by MNNG and H O in fibroblasts and in the presence of p53 ovarian carcinoma cells treated with 2 2 cisplatin undergo apoptosis and that nuclear Caspase-2 is NMDA (N-methyl-D-aspartic acid) in cortical neurons. Moreover, AIF appears to be essential for PARP-1-mediated cell death. It was translocated to the cytosol during this process. By contrast, the also demonstrated that tAIF release depends on PARP-1-mediated absence of p53 led to MC followed by necrosis-like lysis without calpain and Bax activation.58 The critical role of PARP in controlling involvement of Caspase-2. Further, Chk2 inhibition in cells expressing p53 triggers MC in response to DNA damage. However, AIF-mediated cell death is further supported by observations that 61 NMDA-induced AIF translocation and cell death are blocked in at later time points these cells undergo apoptosis (Figure 2). Two conceptual models for how the death response to DNA neurons derived from PARP-null mice as well as in wild-type 62 neurons in the presence of the pharmacological PARP inhibitor damage is regulated were proposed by Borges et al. and termed DPQ. Studies on isolated mouse liver mitochondria and HeLa cells as the ‘integrative surveillance’ and ‘autonomous pathways’. showed that AIF activity is maintained in the presence of the The ‘integrative surveillance’ concept states that cells die when caspase inhibitor zVAD-fmk. Therefore, death mediated by PARP- the DNA damage is irreparable, which is consistent with most of activation and translocation of AIF are examples of caspase- the published data on cellular responses to DNA damage and is independent cell death. widely accepted. Nevertheless, there is still no clear evidence of an Although a number of studies have shown the ability of PARP to irreparable DNA lesion or exact biochemical mechanism for how regulate AIF translocation following different cytotoxic stimuli and the DNA damage signaling network can discriminate between in different cell types, very little is known about the precise reparable versus non-reparable DNA lesions. Yet, the integrative mechanism by which AIF mediates DNA fragmentation upon its suveillance model has remained a reasonable, and seldom translocation to the nucleus and ultimately to the execution challenged, explanation for when and how a damaged cell may of cell death. Examination of the AIF crystal structure has choose to commit suicide. In the alternative concept, termed demonstrated that AIF can bind DNA in a sequence-independent ‘autonomous pathways’, the DNA damage signaling network manner. Structure-based mutagenesis revealed that DNA- activates DNA repair and cell death simultaneously and 63 binding defective mutants of AIF fail to induce cell death while automatically, but the onset of cell death is delayed. One of retaining nuclear translocation. AIF-interacting partners, as the proposed explanations is that new gene expression might be well as components that promote nuclear chromatin break- required for the execution of cell death, and, as accumulation of down in mammalian cells, remain to be identified. The observa- RNA transcripts and protein products naturally takes time, the tion that AIF associates with histone H2AX in the nucleus via death response is delayed. The second possibility is that delay the C-terminal proline-rich binding domain suggests that this occurs due to built-in feedback loops in the death response interaction regulates chromatinolysis and programmed necrosis pathways, so that death occurs only if the negative feedback is by generating an active DNA-degrading complex with overridden. The third possibility involves a requirement for cyclophilin A. The data obtained demonstrated that AIF fails positive feed-forward loops to raise the death signals above a to induce chromatinolysis in H2AX- or cyclophilin A-deficient threshold that can overcome existing survival signals. Thus, in this nuclei.59 Although these data shed new light on the mechanisms model, death is a delayed response that correlates with the regulating programmed necrosis, additional studies are needed to persistence of DNA damage.62 elucidate the key nuclear partner of AIF and clarify its mechanism Overall, as mentioned above, p53 has a central role in a of action in the nucleus. complex signal transduction pathway following DNA damage,

Oncogene (2013) 3789 – 3797 & 2013 Macmillan Publishers Limited DNA damage and cell death O Surova and B Zhivotovsky 3795 regulating the switch between the processes of apoptosis and in vivo assessment methods. Advances in the understanding of cellular senescence. Additionally, cell type appears to be the such complex cell death interplay can be applied, for instance, in a crucial determinant in this process. It is important to note that newly proposed concept in cancer therapy called the concept of senescence can be triggered by much lower stress levels than synthetic lethality.73 According to this concept, the synthetic lethal apoptosis. Some studies point to a cross-talk between apoptosis interactions can be defined as two genetic alterations that occur and cellular senescence, for instance at the level of the in cancer cells and cause cell death when occurring together, anti-apoptotic protein Bcl-2. Overexpression of Bcl-2 in E1A þ while neither alone is lethal. In other words, some ras-transformed rodent fibroblasts in a p38-dependent manner changes in the signaling pathways of cancer cells can be promotes cell-cycle arrest after irradiation and accelerates considered as sensitizing mutations if they make transformed senescence after doxorubicin treatment.64 Caspase inhibition cells dependent on certain molecules or pathway combinations was shown to cause an increased expression of p21waf1 and for survival and are not necessary in normal cells. Thus, such switch the doxorubicin-induced apoptosis to senescence in molecular changes provide excellent therapeutic opportunities if human neuroblastoma SKN-SH cells.65 Nevertheless, the cross- combinations of these pathway can be selectively targeted. This regulation between apoptosis and cellular senescence is far approach can be used to enhance the effects of DNA-damaging from being completely understood. The relationship between agents. It would be interesting to explore the possibility to autophagy, senescence and apoptosis is even more complex and modulate the cell death response and switch between different again involves the tumor-suppressor protein p53 as a master modes to favor a desirable outcome in response to genotoxic regulator of switches between these processes, as described stimuli applying the synthetic lethality concept. The potential of above. Some studies reported that autophagy is induced during such a strategy has to be addressed in further studies. and facilitates senescence or limits damage and delays apoptosis to allow recovery of normal cell function.66,67 In general terms, similar stimuli can induce either apoptosis or autophagy, and CONCLUDING REMARKS decisions can be made by the cell perhaps depending on the Accumulating evidence supports the notion that, depending on variable thresholds for both processes. Autophagy can occur when the severity of treatment in response to DNA damage, cells either apoptosis is inhibited, which was shown, for example, in the can repair the injury and survive normally or, in the case of studies on Bak- and Bax-deficient cells, and vice versa inhibition of inefficient repair, they still can survive while carrying the mutation autophagy may lead to apoptosis. It was shown that autophagy or die. The cell death modality also depends on the severity of delays apoptotic death in breast cancer cells following DNA DNA damage as well as expression of various proteins, which are damage, and downregulation of autophagy proteins, Beclin 1 and involved in the regulation of different cell death programs. The Atg7, unmasked a caspase-dependent apoptotic response in MCF- 68 molecular basis underlying the cross-talk between these programs 7 cells upon camptothecin exposure. Recent findings in non- is currently the subject of intense investigation. Although small cell lung carcinoma (NSCLC) cells suggest that autophagy interaction between some genes and their products essential for suppression leads to inhibition of NSCLC and the switch was described above, there are still many gaps that sensitizes them to cisplatin-induced caspase-dependent and - 69 should be fulfilled to shed full light on this complex process. This independent apoptosis by stimulation of ROS formation. knowledge is important for exploring the possibility to modulate Interestingly, Atg3, an essential regulatory component of the cell death response and switch between different modes to autophagosome biogenesis, was identified as a new substrate favor desirable outcomes in response to DNA damage. The for Caspase-8 during receptor-mediated cell death, providing a 70 characterization and understanding of complex DDR signaling novel link between apoptosis and autophagy. together with finding new targets for engagement of various cell Although the link between autophagy and apoptosis has been death pathways would help us to guide the response to DNA studied to some extent, the link between autophagy and necrosis injury from upstream sensors to favorable downstream response is remaining unclear. In Caenorhabditis elegans, autophagy was outcomes. This would not only help researchers learn more about shown to be required for the necrotic breakdown of nematode the cellular fate after genotoxic insults but more importantly to neurons. Moreover, this work provided evidence that excessive uncover basic mechanisms of how viable and healthy cells autophagy is induced by necrotic cell death, and it showed that recognize signals and respond by committing to cell death. autophagy and lysosomal proteolysis combine to promote cell 71 Presently, we have entered the era of developing new pharma- demise. On the other hand, there are some data indicating that ceuticals that target DNA in order to kill tumor cells and, therefore, in apoptosis-defective cells inactivation of autophagy promotes 72 the clinical advantage as well as the potential adverse effects of necrotic cell death in vitro and in tumors in vivo. Yet, the such therapy must be critically evaluated. interplay between necrosis and autophagy upon DNA damage remains to be investigated in more detail. Thus, depending on the conditions of DNA injury, the different CONFLICT OF INTEREST cellular death outcomes can substitute for each other, which is The authors declare no conflict of interest. important to ensure the elimination of the cells in case the damage cannot be resolved (Figure 2). Both senescence and apoptosis are important tumor-suppressor mechanisms. Addition- ACKNOWLEDGEMENTS ally, autophagy and MC induced by drugs now are considered to make important contributions to the overall loss of tumor cell The work in the author’s laboratory was supported by grants from the Swedish Research Council, the Swedish and the Stockholm Cancer Societies, the Swedish viability. It should be also noted that each type of cellular response Childhood Cancer Foundation, and the FP7 (Apo-Sys) program. OS was supported by can result in a very different clonogenic potential. So, under- a fellowship from the Swedish Institute and Karolinska Institutet. 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