Is NHEJ a tumor suppressor or an aging suppressor?

[Cell Cycle 7:9, 1139-1145; 1 May 2008]; ©2008 Landes Bioscience

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Is NHEJ a tumor suppressor or an aging suppressor? Paul Hasty Department of Molecular Medicine and Institute of Biotechnology; University of Texas Health Science Center; San Antonio, Texas USA

Key words: NHEJ, Ku80, Ku70, p53, apoptosis, cellular senescence, chromosomal rearrangements

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breaks (SSBs) but also an occasional double-strand break (DSB). There are many exogenous agents that can damage DNA and they are found everywhere from water and air to the soil.3 In addition, DNA can be damaged as a consequence of replication errors, spontaneous deamination of cytosine, eroded telomeres or faulty DNA repair intermediates. Cell maintenance pathways that respond to DNA damage are crucial for suppressing tumors and can be classified into two categories, gatekeepers and caretakers.4 Gatekeepers are DNA damage checkpoints that respond to many forms of damage, principally to facilitate the repair or removal of lesions.5 Checkpoint machineries monitor the genome for problems that arise during DNA replication or mitosis, and halt cell cycle progression when such problems are encountered to allow time for the damage to be repaired. If damage is severe or irreparable, these machineries engage either cell death (apoptosis) or cellular senescence pathways. The latter results in a permanent cell cycle arrest.6 These responses are anti-cancer mechanisms, and they utilize classical tumor suppressors that respond to oxidative stress, DNA damage and telomere erosion.7-10 They indirectly reduce mutations by providing cells with optimal conditions for repair, or by eliminating mutant cells (apoptosis).11-13 DNA damage responses to DSBs can also induce cellular senescence, which inhibits tumorigenesis by preventing the growth of mutant cells.14-16 These same or similar responses (apoptosis or senescence) may contribute to aging by reducing the population of healthy cells.17-20 Therefore, apoptosis and cellular senescence are anti-cancer mechanisms needed to ensure longevity, but might also contribute to aging by depleting or altering cells with age.21,22 Caretakers are DNA repair pathways that maintain the genome by correcting DNA damage. There are multiple DNA repair pathways that are uniquely designed to correct specific lesions, with some overlap among them. A variety of excision repair pathways correct lesions restricted to a single DNA strand, including base excision repair (BER),23,24 nucleotide excision repair (NER)25 and mismatch repair (MMR).26 Of these, BER is prominent for repairing ROS-induced DNA damage, NER is important for repairing UV light-induced lesions, and MMR is critical for repairing replicationassociated lesions. In addition, there are several pathways important for repairing DNA DSBs, a particularly cytotoxic lesion, including homologous recombination (HR) and nonhomologous end joining (NHEJ). HR is an error free pathway that utilizes the sister chromatid as a template during S/G2 phase,27,28 whereas NHEJ joins ends together without a template. NHEJ is the dominate pathway in G1–G0 but may also function during S phase.29 Both pathways are

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Tumor suppressors are longevity assurance genes that ensure early life fitness. Genes are defined as tumor suppressors if their mutation predisposes the animal to cancer (a phenotype-based definition). Tumor suppressors fall into two categories: caretakers and gatekeepers. Caretakers suppress cancer by repairing damaged DNA while gatekeepers suppress cancer by halting the cell cycle long enough to repair damaged DNA. If the damage is irreparable, gatekeepers induce either apoptosis or senescence. These responses are deleterious to the cell but protect the organism. p53 is the bestknown gatekeeper because it is mutated in over half of all cancers. Nonhomologous end joining (NHEJ) is considered a caretaker since it repairs DNA double-strand breaks that would otherwise lead to gross chromosomal rearrangements (GCRs). NHEJmutant mice display increased GCRs, but without increased cancer. Instead these mice show early aging. This commentary focuses on the role NHEJ has on aging and cancer. I propose that NHEJ evolved to reduce GCRs and moderate gatekeeper responses that would otherwise cause early aging. Furthermore, NHEJ did not evolve to suppress tumors and any observed tumor suppression is merely circumstantial to unnatural laboratory conditions coupled with human bias that favors defining all DNA repair pathways as caretakers.

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Cell maintenance pathways are longevity assurance mechanisms that sustain an organism long enough to reproduce and propagate. Chief among these mechanisms are those that respond to damaged DNA. DNA is subject to a variety of insults that cause a diverse range of lesions and phenotypic outcomes. These insults may come from exposure to both endogenous and exogenous agents or from defects in DNA metabolism. Common endogenous agents are reactive oxygen species (ROS), which are by-products of oxygen metabolism and are produced in the mitochondria and peroxisomes. ROS are important for multiple biological processes that include cell signaling;1 however, they are also highly reactive due to unpaired electrons that can react with biomolecules including DNA. These reactions cause a wide range of genetic damage,2 mostly base lesions and DNA single-strand Correspondence to: Paul Hasty; Department of Molecular Medicine and Institute of Biotechnology; University of Texas Health Science Center; 15355 Lambda Drive; San Antonio, Texas 78245-3207 USA; Tel.: 210.567.7278; Fax: 210.567.7247; Email: [email protected] Submitted: 02/15/08; Accepted: 02/22/08 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/5807 www.landesbioscience.com

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NHEJ’s impact on aging and cancer

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a poptosis varies between different mutant mice. Mice deleted for Ku70, Ku80, Lig4 or Xrcc4, but not DNA-PKCS display neuronal apoptosis and this condition is more severe after deletion of Lig4/ Xrcc4 compared to Ku70 or Ku80, and causes late embryonic lethality for only the former group. In addition, deletion of Ku70, Ku80 and Lig4/Xrcc4 causes defects in joining both the coding and signal ends during V(D)J recombination, while mutations in DNAPKCS causes only a defect in joining the coding ends. Furthermore, deletion of either DNA-PKCS or Artemis does not cause the same degree of hypersensitivity to ionizing radiation, compared to deletion of other NHEJ proteins.54 Lastly, mice deleted for Ku70, Ku80 or Lig4/Xrcc4, but not DNA-PKCS or Artemis, are small from embryonic development throughout their life. Some of these phenotypic differences may result from toxicity caused by one or more of the remaining NHEJ proteins. For example Lig4-mutant lethality is rescued by Ku80-deletion showing that Ku80 is toxic in the absence of Lig4.55 Thus, there are similarities and differences between mouse models deleted for NHEJ proteins.

NHEJ and Aging

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What is the evidence that NHEJ impacts aging in mice? Mice deleted for Ku70, Ku80 or both exhibit a similar accelerated aging phenotype52,56 that appears to be caused by increased genotoxic stress associated with the repair defect, rather than chronic inflammation associated with scid.57 These mice show a range of gross age-related changes that include kyphosis, rough fur coat, alopecia, paraphimosis, rectal prolapse and suppurative conjunctivitis. These mice also show a variety of histological changes that include osteopenia, early closure of the epiphysis and skin atrophy. Fibroblasts derived from these mice show an early onset of chromosomal abnormalities that include both telomere loss and chromosomal fragments. Importantly, control mice display the same age-related signs, but at a later age. In addition, the life span of Ku70- and Ku80-mutant mice is shorter than control mice and all aging signs are observed in these mutant mice at about the same relative point during their life span. Thus, Ku70- and Ku80-mutant mice exhibit aging phenotypes at about the same point in their life span as control mice do in their (obviously much longer) life span. However, considering only chronological age, Ku70- and Ku80mutant mice exhibit aging earlier than control mice. In addition to Ku70 and Ku80, mice deleted for Xrcc458 and DNA-PKCS59,60 exhibit early aging. Thus, deletion of multiple NHEJ proteins leads to early aging. Do defects in NHEJ actually contribute to normal aging? There is evidence that NHEJ functionally declines with age suggesting that reduced NHEJ proficiency might limit life span and contribute to aging.61 In support of this possibility, as rats age, Ku levels diminish in the testis and Ku70 or Ku80 levels are differentially expressed in various tissues.62 Similarly as humans age, Ku nuclear localization and DNA binding is impaired in blood mononuclear cells63,64 and Ku70, but not Ku80, levels decline in lymphocytes.65 By correlation, Ku levels decline and their cellular distribution is altered as human fibroblasts approach senescence.66 Furthermore, NHEJ function declines in the brains of aging rats67,68 and in Alzheimer’s patients69 and becomes less efficient and more error-prone in senescent cells.70 Thus, NHEJ declines with age supporting the possibility that defective NHEJ contributes to aging.

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frequently used in mammalian cells30 and disruption of either leads to GCRs. Thus, a variety of repair pathways are uniquely designed to repair specific DNA lesions. Germline mutations in DNA repair pathways can lead to heritable disorders that display a range of clinical features specific to the mutated gene. These clinical features include developmental defects and cancer.31,32 With respect to development, mutations in genes such as CS, XPD, FA, NBS, DNA ligase IV and Artemis cause Cockayne syndrome, Trichothiodystrophy, Fanconi anemia, Nijmegen break syndrome (Nbs), LIG4 syndrome and radiosensitive severe combined immunodeficiency (RS-SCID), respectively. These syndromes are associated with microcephaly or dysmorphic facial features and a broad range of other problems. Mutations in Atm and Atld cause progressive ataxia. With respect to cancer, mutations in the NER and MMR pathways cause skin cancer and hereditary nonpolyposis colorectal cancer (HNPCC), respectively. Mutations in Brca1 and Brca2 cause breast and ovarian cancer,33 while mutations in RecQ helicases,34 Nbs35 and FA36 genes cause a variety of cancers. Thus, mutations in DNA repair genes result in both developmental and cancer enhancing phenotypes. Since germline mutations in some DNA repair genes are responsible for hereditary cancer, there seems to be a general assumption that all DNA repair pathways are designed to suppress tumorigenesis, even though only scant evidence supports this assumption for some of them. For example there is little evidence that either BER or NHEJ naturally suppresses tumors outside a laboratory setting. Furthermore, the true impact any DNA repair pathway has on suppressing spontaneous tumors is questionable because malignant tumors rarely if ever harbor genetic mutations in DNA repair genes, in stark contrast to the very common mutations in gatekeepers. Here, I propose that NHEJ may have evolved to reduce GCRs and dampen gatekeeper responses in order to suppress deleterious cellular endpoints that could ultimately impair early life fitness by causing early aging.

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Phenotypes Common to all NHEJ-Mutant Mouse Models

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NHEJ repairs DNA DSBs by joining open ends together. In mammals, at least seven proteins are required: Ku70, Ku80, DNAPKCS, Artemis, Xrcc4, DNA Ligase IV (Lig4) and Xrcc4-like factor.37 Ku70 and Ku80 form a heterodimer called Ku that binds to DNA ends.38 Together with a PI-3 kinase catalytic subunit, DNA-PKCS, Ku forms a holoenzyme referred to as DNA-PK (DNA dependentprotein kinase). Artemis opens hairpins and processes overhangs in a complex with DNA-PKCS and these ends are ligated by the Xrcc4-Lig4 heterodimer in a complex with Xrcc4-like factor.39,40 Mice lacking NHEJ exhibit phenotypes that result from defective repair of DNA DSBs. These phenotypes include hypersensitivity to clastogenic agents and chromosomal rearrangements.41-54 NHEJ also repairs the DSBs formed during the assembly of V(D)J [Variable(Di verse)Joining] segments of antigen receptor genes, which is essential for lymphocyte development; NHEJ-deletion causes failed lymphocyte development resulting in severe combined immunodeficiency (scid). Notably, deletion of any NHEJ component causes a similar phenotype: hypersensitivity to clastogens, GCRs and scid.

Phenotypes Not Common to all NHEJ-Mutant Mouse Models There are also differences in the phenotypes of mice deleted for different NHEJ proteins. For example, the severity of neuronal 1140

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Similar to the aging phenotypes described above, the Ku70and Ku80-mutant mice exhibit an early onset of cancer. However, dissimilar to these aging phenotypes, cancer levels are low compared to controls and are mostly restricted to lymphomas.52 Multiple groups have studied both Ku70- and Ku80-mutant mice and to date no group has reported a high cancer incidence for Ku80-mutant mice.42,56 Several groups, in contrast to our findings,52 reported a high lymphoma incidence in Ku70-mutant mice.45,71 We used the same mice from one of the other reports, but observed a lower cancer incidence.45 It is possible the Ku70-mutant mice from our cohort exhibit lower cancer levels due to early death from another illness; however, this is unlikely since our Ku70-mutants actually live longer than those of the other report (median life spans of 37 vs. 28 weeks).52,71 Thus, there is clear divergence in lymphoma incidence between the Ku70-mutant mice in our study and those from other studies. This discrepancy suggests that divergent genetic backgrounds or environments can impact lymphoma incidence. Even though Ku70-mutant mice show lymphoma at varying levels, this variability is not likely caused by defective general genome maintenance (i.e., NHEJ) since T cell development was not completely blocked in Ku70-mutant mice: double positive thymocytes were present in thymi at low numbers.44 Thus, leaky T cell development, instead of defective NHEJ, likely causes these lymphomas44 and therefore, does not show NHEJ has caretaker function. This is in line with the observation that Ku80-mutant mice do not exhibit either leaky T cell development or high lymphoma levels. It is surprising that NHEJ-deficient mice have a low cancer incidence because DNA repair is considered essential for preventing cancer-causing mutations.31,32 Perhaps Ku-mutant mice exhibit less cancer because they have less time to develop it. This is the logical conclusion when cancer incidence is viewed from the perspective of chronological time because most Ku-mutant mice die before the control mice begin to show cancer. However, this conclusion is at odds with that drawn from the other aging phenotypes shown by these mice: these phenotypes show an increased incidence within the same time frame. In addition, other DNA repair-mutant mouse models do show increased cancer within the life span of Ku70 and Ku80-deficient mice. Thus, the Ku70- and Ku80-mutant mice live long enough to develop cancer at high incidence. It is also possible that Ku-deletion reduces cancer in real time. This is the logical conclusion when aging signs are viewed from the perspective of biological time (a measurement relative to their life span) since control and Ku70- and Ku80-mutants exhibit these aging characteristics at approximately the same point within their life span. However, this possibility is contrary to the notion that NHEJ is important for tumor suppression. Is there direct evidence that NHEJ suppresses tumors in mice? There is no obvious evidence that deletion of NHEJ predisposes mice to cancer in an otherwise wild type background. However, haploinsufficiency of Ku80 in Poly(ADP-ribose) polymerase (PARP1)-mutant mice promotes the development of hepatocellular adenoma and hepatocellular carcinoma.72 PARP-1 binds to DNA ends and catalyzes the synthesis of poly(ADP-ribose) on target proteins and is believed to be important for repairing single-strand breaks. In addition, NHEJ suppress cancer in a p53-mutant background. p53 is a

tumor suppressor in the gatekeeper category that induces apoptosis and cell cycle checkpoints in response to genotoxic stress.73 Deletion of p53 along with most NHEJ proteins causes pro-B cell lymphoma due to open coding ends that lead to translocations linking a c-myc oncogene and IgH locus sequences.49,74,75 These tumors appear very early (within three months) and are very aggressive. Similarly, Artemis/p53-deficient mice also succumb to pro-B cell tumors; however, the majority of Artemis/p53-deficient tumors coamplified IgH and N-myc through an intra- or interchromosome 12 breakagefusion-bridge mechanism.76 Thus, these cancers are not due to general genomic damage but instead to specific DSBs that induce V(D)J recombination.77,78 In addition, deletion of p53 along with either Lig4 or Ku80 predisposes mice to medulloblastoma.78-80 These tumors also occur very early (three-four months). Based on these data some have called Ku80 and NHEJ tumor suppressors;74,75,81 however, this interpretation should be viewed with caution since there are no PAPR-1 or p53 mutant mammals in nature: hence, these interpretations are based on unnatural laboratory conditions. In addition, deletion of Ku80 did not exacerbate T cell lymphoma in p53-mutant mice suggesting that a decrease in repairing both V(D)J recombination-specific DSBs and nonspecific DSBs is not always oncogenic even in p53-mutant cells.78 Is there direct evidence that NHEJ suppresses tumors in humans? There is evidence that single nucleotide polymorphisms (SNPs) in NHEJ genes associate with a variety of cancers in humans.82 For example, glioma,83,84 breast cancer,85 urothelial carcinoma86 and bladder cancer.87 These SNP variants may cause diminished function or over-processing of DNA ends that induces mutations during end joining. There are also alleles that associate with low levels of cancer. At this time it is difficult to know if these SNPs actually influence NHEJ function and oncogenesis. However, in vitro end joining activity seems diminished for SNPs that associate with breast cancer,88 although, there is no biological data that shows any of these SNPs enhance genomic instability or increase cancer risk. Moreover, association of these NHEJ SNP variants to cancer are not always reproduced from one study to the next.83 Thus, it is uncertain they contribute to cancer risk. If these SNPs truly contributed to cancer risk then one would predict to observe mutations in at least some of the NHEJ genes that clearly increase cancer risk as is true for many tumor suppressors. There is no evidence that NHEJ mutations increase cancer risk in humans, perhaps because NHEJ is required for immunocompetence due to its role in V(D)J recombination37 and because Ku80 is essential for survival of human HCT116 cells.89 Thus, null NHEJ mutations would likely be lethal to humans and therefore individuals with homozygote mutations would not be present in the human population. However, homozygosity is not required to find tumor suppressors. Hereditary transmission of tumor susceptibility is often found in heterozygotes, which develop cancer after either loss of heterozygosity or haploinsufficiency (or both). Thus, if NHEJ truly suppressed tumors one would expect to find NHEJ-heterozygous mutants in a cancer-prone human population, as seen for many caretakers (Brca1, Brca1 and MMR genes) and gatekeepers (pRb, p53). Therefore, none of the NHEJ genes fit this basic standard for a tumor suppressor. Even though heterozygotes are not found in the human population, there are Lig4 and Artemis hypomorphic alleles.31 LIG4

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NHEJ and Cancer

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The Impact GCRs have on the NHEJ-Mutant Phenotypes

Potential Co-evolution in Chordates of NHEJ and p53Mediated Cellular Senescence as Major Pathways that Respond to DNA DSBs

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NHEJ is unique compared to many other DNA repair pathways since it is not strongly conserved from lower species to vertebrates. For example, NHEJ along with HR are the two major pathways to repair DNA DSBs in vertebrates. However, HR is the predominant DSB repair pathway in prokaryotes and lower eukaryotes. For this reason NHEJ was discovered first in mammals, then in lower eukaryotes and finally in some, but not all prokaryotes (the reverse order of discovery compared to most other DNA repair pathways including HR).97 In prokaryotes, NHEJ is comprised of only two components, a Ku homodimer along with a ligase and their exact function is not completely understood. In lower eukaryotes like the budding yeast Saccharomyces cerevisiae there are homologues to Ku70, Ku80, Lig4 and Xrcc4 but not to DNA-PKCS and Artemis.98 Furthermore, NHEJ in S. cerevisiae, but not vertebrates, requires the Mre11/Rad50/Xrs2 complex.99 DNA-PKCS is generally restricted to vertebrates, but was recently identified by sequence homology in some arthropods.100 Its exact function in these species is not yet known. Thus, a primitive form of NHEJ exists in some prokaryotes and lower eukaryotes, but NHEJ only emerges as a major pathway for repairing nonspecific DSBs during chordate evolution as seen in fish,101,102 frogs,103 birds104 and mammals.37 Ancestral p53 is found in metazoans including worms, flies, calms, squids, frogs, fish and mammals but not in prokaryotes, yeast or plants.105 In mammals, p53 is well known for suppressing tumors. However, p53 also suppress tumors in response to DNA damage in more primitive vertebrates. For example, in zebrafish NHEJ-deletion induces p53-mediated apoptosis after IR exposure101 and p53(M214K) mutant zebrafish embryos exposed to γ-radiation failed to undergo apoptosis, upregulate p21 and arrest at the G(1)/S checkpoint. These mutant fish developed malignant peripheral nerve sheath tumors by 8.5 months.106 However, p53 clearly does not suppress tumors in worms or flies. Yet, from its earliest origins, p53 mediates DNA damage responses. For example, a p53 homologue in nematodes (Caenorhabditis elegans, CEP-1) and flies (Drosophila melanogaster, dmp53) is important for radiation-induced apoptosis in the germ line.107 However, p21 mediated cellular senescence in response to DNA damage is not observed in flies or worms but is likely restricted to vertebrates.105 This shows a relationship between NHEJ and p53-mediated DNA damage responses in lower vertebrates that correlate DSB repair by NHEJ with p53-mediated p21-induced checkpoints. Therefore, the co-evolution of NHEJ as a major DSB repair pathway and p53-mediated cellular senescence in vertebrates supports the notion that NHEJ is designed to dampen these responses. Could p53-mediated responses that lead to cellular senescence contribute to the aging process? Cellular senescence is known to suppresses tumors but its relationship to aging is less clear.5,10 Studies show that p53 levels influence cellular replication capacity since p53-deletion increases replicative potential108 while p53

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It is possible that many aging phenotypes are due to an accumulation of GCRs that impair cell function with age.93 NHEJ-deleted cells may accumulate GCRs since they are hypersensitive to agents that cause DSBs including ROS.49 Deletion of NHEJ proteins causes a clear increase in GCRs, including amplifications, translocations and telomere end-joining.82 In addition, Ku80 haploinsufficiency increases DNA breaks.48 It is clear NHEJ-deletion elevates breaks and GCRs that may contribute to early aging but it is strange they do not increase cancer incidence.

likely elevated in NHEJ-mutant mice supporting the perspective that NHEJ functions to dampen them. Thus, elevated gatekeeper responses could contribute to the low cancer incidence and early aging phenotypes as previously proposed.56,96

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syndrome causes pancytopenia and developmental defects, whereas Artemis mutations cause radiosensitive severe combined immunodeficiency (RS-SCID). At this time, one in five Lig4 patients has developed cancer, in this case a leukemia that may have arisen from defective V(D)J recombination. In addition, two RS-SCID patients show EBV positive B cell lymphoma.90 EBV impairs DNA damage responses in B cells,91 thus, these cases may be similar to mice deleted for Ku80 and p53, which exhibit high levels of pro-B cell lymphomas dependent on defective V(D)J recombination.78 Thus, it is difficult to determine if defective NHEJ contributed to oncogenesis in these individuals due to low numbers. Even if it did, the tumors may have arisen due to defects in V(D)J recombination, coupled with impaired gatekeeper responses and not due to defects in repairing nonspecific DNA DSBs. In addition, these mutant genes would clearly be removed by selective pressure in a historically natural setting which diminishes any view of a naturally designed-function as a caretaker. There are also patients with mutations in Cernunnos/ XLF that present with severe immunodeficiency, developmental defects and hypersensitivity to clastogenic agents, but at this time no cancer.39,40,92 Thus, there is no compelling evidence at this time that mutations in NHEJ genes cause cancer, giving reason to question the notion that NHEJ genes are caretakers.

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The Impact p53-Mediated Gatekeeper Responses have on the NHEJ-Mutant Phenotype in Mice

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It is possible the NHEJ-mutant mice exhibit low cancer levels and early aging due to inefficiently repaired DNA damage that induces persistent or exaggerated gatekeeper responses. To evaluate this possibility mice deleted for both NHEJ and p53 were observed. Persistent gatekeeper responses influence at least several aspects of the NHEJ-mutant phenotype. For example, mice deleted for Ku70, Ku80, Xrcc4 or Lig4 exhibit neuronal apoptosis94 that is rescued by p53-deletion75 or by expression of a p53 mutant that is defective for apoptosis (p53R172P or p53S18/23A).58,95 Thus, neuronal apoptosis is mediated by the gatekeeper p53. In addition, Ku80- and Xrcc4mutant fibroblasts in tissue culture undergo premature cellular senescence that is dependent on p53.49,75 Cellular senescence is an irreversible proliferation arrest that limits capacity for proliferation of many cells.6 This observation means that cellular senescence is not induced by defective DSB repair or an accumulation of GCRs, but instead by the p53-mediated gatekeeper response to DSBs. Since both apoptosis and cellular senescence suppress oncogenesis,10 it is possible that defects in gatekeepers will substantially elevate cancer in NHEJ-mutant mice. As mentioned above, mice deleted for both NHEJ and p53 show very high levels of pro-B cell lymphoma and medulloblastoma at a very young age. Gatekeeper responses are 1142

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Figure 1. NHEJ assures longevity by suppressing GCRs and gatekeeper checkpoints. NHEJ repairs DNA DSBs that would otherwise activate gatekeeper checkpoints or become a GCR. In the absence of NHEJ, DSBs are inefficiently repaired and may become a GCR or may induce persistent gatekeeper responses that could lead to either apoptosis or cellular senescence. Thus, increased GCRs or persistent gatekeeper responses may cause early aging without increased cancer. This would indicate that NHEJ evolved to become a major DSB repair pathway to prolong youth and fitness by suppressing GCRs and gatekeeper checkpoints. Grey: very rare events. Black: common events.

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overexpression decreases replicative potential and promotes cellular senescence.109 These observations suggest conditions that initiate the p53-dependent G1/S checkpoint, like DNA damage, induce cell cycle arrest in G1 that may ultimately result in cellular senescence, especially if the DNA damage persists. In addition, two mouse models that overexpress a short isoform of p53 that lacks some of the N-terminus, further suggest that some aspects of aging are the consequence of a subset of distinct cellular responses to DNA damage.110,111 These models exhibit elevated cancer resistance, but shortened life span that is accompanied by an early onset of aging phenotypes. For both models full-length p53 is required to observe early aging. Since p53 naturally forms a tetramer, it is likely these truncated isoforms associate with full-length p53. N-terminally truncated p53 isoforms are found in tissue extracts from nontransgenic mice111 and human cells112,113 suggesting they perform an in vivo function. A short isoform stabilizes p53 in the presence of Mdm2 and alters the expression levels of p53-induced gene products.113 Thus, overexpression of this p53 isoform likely increases some aspects of p53 function. Similarly in zebrafish, increased expression of a p53 isoform, Δ113p53, induces the expression of p53-responsive genes to trigger the arrest of the cell cycle but not apoptosis.114 In addition, MDM2 is found in vertebrates including zebrafish but not in flies and worms. However, MDM2 is unable to bind to these truncated p53-isoforms since these binding sites are encoded by the N-terminus.115 Therefore, these mouse models show that activation of p53 by a dominant-N-terminally truncated isoform accelerates the aging process and highlights the importance of p53 isoforms and MDM2-mediated p53 regulation. Thus, the utilization of NHEJ as a major pathway for repairing DSBs co-evolved with p53-mediated cellular senescence and the need for gatekeeper tumor suppressors. This is not true for other DNA repair pathways that are obviously important in prokaryotes (HR, NER, BER, MMR).

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convincingly raise cancer risk. Furthermore, it is difficult to claim NHEJ functions to prevent GCRs as an anti-cancer mechanism since NHEJ-mutant mice exhibit an early onset of chromosomal aberrations but no increase in cancer. Therefore, to place NHEJ into the tumor suppressor category seems unjustified. The phenotype shown by deletion of most NHEJ proteins is early aging with no increase in cancer. Thus, based on phenotype, the more appropriate category would be aging suppressor. Why would NHEJ suppress aging? There are two possibilities that are not mutually exclusive (see Fig. 1). First, NHEJ suppresses GCRs that may accumulate with age and decrease cellular health. Second, NHEJ repairs DNA DSBs that would otherwise induce either apoptosis or cellular senescence by stimulating gatekeepers. In support of this idea, NHEJ deletion causes both GCRs and p53mediated replicative senescence. In addition, there is an intriguing co-evolution of NHEJ becoming a major DSB repair pathway as p53-mediated cellular senescence becomes a major DSB response. According to this model at the early stages of life—NHEJ would repair DNA DSBs to prevent GCRs and to dampen checkpoints to suppress aging. At late stages; however, NHEJ declines (as shown in rodents and humans) such that GCRs and cellular responses increase to promote aging. Current data supports this model, yet more data is needed. For example, it is important to show that NHEJ-deletion can actually ameliorate cancer in chronological time by activating cellular responses. This would convincingly show that NHEJ is not designed to suppress tumors. In addition, NHEJ SNP variants could support this model if they associate with age-related phenotypes in human. These additional investigations would help clarify the role of NHEJ as either a tumor suppressor or an aging suppressor.

Conclusion The term “tumor suppressor” is based purely on phenotype and many genes and pathways easily fit this category since their mutation clearly elevate cancer risk; however, NHEJ is not one of them. Notably, NHEJ-deletion does not elevate cancer risk in otherwise wild type mice and there are no known human mutations that www.landesbioscience.com

Acknowledgements

I thank Drs. Jan Vijg and Judith Campisi for critical review and constructive comments. This work was supported by P01 AG17242, R01 CA76317-05A1, UO1 ES11044. References 1. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007; 39:44-84. 2. Friedberg EC, Walker GC, Siede W. DNA repair and mutagenesis. Washington D.C.: American Society of Microbiology, 1995. 3. White PA. The sources and potential hazards of mutagens in complex environmental matrices-Part II. Mutat Res 2007. 4. Kinzler KW, Vogelstein B. Gatekeepers and caretakers. Nature 1997; 386:761-3. 5. Campisi J, d’Adda di Fagagna F. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol 2007; 8:729-40. 6. Hayflick L. The limited in vitro lifetime of human diploid cell strains. Experimental Cell Research 1965; 37:614-36. 7. Reed JC. Mechanisms of apoptosis avoidance in cancer. Curr Opin Oncol 1999; 11:68-75. 8. Parrinello S, Samper E, Krtolica A, Goldstein J, Melov S, Campisi J. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell Biol 2003; 5:741-7. 9. Sharpless NE, DePinho RA. How stem cells age and why this makes us grow old. Nat Rev Mol Cell Biol 2007; 8:703-13.

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.

42. Nussenzweig A, Chen C, da Costa Soares V, Sanchez M, Sokol K, Nussenzweig MC, Li GC. Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature 1996; 382:551-5. 43. Nussenzweig A, Sokol K, Burgman P, Li L, Li GC. Hypersensitivity of Ku80-deficient cell lines and mice to DNA damage: the effects of ionizing radiation on growth, survival, and development. Proc Natl Acad Sci USA 1997; 94:13588-93. 44. Gu Y, Jin S, Gao Y, Weaver DT, Alt FW. Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination. Proc Natl Acad Sci USA 1997; 94:8076-81. 45. Gu Y, Seidl KJ, Rathbun GA, Zhu C, Manis JP, van der Stoep N, Davidson L, Cheng HL, Sekiguchi JM, Frank K, Stanhope-Baker P, Schlissel MS, Roth DB, Alt FW. Growth retardation and leaky SCID phenotype of Ku70-deficient mice. Immunity 1997; 7:653-65. 46. Gao Y, Sun Y, Frank KM, Dikkes P, Fujiwara Y, Seidl KJ, Sekiguchi JM, Rathbun GA, Swat W, Wang J, Bronson RT, Malynn BA, Bryans M, Zhu C, Chaudhuri J, Davidson L, Ferrini R, Stamato T, Orkin SH, Greenberg ME, Alt FW. A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis. Cell 1998; 95:891-902. 47. Frank KM, Sekiguchi JM, Seidl KJ, Swat W, Rathbun GA, Cheng HL, Davidson L, Kangaloo L, Alt FW. Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV. Nature 1998; 396:173-7. 48. Karanjawala ZE, Grawunder U, Hsieh CL, Lieber MR. The nonhomologous DNA end joining pathway is important for chromosome stability in primary fibroblasts. Curr Biol 1999; 9:1501-4. 49. Lim DS, Vogel H, Willerford DM, Sands AT, Platt KA, Hasty P. Analysis of ku80-mutant mice and cells with deficient levels of p53. Mol Cell Biol 2000; 20:3772-80. 50. Frank KM, Sharpless NE, Gao Y, Sekiguchi JM, Ferguson DO, Zhu C, Manis JP, Horner J, DePinho RA, Alt FW. DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway. Mol Cell 2000; 5:993-1002. 51. Ferguson DO, Sekiguchi JM, Chang S, Frank KM, Gao Y, DePinho RA, Alt FW. The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations. Proc Natl Acad Sci USA 2000; 97:6630-3. 52. Li H, Vogel H, Holcomb VB, Gu Y, Hasty P. Deletion of Ku70, Ku80, or Both Causes Early Aging without Substantially Increased Cancer. Mol Cell Biol 2007; 27:8205-14. 53. Rooney S, Sekiguchi J, Zhu C, Cheng HL, Manis J, Whitlow S, DeVido J, Foy D, Chaudhuri J, Lombard D, Alt FW. Leaky Scid phenotype associated with defective V(D)J coding end processing in Artemis-deficient mice. Mol Cell 2002; 10:1379-90. 54. Rooney S, Alt FW, Lombard D, Whitlow S, Eckersdorff M, Fleming J, Fugmann S, Ferguson DO, Schatz DG, Sekiguchi J. Defective DNA repair and increased genomic instability in Artemis-deficient murine cells. J Exp Med 2003; 197:553-65. 55. Karanjawala ZE, Adachi N, Irvine RA, Oh EK, Shibata D, Schwarz K, Hsieh CL, Lieber MR. The embryonic lethality in DNA ligase IV-deficient mice is rescued by deletion of Ku: implications for unifying the heterogeneous phenotypes of NHEJ mutants. DNA Repair (Amst) 2002; 1:1017-26. 56. Vogel H, Lim DS, Karsenty G, Finegold M, Hasty P. Deletion of Ku86 causes early onset of senescence in mice. Proc Natl Acad Sci USA 1999; 96:10770-5. 57. Holcomb VB, Vogel H, Hasty P. Deletion of Ku80 causes early aging independent of chronic inflammation and Rag-1-induced DSBs. Mech Ageing Dev 2007. 58. Chao C, Herr D, Chun J, Xu Y. Ser18 and 23 phosphorylation is required for p53-dependent apoptosis and tumor suppression. Embo J 2006; 25:2615-22. 59. Espejel S, Klatt P, Menissier-de Murcia J, Martin Caballero J, Flores JM, Taccioli G, de Murcia G, Blasco MA. Impact of telomerase ablation on organismal viability, aging, and tumorigenesis in mice lacking the DNA repair proteins PARP-1, Ku86, or DNA-PKcs. J Cell Biol 2004; 167:627-38. 60. Espejel S, Martin M, Klatt P, Martin-Caballero J, Flores JM, Blasco MA. Shorter telomeres, accelerated ageing and increased lymphoma in DNA-PKcs-deficient mice. EMBO Rep 2004; 5:503-9. 61. Gorbunova V, Seluanov A, Mao Z, Hine C. Changes in DNA repair during aging. Nucleic Acids Res 2007. 62. Um JH, Kim SJ, Kim DW, Ha MY, Jang JH, Kim DW, Chung BS, Kang CD, Kim SH. Tissue-specific changes of DNA repair protein Ku and mtHSP70 in aging rats and their retardation by caloric restriction. Mech Ageing Dev 2003; 124:967-75. 63. Doria G, Barattini P, Scarpaci S, Puel A, Guidi L, Frasca D. Role of immune responsiveness and DNA repair capacity genes in ageing. Ageing Res Rev 2004; 3:143-51. 64. Frasca D, Barattini P, Tirindelli D, Guidi L, Bartoloni C, Errani A, Costanzo M, Tricerri A, Pierelli L, Doria G. Effect of age on DNA binding of the ku protein in irradiated human peripheral blood mononuclear cells (PBMC). Exp Gerontol 1999; 34:645-58. 65. Ju YJ, Lee KH, Park JE, Yi YS, Yun MY, Ham YH, Kim TJ, Choi HM, Han GJ, Lee JH, Lee J, Han JS, Lee KM, Park GH. Decreased expression of DNA repair proteins Ku70 and Mre11 is associated with aging and may contribute to the cellular senescence. Exp Mol Med 2006; 38:686-93. 66. Seluanov A, Danek J, Hause N, Gorbunova V. Changes in the level and distribution of Ku proteins during cellular senescence. DNA Repair (Amst) 2007. 67. Ren K, de Ortiz SP. Non-homologous DNA end joining in the mature rat brain. J Neurochem 2002; 80:949-59. 68. Vyjayanti VN, Rao KS. DNA double strand break repair in brain: reduced NHEJ activity in aging rat neurons. Neurosci Lett 2006; 393:18-22. 69. Shackelford DA. DNA end joining activity is reduced in Alzheimer’s disease. Neurobiol Aging 2006; 27:596-605.

©

20

08

LA

ND

ES

BIO

SC

IEN CE

.D

ON

10. Collado M, Blasco MA, Serrano M. Cellular senescence in cancer and aging. Cell 2007; 130:223-33. 11. Sharpless NE, DePinho RA. Cancer: crime and punishment. Nature 2005; 436:636-7. 12. Collado M, Serrano M. The power and the promise of oncogene-induced senescence markers. Nat Rev Cancer 2006; 6:472-6. 13. Busuttil RA, Rubio M, Dolle ME, Campisi J, Vijg J. Oxygen accelerates the accumulation of mutations during the senescence and immortalization of murine cells in culture. Aging Cell 2003; 2:287-94. 14. Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, Guldberg P, Sehested M, Nesland JM, Lukas C, Orntoft T, Lukas J, Bartek J. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 2005; 434:864-70. 15. Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N, Vassiliou LV, Kolettas E, Niforou K, Zoumpourlis VC, Takaoka M, Nakagawa H, Tort F, Fugger K, Johansson F, Sehested M, Andersen CL, Dyrskjot L, Orntoft T, Lukas J, Kittas C, Helleday T, Halazonetis TD, Bartek J, Gorgoulis VG. Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 2006; 444:633-7. 16. Di Micco R, Fumagalli M, Cicalese A, Piccinin S, Gasparini P, Luise C, Schurra C, Garre M, Nuciforo PG, Bensimon A, Maestro R, Pelicci PG, d’Adda di Fagagna F. Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. Nature 2006; 444:638-42. 17. Campisi J. Aging and cancer: the double-edged sword of replicative senescence. J Am Geriatr Soc 1997; 45:482-8. 18. Campisi J. Cancer, aging and cellular senescence. In Vivo 2000; 14:183-8. 19. Pelicci PG. Do tumor-suppressive mechanisms contribute to organism aging by inducing stem cell senescence? J Clin Invest 2004; 113:4-7. 20. Bree RT, Stenson Cox C, Grealy M, Byrnes L, Gorman AM, Samali A. Cellular longevity: role of apoptosis and replicative senescence. Biogerontology 2002; 3:195-206. 21. Janzen V, Forkert R, Fleming HE, Saito Y, Waring MT, Dombkowski DM, Cheng T, DePinho RA, Sharpless NE, Scadden DT. Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a. Nature 2006; 443:421-6. 22. von Zglinicki T, Saretzki G, Ladhoff J, d’Adda di Fagagna F, Jackson SP. Human cell senescence as a DNA damage response. Mech Ageing Dev 2005; 126:111-7. 23. Barnes DE, Lindahl T. Repair and genetic consequences of endogenous DNA base damage in mammalian cells. Annu Rev Genet 2004; 38:445-76. 24. Almeida KH, Sobol RW. A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification. DNA Repair (Amst) 2007; 6:695-711. 25. de Boer J, Hoeijmakers JH. Nucleotide excision repair and human syndromes. Carcinogenesis 2000; 21:453-60. 26. Kolodner RD, Marsischky GT. Eukaryotic DNA mismatch repair. Curr Opin Genet Dev 1999; 9:89-96. 27. Sung P, Trujillo KM, Van Komen S. Recombination factors of Saccharomyces cerevisiae. Mutat Res 2000; 451:257-75. 28. West SC. Molecular views of recombination proteins and their control. Nat Rev Mol Cell Biol 2003; 4:435-45. 29. Lieber MR, Ma Y, Pannicke U, Schwarz K. Mechanism and regulation of human nonhomologous DNA end-joining. Nat Rev Mol Cell Biol 2003; 4:712-20. 30. Jasin M. Chromosome breaks and genomic instability. Cancer Invest 2000; 18:78-86. 31. O’Driscoll M, Gennery AR, Seidel J, Concannon P, Jeggo PA. An overview of three new disorders associated with genetic instability: LIG4 syndrome, RS-SCID and ATR-Seckel syndrome. DNA Repair (Amst) 2004; 3:1227-35. 32. Hoeijmakers JH. Genome maintenance mechanisms for preventing cancer. Nature 2001; 411:366-74. 33. Fackenthal JD, Olopade OI. Breast cancer risk associated with BRCA1 and BRCA2 in diverse populations. Nat Rev Cancer 2007; 7:937-48. 34. Hanada K, Hickson ID. Molecular genetics of RecQ helicase disorders. Cell Mol Life Sci 2007; 64:2306-22. 35. Demuth I, Digweed M. The clinical manifestation of a defective response to DNA doublestrand breaks as exemplified by Nijmegen breakage syndrome. Oncogene 2007; 26:7792-8. 36. Hinz JM, Nham PB, Salazar EP, Thompson LH. The Fanconi anemia pathway limits the severity of mutagenesis. DNA Repair (Amst) 2006; 5:875-84. 37. Lieber MR, Ma Y, Pannicke U, Schwarz K. The mechanism of vertebrate nonhomologous DNA end joining and its role in V(D)J recombination. DNA Repair (Amst) 2004; 3:817-26. 38. Walker JR, Corpina RA, Goldberg J. Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature 2001; 412:607-14. 39. Buck D, Malivert L, de Chasseval R, Barraud A, Fondaneche MC, Sanal O, Plebani A, Stephan JL, Hufnagel M, le Deist F, Fischer A, Durandy A, de Villartay JP, Revy P. Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly. Cell 2006; 124:287-99. 40. Ahnesorg P, Smith P, Jackson SP. XLF Interacts with the XRCC4-DNA Ligase IV Complex to Promote DNA Nonhomologous End-Joining. Cell 2006; 124:301-13. 41. Zhu C, Bogue MA, Lim DS, Hasty P, Roth DB. Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell 1996; 86:379-89.

1144

Cell Cycle



OT D

IST RIB

UT E

.

98. Dudasova Z, Dudas A, Chovanec M. Non-homologous end-joining factors of Saccharomyces cerevisiae. FEMS Microbiol Rev 2004; 28:581-601. 99. Di Virgilio M, Gautier J. Repair of double-strand breaks by nonhomologous end joining in the absence of Mre11. J Cell Biol 2005; 171:765-71. 100. Dore AS, Drake AC, Brewerton SC, Blundell TL. Identification of DNA-PK in the arthropods. Evidence for the ancient ancestry of vertebrate non-homologous end-joining. DNA Repair (Amst) 2004; 3:33-41. 101. Bladen CL, Lam WK, Dynan WS, Kozlowski DJ. DNA damage response and Ku80 function in the vertebrate embryo. Nucleic Acids Res 2005; 33:3002-10. 102. Bladen CL, Navarre S, Dynan WS, Kozlowski DJ. Expression of the Ku70 subunit (XRCC6) and protection from low dose ionizing radiation during zebrafish embryogenesis. Neurosci Lett 2007; 422:97-102. 103. Labhart P. Ku-dependent nonhomologous DNA end joining in Xenopus egg extracts. Mol Cell Biol 1999; 19:2585-93. 104. Takata M, Sasaki MS, Sonoda E, Morrison C, Hashimoto M, Utsumi H, Yamaguchi-Iwai Y, Shinohara A, Takeda S. Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. Embo J 1998; 17:5497-508. 105. Lu WJ, Abrams JM. Lessons from p53 in non-mammalian models. Cell Death Differ 2006; 13:909-12. 106. Berghmans S, Murphey RD, Wienholds E, Neuberg D, Kutok JL, Fletcher CD, Morris JP, Liu TX, Schulte Merker S, Kanki JP, Plasterk R, Zon LI, Look AT. tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors. Proc Natl Acad Sci USA 2005; 102:407-12. 107. de Nooij JC, Letendre MA, Hariharan IK. A cyclin-dependent kinase inhibitor, Dacapo, is necessary for timely exit from the cell cycle during Drosophila embryogenesis. Cell 1996; 87:1237-47. 108. Harvey M, Sands AT, Weiss RS, Hegi ME, Wiseman RW, Pantazis P, Giovanella BC, Tainsky MA, Bradley A, Donehower LA. In vitro growth characteristics of embryo fibroblasts isolated from p53-deficient mice. Oncogene 1993; 8:2457-67. 109. Sugrue MM, Shin DY, Lee SW, Aaronson SA. Wild-type p53 triggers a rapid senescence program in human tumor cells lacking functional p53. Proc Natl Acad Sci USA 1997; 94:9648-53. 110. Tyner SD, Venkatachalam S, Choi J, Jones S, Ghebranious N, Igelmann H, Lu X, Soron G, Cooper B, Brayton C, Hee Park S, Thompson T, Karsenty G, Bradley A, Donehower LA. p53 mutant mice that display early ageing-associated phenotypes. Nature 2002; 415:45-53. 111. Maier B, Gluba W, Bernier B, Turner T, Mohammad K, Guise T, Sutherland A, Thorner M, Scrable H. Modulation of mammalian life span by the short isoform of p53. Genes Dev 2004; 18:306-19. 112. Courtois S, Verhaegh G, North S, Luciani MG, Lassus P, Hibner U, Oren M, Hainaut P. DeltaN-p53, a natural isoform of p53 lacking the first transactivation domain, counteracts growth suppression by wild-type p53. Oncogene 2002; 21:6722-8. 113. Yin Y, Stephen CW, Luciani MG, Fahraeus R. p53 Stability and activity is regulated by Mdm2-mediated induction of alternative p53 translation products. Nat Cell Biol 2002; 4:462-7. 114. Chen J, Ruan H, Ng SM, Gao C, Soo HM, Wu W, Zhang Z, Wen Z, Lane DP, Peng J. Loss of function of def selectively upregulates Delta113p53 expression to arrest expansion growth of digestive organs in zebrafish. Genes Dev 2005; 19:2900-11. 115. Scrable H, Sasaki T, Maier B. DeltaNp53 or p44: priming the p53 pump. Int J Biochem Cell Biol 2005; 37:913-9.

©

20

08

LA

ND

ES

BIO

SC

IEN CE

.D

ON

70. Seluanov A, Mittelman D, Pereira-Smith OM, Wilson JH, Gorbunova V. DNA end joining becomes less efficient and more error-prone during cellular senescence. Proc Natl Acad Sci USA 2004; 101:7624-9. 71. Li GC, Ouyang H, Li X, Nagasawa H, Little JB, Chen DJ, Ling CC, Fuks Z, CordonCardo C. Ku70: a candidate tumor suppressor gene for murine T cell lymphoma. Mol Cell 1998; 2:1-8. 72. Tong WM, Cortes U, Hande MP, Ohgaki H, Cavalli LR, Lansdorp PM, Haddad BR, Wang ZQ. Synergistic role of Ku80 and poly(ADP-ribose) polymerase in suppressing chromosomal aberrations and liver cancer formation. Cancer Res 2002; 62:6990-6. 73. Meek DW. The p53 response to DNA damage. DNA Repair (Amst) 2004; 3:1049-56. 74. Difilippantonio MJ, Zhu J, Chen HT, Meffre E, Nussenzweig MC, Max EE, Ried T, Nussenzweig A. DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation [see comments]. Nature 2000; 404:510-4. 75. Gao Y, Ferguson DO, Xie W, Manis JP, Sekiguchi J, Frank KM, Chaudhuri J, Horner J, DePinho RA, Alt FW. Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development [see comments]. Nature 2000; 404:897-900. 76. Rooney S, Sekiguchi J, Whitlow S, Eckersdorff M, Manis JP, Lee C, Ferguson DO, Alt FW. Artemis and p53 cooperate to suppress oncogenic N-myc amplification in progenitor B cells. Proc Natl Acad Sci USA 2004; 101:2410-5. 77. Zhu C, Mills KD, Ferguson DO, Lee C, Manis J, Fleming J, Gao Y, Morton CC, Alt FW. Unrepaired DNA breaks in p53-deficient cells lead to oncogenic gene amplification subsequent to translocations. Cell 2002; 109:811-21. 78. Holcomb VB, Vogel H, Marple T, Kornegay RW, Hasty P. Ku80 and p53 suppress medulloblastoma that arise independent of Rag-1-induced DSBs. Oncogene 2006; 25:7159-65. 79. Lee Y, McKinnon PJ. DNA ligase IV suppresses medulloblastoma formation. Cancer Res 2002; 62:6395-9. 80. Yan CT, Kaushal D, Murphy M, Zhang Y, Datta A, Chen C, Monroe B, Mostoslavsky G, Coakley K, Gao Y, Mills KD, Fazeli AP, Tepsuporn S, Hall G, Mulligan R, Fox E, Bronson R, De Girolami U, Lee C, Alt FW. XRCC4 suppresses medulloblastomas with recurrent translocations in p53-deficient mice. Proc Natl Acad Sci USA 2006; 103:7378-83. 81. Roth DB, Gellert M. New guardians of the genome. Nature 2000; 404:823-5. 82. Burma S, Chen BP, Chen DJ. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst) 2006; 5:1042-8. 83. Liu Y, Zhang H, Zhou K, Chen L, Xu Z, Zhong Y, Liu H, Li R, Shugart YY, Wei Q, Jin L, Huang F, Lu D, Zhou L. Tagging SNPs in non-homologous end-joining pathway genes and risk of glioma. Carcinogenesis 2007; 28:1906-13. 84. Liu Y, Zhou K, Zhang H, Shugart YY, Chen L, Xu Z, Zhong Y, Liu H, Jin L, Wei Q, Huang F, Lu D, Zhou L. Polymorphisms of LIG4 and XRCC4 involved in the NHEJ pathway interact to modify risk of glioma. Hum Mutat 2007. 85. Willems P, Claes K, Baeyens A, Vandersickel V, Werbrouck J, De Ruyck K, Poppe B, Van den Broecke R, Makar A, Marras E, Perletti G, Thierens H, Vral A. Polymorphisms in nonhomologous end-joining genes associated with breast cancer risk and chromosomal radiosensitivity. Genes Chromosomes Cancer 2008; 47:137-48. 86. Windhofer F, Krause S, Hader C, Schulz WA, Florl AR. Distinctive differences in DNA double-strand break repair between normal urothelial and urothelial carcinoma cells. Mutat Res 2008; 638:56-65. 87. Figueroa JD, Malats N, Rothman N, Real FX, Silverman D, Kogevinas M, Chanock S, Yeager M, Welch R, Dosemeci M, Tardon A, Serra C, Carrato A, Garcia-Closas R, Castano Vinyals G, Garcia Closas M. Evaluation of genetic variation in the double-strand break repair pathway and bladder cancer risk. Carcinogenesis 2007; 28:1788-93. 88. Bau DT, Mau YC, Ding SL, Wu PE, Shen CY. DNA double-strand break repair capacity and risk of breast cancer. Carcinogenesis 2007; 28:1726-30. 89. Li G, Nelsen C, Hendrickson EA. Ku86 is essential in human somatic cells. Proc Natl Acad Sci USA 2002; 99:832-7. 90. Moshous D, Pannetier C, Chasseval Rd R, Deist Fl F, Cavazzana-Calvo M, Romana S, Macintyre E, Canioni D, Brousse N, Fischer A, Casanova JL, Villartay JP. Partial T and B lymphocyte immunodeficiency and predisposition to lymphoma in patients with hypomorphic mutations in Artemis. J Clin Invest 2003; 111:381-7. 91. O’Nions J, Turner A, Craig R, Allday MJ. Epstein-Barr virus selectively deregulates DNA damage responses in normal B cells but has no detectable effect on regulation of the tumor suppressor p53. J Virol 2006; 80:12408-13. 92. Cantagrel V, Lossi AM, Lisgo S, Missirian C, Borges A, Philip N, Fernandez C, Cardoso C, Figarella-Branger D, Moncla A, Lindsay S, Dobyns WB, Villard L. Truncation of NHEJ1 in a patient with polymicrogyria. Hum Mutat 2007; 28:356-64. 93. Vijg J, Dolle ME. Large genome rearrangements as a primary cause of aging. Mech Ageing Dev 2002; 123:907-15. 94. Gu Y, Sekiguchi J, Gao Y, Dikkes P, Frank K, Ferguson D, Hasty P, Chun J, Alt FW. Defective embryonic neurogenesis in Ku-deficient but not DNA-dependent protein kinase catalytic subunit-deficient mice. Proc Natl Acad Sci USA 2000; 97:2668-73. 95. Van Nguyen T, Puebla-Osorio N, Pang H, Dujka ME, Zhu C. DNA damage-induced cellular senescence is sufficient to suppress tumorigenesis: a mouse model. J Exp Med 2007; 204:1453-61. 96. Hasty P. The impact of DNA damage, genetic mutation and cellular responses on cancer prevention, longevity and aging: observations in humans and mice. Mech Ageing Dev 2005; 126:71-7. 97. Bowater R, Doherty AJ. Making ends meet: repairing breaks in bacterial DNA by nonhomologous end-joining. PLoS Genet 2006; 2:8.

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