ATM mediated phosphorylation of CHD4 contributes to genome maintenance

Background: In order to maintain cellular viability and genetic integrity cells must respond quickly following the induction of cytotoxic double strand DNA breaks (DSB). This response requires a number of processes including stabilisation of the DSB, signalling of the break and repair. It is becoming increasingly apparent that one key step in this process is chromatin remodelling. Results: Here we describe the chromodomain helicase DNA-binding protein (CHD4) as a target of ATM kinase. We show that ionising radiation (IR)-induced phosphorylation of CHD4 affects its intranuclear organization resulting in increased chromatin binding/retention. We also show assembly of phosphorylated CHD4 foci at sites of DNA damage, which might be required to fulfil its function in the regulation of DNA repair. Consistent with this, cells overexpressing a phospho-mutant version of CHD4 that cannot be phosphorylated by ATM fail to show enhanced chromatin retention after DSBs and display high rates of spontaneous damage. Conclusion: These results provide insight into how CHD4 phosphorylation might be required to remodel chromatin around DNA breaks allowing efficient DNA repair to occur.

Temporally distinct roles of ATM and ROS in genotoxic- stress-dependent induction and maintenance of cellular senescence

Cells exposed to genotoxic stress induce cellular senescence through a DNA damage response (DDR) pathway regulated by ATM kinase and reactive oxygen species (ROS). Here, we show that the regulatory roles for ATM kinase and ROS differ during induction and maintenance of cellular senescence. Cells treated with different genotoxic agents were analyzed using specific pathway markers and inhibitors to determine that ATM kinase activation is directly proportional to the dose of the genotoxic stress and that senescence initiation is not dependent on ROS or the p53 status of cells. Cells in which ROS was quenched still activated ATM and initiated the DDR when insulted, and progressed normally to senescence. By contrast, maintenance of a viable senescent state required the presence of ROS as well as activated ATM. Inhibition or removal of either of the components caused cell death in senescent cells, through a deregulated ATM-ROS axis. Overall, our work demonstrates existence of an intricate temporal hierarchy between genotoxic stress, DDR and ROS in cellular senescence. Our model reports the existence of different stages of cellular senescence with distinct regulatory networks.

UV-induced Hyperphosphorylation of Replication Protein A Depends on DNA Replication and Expression of ATM Protein

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4–8 h) to UV radiation (10–30 J/m2). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

Functional consequences of sequence alterations in the ATM gene

The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is a high molecular weight, protein (similar to350 kDa) containing a C-terminal protein kinase domain and a number of other putative domains not yet functionally defined. The majority of ATM gene mutations in A-T patients are truncating, resulting in prematurely terminated products that are highly unstable. Missense mutations within the kinase domain and elsewhere in the molecule alter the stability of the protein and lead to loss of protein kinase activity. Only rarely are patients observed with two missense mutations and this gives rise to a milder disease phenotype. Evidence for a dominant interfering effect on normal ATM kinase activity has been reported in cell lines transfected with missense mutant ATM and in cell lines from some A-T heterozygotes. The dominant negative effect of mutant ATM is manifested by an enhancement of cellular radiosensitivity and may be responsible for the cancer predisposition observed in carriers of ATM missense mutations. In this review, we explore the domain structure of the ATM molecule, sites of interaction with other proteins and the consequences of specific amino acid changes on function. (C) 2003 Elsevier B.V. All rights reserved.

Involvement of novel autophosphorylation sites in ATM activation

ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.

Single-stranded DNA-binding protein hSSB1 is critical for genomic stability

Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquitous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair1. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein–protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer1. Here we describe a second human SSB (hSSB1), with a domain organization closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co-localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences diverse endpoints in the cellular DNA damage response.

Krüppel-associated box (KRAB)-associated co-repressor (KAP-1) Ser-473 phosphorylation regulates heterochromatin protein 1 (HP1- ) mobilization and DNA repair in heterochromatin

The DNA damage response encompasses a complex series of signaling pathways that function to regulate and facilitate the repair of damaged DNA. Recent studies have shown that the repair of transcriptionally inactive chromatin, named heterochromatin, is dependent upon the phosphorylation of the co-repressor, Krüppel-associated box (KRAB) domain-associated protein (KAP-1), by the ataxia telangiectasia-mutated (ATM) kinase. Co-repressors, such as KAP-1, function to regulate the rigid structure of heterochromatin by recruiting histone-modifying enzymes, such HDAC1/2, SETDB1, and nucleosome-remodeling complexes such as CHD3. Here, we have characterized a phosphorylation site in the HP1-binding domain of KAP-1, Ser-473, which is phosphorylated by the cell cycle checkpoint kinase Chk2. Expression of a nonphosphorylatable S473A mutant conferred cellular sensitivity to DNA-damaging agents and led to defective repair of DNA double-strand breaks in heterochromatin. In addition, cells expressing S473A also displayed defective mobilization of the HP1-β chromodomain protein. The DNA repair defect observed in cells expressing S473A was alleviated by depletion of HP1-β, suggesting that phosphorylation of KAP-1 on Ser-473 promotes the mobilization of HP1-β from heterochromatin and subsequent DNA repair. These results suggest a novel mechanism of KAP-1-mediated chromatin restructuring via Chk2-regulated HP1-β exchange from heterochromatin, promoting DNA repair.

DNA damage recognition in the normal epithelium of human prostate and seminal vesicles

Prostate cancer is one of the most prevalent cancer types in men. The development of prostate tumors is known to require androgen exposure, and several pathways governing cell growth are deregulated in prostate tumorigenesis. Recent genetic studies have revealed that complex gene fusions and copy - number alterations are frequent in prostate cancer, a unique feature among solid tumors. These chromosomal aberrations are though to arise as a consequence of faulty repair of DNA double strand breaks (DSB). Most repair mechanisms have been studied in detail in cancer cell lines, but how DNA damage is detected and repaired in normal differentiated human cells has not been widely addressed. The events leading to the gene fusions in prostate cancer are under rigorous studies, as they not only shed light on the basic pathobiologic mechanisms but may also produce molecular targets for prostate cancer treatment and prevention. Prostate and seminal vesicles are part of the male reproductive system. They share similar structure and function but differ dramatically in their cancer incidence. Approximately fifty primary seminal vesicle carcinomas have been reported worldwide. Surprisingly, only little is known on why seminal vesicles are resistant to neoplastic changes. As both tissues are androgen dependent, it is a mystery that androgen signaling would only lead to tumors in prostate tissue. In this work, we set up novel ex vivo human tissue culture models of prostate and seminal vesicles, and used them to study how DNA damage is recognized in normal epithelium. One of the major DNA - damage inducible pathways, mediated by the ATM kinase, was robustly activated in all main cell types of both tissues. Interestingly, we discovered that secretory epithelial cells had less histone variant H2A.X and after DNA damage lower levels of H2AX were phosphorylated on serine 139 (γH2AX) than in basal or stromal cells. γH2AX has been considered essential for efficient DSB repair, but as there were no significant differences in the γH2AX levels between the two tissues, it seems more likely that the role of γH2AX is less important in postmitotic cells. We also gained insight into the regulation of p53, an important transcription factor that protects genomic integrity via multiple mechanisms, in human tissues. DSBs did not lead to a pronounced activation of p53, but treatments causing transcriptional stress, on the other hand, were able to launch a notable p53 response in both tissue types. In general, ex vivo culturing of human tissues provided unique means to study differentiated cells in their relevant tissue context, and is suited for testing novel therapeutic drugs before clinical trials. In order to study how prostate and seminal vesicle epithelial cells are able to activate DNA damage induced cell cycle checkpoints, we used primary cultures of prostate and seminal vesicle epithelial cells. To our knowledge, we are the first to report isolation of human primary seminal vesicle cells. Surprisingly, human prostate epithelial cells did not activate cell cycle checkpoints after DSBs in part due to low levels of Wee1A, a kinase regulating CDK activity, while primary seminal vesicle epithelial cells possessed proficient cell cycle checkpoints and expressed high levels of Wee1A. Similarly, seminal vesicle cells showed a distinct activation of the p53 - pathway after DSBs that did not occur in prostate epithelial cells. This indicates that p53 protein function is under different control mechanisms in the two cell types, which together with proficient cell cycle checkpoints may be crucial in protecting seminal vesicles from endogenous and exogenous DNA damaging factors and, as a consequence, from carcinogenesis. These data indicate that two very similar organs of male reproductive system do not respond to DNA damage similarly. The differentiated, non - replicating cells of both tissues were able to recognize DSBs, but under proliferation human prostate epithelial cells had deficient activation of the DNA damage response. This suggests that prostate epithelium is most vulnerable to accumulating genomic aberrations under conditions where it needs to proliferate, for example after inflammatory cellular damage.

BRCA1 and its phosphorylation involved in caffeine-inhibitable event upstream of G2 checkpoint

Caffeine, which specifically inhibits ATM/ATR kinases, efficiently abrogates the ionizing radiation (IR)-induced G2 arrest and increases the sensitivity of various tumor cells to IR. Mechanisms for the effect of caffeine remain to be elucidated. As a target of ATM/ATR kinases, BRCA1 becomes activated and phosphorylated in response to IR. Thus, in this work, we investigated the possible role of BRCA1 in the effect of caffeine on G2 checkpoint and observed how BRCA1 phosphorylation was regulated in this process. For these purposes, the BRCA1 protein level and the phosphorylation states were analyzed by Western blotting by using an antibody against BRCA1 and phospho-specific antibodies against Ser-1423 and Ser-1524 residues in cells exposed to a combination of IR and caffeine. The results showed that caffeine down-regulated IR-induced BRCA1 expression and specifically abolished BRCA1 phosphorylation of Ser-1524, which was followed by an override of G2 arrest by caffeine. In addition, the ability of BRCA1 to transactivate p21 may be required for MCF-7 but not necessary for Hela response to caffeine. These data suggest that BRCA1 may be a potential target of caffeine. BRCA1 and its phosphorylation are most likely to be involved in the caffeine-inhibitable event upstream of G2 arrest.

Kruppel-associated Box (KRAB)-associated co-repressor (KAP-1) Ser-473 phosphorylation regulates heterochromatin protein 1β (HP1-β) mobilization and DNA repair in heterochromatin

The DNA damage response encompasses a complex series of signaling pathways that function to regulate and facilitate the repair of damaged DNA. Recent studies have shown that the repair of transcriptionally inactive chromatin, named heterochromatin, is dependent upon the phosphorylation of the co-repressor, Krüppel-associated box (KRAB) domain-associated protein (KAP-1), by the ataxia telangiectasia-mutated (ATM) kinase. Co-repressors, such as KAP-1, function to regulate the rigid structure of heterochromatin by recruiting histone-modifying enzymes, such HDAC1/2, SETDB1, and nucleosome-remodeling complexes such as CHD3. Here, we have characterized a phosphorylation site in the HP1-binding domain of KAP-1, Ser-473, which is phosphorylated by the cell cycle checkpoint kinase Chk2. Expression of a nonphosphorylatable S473A mutant conferred cellular sensitivity to DNA-damaging agents and led to defective repair of DNA double-strand breaks in heterochromatin. In addition, cells expressing S473A also displayed defective mobilization of the HP1-ß chromodomain protein. The DNA repair defect observed in cells expressing S473A was alleviated by depletion of HP1-ß, suggesting that phosphorylation of KAP-1 on Ser-473 promotes the mobilization of HP1-ß from heterochromatin and subsequent DNA repair. These results suggest a novel mechanism of KAP-1-mediated chromatin restructuring via Chk2-regulated HP1-ß exchange from heterochromatin, promoting DNA repair.

Single-stranded DNA-binding protein hSSB1 is critical for genomic stability

Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquitous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein-protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer. Here we describe a second human SSB (hSSB1), with a domain organization closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co-localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences diverse endpoints in the cellular DNA damage response.

Dysfunction of Endothelial Progenitor Cells from Smokers and Chronic Obstructive Pulmonary Disease Patients Due to Increased DNA Damage and Senescence

Cardiovascular disease (CVD) is a major cause of death in smokers, particularly in those with chronic obstructive pulmonary disease (COPD). Circulating endothelial progenitor cells (EPC) are required for endothelial homeostasis, and their dysfunction contributes to CVD. To investigate EPC dysfunction in smokers, we isolated and expanded blood outgrowth endothelial cells (BOEC) from peripheral blood samples from healthy nonsmokers, healthy smokers, and COPD patients. BOEC from smokers and COPD patients showed increased DNA double-strand breaks and senescence compared to nonsmokers. Senescence negatively correlated with the expression and activity of sirtuin-1 (SIRT1), a protein deacetylase that protects against DNA damage and cellular senescence. Inhibition of DNA damage response by silencing of ataxia telangiectasia mutated (ATM) kinase resulted in upregulation of SIRT1 expression and decreased senescence. Treatment of BOEC from COPD patients with the SIRT1 activator resveratrol or an ATM inhibitor (KU-55933) also rescued the senescent phenotype. Using an in vivo mouse model of angiogenesis, we demonstrated that senescent BOEC from COPD patients are dysfunctional, displaying impaired angiogenic ability and increased apoptosis compared to cells from healthy nonsmokers. Therefore, this study identifies epigenetic regulation of DNA damage and senescence as pathogenetic mechanisms linked to endothelial progenitors' dysfunction in smokers and COPD patients. These defects may contribute to vascular disease and cardiovascular events in smokers and could therefore constitute therapeutic targets for intervention. 

Identification du rôle et des modifications post-traductionnelles modulant l’export nucléaire de l’hélicase virale E1 au cours du cycle de réplication du virus du papillome humain

Les virus du papillome humain (VPH) sont de petits virus à ADN double brin infectant les épithéliums de la peau et des muqueuses. La réplication nécessaire au maintien de leur génome dans les cellules infectées dépend des protéines virales E1 et E2. Au cours de la réplication, E1 est recrutée à l’origine de réplication par E2 afin d’être assemblée en doubles hexamères capables de dérouler l’ADN. E1 contient un domaine C-terminal responsable de l’activité ATPase/hélicase, un domaine central de liaison à l’origine et une région N-terminale régulant la réplication in vivo. Cette région contient des signaux de localisation et d’export nucléaire qui modulent le transport intracellulaire de E1. Chez le virus du papillome bovin (VPB), il a été proposé que ce transport est régulé par la sumoylation de E1. Finalement, la région N-terminale de E1 contient un motif de liaison aux cyclines permettant son interaction avec la cycline E/A-Cdk2. La phosphorylation de E1 par cette dernière régule différemment l’export nucléaire des protéines E1 du VPB et du VPH. Dans la première partie de cette étude, nous avons démontré que bien que la protéine E1 des VPH interagit avec Ubc9, l’enzyme de conjugaison de la voie de sumoylation, cette voie n’est pas requise pour son accumulation au noyau. Dans la seconde partie, nous avons déterminé que l’accumulation nucléaire de E1 est plutôt régulée pas sa phosphorylation. En fait, nous avons démontré que l’export nucléaire de E1 est inhibé par la phosphorylation de sérines conservées de la région N-terminale de E1 par Cdk2. Puis, nous avons établi que l’export nucléaire de E1 n’est pas nécessaire à l’amplification du génome dans les kératinocytes différenciés mais qu’il est requis pour le maintien du génome dans les kératinocytes non différenciés. En particulier, nous avons découvert que l’accumulation nucléaire de E1 inhibe la prolifération cellulaire en induisant un arrêt du cycle cellulaire en phase S et que cet effet anti-prolifératif est contrecarrée par l’export de E1 au cytoplasme. Dans la troisième partie de cette étude, nous avons démontré que l’arrêt cellulaire induit par E1 dépend de sa liaison à l’ADN et à l’ATP, et qu’il est accompagné par l’activation de la voie de réponse aux dommages à l’ADN dépendante de ATM (Ataxia Telangiectasia Mutated). Ces deux événements semblent toutefois distincts puisque la formation d’un complexe E1-E2 réduit l’activation de la voie de réponse aux dommages par E1 sans toutefois prévenir l’arrêt de cycle cellulaire. Finalement, nous avons démontré que la réplication transitoire de l’ADN viral peut avoir lieu dans des cellules arrêtées en phase S, indépendamment de l’activation de la voie de réponse aux dommages à l’ADN et de la kinase ATM. Globalement, nos résultats démontrent que l’export nucléaire de E1 est régulé par sa phosphorylation et non par sa sumoylation. Ils démontrent également que l’export nucléaire de E1 est essentiel au maintien du génome dans les kératinocytes, possiblement parce qu’il prévient l’inhibition de la prolifération cellulaire et l’activation de la voie de réponse aux dommages à l’ADN en limitant l’accumulation de E1 au noyau.

Chk1 phosphorylation of Metnase enhances DNA repair but inhibits replication fork restart

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylating downstream effectors. Although there has been a concerted effort to identify effectors of Chk1 activity, underlying mechanisms of effector action are still being identified. Metnase (also called SETMAR) is a SET and transposase domain protein that promotes both DNA double-strand break (DSB) repair and restart of stalled replication forks. In this study, we show that Metnase is phosphorylated only on Ser495 (S495) in vivo in response to DNA damage by ionizing radiation. Chk1 is the major mediator of this phosphorylation event. We had previously shown that wild-type (wt) Metnase associates with chromatin near DSBs and methylates histone H3 Lys36. Here we show that a Ser495Ala (S495A) Metnase mutant, which is not phosphorylated by Chk1, is defective in DSB-induced chromatin association. The S495A mutant also fails to enhance repair of an induced DSB when compared with wt Metnase. Interestingly, the S495A mutant demonstrated increased restart of stalled replication forks compared with wt Metnase. Thus, phosphorylation of Metnase S495 differentiates between these two functions, enhancing DSB repair and repressing replication fork restart. In summary, these data lend insight into the mechanism by which Chk1 enhances repair of DNA damage while at the same time repressing stalled replication fork restart. Oncogene (2012) 31, 4245-4254; doi:10.1038/onc.2011.586; published online 9 January 2012

Processing of O 6 - methylguanine in mammalian cells

DNA methylating compounds are widely used as anti-cancer chemotherapeutics. The pharmaceutical critical DNA lesion induced by these drugs is O6-methylguanine (O6MeG). O6MeG is highly mutagenic and genotoxic, by triggering apoptosis. Despite the potency of O6MeG to induce cell death, the mechanism of O6MeG induced toxicity is still poorly understood. Comparing the response of mouse fibroblasts wild-type (wt) and deficient for ataxia telangiectasia mutant protein (ATM), a kinase responsible for both the recognition and the signalling of DNA double-strand breaks (DSBs), it was shown that ATM deficient cells are more sensitive to the methylating agents N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), methyl methansulfonate (MMS) and the anti-cancer drug temozolomide, in both colony formation and apoptosis assays. This clearly shows that DSBs are involved in O6MeG toxicity. By inactivating the O6MeG repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) with the specific inhibitor O6-benzylguanine (O6BG), ATM wt and deficient cells became more sensitive to MNNG and MMS. The opposite effect was observed when over-expressing MGMT in ATM -/- cells. The results show that O6MeG is the critical DNA lesion causing death in ATM cells following MNNG treatment, and is partially responsible for the toxicity observed following MMS treatment. Furthermore, by inhibiting the ATM kinase activity with caffeine, it was shown that the resistance of wt cells to MNNG was due to the kinase activity of ATM, as wt cells underwent more apoptosis following methylating agent treatment in the presence of caffeine. Apoptosis and caspase-3 activation were late events, starting 48h after treatment. This lends support to the model where O6MeG lesions are converted into DSBs during replication. As ATM wt and deficient cells showed similar G2/M blockage and Chk1 activation following MNNG and MMS treatment, it was concluded that the protective effect of ATM is not due to cell cycle progression control. The hypersensitivity of ATM deficient cells was accompanied by their inability to activate the anti-apoptotic NFkB pathway. In a second part of this study, it was shown that the inflammatory cytokine IL-1 up-regulates the DNA repair gene apurinic endonuclease 2 (APEX2). Up-regulation of APEX2 occurred by transcriptional regulation as it was abrogated by actinomycin D. APEX2 mRNA accumulation was accompanied by increase in APEX2 protein level. IL-1 induced APEX2 expression as well as transfection of cells with APEX2 cDNA positively correlated with a decrease in apoptosis after treatment with genotoxic agents, particularly affecting cell death after H2O2. This indicates an involvement of APEX2 in the BER pathway in cells responding to IL-1.