p53 coordinates base excision repair to prevent genomic instability.

DNA constantly undergoes chemical modification due to endogenous and exogenous mutagens. The DNA base excision repair (BER) pathway is the frontline mechanism handling the majority of these lesions, and primarily involves a DNA incision and subsequent resealing step. It is imperative that these processes are extremely well-coordinated as unrepaired DNA single strand breaks (SSBs) can be converted to DNA double strand breaks during replication thus triggering genomic instability. However, the mechanism(s) governing the BER process are poorly understood. Here we show that accumulation of unrepaired SSBs triggers a p53/Sp1-dependent downregulation of APE1, the endonuclease responsible for the DNA incision during BER. Importantly, we demonstrate that impaired p53 function, a characteristic of many cancers, leads to a failure of the BER coordination mechanism, overexpression of APE1, accumulation of DNA strand breaks and results in genomic instability. Our data provide evidence for a previously unrecognized mechanism for coordination of BER by p53, and its dysfunction in p53-inactivated cells.

Lack of aprataxin impairs mitochondrial functions via downregulation of the APE1/NRF1/NRF2 pathway.

Ataxia oculomotor apraxia type 1 (AOA1) is an autosomal recessive disease caused by mutations in APTX, which encodes the DNA strand-break repair protein aprataxin (APTX). CoQ10 deficiency has been identified in fibroblasts and muscle of AOA1 patients carrying the common W279X mutation, and aprataxin has been localized to mitochondria in neuroblastoma cells, where it enhances preservation of mitochondrial function. In this study, we show that aprataxin deficiency impairs mitochondrial function, independent of its role in mitochondrial DNA repair. The bioenergetics defect in AOA1-mutant fibroblasts and APTX-depleted Hela cells is caused by decreased expression of SDHA and genes encoding CoQ biosynthetic enzymes, in association with reductions of APE1, NRF1 and NRF2. The biochemical and molecular abnormalities in APTX-depleted cells are recapitulated by knockdown of APE1 in Hela cells and are rescued by overexpression of NRF1/2. Importantly, pharmacological upregulation of NRF1 alone by 5-aminoimidazone-4-carboxamide ribonucleotide does not rescue the phenotype, which, in contrast, is reversed by the upregulation of NRF2 by rosiglitazone. Accordingly, we propose that the lack of aprataxin causes reduction of the pathway APE1/NRF1/NRF2 and their target genes. Our findings demonstrate a critical role of APTX in transcription regulation of mitochondrial function and the pathogenesis of AOA1 via a novel pathomechanistic pathway, which may be relevant to other neurodegenerative diseases.

An in vitro study ascertaining the role of H2O2 and glucose oxidase in modulation of antioxidant potential and cancer cell survival mechanisms in glioblastoma U-87 MG cells.

Glial cells protect themselves from the elevated reactive oxygen species (ROS) via developing unusual mechanisms to maintain the genomic stability, and reprogramming of the cellular antioxidant system to cope with the adverse effects. In the present study non-cytotoxic dose of oxidants, H2O2 (100 µM) and GO (10 µU/ml) was used to induce moderate oxidative stress via generating ROS in human glioblastoma cell line U-87 MG cells, which showed a marked increase in the antioxidant capacity as studied by measuring the modulation in expression levels and activities of superoxide dismutase (SOD1 and SOD2) and catalase (CAT) enzymes, and the GSH content. However, pretreatment (3 h) of Curcumin and Quercetin (10 µM) followed by the treatment of oxidants enhanced the cell survival, and the levels/activities of the antioxidants studied. Oxidative stress also resulted in an increase in the nitrite levels in the culture supernatants, and further analysis by immunocytochemistry showed an increase in iNOS expression. In addition, phytochemical pretreatment decreased the nitrite level in the culture supernatants of oxidatively stressed U-87 MG cells. Elevated ROS also increased the expression of COX-2 and APE1 enzymes and pretreatment of Curcumin and Quercetin decreased COX-2 expression and increased APE1 expression in the oxidatively stressed U-87 MG cells. The immunocytochemistry also indicates for APE1 enhanced stress-dependent subcellular localization to the nuclear compartment, which advocates for enhanced DNA repair and redox functions of APE1 towards survival of U-87 MG cells. It can be concluded that intracellular oxidants activate the key enzymes involved in antioxidant mechanisms, NO-dependent survival mechanisms, and also in the DNA repair pathways for glial cell survival in oxidative-stress micro-environment.

Differential expression of APE1 and APE2 in germinal centers promotes error-prone repair and A:T mutations during somatic hypermutation.

Somatic hypermutation (SHM) of antibody variable region genes is initiated in germinal center B cells during an immune response by activation-induced cytidine deaminase (AID), which converts cytosines to uracils. During accurate repair in nonmutating cells, uracil is excised by uracil DNA glycosylase (UNG), leaving abasic sites that are incised by AP endonuclease (APE) to create single-strand breaks, and the correct nucleotide is reinserted by DNA polymerase ß. During SHM, for unknown reasons, repair is error prone. There are two APE homologs in mammals and, surprisingly, APE1, in contrast to its high expression in both resting and in vitro-activated splenic B cells, is expressed at very low levels in mouse germinal center B cells where SHM occurs, and APE1 haploinsufficiency has very little effect on SHM. In contrast, the less efficient homolog, APE2, is highly expressed and contributes not only to the frequency of mutations, but also to the generation of mutations at A:T base pair (bp), insertions, and deletions. In the absence of both UNG and APE2, mutations at A:T bp are dramatically reduced. Single-strand breaks generated by APE2 could provide entry points for exonuclease recruited by the mismatch repair proteins Msh2-Msh6, and the known association of APE2 with proliferating cell nuclear antigen could recruit translesion polymerases to create mutations at AID-induced lesions and also at A:T bp. Our data provide new insight into error-prone repair of AID-induced lesions, which we propose is facilitated by down-regulation of APE1 and up-regulation of APE2 expression in germinal center B cells.

Endothelial cell tumor growth is Ape/ref-1 dependent.

Tumor-forming endothelial cells have highly elevated levels of Nox-4 that release H2O2 into the nucleus, which is generally not compatible with cell survival. We sought to identify compensatory mechanisms that enable tumor-forming endothelial cells to survive and proliferate under these conditions. Ape-1/ref-1 (Apex-1) is a multifunctional protein that promotes DNA binding of redox-sensitive transcription factors, such as AP-1, and repairs oxidative DNA damage. A validated mouse endothelial cell (EOMA) tumor model was used to demonstrate that Nox-4-derived H2O2 causes DNA oxidation that induces Apex-1 expression. Apex-1 functions as a chaperone to keep transcription factors in a reduced state. In EOMA cells Apex-1 enables AP-1 binding to the monocyte chemoattractant protein-1 (mcp-1) promoter and expression of that protein is required for endothelial cell tumor formation. Intraperitoneal injection of the small molecule inhibitor E3330, which specifically targets Apex-1 redox-sensitive functions, resulted in a 50% decrease in tumor volume compared with mice injected with vehicle control (n = 6 per group), indicating that endothelial cell tumor proliferation is dependent on Apex-1 expression. These are the first reported results to establish Nox-4 induction of Apex-1 as a mechanism promoting endothelial cell tumor formation.

Polymorphisms in NEIL-2, APE-1, CYP2E1 and MDM2 Genes are Independent Predictors of Gastric Cancer Risk in a Northern Jiangsu Population (China).

China is one of the countries with the highest incidence of gastric cancer, and accounts for over 40% of all new gastric cancer cases in the world. Genetic factors as well as environmental factors play a role in development of gastric cancer. To investigate the independent roles of single nucleotide polymorphisms (SNPs) in base excision repair (BER) genes (APE1 and NEIL2), carcinogen metabolism gene (CYP2E1) and tumor suppressor pathway gene (MDM2) for gastric cancer susceptibility in a Chinese population, we conducted a hospital based case-control study to evaluate the potential association between these polymorphisms and susceptibility to gastric cancer in a Northern Jiangsu population. We also associated the NEIL-2 mRNA expression with the studied NEIL2 SNP genotypes to assess whether the genotypes have influence on the NEIL2 mRNA (hence protein) expression. Five SNPs, APE 1 (rs2275008), NEIL 2 (rs804270), MDM2 (rs2279744), and CYP 2E1 (rs2480256 and rs2031920), were genotyped by TaqMan assays in 105 gastric cancer cases and 118 controls. Genotype frequency distribution showed that the APE 1 SNP (rs2275008), NEIL 2 SNP (rs804270), MDM2 SNP (rs2279744), and CYP 2E1 SNP (rs2031920) had more mutant alleles in gastric cancer cases than controls (76.19, 68.57, 54.29, and 43.81%, respectively), while CYP 2E1 SNP (rs2480256) had large percentage of both alleles (43.81%). Risk analysis revealed that there was increased risk for gastric cancer in subjects with mutant alleles in APE 1 (rs2275008: OR 5.49, 95% CI = 2.6-5.7, p <.0001), NEIL 2 (rs804270: OR 2.3, 95% CI = 1.22-4.3, p=0.01), MDM2 (rs2279744: OR 14.65, 95% CI = 5.63-8.15, p < .0001), and CYP 2E1 (rs2031920: OR 8.385, 95% CI = 3.2-5.3, p < .0001) SNPs. Moreover, the NEIL2 mRNA expression analysis showed that there was significant differential expression of NEIL2 mRNA among the randomly tested NEIL2 genotypes (p = 0.005), with low expression seen in variant genotypes than in other genotypes. In conclusion, variant alleles in the NEIL2 (rs804270), APE1 (rs2275008), CYP2E1 (rs2031920) and MDM2 (rs2279744) SNPs may independently influence susceptibility to gastric cancer in a Northern Jiangsu Chinese population. The genotypes may also independently influence their respective gene mRNA expression, as seen in our study, where there was differential expression of the NEIL2 mRNA among the genotypes, with low NEIL2 mRNA expression seen in the variant genotype.

Mitochondrial 8-oxoguanine glycosylase decreases mitochondrial fragmentation and improves mitochondrial function in H9C2 cells under oxidative stress conditions.

The mitochondrial DNA base modification 8-hydroxy 2'-deoxyguanine (8-OHdG) is one of the most common DNA lesions induced by reactive oxygen species (ROS) and is considered an index of DNA damage. High levels of mitochondrial 8-OHdG have been correlated with increased mutation, deletion, and loss of mitochondrial (mt) DNA, as well as apoptosis. 8-Oxoguanosine DNA glycosylase-1 (OGG1) recognizes and removes 8-OHdG to prevent further DNA damage. We evaluated the effects of OGG1 on mtDNA damage, mitochondrial function, and apoptotic events induced by oxidative stress using H9C2 cardiac cells treated with menadione and transduced with either Adv-Ogg1 or Adv-Control (empty vector). The levels of mtDNA 8-OHdG and the presence of apurinic/apyrimidinic (AP) sites were decreased by 30% and 35%, respectively, in Adv-Ogg1 transduced cells (P < 0.0001 and P < 0.005, respectively). In addition, the expression of base excision repair (BER) pathway members APE1 and DNA polymerase γ was upregulated by Adv-Ogg1 transduction. Cells overexpressing Ogg1 had increased membrane potential (P < 0.05) and decreased mitochondrial fragmentation (P < 0.005). The mtDNA content was found to be higher in cells with increased OGG1 (P < 0.005). The protein levels of fission and apoptotic factors such as DRP1, FIS1, cytoplasmic cytochrome c, activated caspase-3, and activated caspase-9 were lower in Adv-Ogg1 transduced cells. These observations suggest that Ogg1 overexpression may be an important mechanism to protect cardiac cells against oxidative stress damage.

Expression, functionality, and localization of apurinic/apyrimidinic endonucleases in replicative and non-replicative forms of Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas disease. The parasite has to overcome oxidative damage by ROS/RNS all along its life cycle to survive and to establish a chronic infection. We propose that T. cruzi is able to survive, among other mechanisms of detoxification, by repair of its damaged DNA through activation of the DNA base excision repair (BER) pathway. BER is highly conserved in eukaryotes with apurinic/apirimidinic endonucleases (APEs) playing a fundamental role. Previous results showed that T. cruzi exposed to hydrogen peroxide and peroxinitrite significantly decreases its viability when co-incubated with methoxyamine, an AP endonuclease inhibitor. In this work the localization, expression and functionality of two T. cruzi APEs (TcAP1, Homo sapiens APE1 orthologous and TcAP2, orthologous to Homo sapiens APE2 and to Schizosaccaromyces pombe Apn2p) were determined. These enzymes are present and active in the two replicative parasite forms (epimastigotes and amastigotes) as well as in the non-replicative, infective trypomastigotes. TcAP1 and TcAP2 are located in the nucleus of epimastigotes and their expression is constitutive. Epimastigote AP endonucleases as well as recombinant TcAP1 and TcAP2 are inhibited by methoxyamine. Overexpression of TcAP1 increases epimastigotes viability when they are exposed to acute ROS/RNS attack. This protective effect is more evident when parasites are submitted to persistent ROS/RNS exposition, mimicking nature conditions. Our results confirm that the BER pathway is involved in T. cruzi resistance to DNA oxidative damage and points to the participation of DNA AP endonucleases in parasite survival.

Clinicopathological significance of human apurinic/apyrimidinic endonuclease 1 (APE1) expression in oestrogen-receptor-positive breast cancer.

Oestrogen metabolites can induce oxidative DNA base damage and generate potentially mutagenic apurinic sites (AP sites) in the genomic DNA. If unrepaired, mutagenic AP sites could drive breast cancer pathogenesis and aggressive phenotypes. Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA base excision repair (BER) protein and essential for processing AP sites generated either directly by oestrogen metabolites or during BER of oxidative base damage. Our hypothesis is that altered APE1 expression may be associated with aggressive tumour biology and impact upon clinical outcomes in breast cancer. In the current study, we have investigated APE1 protein expression in a large cohort of breast cancers (n = 1285) and correlated to clinicopathological features and survival outcomes. Low APE1 protein expression was associated with high histological grade (p < 0.000001), high mitotic index (p < 0.000001), glandular de-differentiation (p < 0.000001), pleomorphism (p = 0.003), absence of hormonal receptors (ER-/PgR-/AR-) (p < 0.0001) and presence of triple negative phenotype (p = 0.001). Low APE1 protein expression was associated with loss of BRCA1, low XRCC1, low FEN1, low SMUG1 and low pol ß (ps < 0.0001). High MIB1 (p = 0.048), bcl-2 negativity (p < 0.0001) and low TOP2A (p < 0.0001) were likely in low APE1 tumours. In the ER-positive sub-group, specifically, low APE1 remains significantly associated with high histological grade, high mitotic index, glandular de-differentiation (ps < 0.00001) and poor breast cancer specific survival (p = 0.007). In the ER-positive cohort that received adjuvant endocrine therapy, low APE1 protein expression is associated with poor survival (p = 0.006). In multivariate analysis, low APE1 remains independently associated with poor survival in ER-positive tumours (p = 0.048). We conclude that low APE1 expression may have prognostic and predictive significance in ER-positive breast cancers.

Protein arginine methyltransferase 7 regulates cellular response to DNA damage by methylating promoter histones H2A and H4 of the polymerase δ catalytic subunit gene, POLD1.

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage.

Induction of base excision repair enzymes NTH1 and APE1 in rat spleen following aniline exposure.

Mechanisms by which aniline exposure elicits splenotoxicity, especially a tumorigenic response, are not well-understood. Earlier, we have shown that aniline exposure leads to oxidative DNA damage and up-regulation of OGG1 and NEIL1/2 DNA glycosylases in rat spleen. However, the contribution of endonuclease III homolog 1 (NTH1) and apurinic/apyrimidinic endonuclease 1 (APE1) in the repair of aniline-induced oxidative DNA damage in the spleen is not known. This study was, therefore, focused on examining whether NTH1 and APE1 contribute to the repair of oxidative DNA lesions in the spleen, in an experimental condition preceding tumorigenesis. To achieve this, male SD rats were subchronically exposed to aniline (0.5 mmol/kg/day via drinking water for 30 days), while controls received drinking water only. By quantitating the cleavage products, the activities of NTH1 and APE1 were assayed using substrates containing thymine glycol (Tg) and tetrahydrofuran, respectively. Aniline treatment led to significant increases in NTH1- and APE1-mediated BER activity in the nuclear extracts of spleen of aniline-treated rats compared to the controls. NTH1 and APE1 mRNA expression in the spleen showed 2.9- and 3.2-fold increases, respectively, in aniline-treated rats compared to the controls. Likewise, Western blot analysis showed that protein expression of NTH1 and APE1 in the nuclear extracts of spleen from aniline-treated rats was 1.9- and 2.7-fold higher than the controls, respectively. Immunohistochemistry indicated that aniline treatment also led to stronger immunoreactivity for both NTH1 and APE1 in the spleens, confined to the red pulp areas. These results, thus, show that aniline exposure is associated with induction of NTH1 and APE1 in the spleen. The increased repair activity of NTH1 and APE1 could be an important mechanism for the removal of oxidative DNA lesions. These findings thus identify a novel mechanism through which NTH1 and APE1 may regulate the repair of oxidative DNA damage in aniline-induced splenic toxicity.

Inhibiting the redox function of APE1 suppresses cervical cancer metastasis via disengagement of ZEB1 from E-cadherin in EMT.

BACKGROUND: Metastasis is a major challenge in cervical cancer treatment. Previous studies have shown that the dual functional protein apurinic/apyrimidinic endonuclease 1 (APE1) promotes tumor metastasis and is overexpressed in cervical cancer. However, the biological role and mechanism of APE1 in cervical cancer metastasis have rarely been studied. METHODS: We used gene set enrichment analysis (GSEA) to determine the APE1-related signaling pathways in cervical cancer. To investigate the role and mechanism of APE1 in cervical cancer metastasis and invasion, immunohistochemistry, immunofluorescence, western blotting, secondary structure prediction, coimmunoprecipitation, luciferase reporter, and electrophoretic mobility shift assays were performed. The inhibitory effects of the APE1 redox function inhibitor APX3330 on cervical cancer metastasis were evaluated using animal models. RESULTS: Clinical data showed that high expression of APE1 was associated with lymph node metastasis in cervical cancer patients. GSEA results showed that APE1 was associated with epithelial to mesenchymal transition (EMT) in cervical cancer. Ectopic expression of APE1 promoted EMT and invasion of cervical cancer cells, whereas inhibition of APE1 suppressed EMT and invasion of cervical cancer cells in a redox function-dependent manner. Notably, APE1 redox function inhibitor APX3330 treatment dramatically suppressed cervical cancer cell lymph node and distant metastasis in vivo. Furthermore, we found that APE1 enhanced the interaction between ZEB1 and the E-cadherin promoter by binding to ZEB1, thereby suppressing the expression of E-cadherin, a negative regulator of EMT. CONCLUSION: Our findings help to elucidate the role played by APE1 in cervical cancer metastasis and targeting APE1 redox function may be a novel strategy for inhibiting cervical cancer metastasis.

Downregulation of APE1/Ref-1 is involved in the senescence of mesenchymal stem cells.

The senescence of human mesenchymal stem cells (hMSCs) causes disruption of tissue and organ maintenance, and is thus an obstacle to stem cell-based therapies for disease. Although some researchers have studied changes in the characteristics of hMSCs (decreases in differentiation ability and self-renewal), comparing young and old ages, the mechanisms of stem cell senescence have not yet been defined. In this study, we developed a growth curve for human bone marrow derived MSCs (hBMSCs) which changes into a hyperbolic state after passage number 7. Senescence associated beta-galactosidase (SA beta-gal) staining of hBMSCs showed 10% in passage 9 and 45% in passage 11. We detected an increase in endogenous superoxide levels during senescence that correlated with senescence markers (SA beta-gal, hyperbolic growth curve). Interestingly, even though endogenous superoxide increased in a replicative senescence model, the expression of APE1/Ref-1, which is sensitive to intracellular redox state, decreased. These effects were confirmed in a stress-induced senescence model by exogenous treatment with H(2)O(2). This change is related to the p53 activity that negatively regulates APE1/Ref-1. p21 expression levels, which represent p53 activity, were transiently increased in passage 9, meaning that they correlated with the expression of APE1/Ref-1. Overexpression of APE1/Ref-1 suppressed superoxide production and decreased SA beta-gal in hBMSCs. In conclusion, intracellular superoxide accumulation appears to be the main cause of the senescence of hBMSCs, and overexpression of APE1/Ref-1 can rescue cells from the senescence phenotype. Maintaining characteristics of hBMSCs by regulating intracellular reactive oxygen species production can contribute to tissue regeneration and to improved cell therapy.

Genomic alterations and abnormal expression of APE2 in multiple cancers.

Although APE2 plays essential roles in base excision repair and ATR-Chk1 DNA damage response (DDR) pathways, it remains unknown how the APE2 gene is altered in the human genome and whether APE2 is differentially expressed in cancer patients. Here, we report multiple-cancer analyses of APE2 genomic alterations and mRNA expression from cancer patients using available data from The Cancer Genome Atlas (TCGA). We observe that APE2 genomic alterations occur at ~17% frequency in 14 cancer types (n = 21,769). Most frequent somatic mutations of APE2 appear in uterus (2.89%) and skin (2.47%) tumor samples. Furthermore, APE2 expression is upregulated in tumor tissue compared with matched non-malignant tissue across 5 cancer types including kidney, breast, lung, liver, and uterine cancers, but not in prostate cancer. We also examine the mRNA expression of 13 other DNA repair and DDR genes from matched samples for 6 cancer types. We show that APE2 mRNA expression is positively correlated with PCNA, APE1, XRCC1, PARP1, Chk1, and Chk2 across these 6 tumor tissue types; however, groupings of other DNA repair and DDR genes are correlated with APE2 with different patterns in different cancer types. Taken together, this study demonstrates alterations and abnormal expression of APE2 from multiple cancers.

Intracellular signaling pathways regulating radioresistance of human prostate carcinoma cells.

Radiation therapy plays an important role in the management of prostate carcinoma. However, the problem of radioresistance and molecular mechanisms by which prostate carcinoma cells overcome cytotoxic effects of radiation therapy remains to be elucidated. In order to investigate possible intracellular mechanisms underlying the prostate carcinoma recurrences after radiotherapy, we have established three radiation-resistant prostate cancer cell lines, LNCaP-IRR, PC3-IRR, and Du145-IRR derived from the parental LNCaP, PC3, and Du145 prostate cancer cells by repetitive exposure to ionizing radiation. LNCaP-IRR, PC3-IRR, and Du145-IRR cells (prostate carcinoma cells recurred after radiation exposure (IRR cells)) showed higher radioresistance and cell motility than parental cell lines. IRR cells exhibited higher levels of androgen and epidermal growth factor (EGF) receptors and activation of their downstream pathways, such as Ras-mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K)-Akt and Jak-STAT. In order to define additional mechanisms involved in the radioresistance development, we determined differences in the proteome profile of parental and IRR cells using 2-D DIGE followed by computational image analysis and MS. Twenty-seven proteins were found to be modulated in all three radioresistant cell lines compared to parental cells. Identified proteins revealed capacity to interact with EGF and androgen receptors related signal transduction pathways and were involved in the regulation of intracellular routs providing cell survival, increased motility, mutagenesis, and DNA repair. Our data suggest that radioresistance development is accompanied by multiple mechanisms, including activation of cell receptors and related downstream signal transduction pathways. Identified proteins regulated in the radioresistant prostate carcinoma cells can significantly intensify activation of intracellular signaling that govern cell survival, growth, proliferation, invasion, motility, and DNA repair. In addition, such analyses may be utilized in predicting cellular response to radiotherapy.

Alteration of APE1/ref-1 expression in non-small cell lung cancer: the implications of impaired extracellular superoxide dismutase and catalase antioxidant systems.

PURPOSE: Apurinic/apyrimidinic endonuclease1/ref-1(APE1/ref-1) is a key enzyme in the base excision repair and in transcriptional modulation against oxidative stress. We investigated the altered expression of APE1/ref-1 and antioxidant systems in lung cancer. PATIENTS AND METHODS: Tumor specimens from 48 patients with operable non-small cell lung cancer were obtained from 2004 to 2006. Immunohistochemistry, Western blot, lipid peroxidation and superoxide production were performed on the tumor samples and a cultured H460 cell line. RESULTS: APE1/ref-1 was mainly localized to the nucleus in the non-tumor regions of the lung cancer tissue specimens. However, nuclear and cytoplasmic expressions of APE1/ref-1 in the lung cancers were markedly up-regulated in the non-small cell lung cancer (NSCLC) specimens including squamous cell and adenocarcinoma specimens. Extracellular superoxide dismutase (ECSOD) and catalase were down-regulated and manganese superoxide dismutase (MnSOD) was up-regulated in the tumor regions of the NSCLC. Tumor regions of the NSCLC showed higher superoxide production and lipid peroxidation levels than non-tumor regions. In the lung adenocarcinoma cell line, H460, treatment with hydrogen peroxide in the presence of a catalase inhibitor, aminotriazole, increased APE1/ref-1 expression, suggesting oxidative stress might have contributed to the induction of APE1/ref-1. CONCLUSION: The results of this study suggest that APE1/ref-1 is up-regulated in the tumor regions of NSCLC. Altered expression of antioxidant systems lead to enhanced production of superoxide production and lipid peroxidation, which can induce APE1/ref-1 in the tumor regions of NSCLS.

Celecoxib inhibits apurinic/apyrimidinic endonuclease-1 expression and prevents gastric cancer in Helicobacter pylori-infected mongolian gerbils.

BACKGROUND AND AIMS: The aim of this study was to see whether administration of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, could prevent the development of gastric cancer via inhibition of apurinic/apyrimidinic endonuclease-1 (APE-1) expression induced by Helicobacter pylori infection. METHODS: 70 Mongolian gerbils were divided into 6 groups. Group 1 gerbils served as controls (n = 6). Ten gerbils were given N-methyl-N-nitrosourea (MNU), 30 ppm, 5 times biweekly (group 2). Short-term H. pylori infection was induced in 6 gerbils which were sacrificed 8 weeks after H. pylori infection (group 3). Long-term H. pylori infection was induced in 6 other gerbils which were sacrificed 44 weeks after H. pylori infection (group 4). Twenty gerbils were given MNU pretreatment 5 times biweekly and long-term H. pylori infection (group 5). In addition, after H. pylori inoculation, group 6 gerbils also received celecoxib with their diet for 26 weeks. APE-1 expression alone or with COX-2 in gastric tissues was evaluated by Western blot and immunohistological analysis. Myeloperoxidase (MPO) activity and thiobarbituric-acid-reactive substance (TBARS) levels were also evaluated. RESULTS: APE-1 was localized in gastric epithelial cells and mesenchymal cells including macrophages in H. pylori-infected gerbils. The numbers of APE-1-positive cells in group 4 and 5 were significantly increased compared to those of group 3. Celecoxib treatment significantly reduced MPO activity, TBARS levels and the incidence of gastric cancer. APE-1 and IkappaBalpha phosphorylation levels were significantly increased in MNU-pretreated H. pylori-infected gerbils compared to those in MNU-only gerbils. Celecoxib significantly reduced APE-1 and IkappaBalpha phosphorylation levels in MNU-pretreated H. pylori-infected gerbils. COX-2 and APE-1 were coexpressed in the macrophages of H. pylori-infected gerbils. CONCLUSION: Celecoxib prevented gastric cancer in MNU-pretreated H. pylori-infected gerbils with a reduction in APE-1 expression thereby suggesting the implication of APE-1 in gastric carcinogenesis in this model.

APE1/Ref-1 promotes the effect of angiotensin II on Ca2+ -activated K+ channel in human endothelial cells via suppression of NADPH oxidase.

The effects of angiotensin II (Ang II) on whole-cell large conductance Ca(2+)-activated K(+) (BK(Ca)) currents was investigated in control and Apurinic/apyrimidinic endonuclease1/redox factor 1 (APE1/Ref-1)-overexpressing human umbilical vein endothelial cells (HUVECs). Ang II blocked the BK(Ca) current in a dose-dependent fashion, and this inhibition was greater in APE1/Ref-1-overexpressing HUVECs than in control HUVECs (half-inhibition values of 102.81+/-9.54 nM and 11.34+/-0.39 nM in control and APE1/Ref-1-overexpressing HUVECs, respectively). Pretreatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) or knock down of NADPH oxidase (p22 phox) using siRNA increased the inhibitory effect of Ang II on the BK(Ca) currents, similar to the effect of APE1/Ref-1 overexpression. In addition, application of Ang II increased the superoxide and hydrogen peroxide levels in the control HUVECs but not in APE1/Ref-1-overexpressing HUVECs. Furthermore, direct application of hydrogen peroxide increased BK(Ca) channel activity. Finally, the inhibitory effect of Ang II on the BK(Ca) current was blocked by an antagonist of the Ang II type 1 (AT(1)) receptor in both control and APE1/Ref-1-overexpressing HUVECs. From these results, we conclude that the inhibitory effect of Ang II on BK(Ca) channel function is NADPH oxidase-dependent and may be promoted by APE1/Ref-1.

Lead facilitates foci formation in a Balb/c-3T3 two-step cell transformation model: role of Ape1 function.

Several possible mechanisms have been examined to gain an understanding on the carcinogenic properties of lead, which include among others, mitogenesis, alteration of gene expression, oxidative damage, and inhibition of DNA repair. The aim of the present study was to explore if low concentrations of lead, relevant for human exposure, interfere with Ape1 function, a base excision repair enzyme, and its role in cell transformation in Balb/c-3T3. Lead acetate 5 and 30 µM induced APE1 mRNA and upregulation of protein expression. This increase in mRNA expression is consistent throughout the chronic exposure. Additionally, we also found an impaired function of Ape1 through molecular beacon-based assay. To evaluate the impact of lead on foci formation, a Balb/c-3T3 two-step transformation model was used. Balb/c-3T3 cells were pretreated 1 week with low concentrations of lead before induction of transformation with n-methyl-n-nitrosoguanidine (MNNG) (0.5 µg/mL) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (0.1 µg/mL) (a classical two-step protocol). Morphological cell transformation increased in response to lead pretreatment that was paralleled with an increase in Ape1 mRNA and protein overexpression and an impairment of Ape1 activity and correlating with foci number. In addition, we found that lead pretreatment and MNNG (transformation initiator) increased DNA damage, determined by comet assay. Our data suggest that low lead concentrations (5, 30 µM) could play a facilitating role in cellular transformation, probably through the impaired function of housekeeping genes such as Ape1, leading to DNA damage accumulation and chromosomal instability, one of the most important hallmarks of cancer induced by chronic exposures.

APE1/Ref-1 redox function contributes to inflammatory pain sensitization.

Inflammatory pain is a complex and multifactorial disorder. Apurinic/apyrimidinic endonuclease 1 (APE1), also called Redox Factor-1 (Ref-1), is constitutively expressed in the central nervous system and regulates various cellular functions including oxidative stress. In the present study, we investigated APE1 modulation and associated pain behavior changes in the complete Freund's adjuvant (CFA) model of inflammatory pain in rats. In addition we tested the anti-inflammatory effects of E3330, a selective inhibitor of APE1-redox activity, in CFA pain condition. We demonstrate that APE1 expression and subcellular distribution are significantly altered in rats at 4 days post CFA injection. We observed around 30% reduction in the overall APE1 mRNA and protein levels. Interestingly, our data point to an increased nuclear accumulation in the inflamed group as compared to the sham group. E3330 inhibitor injection in CFA rats normalized APE1 mRNA expression and changed its distribution toward cytosolic accumulation. Furthermore, intrathecal injection of E3330 decreased inflammation (i.e. reduced IL-6 expression) and alleviated pain, as assessed by measuring the paw withdrawal threshold with the von Frey test. In conclusion, our data indicate that changes in APE1 expression and sub-cellular distribution are implicated in inflammatory pain mechanisms mediated by APE1 redox functions. Further studies are required to elucidate the exact function of APE1 in inflammatory pain processes.