Role of APE1 in differentiated neuroblastoma SH-SY5Y cells in response to oxidative stress: Use of APE1 small molecule inhibitors to delineate APE1 functions

Oxidative DNA damage has been implicated in a number of central nervous system pathologies. The base excision repair (BER) pathway is one of the most important cellular protection mechanisms that respond to oxidative DNA damage. Human apurinic (apyrimidinic) endonuclease/redox effector factor (APE1/Ref-1 or APE1) is an essential enzyme in the BER pathway and is expressed in both mitotic and post-mitotic cells in humans. In neurons, a reduction of APE1 expression increases chemotherapy-induced cytotoxicity, while overexpression of APE1 protects cells against the cytotoxicity. However, given the multiple functions of APE1, knockdown of total APE1 is not completely informative of whether it is the redox or DNA repair activity, or interactions with other proteins. Therefore, the use of selective small molecules that can block each function independent of the other is of great benefit in ascertaining APE1 function in post-mitotic cells. In this study, we chose differentiated SH-SY5Y cells as our post-mitotic cell line model to investigate whether a drug-induced decrease in APE1 DNA repair or redox activity contributes to the growth and survival of post-mitotic cells under oxidative DNA damaging conditions. Here, we demonstrate that overexpression of WT-APE1 or C65-APE1 (repair competent) results in significant increase in cell viability after exposure to H₂O₂. However, the 177/226-APE1 (repair deficient) did not show a protective effect. This phenomenon was further confirmed by the use of methoxyamine (MX), which blocks the repair activity of APE1 that results in enhanced cell killing and apoptosis in differentiated SH-SY5Y cells and in neuronal cultures after oxidative DNA damaging treatments. Blocking APE1 redox function by a small molecule inhibitor, BQP did not decrease viability of SH-SY5Y cells or neuronal cultures following oxidative DNA damaging treatments. Our results demonstrate that the DNA repair function of APE1 contributes to the survival of nondividing post-mitotic cells following oxidative DNA damage.     

Expression of apurinic/apyrimidinic endonuclease-1 (APE-1) in H. pylori-associated gastritis, gastric adenoma, and gastric cancer

Background and Aim: Apurinic/apyrimidinic endonuclease‐1 (APE‐1) is a key enzyme in DNA base excision repair (BER), linked to cancer chemosensitivity. However, little is known about the localization of APE‐1 in Helicobacter pylori‐infected gastric mucosa or its role in the development of gastric cancer. To investigate the role of APE‐1 in the development of gastric cancer, we examined APE‐1 expression and localization in cultured cells and gastric biopsies from patients with H. pylori‐infected gastritis or gastric adenoma, and from surgically resected gastric cancer. Methods: APE‐1 mRNA and protein expression were determined in H. pylori (CagA+) water‐extract protein (HPWEP)‐stimulated MKN‐28 cells, gastric adenocarcinoma cell‐line (AGS) cells, and human peripheral macrophages by real‐time polymerase chain reaction and Western blot analysis. APE‐1 expression and 8‐OHdG as a measure of oxidative DNA damage were evaluated by immunostaining. Localization of APE‐1 and IκBα phosphorylation in gastric adenoma and gastric cancer tissues were evaluated by single‐ and double‐label immunohistochemistry. Results: In studies in vitro, HPWEP‐stimulation significantly increased APE‐1 mRNA expression levels in both MKN‐28 cells and human peripheral macrophages. Hypo/reoxygenation treatment significantly increased APE‐1 protein expression in HPWEP‐stimulated MKN‐28 cells. HPWEP stimulation significantly increased both APE‐1 expression and IκBα phosphorylation levels in MKN‐28 and AGS cells. In human tissues, APE‐1 expression in H. pylori‐infected gastritis without goblet cell metaplasia was significantly increased as compared to that in tissues from uninfected subjects. Eradication therapy significantly reduced both APE‐1 and 8‐OHdG expression levels in the gastric mucosa. APE‐1 expression was mainly localized in epithelial cells within gastric adenoma and in mesenchymal cells of gastric cancer tissues. APE‐1 expression in gastric cancer tissues was significantly reduced compared to that in H. pylori‐infected gastric adenoma, while 8‐OHdG index and IκBα phosphorylation levels did not differ between these two neoplastic tissue types. Co‐localization of APE‐1 and IκBα phosphorylation was observed not in gastric cancer cells but in gastric adenoma cells. Conclusion: H. pylori infection is associated with increased APE‐1 expression in human cell lines and in gastric tissues from subjects with gastritis and gastric adenomas. The observed distinct expression patterns of APE‐1 and 8‐OHdG in gastric adenoma and gastric cancer tissues may provide insight into the progression of these conditions and warrants further investigation.     

Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage

The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein, plays a central role in the repair of oxidative base damage via the DNA base excision repair (BER) pathway. The mammalian AP-endonuclease (APE1) overexpression is often observed in tumor cells, and confers resistance to various anticancer drugs; its downregulation sensitizes tumor cells to those agents via induction of apoptosis. Here we show that wild type (WT) but not mutant p53 negatively regulates APE1 expression. Time-dependent decrease was observed in APE1 mRNA and protein levels in the human colorectal cancer line HCT116 p53^(+/+), but not in the isogenic p53 null mutant after treatment with camptothecin, a DNA topoisomerase I inhibitor. Furthermore, ectopic expression of WTp53 in the p53 null cells significantly reduced both endogenous APE1 and APE1 promoter-dependent luciferase expression in a dose-dependent fashion. Chromatin immunoprecipitation assays revealed that endogenous p53 is bound to the APE1 promoter region that includes a Sp1 site. We show here that WTp53 interferes with Sp1 binding to the APE1 promoter, which provides a mechanism for the downregulation of APE1. Taken together, our results demonstrate that WTp53 is a negative regulator of APE1 expression, so that repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of apoptosis in response to DNA damage.     

V-sis induces Egr-1 expression by a pathway mediated by c-Ha-ras

The early growth response gene, Egr-1, is up-regulated transiently by mitogens and many other simuli in all cells tested. Using NIH3T3 cells conditionally expressing v-sis from a metallothionein promoter, we show that the addition of Zn²⁺ stimulates the production of PDGF-B(v-sis) and elicits the expression of Egr-1 in a dose-dependent and time-regulated manner. The signal is likely independent of protein kinase C, but depends on tyrosine kinase and other kinase activities and is mediated by c-Ha-Ras since the presence of dominant-negative mutants of Ras and Raf abrogates the induction of Egr-1 expression by Zn²⁺. Transiently activated Ras expression in NIH3T3 cells also stimulates the transient expression of Egr-1, but cells that constitutively express Rass do not have elevated levels of Egr-1. Transient assays also demonstrated that Zn²⁺ or activated Ras expression stimulated the activity of a 950 bp Egr-1 promoter-reporter gene construct and this is abrogated in the presence of mutant Ras and Raf. The accumulated data show that Egr-1 gene expression is regulated by multiple mechanisms, as would be needed for putative role in Cell proliferation, in suppression of transformation and in differentiation.     

Egr-1 and serum response factor are involved in growth factors- and serum-mediated induction of E2-EPF UCP expression that regulates the VHL-HIF pathway

E2-EPF ubiquitin carrier protein (UCP) has been shown to be highly expressed in common human cancers and target von Hippel-Lindau (VHL) for proteosomal degradation in cells, thereby stabilizing hypoxia-inducible factor (HIF)-1alpha. Here, we investigated cellular factors that regulate the expression of UCP gene. Promoter deletion assay identified binding sites for early growth response-1 (Egr-1) and serum response factor (SRF) in the UCP promoter. Hepatocyte or epidermal growth factor (EGF), or phorbol 12-myristate 13-acetate induced UCP expression following early induction of Egr-1 expression in HeLa cells. Serum increased mRNA and protein levels of SRF and UCP in the cell. By electrophoretic mobility shift and chromatin immunoprecipitation assays, sequence-specific DNA-binding of Egr-1 and SRF to the UCP promoter was detected in nuclear extracts from HeLa cells treated with EGF and serum, respectively. Overexpression of Egr-1 or SRF increased UCP expression. RNA interference-mediated depletion of endogenous Egr-1 or SRF impaired EGF- or serum-mediated induction of UCP expression, which was required for cancer cell proliferation. Systemic delivery of EGF into mice also increased UCP expression following early induction of Egr-1 expression in mouse liver. The induced UCP expression by the growth factors or serum increased HIF-1alpha protein level under non-hypoxic conditions, suggesting that the Egr-1/SRF-UCP-VHL pathway is in part responsible for the increased HIF-1alpha protein level in vitro and in vivo. Thus, growth factors and serum induce expression of Egr-1 and SRF, respectively, which in turn induces UCP expression that positively regulates cancer cell growth.     

IGF2BP1

The oncofetal RNA-binding protein IGF2BP1 (IGF2 mRNA binding protein 1) controls the cytoplasmic fate of specific target mRNAs including ACTB and CD44. During neural development, IGF2BPs promote neurite protrusion and the migration of neuronal crest cells. In tumor-derived cells, IGF2BP1 enhances the formation of lamellipodia and invadopodia. Accordingly, the de novo synthesis of IGF2BP1 observed in primary malignancies was reported to correlate with increased metastasis and an overall poor prognosis. However, if and how the protein enhances metastasis remains controversial. In recent studies, we reveal that IGF2BP1 promotes the directed migration of tumor-derived cells in vitro by controlling the expression of MAPK4 and PTEN. The IGF2BP1-facilitated inhibition of MAPK4 mRNA translation interferes with MK5-directed phosphorylation of the heat shock protein 27 (HSP27). This limits G-actin sequestering by phosphorylated HSP27, enhances cell adhesion and elevates the velocity of tumor cell migration. Concomitantly, IGF2BP1 promotes the expression of PTEN by interfering with PTEN mRNA turnover. This results in a shift of cellular PtdIns(3,4,5)P₃/PtdIns(4,5)P₂ ratios and enhances RAC1-dependent cell polarization which finally promotes the directionality of tumor cell migration. These findings identify IGF2BP1 as a potent oncogenic factor that regulates the adhesion, migration and invasiveness of tumor cells by modulating intracellular signaling.     

Tumor suppressor Ing1b facilitates DNA repair and prevents oxidative stress induced cell death

Inhibitor of growth (ING) family of proteins are known to coordinate with histone acetyltransferases and regulate the key events of cell cycle and DNA repair. Previous work from our lab showed that Ing1b regulated the nucleotide excision repair by facilitating histone acetylation and subsequent chromatin relaxation. Further, it was also shown that Ing1b protected the cells from genomic instability induced cell death by promoting ubiquitination of proliferating cell nuclear antigen (PCNA). In the present study we explored the role of Ing1b in the repair of oxidized DNA and prevention of oxidative stress induced genotoxic cell death. Using HCT116 cells we show that Ing1b protein expression is induced by treatment with H₂O₂. Ing1b lacking cells showed decreased ability to repair the oxidized DNA. PCNA monoubiquitination, a critical event of DNA repair was blunted in Ing1b knock down cells and augmented in Ing1b over expressing cells. Moreover, oxidative stress induced cell death was higher in cells lacking Ing1b whereas it was lower in Ing1b over expressing cells. Finally we show that inhibition of histone deacetylases, rescued the Ing1b knock down cells from cytotoxic effects of H₂O₂ treatment.     

Further in vivo evidence implying DNA apurinic/apyrimidinic endonuclease activity in Trypanosoma cruzi oxidative stress survival

Trypanosoma cruzi is under the attack of reactive species produced by its mammalian and insect hosts. To survive, it must repair its damaged DNA. We have shown that a base excision DNA repair (BER)-specific parasite TcAP1 endonuclease is involved in the resistance to H₂O₂. However, a putative TcAP1 negative dominant form impairing TcAP1 activity in vitro did not show any in vivo effect. Here, we show that a negative dominant form of the human APE1 apurinic/apyrimidinic (AP) endonuclease (hAPE1DN) induces a decrease in epimastigote and metacyclic trypomastigote viability when parasites were exposed to H₂O₂. Those results confirm that TcAP1 AP endonuclease activity plays an important role in epimastigote and in infective metacyclic trypomastigote oxidative DNA damage resistance leading to parasite persistence in the insect and mammalian hosts. All along its biological cycle and in its different cellular forms, T. cruzi, the etiological parasite agent of Chagas’ disease, is under the attack of reactive species produced by its mammalian and insect hosts. To survive, T. cruzi must repair their oxidative damaged DNA. We have previously shown that a specific parasite TcAP1 AP endonuclease of the BER is involved in the T. cruzi resistance to oxidative DNA damage. We have also demonstrated that epimastigotes and cell-derived trypomastigotes parasite forms expressing a putative TcAP1 negative dominant form (that impairs the TcAP1 activity in vitro), did not show any in vivo effect in parasite viability when exposed to oxidative stress. In this work, we show the expression of a negative dominant form of the human APE1 AP endonuclease fused to a green fluorescent protein (GFP; hAPE1DN-GFP) in T. cruzi epimastigotes. The fusion protein is found both in the nucleus and cytoplasm of noninfective epimastigotes but only in the nucleus in metacyclic and cell-derived trypomastigote infective forms. Contrarily to the TcAP1 negative dominant form, the ectopic expression of hAPE1DN-GFP induces a decrease in epimastigote and metacyclic trypomastigote viability when parasites were exposed to increasing H₂O₂ concentrations. No such effect was evident in expressing hAPE1DN-GFP cell-derived trypomastigotes. Although the viability of both wild-type infective trypomastigote forms diminishes when parasites are submitted to acute oxidative stress, the metacyclic forms are more resistant to H₂O₂ exposure than cell-derived trypomastigotes.Those results confirm that the BER pathway and particularly the AP endonuclease activity play an important role in epimastigote and metacyclic trypomastigote oxidative DNA damage resistance leading to parasite survival and persistence inside the mammalian and insect host cells.     

Egr-1和PTEN在皮肤鳞癌中的表达和临床意义

目的:探讨Egr-1和PTEN在皮肤鳞癌中的表达和临床意义。方法:收集2014年1月到2015年12月100例在我院经病理学诊断为皮肤鳞状细胞癌患者以及100例健康对照的组织样本,采用免疫组化法SP法检测在皮肤鳞癌中Egr-1和PTEN的表达,并分析其与患者肿瘤转移的关系。结果:皮肤鳞状细胞癌组织中Egr-1和PTEN的表达明显高于健康对照组织(P0.05);发生远处转移的皮肤鳞状细胞癌组织中Egr-1水平明显高于未转移者(P0.05)PTEN的表达显著低于未发生远处转移的患者(P0.05)。结论:Egr-1和PTEN在皮肤鳞状细胞癌患者中表达明显增高,并与癌组织远处转移密切相关,二者可能作为皮肤鳞癌诊断和预后评估的参考指标。     

Apurinic/apyrimidinic endonuclease 1, the sensitive marker for DNA deterioration in dextran sulfate sodium-induced acute colitis

Abstract

Mutations in mismatch repair (MMR) genes are commonly associated with the development of colorectal cancer. Additionally, base excision repair, which involves apurinic/apyrimidinic endonuclease 1 (APE1), recognizes and eliminates oxidative DNA damage. Here, we investigated the possible roles of APE1 in dextran sulfate sodium (DSS)-induced acute colitis using the young rat model. Four-week-old Sprague–Dawley rats were administered 2% DSS in drinking water for 1 week. MMR and APE1 expression levels were assessed by western blotting and immunohistochemistry. Following DSS treatment, growth of young rats failed and the animals had loose stools. Together with the histological changes associated with acute colitis, APE1 and MSH2 levels increased significantly at 3 and 5 days after DSS treatment, respectively. The difference between APE1 and MSH2 expression was significant. DSS-induced DNA damage and subsequent repair activity were evaluated by staining for 8-hydroxy-deoxyguanosine (8-OHdG) and APE1, respectively; 8-OHdG immunoreactivity increased throughout the colonic mucosa, while APE1 levels in the surface epithelium increased at an earlier timepoint. Taken together, our data suggest that changes in APE1 expression after DSS treatment occurred earlier and were more widespread than changes in MMR expression, suggesting that APE1 is more sensitive for prediction of DNA deterioration in DSS-induced colitis.     

Regulation of Rac1 and Reactive Oxygen Species Production in Response to Infection of Gastrointestinal Epithelia

Generation of reactive oxygen species (ROS) during infection is an immediate host defense leading to microbial killing. APE1 is a multifunctional protein induced by ROS and after induction, protects against ROS-mediated DNA damage. Rac1 and NAPDH oxidase (Nox1) are important contributors of ROS generation following infection and associated with gastrointestinal epithelial injury. The purpose of this study was to determine if APE1 regulates the function of Rac1 and Nox1 during oxidative stress. Gastric or colonic epithelial cells (wild-type or with suppressed APE1) were infected with Helicobacter pylori or Salmonella enterica and assessed for Rac1 and NADPH oxidase-dependent superoxide production. Rac1 and APE1 interactions were measured by co-immunoprecipitation, confocal microscopy and proximity ligation assay (PLA) in cell lines or in biopsy specimens. Significantly greater levels of ROS were produced by APE1-deficient human gastric and colonic cell lines and primary gastric epithelial cells compared to control cells after infection with either gastric or enteric pathogens. H. pylori activated Rac1 and Nox1 in all cell types, but activation was higher in APE1 suppressed cells. APE1 overexpression decreased H. pylori-induced ROS generation, Rac1 activation, and Nox1 expression. We determined that the effects of APE1 were mediated through its N-terminal lysine residues interacting with Rac1, leading to inhibition of Nox1 expression and ROS generation. APE1 is a negative regulator of oxidative stress in the gastrointestinal epithelium during bacterial infection by modulating Rac1 and Nox1. Our results implicate APE1 in novel molecular interactions that regulate early stress responses elicited by microbial infections.     

Neurons Efficiently Repair Glutamate-induced Oxidative DNA Damage by a Process Involving CREB-mediated Up-regulation of Apurinic Endonuclease 1

Glutamate, the major excitatory neurotransmitter in the brain, activates receptors coupled to membrane depolarization and Ca²⁺ influx that mediates functional responses of neurons including processes such as learning and memory. Here we show that reversible nuclear oxidative DNA damage occurs in cerebral cortical neurons in response to transient glutamate receptor activation using non-toxic physiological levels of glutamate. This DNA damage was prevented by intracellular Ca²⁺ chelation, the mitochondrial superoxide dismutase mimetic MnTMPyP (Mn-5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine chloride tetrakis(methochloride)), and blockade of the permeability transition pore. The repair of glutamate-induced DNA damage was associated with increased DNA repair activity and increased mRNA and protein levels of apurinic endonuclease 1 (APE1). APE1 knockdown induced accumulation of oxidative DNA damage after glutamate treatment, suggesting that APE1 is a key repair protein for glutamate-induced DNA damage. A cAMP-response element-binding protein (CREB) binding sequence is present in the Ape1 gene (encodes APE1 protein) promoter and treatment of neurons with a Ca²⁺/calmodulin-dependent kinase inhibitor (KN-93) blocked the ability of glutamate to induce CREB phosphorylation and APE1 expression. Selective depletion of CREB using RNA interference prevented glutamate-induced up-regulation of APE1. Thus, glutamate receptor stimulation triggers Ca²⁺- and mitochondrial reactive oxygen species-mediated DNA damage that is then rapidly repaired by a mechanism involving Ca²⁺-induced, CREB-mediated APE1 expression. Our findings reveal a previously unknown ability of neurons to efficiently repair oxidative DNA lesions after transient activation of glutamate receptors.