APE1 Protein Research Articles

Distinct regulation of ATM signaling by DNA single-strand breaks and APE1

In response to DNA double-strand breaks or oxidative stress, ATM-dependent DNA damage response (DDR) is activated to maintain genome integrity. However, it remains elusive whether and how DNA single-strand breaks (SSBs) activate ATM. Here, we provide direct evidence in Xenopus egg extracts that ATM-mediated DDR is activated by a defined SSB structure. Our mechanistic studies reveal that APE1 promotes the SSB-induced ATM DDR through APE1 exonuclease activity and ATM recruitment to SSB sites. APE1 protein can form oligomers to activate the ATM DDR in Xenopus egg extracts in the absence of DNA and can directly stimulate ATM kinase activity in vitro. Our findings reveal distinct mechanisms of the ATM-dependent DDR activation by SSBs in eukaryotic systems and identify APE1 as a direct activator of ATM kinase.

ABCA1 deficiency contributes to podocyte pyroptosis priming via the APE1/IRF1 axis in diabetic kidney disease

Decreased ATP Binding Cassette Transporter A1 (ABCA1) expression and caspase-4-mediated noncanonical inflammasome contribution have been described in podocytes in diabetic kidney disease (DKD). To investigate a link between these pathways, we evaluated pyroptosis-related mediators in human podocytes with stable knockdown of ABCA1 (siABCA1) and found that mRNA levels of IRF1, caspase-4, GSDMD, caspase-1 and IL1β were significantly increased in siABCA1 compared to control podocytes and that protein levels of caspase-4, GSDMD and IL1β were equally increased. IRF1 knockdown in siABCA1 podocytes prevented increases in caspase-4, GSDMD and IL1β. Whereas TLR4 inhibition did not decrease mRNA levels of IRF1 and caspase-4, APE1 protein expression increased in siABCA1 podocytes and an APE1 redox inhibitor abrogated siABCA1-induced expression of IRF1 and caspase-4. RELA knockdown also offset the pyroptosis priming, but ChIP did not demonstrate increased binding of NFκB to IRF1 promoter in siABCA1 podocytes. Finally, the APE1/IRF1/Casp1 axis was investigated in vivo. APE1 IF staining and mRNA levels of IRF1 and caspase 11 were increased in glomeruli of BTBR ob/ob compared to wildtype. In conclusion, ABCA1 deficiency in podocytes caused APE1 accumulation, which reduces transcription factors to increase the expression of IRF1 and IRF1 target inflammasome-related genes, leading to pyroptosispriming.

APE2 Promotes AID-Dependent Somatic Hypermutation in Primary B Cell Cultures That Is Suppressed by APE1.

Somatic hypermutation (SHM) is necessary for Ab diversification and involves error-prone DNA repair of activation-induced cytidine deaminase-induced lesions in germinal center (GC) B cells but can also cause genomic instability. GC B cells express low levels of the DNA repair protein apurinic/apyrimidinic (AP) endonuclease (APE)1 and high levels of its homolog APE2. Reduced SHM in APE2-deficient mice suggests that APE2 promotes SHM, but these GC B cells also exhibit reduced proliferation that could impact mutation frequency. In this study, we test the hypothesis that APE2 promotes and APE1 suppresses SHM. We show how APE1/APE2 expression changes in primary murine spleen B cells during activation, impacting both SHM and class-switch recombination (CSR). High levels of both APE1 and APE2 early after activation promote CSR. However, after 2 d, APE1 levels decrease steadily with each cell division, even with repeated stimulation, whereas APE2 levels increase with each stimulation. When GC-level APE1/APE2 expression was engineered by reducing APE1 genetically (apex1+/-) and overexpressing APE2, bona fide activation-induced cytidine deaminase-dependent VDJH4 intron SHM became detectable in primary B cell cultures. The C terminus of APE2 that interacts with proliferating cell nuclear Ag promotes SHM and CSR, although its ATR-Chk1-interacting Zf-GRF domain is not required. However, APE2 does not increase mutations unless APE1 is reduced. Although APE1 promotes CSR, it suppresses SHM, suggesting that downregulation of APE1 in the GC is required for SHM. Genome-wide expression data compare GC and cultured B cells and new models depict how APE1 and APE2 expression and protein interactions change during B cell activation and affect the balance between accurate and error-prone repair during CSR and SHM.

APE1 assembles biomolecular condensates to promote the ATR-Chk1 DNA damage response in nucleolus.

Multifunctional protein APE1/APEX1/HAP1/Ref-1 (designated as APE1) plays important roles in nuclease-mediated DNA repair and redox regulation in transcription. However, it is unclear how APE1 regulates the DNA damage response (DDR) pathways. Here we show that siRNA-mediated APE1-knockdown or APE1 inhibitor treatment attenuates the ATR–Chk1 DDR under stress conditions in multiple immortalized cell lines. Congruently, APE1 overexpression (APE1-OE) activates the ATR DDR under unperturbed conditions, which is independent of APE1 nuclease and redox functions. Structural and functional analysis reveals a direct requirement of the extreme N-terminal motif within APE1 in the assembly of distinct biomolecular condensates in vitro and DNA/RNA-independent activation of the ATR DDR. Overexpressed APE1 co-localizes with nucleolar NPM1 and assembles biomolecular condensates in nucleoli in cancer but not non-malignant cells, which recruits ATR and activator molecules TopBP1 and ETAA1. APE1 protein can directly activate ATR to phosphorylate its substrate Chk1 in in vitro kinase assays. W119R mutant of APE1 is deficient in nucleolar condensation, and is incapable of activating nucleolar ATR DDR in cells and ATR kinase in vitro. APE1-OE-induced nucleolar ATR DDR activation leads to compromised ribosomal RNA transcription and reduced cell viability. Taken together, we propose distinct mechanisms by which APE1 regulates ATR DDR pathways.

EVOLUTIONARY HISTORY OF THE APEX1 GENE: FROM VERTEBRATES TO SICKLE CELL ANEMIA

In sickle cell anemia, it is known that the APEX1 gene, which encodes the APE1 protein, plays several roles, including DNA strand repair and redox functions, and it is highly expressed during the inflammatory process. The present study sought to investigate how conserved the APEX1 gene is in vertebrates. For this, the coding sequences of the APEX1 gene were obtained by searching genomic databases at the NCBI, with 14 sequences ( Pan troglodytes, Macaca mulatta, Homo sapiens, Bos taurus, Cervos elaphaus, Gallus gallus, Lagopus muta, Petromyson marinus, Felis catus, Rattus norvengicus, Alosa alosa, Dermochelys coriacea, Caretta caretta, Trachemys scriptaelegans) being chosen, using up to 300 amino acids as a selection criterion. After the sequences of the species were aligned using MAFFT v7.407 software, the quality of the sequences was verified and the aligned regions without divergence information were removed using Gblocks. Following this, phylogenetic trees were constructed using the MEGA-X software using the Maximum Likelihood method with 1,000 bootstraps. The generated phylogenies were edited using the FigTree version 1.44 program. A similarity in gene conservation has been observed in Primate species ( Pan troglodytes, Macaca mulatta , and Homo sapiens ). There was a grouping in the same clade of the species, Bos taurus and Cervus elaphus ; the same happened with the order Galliforme ( Gallus gallus and Lagopus muta ) and among the studied reptile species. The vertebrate that presented the least evolutionarily conserved gene was the marine lamprey ( Petromyson marinus) . According to the findings, APEX1 is linked to the oxidative stress pathway in diseases such as sickle cell anemia, preventing further oxidative damage and regulating transcription factors involved in the redox process. Interesting to note is the existence of sicklings of erythrocytes in deer; however, the genetic basis of the phenomenon is still unknown. It is, however, evident that sickling and the APEX1 gene may be related in an adaptive manner. There was a genetic distance of seventy for the APEX1 gene in Felis catus , which suggests that the presence of sickle erythrocytes is not related to the type of hemoglobin since feline hemoglobin contains six additional sulfhydryl groups compared to human hemoglobin. As for reptiles, grouped into a single clade, it is known that they may have sickled erythrocytes, however, unlike Primates, these are nucleated. Unlike the other groups, the capillary diameter in reptiles is greater, which in turn could favor a decrease in vaso-occlusive processes. The inflammatory process in vertebrates is accompanied by a series of cellular and molecular mechanisms that can trigger different costs, which are optimized by the forces of natural selection. The sickling of erythrocytes, which can culminate in inflammatory events, is common in some groups of the animal kingdom. It increases the expression of the APEX1 gene, which has been shown to be evolutionarily more conserved in animals with similar erythrocyte sickling mechanisms, suggesting that it has evolved convergently to protect against oxidative stress.

Knockout and Inhibition of Ape1: Roles of Ape1 in Base Excision DNA Repair and Modulation of Gene Expression.

Apurinic/apyrimidinic endonuclease 1/redox effector-1 (Ape1/Ref-1) is the major apurinic/apyrimidinic (AP) endonuclease in mammalian cells. It functions mainly in the base excision repair pathway to create a suitable substrate for DNA polymerases. Human Ape1 protein can activate some transcription factors to varying degrees, dependent on its N-terminal, unstructured domain, and some of the cysteines within it, apparently via a redox mechanism in some cases. Many cancer studies also suggest that Ape1 has potential for prognosis in terms of the protein level or intracellular localization. While homozygous disruption of the Ape1 structural gene APEX1 in mice causes embryonic lethality, and most studies in cell culture indicate that the expression of Ape1 is essential, some recent studies reported the isolation of viable APEX1 knockout cells with only mild phenotypes. It has not been established by what mechanism the Ape1-null cell lines cope with the endogenous DNA damage that the enzyme normally handles. We review the enzymatic and other activities of Ape1 and the recent studies of the properties of the APEX1 knockout lines. The APEX1 deletions in CH12F3 and HEK293 FT provide an opportunity to test for possible off-target effects of Ape1 inhibition. For this work, we tested the Ape1 endonuclease inhibitor Compound 3 and the redox inhibitor APX2009. Our results confirmed that both APEX1 knockout cell lines are modestly more sensitive to killing by an alkylating agent than their Ape1-proficient cells. Surprisingly, the knockout lines showed equal sensitivity to direct killing by either inhibitor, despite the lack of the target protein. Moreover, the CH12F3 APEX1 knockout was even more sensitive to Compound 3 than its APEX1+ counterpart. Thus, it appears that both Compound 3 and APX2009 have off-target effects. In cases where this issue may be important, it is advisable that more specific endpoints than cell survival be tested for establishing mechanism.

Abstract 2366: Inhibition of Ref-1/APE1 redox activity with APX3330 enhances Ref-1/APE1 protein unfolded conformation in human PDAC cells

Abstract Ref-1/APE1 (Redox factor-1, Ref-1; Apurinic/apyrimidinic endonuclease 1, APE1) is a redox signaling enzyme that biochemically converts several transcription factors (TFs), such as NF-kB, STAT3, HIF-1α from an oxidized to a reduced state, allowing TFs to bind DNA and activate various biological responses such as cell growth and inflammation. Ref-1 and its transcriptional targets are highly active in Pancreatic Ductal Adenocarcinoma (PDAC) which often presents with tumor metastasis and therapy resistance. The redox activity of Ref-1 is determined by the redox status of the relevant cysteine residues (Cys), particularly Cys residue 65 (C65), which is critical among seven Cys residues for Ref-1’s redox activity. Using CRISPR editing in PDAC cells, we changed the C65 to Ala (C65A) in Ref-1 to evaluate the effects of loss of Ref-1 redox activity on cell signaling pathways and how Ref-1 activity regulates critical transcription factors. Using Ref-1 redox inhibitor, APX3330, we previously investigated interactions between Ref-1 and APX3330 in a cell-free system in which Ref-1 underwent conformational changes in the presence of APX3330. This resulted in complete exposure of all seven Cys residues causing oxidation of the Ref-1 protein and redox signaling inactivity. However, how these events translate in the cell has not been completely established. Using a gel-based mobility shift assay, we examined the redox-modified Cys residues of Ref-1 in response to APX3330 in PDAC cell lines over time. We observed a gradual increase in labeling of Cys in Ref-1 following APX3330 treatment between 10min to 6h which returned to control levels at 48h in both wild-type control (Cas9) and C65A-expressing cells. This reduction in the redox status of Ref-1 was likely due to unfolding alteration like the cell-free system. To confirm this, both cell lines were treated with N-ethylmaleimide (NEM) prior to cell lysing, which blocks free thiols in the protein. We detected dramatic decreases in reduced forms of Ref-1 after APX3330 treatment (between 10min-6h) in both cell lines, demonstrating that C65 is not essential for the unfolded conformational changes of Ref-1. However, C65A-Ref-1 protein demonstrated a distinctly different redox pattern upon APX3330 treatment in comparison to WT Ref-1 in the Cas9 control line. C65A-expressing cells also had reduced mitochondrial function compared to Cas9 control PDAC cells. In vivo studies demonstrated significant reduction in tumor size, weight, and growth in C65A PDAC cells compared to the Cas9 control lines, further demonstrating the critical role of C65 in the Ref-1 redox signaling and downstream pathways including metabolism and proliferation. In conclusion, we demonstrate direct interactions between Ref-1 and APX3330 in PDAC cells and subsequent biological results confirm a critical involvement of Ref-1-C65 in the redox signaling and tumor phenotype. Citation Format: Mahmut Mijit, Olivia Babb, Silpa Gampala, Randall Wireman, Millie M. Georgiadis, Melissa L. Fishel, Mark R. Kelley. Inhibition of Ref-1/APE1 redox activity with APX3330 enhances Ref-1/APE1 protein unfolded conformation in human PDAC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2366.

DNA Repair Protein APE1 Degrades Dysfunctional Abasic mRNA in Mitochondria Affecting Oxidative Phosphorylation

APE1 is a multifunctional protein which plays a central role in the maintenance of nuclear and mitochondrial genomes repairing DNA lesions caused by oxidative and alkylating agents. In addition, it works as a redox signaling protein regulating gene expression by interacting with many transcriptional factors. Apart from these canonical activities, recent studies have shown that APE1 is also enzymatically active on RNA molecules. The present study unveils for the first time a new role of the mitochondrial form of APE1 protein in the metabolism of RNA in mitochondria. Our data demonstrate that APE1 is associated with mitochondrial messenger RNA and exerts endoribonuclease activity on abasic sites. Loss of APE1 results in the accumulation of damaged mitochondrial mRNA species, determining impairment in protein translation and reduced expression of mitochondrial-encoded proteins, finally leading to less efficient mitochondrial respiration. Altogether, our data demonstrate that APE1 plays an active role in the degradation of the mitochondrial mRNA and has a profound impact on mitochondrial well-being.

Molecular Mechanisms Regulating the DNA Repair Protein APE1: A Focus on Its Flexible N-Terminal Tail Domain.

APE1 (DNA (apurinic/apyrimidinic site) endonuclease 1) is a key enzyme of one of the major DNA repair routes, the BER (base excision repair) pathway. APE1 fulfils additional functions, acting as a redox regulator of transcription factors and taking part in RNA metabolism. The mechanisms regulating APE1 are still being deciphered. Structurally, human APE1 consists of a well-characterized globular catalytic domain responsible for its endonuclease activity, preceded by a conformationally flexible N-terminal extension, acquired along evolution. This N-terminal tail appears to play a prominent role in the modulation of APE1 and probably in BER coordination. Thus, it is primarily involved in mediating APE1 localization, post-translational modifications, and protein–protein interactions, with all three factors jointly contributing to regulate the enzyme. In this review, recent insights on the regulatory role of the N-terminal region in several aspects of APE1 function are covered. In particular, interaction of this region with nucleophosmin (NPM1) might modulate certain APE1 activities, representing a paradigmatic example of the interconnection between various regulatory factors.

Glyceraldehyde‐3‐phosphate dehydrogenase protects smooth muscle cells against oxidative/genotoxic stress by activation of the apurinic/apyrimidinic endonuclease I pathway

The role of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a potential regulator of cell death was initially interrogated by our laboratory as it pertains to the protection and stimulation of DNA repair that comes from the direct binding of nuclear GAPDH to apurinic/apyrimidinic endonuclease 1 (Ape1). To further elucidate this mechanism, we set to perturbate the GAPDH system in vascular smooth muscle cells (SMC) using a lentiviral GAPDH-overexpressing vector and cells were treated with etoposide (10-240uM), a pro-oxidant and genotoxic compound. Basal GAPDH and Ape1 protein levels were measured via Western Blotting. Etoposide (20 uM) decreased GAPDH and Ape1 protein levels at 54±7% and 42±7%, respectively. Etoposide increased phospho-histone H2A.X (S139) (double-strand DNA damage marker), and cleaved caspase-3 (Asp 175) (apoptosis marker) levels by 170±17% and 230±15%, respectively. Etoposide induced cell apoptosis in dose-dependent manner (Cell Death Elisa). Etoposide induced appearance of oxidized GAPDH in cell nuclear and cytosolic compartments (immunofluorescence with oxidized GAPDH antibody, 4A1 clone) and Etoposide also markedly suppressed GAPDH-Ape1 binding in the cell nucleus (proximity ligation assay). Cell transfection with GAPDH-overexpressing virus increased GAPDH protein, decreased Etoposide-induced cytotoxicity (live cell assay with Incucyte Sx5/Cytotox Red) and suppressed Etoposide-elicited caspase3/7 activation (Incucyte Caspase 3/7 Red dye). GAPDH overexpression reduced Etoposide-induced apoptosis and decreased phospho-histone H2A.X levels. In summary, we have created an efficient oxidative/genotoxic stress model in vascular SMC to analyze DNA damage and its mechanistic role in cell death and apoptosis-associated pathologies. We show that Etoposide induced a potent cytotoxic/genotoxic stress in SMC including DNA damage, and induction of cell apoptosis and these effects were associated with oxidation of GAPDH, reduction in GAPDH-Ape1 binding and marked Ape1 endonuclease downregulation. GAPDH overexpression prevented Etoposide-induced cytotoxicity and apoptosis potentially via stimulation of Ape1/DNA repair pathway.

Activation of NRF2 by APE1/REF1 is redox-dependent in Barrett's related esophageal adenocarcinoma cells

BackgroundChronic gastroesophageal reflux disease (GERD) is a major risk factor for the development of metaplastic Barrett's esophagus (BE) and its progression to esophageal adenocarcinoma (EAC). Uncontrolled accumulation of reactive oxygen species (ROS) in response to acidic bile salts (ABS) in reflux conditions can be lethal to cells. In this study, we investigated the role of APE1/REF1 in regulating nuclear erythroid factor-like 2 (NRF2), the master antioxidant transcription factor, in response to reflux conditions. ResultsWe found that APE1 protein was critical for protecting against cellular ROS levels, oxidative DNA damage, double strand DNA breaks, and cell death in response to conditions that mimic reflux. Analysis of cell lines and de-identified tissues from patients with EAC demonstrated overexpression of both APE1 and NRF2 in EAC cells, as compared to non-neoplastic esophageal cells. Using reflux conditions, we detected concordant and prolonged increases of APE1 and NRF2 protein levels for several hours, following transient short exposure to ABS (20 min). NRF2 transcription activity, as measured by ARE luciferase reporter, and expression of its target genes (HO-1 and TRXND1) were similarly increased in response to ABS. Using genetic knockdown of APE1, we found that APE1 was required for the increase in NRF2 protein stability, nuclear localization, and transcription activation in EAC. Using knockdown of APE1 with reconstitution of wild-type and a redox-deficient mutant (C65A) of APE1, as well as pharmacologic APE1 redox inhibitor (E3330), we demonstrated that APE1 regulated NRF2 in a redox-dependent manner. Mechanistically, we found that APE1 is required for phosphorylation and inactivation of GSK-3β, an important player in the NRF2 degradation pathway. ConclusionAPE1 redox function was required for ABS-induced activation of NRF2 by regulating phosphorylation and inactivation of GSK-3β. The APE1-NRF2 network played a critical role in protecting esophageal cells against ROS and promoting cell survival under oxidative reflux conditions.

High nuclear expression of APE1 correlates with unfavorable prognosis and promotes tumor growth in hepatocellular carcinoma.

APE1 is a multifunctional protein that plays important roles in cancer development. However, the association between APE1 expression and the clinicopathological parameters of HCC patients has not been fully characterized. In this study, bioinformatics analysis of APE1 was performed in several databases, including the TCGA, GeneCard, Human Protein Atlas and Ualcan databases. The relationship between APE1 mRNA expression and several attributes of liver cancer patients in TCGA was investigated. Then, the protein expression of APE1 was detected by IHC analysis in 95 HCC samples and the association between APE1 expression and the clinicopathological parameters of HCC patients was explored. GSEA-KEGG analysis was performed to predict the potential signaling pathways that associated with APE1 expression. Then the siRNA-mediated knockdown model of APE1 was constructed in HCC cell line to further detect the detailed function of APE1 in HCC development in vitro and in vivo. The results of the bioinformatics analysis showed that APE1 expression was primarily located in the cell nucleus. APE1 mRNA expression was substantially correlated with pathological grade and T status in TCGA database. Elevated APE1 expression was observed in HCC samples and was associated with unfavorable survival time in liver cancer patients. IHC data demonstrated that the nuclear expression of APE1 in HCC tissues was significantly higher than that in noncancerous tissues. The expression level of the APE1 protein in HCC was strongly associated with tumor diameter and overall survival. Survival analysis indicated that APE1 nuclear expression is an independent prognostic marker for the overall survival of HCC patients. GSEA-KEGG results confirmed that APE1 associated with the base excision repair signaling pathway. The data of phenotypic experiments indicated that APE1 remarkably promoted tumor growth both in HCC cells and xenografts. The findings firstly imply that nuclear expression of APE1 is a valuable prognostic marker for HCC. APE1 significantly facilitate HCC development and targeting APE1 may be a promising strategy for HCC treatment.

Xeroderma Pigmentosum C (XPC) Mutations in Primary Fibroblasts Impair Base Excision Repair Pathway and Increase Oxidative DNA Damage.

Xeroderma Pigmentosum C (XPC) is a multi-functional protein that is involved not only in the repair of bulky lesions, post-irradiation, via nucleotide excision repair (NER) per se but also in oxidative DNA damage mending. Since base excision repair (BER) is the primary regulator of oxidative DNA damage, we characterized, post-Ultraviolet B-rays (UVB)-irradiation, the detailed effect of three different XPC mutations in primary fibroblasts derived from XP-C patients on mRNA, protein expression and activity of different BER factors. We found that XP-C fibroblasts are characterized by downregulated expression of different BER factors including OGG1, MYH, APE1, LIG3, XRCC1, and Polβ. Such a downregulation was also observed at OGG1, MYH, and APE1 protein levels. This was accompanied with an increase in DNA oxidative lesions, as evidenced by 8-oxoguanine levels, immediately post-UVB-irradiation. Unlike in normal control cells, these oxidative lesions persisted over time in XP-C cells having lower excision repair capacities. Taken together, our results indicated that an impaired BER pathway in XP-C fibroblasts leads to longer persistence and delayed repair of oxidative DNA damage. This might explain the diverse clinical phenotypes in XP-C patients suffering from cancer in both photo-protected and photo-exposed areas. Therapeutic strategies based on reinforcement of BER pathway might therefore represent an innovative path for limiting the drawbacks of NER-based diseases, as in XP-C case.

Priming alleviates high temperature induced oxidative DNA damage and repair using Apurinic/apyrimidinic endonuclease (Ape1L) homologue in wheat (Triticum aestivum L.)

Crop plants require an optimum range of temperature for normal growth and development however high temperature can adversely affect the plants, induce oxidative stress and disintegrate biomolecules especially DNA and proteins. In wheat, high temperature stress (35–40 °C) during ripening stage hampers the yield tremendously. In this study, we assessed high temperature (HT) induced oxidative stress, subsequent DNA damage and role of priming in stress tolerance by analyzing DNA repair enzyme Triticum aestivum AP endonuclease (TaApe1L). Sixteen days old seedlings of wheat varieties PBW 550 and PBW 343 were primed with mild drought and exposed to HT (38 °C) for 2, 4, and 6 h. Hydrogen peroxide (H2O2) was used as oxidative stress marker and quantified on regular time intervals. DNA damage was analyzed by DNA laddering and TaApe1L gene expression was analyzed using RT PCR and western blotting. Phylogenetic analysis of Ape1 revealed presence of some key amino acids that are evolutionary conserved. A significant increase in H2O2 content was observed after 6 h of exposure especially in PBW 343. Similarly, the DNA damage was also increased with HT exposure especially in PBW 343. The TaApe1L mRNA expression increased after priming in both the varieties after 4 h. But APE1 protein expression was higher in PBW 343, which can be correlated with DNA damage and repair. Lastly, it can be concluded that there is varietal difference in the HT sensitivity but 6 h exposure was detrimental to both the varieties. Also, drought priming improved HT tolerance by over expressing APE1.

Evaluation of antioxidant network proteins as novel prognostic biomarkers for head and neck cancer patients.

Recurrence rates for head and neck squamous cell carcinoma (HNSCC) approach 50% at 5years. Current staging fails to identify patients with a worse prognosis who might benefit from intensified treatment, which warrants improved prognostic biomarkers. The purpose of this retrospective case study is to identify potential prognostic biomarkers in patients with HNSCC including APE1 (DNA repair/redox gene regulator), NRF2 and PPARGC1A (redox gene regulators), SOD3 and DCN (antioxidant proteins). Differential protein expression between benign, carcinoma in situ (CIS), and invasive HNSCC tissue specimens from 77 patients was assessed using immunohistochemistry. Protein expression was analyzed with multivariate, pair-wise, and Kaplan-Meier survival analyses to identify potential prognostic biomarkers. Utilizing The Cancer Genome Atlas's transcriptome database, pair-wise and survival analysis was performed to identify potential prognostic biomarkers. APE1, NRF2, PPARGC1A, SOD3, and DCN expression in HNSCC in relation to, lymph node invasion, and patient survival were examined. Elevated APE1 protein expression in CIS corresponded with reduced survival (p=0.0243). Increased APE1 gene expression in stage T4a HNSCC was associated with reduced patient survival (p<0.015). Increased PPARGC1A in invasive tumor correlated with reduced survival (p=0.0281). Patients with lymph node invasion at diagnosis had significantly increased APE1 protein in the primary sites (p<0.05). Patients with poorly differentiated invasive tumors had reduced PPARGC1A in CIS proximal to the invasive tumor and had elevated DCN and SOD3 in proximal benign tissue (p<0.05). The expression of APE1, DCN, and SOD3 is a potential prognostic signature that identifies patients with worsened survival.

A phase I study of the APE1 protein inhibitor APX3330 in patients with advanced solid tumors.

3097 Background: APX3330 is an orally administered anti-cancer, anti-CIPN agent targeting the APE1 protein. APE1 maintains NFkB, STAT3, AP-1 and HIF-1a in a reduced form, acting as a regulator of transcription factors. A dual function protein, APE1 also plays a role in protecting against oxidative DNA damage in neurons. APX3330 is a highly selective inhibitor of APE1 redox function in tumors that enhances the neuronal protection function of APE1. Methods: We report on study NCT03375086 evaluating APX3330 in patients with incurable malignancies. Eligibility required adequate organ function, PS 0-2 and tumors not amenable to curative therapy. 1° and 2° objectives included determining the recommended phase 2 dose (RP2D), the safety and PK/PD profiles of APX3330 and reporting any RECIST anti-tumor activity. Patients received APX3330 b.i.d, in 21-day cycles. AE evaluation included 1 pt/cohort until the occurrence of ≥ G2 toxicity at which time the study proceeded in a 3+3 design. Additional patient were also recruited in cohorts in order to attain PK/PD and biopsy samples. Results: Between 2/18 and 8/18, 19 subjects (13M, 6F) with median age of 69 y started therapy. Dose (mg/d) escalation and number of patients treated (n) per each cohort proceeded as follows: 240 mg (1), 360 (4), 480 (2), 600 (6) and 720 (6). APX3330 was well tolerated at dose levels from 240-600 mg/d. The most frequent treatment-related adverse event (all grades) was G1 fatigue. A G3 rash occurred in two subjects at the 720 mg level defining 600 mg/d as the RP2D for further development. Six subjects had disease stabilization for ≥ 4 cycles, and of these, four subjects with the following diagnosis, RECIST response and days on study included: (CRC, PR, 356), (Endometrial, SD, 316), (Melanoma, SD, 245), (Prostate, SD, 246). Final PK and PD data, including proteomic, transcriptome, APE1 serum levels and CTC analyses are pending and will be reported at the conference. Conclusions: APX3330 is an orally administered inhibitor of APE1. This phase I study identified 600 mg PO daily as the RP2D for further development. RECIST evaluation identified signs of clinical activity in this un-selected population of patients with advanced cancer. PD analyses indicate APX3330 mediated targeting of the APE1 protein. Clinical trial information: NCT03375086.

Effects of siRNA Targeting Interference of APE1 Gene on Proliferation and Apoptosis of Glioma Cells

This paper explored the application of RNA in cancer gene therapy. Second, the effect of the recombinant plasmid APE1 siRNA on the expression of APE1 protein was observed. In addition, the proliferation of cells in apoptosis and the radiosensitivity of human glioma U251 cells were also discussed. U251 cells were divided into control group, negative group and APE1siRNA group for culturing. There were two holes in each group, and independent experiments were repeated three times. The expression of APE1 protein in U251 cells was detected by Western blot experiments for siRNA transfection. The siRNA cells were transfected, while another group of FAM fluorescent labeled non-sense sequence siRNA was transfected to determine the transfection rate and extract the total protein of the cell. BCA protein assay kit was used for determining the concentration. Meanwhile, 50g sample and SDS-polypropylene diamine gel were prepared. Coomassie blue staining was used to observe the protein electrophoresis, after which the protein on the gel was transferred to the PVDF membrane and then developed in a chemiluminescent detection system with specific antibodies. The β -action test was used as an internal controller, while the optical density scanner was used to detect the gray value of the target strip. The relative expression of target protein was equal to the target band gray value or the same sample reference value of gray. MTT method was used to detect the cell growth curve experiments, while AO or EB method was used to detect the apoptosis rate of glioma cells. SPSS17.0 was used for statistical analysis. The results show that the APE1 siRNA expression plasmid was successfully introduced into human glioma cells by RNA interference, which can reduce the expression of APE1 in cells. It was confirmed that the reduction of APE1 protein could inhibit proliferation and promote the apoptosis of glioma cells. Third, APE1 targeting interference may be an effective gene therapy strategy for human glioma, which can provide an experimental basis for the clinical gene therapy of glioma.

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 30μM 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 1week with low concentrations of lead before induction of transformation with n-methyl-n-nitrosoguanidine (MNNG) (0.5μg/mL) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (0.1μg/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, 30μM) 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.

The role of the N-terminal domain of human apurinic/apyrimidinic endonuclease 1, APE1, in DNA glycosylase stimulation

The base excision repair (BER) pathway consists of sequential action of DNA glycosylase and apurinic/apyrimidinic (AP) endonuclease necessary to remove a damaged base and generate a single-strand break in duplex DNA. Human multifunctional AP endonuclease 1 (APE1, a.k.a. APEX1, HAP-1, or Ref-1) plays essential roles in BER by acting downstream of DNA glycosylases to incise a DNA duplex at AP sites and remove 3′-blocking sugar moieties at DNA strand breaks. Human 8-oxoguanine-DNA glycosylase (OGG1), methyl-CpG-binding domain 4 (MBD4, a.k.a. MED1), and alkyl-N-purine-DNA glycosylase (ANPG, a.k.a. Aag or MPG) excise a variety of damaged bases from DNA. Here we demonstrated that the redox-deficient truncated APE1 protein lacking the first N-terminal 61 amino acid residues (APE1-NΔ61) cannot stimulate DNA glycosylase activities of OGG1, MBD4, and ANPG on duplex DNA substrates. Electron microscopy imaging of APE1–DNA complexes revealed oligomerization of APE1 along the DNA duplex and APE1-mediated DNA bridging followed by DNA aggregation. APE1 polymerizes on both undamaged and damaged DNA in cooperative mode. Association of APE1 with undamaged DNA may enable scanning for damage; however, this event reduces effective concentration of the enzyme and subsequently decreases APE1-catalyzed cleavage rates on long DNA substrates. We propose that APE1 oligomers on DNA induce helix distortions thereby enhancing molecular recognition of DNA lesions by DNA glycosylases via a conformational proofreading/selection mechanism. Thus, APE1-mediated structural deformations of the DNA helix stabilize the enzyme–substrate complex and promote dissociation of human DNA glycosylases from the AP site with a subsequent increase in their turnover rate. Significance StatementThe major human apurinic/apyrimidinic (AP) endonuclease, APE1, stimulates DNA glycosylases by increasing their turnover rate on duplex DNA substrates. At present, the mechanism of the stimulation remains unclear. We report that the redox domain of APE1 is necessary for the active mode of stimulation of DNA glycosylases. Electron microscopy revealed that full-length APE1 oligomerizes on DNA possibly via cooperative binding to DNA. Consequently, APE1 shows DNA length dependence with preferential repair of short DNA duplexes. We propose that APE1-catalyzed oligomerization along DNA induces helix distortions, which in turn enable conformational selection and stimulation of DNA glycosylases. This new biochemical property of APE1 sheds light on the mechanism of redox function and its role in DNA repair.

APE1/Ref-1 knockdown in pancreatic ductal adenocarcinoma - characterizing gene expression changes and identifying novel pathways using single-cell RNA sequencing.

Apurinic/apyrimidinic endonuclease 1/redox factor‐1 (APE1/Ref‐1 or APE1) is a multifunctional protein that regulates numerous transcription factors associated with cancer‐related pathways. Because APE1 is essential for cell viability, generation of APE1‐knockout cell lines and determining a comprehensive list of genes regulated by APE1 has not been possible. To circumvent this challenge, we utilized single‐cell RNA sequencing to identify differentially expressed genes (DEGs) in relation to APE1 protein levels within the cell. Using a straightforward yet novel statistical design, we identified 2837 genes whose expression is significantly changed following APE1 knockdown. Using this gene expression profile, we identified multiple new pathways not previously linked to APE1, including the EIF2 signaling and mechanistic target of Rapamycin pathways and a number of mitochondrial‐related pathways. We demonstrate that APE1 has an effect on modifying gene expression up to a threshold of APE1 expression, demonstrating that it is not necessary to completely knockout APE1 in cells to accurately study APE1 function. We validated the findings using a selection of the DEGs along with siRNA knockdown and qRT‐PCR. Testing additional patient‐derived pancreatic cancer cells reveals particular genes (ITGA1,TNFAIP2,COMMD7,RAB3D) that respond to APE1 knockdown similarly across all the cell lines. Furthermore, we verified that the redox function of APE1 was responsible for driving gene expression of mitochondrial genes such as PRDX5 and genes that are important for proliferation such as SIPA1 and RAB3D by treating with APE1 redox‐specific inhibitor, APX3330. Our study identifies several novel genes and pathways affected by APE1, as well as tumor subtype specificity. These findings will allow for hypothesis‐driven approaches to generate combination therapies using, for example, APE1 inhibitor APX3330 with other approved FDA drugs in an innovative manner for pancreatic and other cancer treatments.