OBJECTIVE: To develop a simple, sensitive and robust method for rapid detection of human apurinic/apyrimidinic endonuclease 1 (APE1) in various biological samples. METHODS: An abasic site-containing DNA probe with a sequence of 5'-T*T*C*C*T*C*T(ROX)AGAGXCGTT (BHQ2)C*A*C*T*G*T*AGTTTATA*C*A*G*T*GAATCTCTCTAG*T*C*T-3' ["X" represents AP site; The phosphorothioated nucleotides (at 3' side) are indicated with an asterisk after the nucleotides; ROX is 6-carboxy-X-rhodamine and BHQ2 is Black Hole quencher 2] was synthesized and used for the detection. In the presence of APE1, the DNA probe could be specifically hydrolyzed by the enzyme and release the fluorophore, resulting in strong fluorescence emission. The activity of APE1 was determined according to the rate of increase in fluorescence intensity. In this work, we modified the reaction buffer and significantly improved the performance of the method. Moreover, the method was further extended to measure the contents of APE1 in the protein extraction from peripheral blood mononuclear cells (PBMCs) extracted from human whole blood samples by density gradient centrifugation. The assay was also applied to measure the activity of APE1 in human serum samples. RESULTS: With a new reaction buffer composed of 0.04% (V/V) Triton X-100, 50 mmol/L KAc, 20 mmol/L Tris-Ac, 10 mmol/L Mg(Ac)2 and 1 mmol/L dithiothreitol (DTT), the method achieved a detection limit of 0.005 U/mL (3 pg/mL) and a linear response ranging from 6 pg/mL to 1.2 ng/mL. The contents of APE1 in the protein extraction from PBMCs of eight blood samples were measured to be in the range from 0.061 to 0.40 ng/μg protein, with an average of 0.16 ng/μg protein. The recovery was 98%±5% (n=3). The levels of APE1 in the sera from 102 normal individuals (51 male and 51 female, age range: 59-75 years) were observed to be from 0.13 to 0.34 ng/mL, with a recovery of 96%±15% (n=3). CONCLUSION The new fluorescence assay was simple, rapid and sensitive, providing a practical tool to measure the activity of APE1 in serum samples and cell extracts. It also holds great potential in measurement of APE1 in many other biological samples for clinical test and laboratory research.
Aged ; DNA Probes ; DNA-(Apurinic or Apyrimidinic Site) Lyase ; Female ; Fluorescence ; Humans ; Leukocytes, Mononuclear ; Male ; Middle Aged

BACKGROUND: The main manifestations of radiation pneumonitis are injury of alveolar epithelial and endothelial cells, abnormal expression of cytokines, abnormal proliferation of fibroblasts and synthesis of fibrous matrix. The occurrence of radiation pneumonitis is associated with multiplecytokine level abnormality. These cytokines can also be used as bio-markers to predict the occurrence of radiation pneumonitis. This study was to evaluate the correlation between the change of apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ape1/Ref-1), intercellular adhesion molecules 1 (ICAM-1) and interleukin-17A (IL-17A) before and after radiotherapy and radiation pneumonitis for local advanced non-small cell lung cancer (NSCLC) patients with concurrent chemoradiotherapy. METHODS: NSCLC patients (68 cases) were treated with concurrent radiotherapy and chemotherapy, every patient's normal tissue were controlled with a same radation dose. 68 local advanced NSCLC patients with concurrent chemoradiotherapy were detected the levels of Ape1/Ref-1, ICAM-1 and IL-17A in serum by ELISA before radiotherapy and in the 14th week after radiotherapy. Acute and advanced radiation pulmonary injury was graded according to Radiation Therapy Oncology Group/European Organization For Research and Treatment (RTOG/EORTC) diagnostic and grading criteria. Grade 2 or more radiation pneumonitis was taken as the main end point. RESULTS: Eighteen cases out of 68 developed radiation pneumonitis, 50 of 68 cases have no radiation pneumonia development. There was no significant change of Ape1/Ref-1 levels before and after radiotherapy in radiation pneumonitis group (P>0.05). There was no significant change of Ape1/Ref-1 concentration in serum after radiotherapy between radiation pneumonitis group and non-radiation pneumonitis group (P>0.05). Compared with before radiotherapy, upregulation degree of ICAM-1 levels in radiation pneumonitis group was significantly higher than that in non- radiation pneumonitis group (P<0.05). There was no significant change of IL-17A concentration before and after radiotherapy in radiation pneumonitis group, but after radiotherapy IL-17A concentration in serum were remarkably higher than that in non-radiation pneumonitis group (P<0.05). Correlation analysis found that the change of ICAM-1 before and after radiotherapy has no obvious correlation with the incidence of radiation pneumonitis, and IL-17A change has obvious correlation with the incidence of radiation pneumonitis. CONCLUSIONS On the basis of strictly controlling radiation dose on normal tissue, IL-17A in serum could be the predictive factors of radiation pneumonitis for local advanced NSCLC patients with concurrent chemoradiotherapy.
Aged ; Carcinoma, Non-Small-Cell Lung ; blood ; drug therapy ; radiotherapy ; Chemoradiotherapy ; adverse effects ; DNA-(Apurinic or Apyrimidinic Site) Lyase ; blood ; Female ; Humans ; Intercellular Adhesion Molecule-1 ; blood ; Interleukin-17 ; blood ; Male ; Middle Aged ; Radiation Pneumonitis ; blood ; etiology

Cytosol ; metabolism ; DNA-(Apurinic or Apyrimidinic Site) Lyase ; metabolism ; Endoplasmic Reticulum ; metabolism ; Humans ; Receptors, sigma ; metabolism

Cell Membrane ; metabolism ; Clathrin ; metabolism ; DNA-(Apurinic or Apyrimidinic Site) Lyase ; metabolism ; Endocytosis ; HeLa Cells ; Humans ; Interleukin-2 Receptor alpha Subunit ; metabolism

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that plays a central role in the cellular response to DNA damage and redox regulation against oxidative stress. APE1/Ref-1 functions in the DNA base excision repair pathway, the redox regulation of several transcription factors, and the control of intracellular redox status through the inhibition of reactive oxygen species (ROS) production. APE1/Ref-1 is predominantly localized in the nucleus; however, its subcellular localization is dynamically regulated and it may be found in the mitochondria or elsewhere in the cytoplasm. Studies have identified a nuclear localization signal and a mitochondrial target sequence in APE1/Ref-1, as well as the involvement of the nuclear export system, as determinants of APE1/Ref-1 subcellular distribution. Recently, it was shown that APE1/Ref-1 is secreted in response to hyperacetylation at specific lysine residues. Additionally, post-translational modifications such as phosphorylation, S-nitrosation, and ubiquitination appear to play a role in fine-tuning the activities and subcellular localization of APE1/Ref-1. In this review, we will introduce the multifunctional role of APE1/Ref-1 and its potential usefulness as a therapeutic target in cancer and cardiovascular disease.
Active Transport, Cell Nucleus ; Biomarkers ; Cardiovascular Diseases ; Cytoplasm ; DNA ; DNA Damage ; DNA Repair ; DNA-(Apurinic or Apyrimidinic Site) Lyase ; Lysine ; Mitochondria ; Nuclear Localization Signals ; Oxidation-Reduction ; Oxidative Stress ; Phosphorylation ; Protein Processing, Post-Translational ; Reactive Oxygen Species ; Transcription Factors ; Ubiquitin ; Ubiquitination

OBJECTIVETo evaluate the effects of polymorphisms in XRCC1 and APE1 genes on vinyl chloride (VC)-induced chromosomal damage in peripheral lymphocytes.

METHODSIn this study, 317 workers occupationally exposed to VC were recruited from a factory in Shandong Province, China. The micronucleus (MN) frequency in peripheral lymphocytes was used as an indicator of chromosomal damage. Polymerase chain reaction-restriction fragment length polymorphism and created restriction site combined with restriction fragment length polymorphism were used to determine the five single nucleotide polymorphisms in XRCC1 and APE1 genes in the base excision repair pathway. The association of chromosomal damage with these polymorphisms and the haplotype of XRCC1 was analyzed using Poisson regression and PHASE 2.0.2.

RESULTSIt was found that among the VC-exposed workers, individuals with XRCC1 polymorphisms (-77C/T, Arg194Trp, Arg280His, and Arg399Gln) had a significantly higher MN frequency than those with homozygous wild-type genotypes, with frequency ratios (FR) as follows, respectively: FR = 1.21, 95%CI: 1.05∼1.39 (P < 0.05); FR = 1.14, 95%CI: 1.00∼1.38 (P < 0.05); FR = 1.26, 95%CI: 1.11∼1.44 (P < 0.05); FR = 1.23, 95%CI: 1.08∼1.46 (P < 0.05). APE1 Asp148Glu was found of no significant relationship with MN frequency. Haplotype analysis of XRCC1 demonstrated that the MN frequencies in subjects with CTAA/CTAA and CCAA/CTAA were significantly higher than that in those with TCGG/TCGG (FR = 1.19, 95%CI: 1.02∼1.32, P < 0.05; FR = 1.41, 95%CI: 1.02∼1.87, P < 0.05). Furthermore, association was found between accumulated exposure to VC and XRCC1 polymorphisms (-77C/T, Arg194Trp, Arg280His, and Arg399Gln) after adjustment for age, sex, drinking, and smoking.

CONCLUSIONVC can induce chromosomal damage even when the exposure level is lower than the national occupational health standard of China (PC-TWA: 10 mg/m(3)); the polymorphisms in XRCC1 and APE1 are associated with chromosomal damage induced by VC.

Adult ; DNA-(Apurinic or Apyrimidinic Site) Lyase ; genetics ; DNA-Binding Proteins ; genetics ; Female ; Haplotypes ; Humans ; Male ; Micronuclei, Chromosome-Defective ; Middle Aged ; Occupational Exposure ; adverse effects ; Polymorphism, Restriction Fragment Length ; Vinyl Chloride ; poisoning ; X-ray Repair Cross Complementing Protein 1 ; Young Adult

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme involved in the base excision repair (BER) pathway, which repairs oxidative base damage caused by endogenous and exogenous agents. APE1 acts as a reductive activator of many transcription factors (TFs) and has also been named redox effector factor 1, Ref-1. For example, APE1 activates activator protein-1, nuclear factor kappa B, hypoxia-inducible factor 1alpha, paired box gene 8, signal transducer activator of transcription 3 and p53, which are involved in apoptosis, inflammation, angiogenesis and survival pathways. APE1/Ref-1 maintains cellular homeostasis (redox) via the activation of TFs that regulate various physiological processes and that crosstalk with redox balancing agents (for example, thioredoxin, catalase and superoxide dismutase) by controlling levels of reactive oxygen and nitrogen species. The efficiency of APE1/Ref-1's function(s) depends on pairwise interaction with participant protein(s), the functions regulated by APE1/Ref-1 include the BER pathway, TFs, energy metabolism, cytoskeletal elements and stress-dependent responses. Thus, APE1/Ref-1 acts as a 'hub-protein' that controls pathways that are important for cell survival. In this review, we will discuss APE1/Ref-1's versatile nature in various human etiologies, including neurodegeneration, cancer, cardiovascular and other diseases that have been linked with alterations in the expression, subcellular localization and activities of APE/Ref-1. APE1/Ref-1 can be targeted for therapeutic intervention using natural plant products that modulate the expression and functions of APE1/Ref-1. In addition, studies focusing on translational applications based on APE1/Ref-1-mediated therapeutic interventions are discussed.
Animals ; DNA Damage ; DNA Repair ; DNA-(Apurinic or Apyrimidinic Site) Lyase/analysis/genetics/*metabolism ; Humans ; *Molecular Targeted Therapy/methods ; Neoplasms/*drug therapy/genetics/*metabolism ; Neurodegenerative Diseases/*drug therapy/genetics/*metabolism ; Oxidative Stress ; Phytochemicals/pharmacology/*therapeutic use ; Polymorphism, Genetic ; Protein Interaction Maps

Osteosarcoma is a genetically unstable malignancy that most frequently occurs in children and young adults. The lack of progress in managing this devastating disease in the clinic has prompted international researchers to collaborate to profile key genomic alterations that define osteosarcoma. A team of researchers and clinicians from China, Finland, and the United States investigated human osteosarcoma by integrating transcriptome sequencing (RNA-seq), high-density genome-wide array comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH), reverse transcription-polymerase chain reaction (RT-PCR), Sanger sequencing, cell culture, and molecular biological approaches. Systematic analysis of genetic/genomic alterations and further functional studies have led to several important findings, including novel rearrangement hotspots, osteosarcoma-specific LRP1-SNRNP25 and KCNMB4-CCND3 fusion genes, VEGF and Wnt signaling pathway alterations, deletion of the WWOX gene, and amplification of the APEX1 and RUNX2 genes. Importantly, these genetic events associate significantly with pathogenesis, prognosis, progression, and therapeutic activity in osteosarcoma, suggesting their potential impact on improved managements of human osteosarcoma. This international initiative provides opportunities for developing new treatment modalities to conquer osteosarcoma.
Adult ; Bone Neoplasms ; Child ; China ; Comparative Genomic Hybridization ; DNA-(Apurinic or Apyrimidinic Site) Lyase ; Genomics ; Humans ; In Situ Hybridization, Fluorescence ; Molecular Targeted Therapy ; Osteosarcoma ; genetics ; therapy ; Prognosis ; Wnt Signaling Pathway ; Young Adult

OBJECTIVETo investigate the prognostic values of HIF-1α, APE1, VEGF, and COX-2 protein expressions and their predictive value of tumor necrosis rate and prognosis in osteosarcoma, as well as their interrelationships.

METHODSFormalin-fixed paraffin-embedded tissue samples were obtained from patients with osteosarcoma. Immunohistochemical assay was performed in pre-chemotherapy samples to determine the HIF-1α, VEGF, APE1, and COX-2 protein expression levels. Hematoxylin-eosin staining was used in post-operative samples to determine the tumor necrosis rate. Univariate and multivariate analyses were used to assess the impact of protein expression on prognosis.

RESULTSTumor tissues were obtained from 49 patients. Their median follow up was 29 months. HIF-1α was significantly correlated to every protein we tested: VEGF (P = 0.032), APE1 (P < 0.001), and COX-2 (P < 0.001). HIF-1α protein expression had a significant impact on disease free survival (P = 0.006). Expression of HIF-1α had a sensitivity of 64.7% and a specificity of 71.9% for poor pathological response (< 90% of tumor necrosis) versus good pathological response to chemotherapy (≥ 90% necrosis).

CONCLUSIONExpression of HIF-1α is a predictor of tumor response to neoadjuvant chemotherapy and outcome in osteosarcoma and is correlated with VEGF, APE1, and COX-2 expression.

Adolescent ; Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Bone Neoplasms ; drug therapy ; metabolism ; pathology ; Chemotherapy, Adjuvant ; Child ; Cyclooxygenase 2 ; metabolism ; DNA-(Apurinic or Apyrimidinic Site) Lyase ; metabolism ; Disease-Free Survival ; Female ; Follow-Up Studies ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Male ; Middle Aged ; Neoadjuvant Therapy ; Neoplasm Staging ; Osteosarcoma ; drug therapy ; metabolism ; pathology ; Vascular Endothelial Growth Factor A ; metabolism ; Young Adult

Apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1), as a type of multifunctional protein, plays an essential role in the base excision repair (BER) pathway, which is responsible for the repair of DNA caused by oxidative and alkylation damage. As importantly, APE/Ref-1 also functions as a redox factor maintaining transcription factors in an active reduced state. APE/Ref-1 stimulates the DNA-binding activity of numerous transcription factors that are involved in cancer promotion and progression, such as AP-1 (Fos/Jun), NF-kappaB, HIF-1alpha, p53, and others. Based on the structures and functions of APE1/Ref-1, we will provide an overview of its activities and explore the budding clinical use of this protein as a target in cancer treatment, and propose that APE/Ref-1 has a great potential for application in clinical research.
Apoptosis ; DNA Repair ; genetics ; DNA-(Apurinic or Apyrimidinic Site) Lyase ; antagonists & inhibitors ; genetics ; metabolism ; physiology ; Drug Delivery Systems ; Drug Resistance, Neoplasm ; Humans ; Neoplasms ; drug therapy ; metabolism ; radiotherapy ; Oxidation-Reduction ; Polymorphism, Single Nucleotide ; Precancerous Conditions ; metabolism ; Radiation Tolerance