RESUMEN
To achieve highly sensitive and reliable detection of apurinic/apyrimidinic endonuclease 1 (APE1), a critical cancer diagnostic biomarker, we designed a DNA walker-based dual-mode biosensor, utilizing cellular endogenous dual enzymes (APE 1 and Flap endonuclease 1 (FEN 1)) to collaborate in activating and propelling DNA walker motion on DNA-functionalized Au nanoparticles. Incorporating both fluorescence and electrochemical detection modes, this system leverages signal amplification from DNA walker movement and cascade amplification through tandem hybridization chain reactions (HCR), achieving highly sensitive detection of APE 1. In the fluorescence mode, continuous DNA walker movement, initiated by APE1 and driven by FEN1, generates a robust signal response within a concentration range of 0.01-500 U mL-1, presenting a good linearity in the concentration range of 0.01-10 U mL-1, with a detection limit of 0.01 U mL-1. In the electrochemical detection module, the cascade upstream DNA walker and downstream HCR dual signal amplification strategy further enhances the sensitivity of APE1 detection, extending the linear range to 0.01-50 U mL-1 and reducing the detection limit to 0.002 U mL-1. Rigorous validation demonstrates the biosensor's specificity and anti-interference capability against multiple enzymes. Moreover, it effectively distinguishes cancer cells from normal cell lysates, exhibiting excellent stability and consistency in the dual-modes. Overall, our findings underscore the efficacy of the developed dual-mode biosensor for detecting APE1 in serum and cell lysates samples, indicating its potential for clinical applications in disease diagnosis.
Asunto(s)
Técnicas Biosensibles , ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN , Endonucleasas de ADN Solapado , Oro , Límite de Detección , Nanopartículas del Metal , Técnicas Biosensibles/métodos , Humanos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , ADN/química , Endonucleasas de ADN Solapado/química , Endonucleasas de ADN Solapado/metabolismo , Nanopartículas del Metal/química , Oro/química , Técnicas Electroquímicas/métodosRESUMEN
Apurinic/apyrimidinic endonuclease 1 (APE1), as a vital base excision repair enzyme, is essential for maintaining genomic integrity and stability, and its abnormal expression is closely associated with malignant tumors. Herein, we constructed an electrochemiluminescence (ECL) biosensor for detecting APE1 activity by combining nanoconfined ECL silver nanoclusters (Ag NCs) with X-shaped DNA recognizer-triggered cascade amplification. Specifically, the Ag NCs were prepared and confined in the glutaraldehyde-cross-linked chitosan hydrogel network using the one-pot method, resulting in a strong ECL response and exceptional stability in comparison with discrete Ag NCs. Furthermore, the self-assembled X-shaped DNA recognizers were designed for APE1 detection, which not only improved reaction kinetics due to the ordered arrangement of recognition sites but also achieved high sensitivity by utilizing the recognizer-triggered cascade amplification of strand displacement amplification (SDA) and DNAzyme catalysis. As expected, this biosensor achieved sensitive ECL detection of APE1 in the range of 1.0 × 10-3 U·µL-1 to 1.0 × 10-10 U·µL-1 with the detection limit of 2.21 × 10-11 U·µL-1, rendering it a desirable approach for biomarker detection.
Asunto(s)
Técnicas Biosensibles , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Técnicas Electroquímicas , Mediciones Luminiscentes , Nanopartículas del Metal , Plata , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , Plata/química , Humanos , Nanopartículas del Metal/química , Técnicas Electroquímicas/métodos , Mediciones Luminiscentes/métodos , Técnicas Biosensibles/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ADN/química , Límite de Detección , ADN Catalítico/química , ADN Catalítico/metabolismoRESUMEN
APE1 (apurinic/apyrimidinic endodeoxyribonuclease 1) is a central enzyme of the base excision repair (BER) pathway playing a pivotal role in protecting mammalian cells against genotoxins and in safeguarding genome stability. Recently, we demonstrated the APE1 ability to process abasic ribonucleotides embedded in DNA. Here, we provide a pipeline of protocols to quantify endodeoxyribonuclease activity by APE1 on these substrates, by using recombinant protein and whole-cell extracts. The repair capacity is measured by using fluorescent oligonucleotide substrates, which are then separated by polyacrylamide gel electrophoresis and detected by imaging scanning. The specificity of APE1 action is demonstrated using specific APE1 enzymatic inhibitors.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa , Pruebas de Enzimas , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Pruebas de Enzimas/métodos , Ribosa/metabolismo , Técnicas In VitroRESUMEN
Human apurinic/pyrimidine endonuclease 1 (APE1) played a critical role in the occurrence, progress and prognosis of tumors through overexpression and subcellular localization. Thus, it has become an important target for enhancing the sensitivity of tumor cells to radiotherapy and chemotherapy. Therefore, detecting and imaging its intracellular activity is of great significance for inhibitor discovery, cancer diagnosis and therapy. In this work, using DNA-based nanoprobe, we developed a new method for monitor intracellular APE1 activity. The detecting system was consisted by single fluorophore labeled hairpin probe and reduced graphene oxide (rGO). The in vitro result showed that a liner response of the detection method ranged from 0.02 U/mL to 2 U/mL with a limit of detection of 0.02 U/mL. Furthermore, this strategy possessing high specificity was successfully applied for APE1-related inhibitor screening using intracellular fluorescence imaging. Panaxytriol, an effective inhibitor of APE1 activity, was screened from traditional Chinese medicine (TCM) and its effect on APE1 activity was monitored in real time in A549 cells. In summary, this sensitive and specific APE1 detection technology is expected to provide an assistance for APE1-related inhibitor screening and diseases diagnosis.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , ADN/química , Grafito/química , Nanopartículas/química , Células A549 , ADN-(Sitio Apurínico o Apirimidínico) Liasa/antagonistas & inhibidores , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Evaluación Preclínica de Medicamentos , Enediinos/farmacología , Alcoholes Grasos/farmacología , Humanos , Imagen Óptica , Factores de TiempoRESUMEN
The dynamic variation of the expression profile and spatial landscape of multiple enzymes are crucial factors influencing tumor progression and drug treatment. However, the comprehensive analysis of these events has been hampered by the limitations of existing imaging technologies. Here we report a cooperatively activatable, DNA-based fluorescent reporter programmed to detect the correlated activity of dual enzymes, telomerase (TE) and apurinic/apyrimidinic endonuclease 1 (APE1), both in vitro and in vivo. The conformational change of the DNA probe can be orthogonally triggered through TE-induced DNA elongation and APE1-mediated specific cleavage, producing a fluorescent signal for imaging the activity of the two enzymes in an AND-gated manner. Furthermore, we demonstrate the capability of the system for specific tumor imaging through "dual lock-and-key" strategy, and visualizing the correlated enzymatic activities during drug treatment of cancer.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , ADN/química , Colorantes Fluorescentes/química , Línea Celular , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Humanos , Imagen ÓpticaRESUMEN
Understanding of the functions of enzymes in diverse cellular processes is important, but the design of sensors with controllable localization for in situ imaging of subcellular levels of enzymatic activity is particularly challenging. We introduce herein a spatiotemporally controlled sensor technology that permits in situ localization and photoactivated imaging of human apurinic/apyrimidinic endonucleaseâ 1 (APE1) within an intracellular organelle of choice (e.g., mitochondria or nucleus). The hybrid sensor platform is constructed by photoactivatable engineering of a DNA-based fluorescent probe and further combination with an upconversion nanoparticle and a specific organelle localization signal. Controlled localization and NIR-light-mediated photoactivation of the sensor "on demand" effectively constrains the imaging signal to the organelle of interest, with improved subcellular resolution. We further demonstrate the application of the nanosensors for the imaging of subcellular APE1 translocation in response to oxidative stress in live cells.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , ADN/química , Colorantes Fluorescentes/química , Nanopartículas/química , Orgánulos/química , Animales , Técnicas Biosensibles , Línea Celular , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Células HeLa , Humanos , Ratones , Neoplasias Experimentales/diagnóstico por imagen , Imagen Óptica , Procesos FotoquímicosRESUMEN
Quantification of stable isotope tracers after metabolic labeling provides a snapshot of the dynamic state of living cells and tissue. A form of imaging mass spectrometry quantifies isotope ratios with a lateral resolution <50 nm, using a methodology that we refer to as multi-isotope imaging mass spectrometry (MIMS). Despite lateral resolution exceeding diffraction-limited light microscopy, lack of contrast has largely limited use of MIMS to large or specialized subcellular structures, such as the nucleus and stereocilia. In this study, we repurpose the engineered peroxidase APEX2 as the first genetically encoded marker for MIMS. Coupling APEX2 labeling of lysosomes and metabolic labeling of protein, we identify that individual lysosomes exhibit substantial heterogeneity in protein age, which is lost in iPSC-derived neurons lacking the lysosomal protein progranulin. This study expands the practical use of MIMS for cell biology by enabling measurements of metabolic function from stable isotope labeling within individual organelles in situ.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Marcaje Isotópico/métodos , Lisosomas/metabolismo , Espectrometría de Masas/métodos , Neuronas/metabolismo , Orgánulos/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , Células HeLa , Humanos , Células Madre Pluripotentes Inducidas/citología , Neuronas/citología , ProteolisisRESUMEN
BACKGROUND: Reactive oxygen species (ROS) produce different lesions in DNA by ROS-induced DNA damage. Detection and quantification of 8-oxo-7,8-dihydroguanine (8-oxoG) within cells are important for study. Human ribosomal protein S3 (hRpS3) has a high binding affinity to 8-oxoG. In this study, we developed an imaging probe to detect 8-oxoG using a specific peptide from hRpS3. Transactivator (TAT) proteins are known to have cell-penetrating properties. Therefore, we developed a TAT-S3 probe by attaching a TAT peptide to our imaging probe. RESULTS: A DNA binding assay was conducted to confirm that our probe bound to 8-oxoG and apurinic/apyrimidinic (AP) sites. We confirmed that the TAT-S3 probe localized in the mitochondria, without permeabilization, and fluoresced in H2O2-treated HeLa cells and zebrafish embryos. Treatment with Mitoquinone (MitoQ), a mitochondria-targeted antioxidant, reduced TAT-S3 probe fluorescence. Additionally, treatment with O8, an inhibitor of OGG1, increased probe fluorescence. A competition assay was conducted with an aldehyde reaction probe (ARP) and methoxyamine (MX) to confirm binding of TAT-S3 to the AP sites. The TAT-S3 probe showed competitive binding to AP sites with ARP and MX. CONCLUSIONS: These results revealed that the TAT-S3 probe successfully detected the presence of 8-oxoG and AP sites in damaged cells. The TAT-S3 probe may have applications for the detection of diseases caused by reactive oxygen species.
Asunto(s)
ADN/análisis , Colorantes Fluorescentes , Guanina/análogos & derivados , Animales , Sitios de Unión , ADN/química , Daño del ADN , ADN Mitocondrial , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Citometría de Flujo , Colorantes Fluorescentes/síntesis química , Guanina/análisis , Guanina/metabolismo , Células HeLa , Humanos , Microscopía Confocal , Mitocondrias/patología , Unión Proteica , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Transactivadores/química , Pez CebraRESUMEN
A novel electrochemical biosensing method that can take into account both immunoassay and enzyme activity analysis was reported in this work for determination of the enzymatically active human apurinic/apyrimidinic endonuclease 1 (APE1). The basic principle is to design and construct a DNA catalytic hairpin assembly (CHA) triggered by APE1 catalysis in enzyme activity analysis, and the assembled DNAs are labeled with electrochemically active CdS and PbS quantum dots to output electrochemical signals. In this system, the signal generation needs to satisfy both the conditions of immunological recognition and enzymatic catalysis, providing a basis for accurate analysis of active APE1. Results show that this method can reflect the regulation of the enzyme activity and can also distinguish APE1 from its isozymes with the same enzyme activity. The concept and successful implementation of this integrated system will contribute to the research and application of APE1 in biomedicine, and provide a reference for the accurate analysis of other enzymes.
Asunto(s)
Anticuerpos Inmovilizados/química , Técnicas Biosensibles/métodos , ADN Catalítico/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , Puntos Cuánticos/química , Compuestos de Cadmio/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Pruebas de Enzimas/métodos , Humanos , Inmunoensayo/métodos , Plomo/química , Sulfuros/químicaRESUMEN
Mitochondria, the centers of energy production, are highly organized with inner membranes, cristae and outer membranes. The mitochondrial architecture determines their functions in all cellular processes. Changes in the mitochondrial ultrastructure are tightly related to a wide variety of diseases. MGARP, a mitochondria-localized protein, was predicted by bioinformatics and confirmed by cellular and biochemical methods to be located in mitochondria, but there is no direct and clear evidence for its precise location. This report demonstrates the precise ultrastructural location of MGARP within mitochondria by the ascorbate peroxidase 2 (APEX2) system in combination with electron microscopy (EM). EM revealed that more MGARP is located in the inner/cristae membranes, with its C-terminus at the inner faces of the intramembrane spaces, than in the outer membranes. MGARP overexpression caused both mitochondrial remodeling and cristae shaping, leading to the collapse of the mitochondrial network. The mitochondrial morphologies in MGARP-overexpressing cells were diverse; the cells became round or short, and their cristae were deformed and became discontinuous or circular. An engineered MGARP mutant deficient in its transmembrane domain no longer localized to the mitochondria and lost its effects on mitochondrial structure, confirming that the localization of MGARP in the mitochondria depends on its structural integrity. Collectively, our findings define the location of MGARP within the mitochondria, which is associated with its functional implications for the architecture and organization of mitochondria.
Asunto(s)
Proteínas de la Membrana/análisis , Membranas Mitocondriales/ultraestructura , Proteínas Mitocondriales/análisis , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , Endonucleasas/análisis , Células HeLa , Humanos , Microscopía Electrónica , Membranas Mitocondriales/química , Enzimas Multifuncionales/análisisRESUMEN
To analyze the expression trends and clinical significance of Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1/Ref-1) and Nucleophosmin (NPM1) proteins in high-grade serous ovarian adenocarcinoma (HGSC). The expressions of APE1/Ref-1 and NPM1 proteins in 94 patients with HGSC were determined using the immunohistochemical (IHC) method, and their relationships with clinicopathological features were analyzed by the χ2 test or Fisher's exact test. The follow-up data, Cox proportional hazards univariate and multivariate survival analyses were integrated to evaluate the prognostic factors affecting patients with HGSC. In the normal fallopian tubes, APE1/Ref-1 and NPM1 protein were mainly distributed in the nuclear. The HGSC experienced changes in the cellular localization of APE1/Ref-1 and NPM1 protein expressions, which were abnormally expressed in the cytoplasm. The rates of abnormal cytoplasmic expression of APE1/Ref-1 and NPM1 proteins in 94 patients with HGSC were 69.1% and 73.4%, respectively, which were significantly higher than the normal fallopian tube tissues (p < 0.05). The abnormal cytoplasmic APE1/Ref-1 and NPM1 are significantly correlated with the lymph node metastasis, chemosensitivity, FIGO staging, and prognosis. The COX multivariate survival analysis showed that the abnormal expression of APE1/Ref-1 protein, FIGO staging, and lymph node metastasis are independent prognostic factors. Collectively, the abnormal cytoplasmic APE1/Ref-1 and NPM1 proteins are associated with the oncogenic progression and chemoresistance of HGSC, and predict a poor prognosis.
Asunto(s)
Adenocarcinoma/diagnóstico , Adenocarcinoma/patología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , Proteínas Nucleares/análisis , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/patología , Adulto , Anciano , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Proteínas Nucleares/genética , Nucleofosmina , PronósticoRESUMEN
Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential protein crucial for repair of oxidized DNA damage not only in genomic DNA but also in mitochondrial DNA. Parkin, a tumor suppressor and Parkinson's disease (PD) associated gene, is an E3 ubiquitin ligase crucial for mitophagy. Although DNA damage is known to induce mitochondrial stress, Parkin's role in regulating DNA repair proteins has not been elucidated. In this study, we examined the possibility of Parkin-dependent ubiquitination of APE1. Ectopically expressed APE1 was degraded by Parkin and PINK1 via polyubiquitination in mouse embryonic fibroblast cells. PD-causing mutations in Parkin and PINK1 abrogated APE1 ubiquitination. Interaction of APE1 with Parkin was observed by co-immunoprecipitation, proximity ligation assay, and co-localization in the cytoplasm. N-terminal deletion of 41 amino acid residues in APE1 significantly reduced the Parkin-dependent APE1 degradation. These results suggested that Parkin directly ubiquitinated N-terminal Lys residues in APE1 in the cytoplasm. Modulation of Parkin and PINK1 activities under mitochondrial or oxidative stress caused moderate but statistically significant decrease of endogenous APE1 in human cell lines including SH-SY5Y, HEK293, and A549 cells. Analyses of glioblastoma tissues showed an inverse relation between the expression levels of APE1 and Parkin. These results suggest that degradation of endogenous APE1 by Parkin occur when cells are stressed to activate Parkin, and imply a role of Parkin in maintaining the quality of APE1, and loss of Parkin may contribute to elevated APE1 levels in glioblastoma. © 2016 Wiley Periodicals, Inc.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Células A549 , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , Células HEK293 , Humanos , Mapas de Interacción de Proteínas , Proteínas Quinasas/análisis , Proteínas Quinasas/metabolismo , Proteolisis , Ubiquitina-Proteína Ligasas/análisisRESUMEN
Human apurinic/apyrimidinic endonuclease/redox effector factor 1 (APE1) is an essential DNA repair protein. Herein, we demonstrate that avidin-oriented abasic site-containing DNA strands (AP-DNA) on the surface of silica coated magnetic nanoparticles (SiMNP) can selectively respond to APE1 while resist the digestion by other nucleases. Mechanism studies have revealed that avidin may serve as an organizer protein and recruit APE1 to the DNA substrates on the nanoparticles via strong and specific interactions. Taking advantage of this newly disclosed property, we for the first time successfully displayed the intracellular activities of APE1 in living cells by fluorescence imaging. The avidin organized AP-DNA-SiMNP assembly holds great potential for enzyme-mediated release of drugs inside tumor cells which often contain higher levels of APE1 than normal cells.
Asunto(s)
Sondas de ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , Nanopartículas del Metal , Avidina , División del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Desoxirribonucleasa I/análisis , Desoxirribonucleasa I/metabolismo , Células HeLa , Humanos , Imagen ÓpticaRESUMEN
To evaluate the expression of markers correlated with cellular senescence and DNA damage (8-hydroxy-2'-deoxy-guanosine (8-OHdG), p53, p21, APE1/Ref-1 (APE1), interleukin (IL-6 and IL-8) in placentas from healthy and pathologic pregnancies. This retrospective study considered a placental tissue micro-array containing 92 controls from different gestational ages and 158 pathological cases including preeclampsia (PE), HELLP syndrome (hemolysis, elevated liver enzymes, low platelet count), small for gestational age (SGA) fetuses, and intrauterine growth restriction (IUGR) occurring at different gestational ages. In this study, we demonstrated a significant influence of gestational age on the expression in the trophoblast of 8-OHdG, p53, p21, APE1, and IL-6. In placentas of cases affected by PE, HELLP, or IUGR, there was an increased expression of 8-OHdG, p53, APE1, and IL-6 compared to controls (only IL-8 was significantly decreased in cases). In both groups of pathology between 22- and 34-week gestation and after 34-week gestation, APE1 levels were higher in the trophoblast of women affected by hypertensive disorders of pregnancy than women carrying an IUGR fetus. The cytoplasmic expression of 8-OHdG was increased in placentas in IUGR cases compared to PE or HELLP pregnancies. In cases after 34-week gestation, p21 was higher in SGA and IUGR than in controls and late PE. Moreover, p53 was increased after 34-week gestation in IUGR pregnancies. Placentas from pathological pregnancies had an altered expression of 8-OHdG, p53, p21, APE1, IL-6, and IL-8. The alterations of intracellular pathways involving these elements may be the cause or the consequence of placental dysfunction, but in any case reflect an impaired placental function, possibly due to increased aging velocity in pathologic cases.
Asunto(s)
Senescencia Celular , Modelos Biológicos , Estrés Oxidativo , Placenta/metabolismo , Placenta/patología , Análisis de Matrices Tisulares , Adulto , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Femenino , Humanos , Inmunohistoquímica , Interleucina-6/análisis , Interleucina-6/metabolismo , Interleucina-8/análisis , Interleucina-8/metabolismo , Embarazo , Proteínas Proto-Oncogénicas p21(ras)/análisis , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Estudios Retrospectivos , Proteína p53 Supresora de Tumor/análisis , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
A novel DNA structure containing a 3' internal-loop modified abasic site has been constructed which enables effective differentiation between apurinic/apyrimidinic endonuclease (APE1) and nonspecific endonuclease (DNase I). When this unique substrate structure is employed, a double-loop frayed-end chimeric fluorescent probe is successfully developed for quantitative measurement of the activity of APE1 in biological samples without the need of additional cleanup or preconcentration steps. The method is simple and rapid and has a single-step with a linear working range between 0.1 and 5.0 U/mL and a lower limit of detection of 0.1 U/mL. It holds great potential in real-time monitoring of the variation of intracellular and extracellular APE1, which will be very useful for further understanding of the DNA repair pathways in different organisms.
Asunto(s)
Sondas de ADN/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , Pruebas de Enzimas/instrumentación , Pruebas de Enzimas/métodos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/sangre , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Colorantes Fluorescentes/química , Humanos , Estándares de Referencia , Factores de TiempoRESUMEN
Apurinic/apyrimidinic endonuclease 1 (APE1) is a DNA repair protein and plays other important roles. Increased levels of APE1 in cancer have been reported. However, available methods for measuring APE1 levels are indirect and not quantitative. We previously developed an approach using liquid chromatography and tandem mass spectrometry with isotope dilution to accurately measure APE1 levels. Here, we applied this methodology to measure APE1 levels in normal and cancerous human breast tissues. Extreme expression of APE1 in malignant tumors was observed, suggesting that breast cancer cells may require APE1 for survival. Accurate measurement of APE1 may be essential for the development of novel treatment strategies and APE1 inhibitors as anticancer drugs.
Asunto(s)
Neoplasias de la Mama/patología , Mama/patología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , Secuencia de Aminoácidos , Neoplasias de la Mama/química , Línea Celular Tumoral , Cromatografía Liquida , Femenino , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Espectrometría de Masas en TándemRESUMEN
The key role of DNA repair in removing DNA damage and minimizing mutations makes it an attractive target for cancer risk assessment and prevention. Here we describe the development of a robust assay for apurinic/apyrimidinic (AP) endonuclease 1 (APE1; APEX1), an essential enzyme involved in the repair of oxidative DNA damage. APE1 DNA repair enzymatic activity was measured in peripheral blood mononuclear cell protein extracts using a radioactivity-based assay, and its association with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean APE1 enzyme activity in case patients was 691 [95% confidence interval (CI) = 655-727] units/ng protein, significantly lower than in control subjects (mean = 793, 95% CI = 751-834 units/ng protein, P = 0.0006). The adjusted odds ratio for lung cancer associated with 1 SD (211 units) decrease in APE1 activity was 2.0 (95% CI = 1.3-3.1; P = 0.002). Comparison of radioactivity- and fluorescence-based assays showed that the two are equivalent, indicating no interference by the fluorescent tag. The APE1Asp148Glu SNP was associated neither with APE1 enzyme activity nor with lung cancer risk. Taken together, our results indicate that low APE1 activity is associated with lung cancer risk, consistent with the hypothesis that 'bad DNA repair', rather than 'bad luck', is involved in cancer etiology. Such assays may be useful, along with additional DNA repair biomarkers, for risk assessment of lung cancer and perhaps other cancers, and for selecting individuals to undergo early detection techniques such as low-dose CT.
Asunto(s)
Reparación del ADN/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/epidemiología , Estudios de Casos y Controles , Daño del ADN/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Femenino , Fluorescencia , Predisposición Genética a la Enfermedad , Humanos , Leucocitos Mononucleares/citología , Pulmón/enzimología , Pulmón/patología , Neoplasias Pulmonares/genética , Masculino , Polimorfismo de Nucleótido Simple , RiesgoRESUMEN
In this paper, the Au nanoparticles decorated graphene nanosheets (AuNPs/Gr) were prepared as nanocarriers using ionic liquid (IL) as linker reagent. Then the alkaline phosphatase (ALP) and the ferrocene tagged detection antibodies (Fc-Ab2) were loaded on the IL doped AuNPs/Gr as a trace label for ultrasensitive measurements of human apurinic/apyrimidinic endonuclease 1 (APE1), which is a multifunctional protein in the DNA base excision repair pathway relating to various types of cancer. Several labeling protocols were investigated for the determination of the APE1 protein concentration and improved analytical features were obtained with the proposed carriers of IL doped AuNPs/Gr which were labeled with Fc-Ab2 and ALP (ALP/Fc-Ab2/AuNPs/IL/Gr). The reason may be that the IL doped AuNPs/Gr carriers (AuNPs/IL/Gr) could not only enhance the immobilized amount of ALP and Fc-Ab2, but also promote the electron transfer rate. Thus, through the specific recognition of antigen-antibody, numerous ALP/Fc-Ab2/AuNPs/IL/Gr, which are captured onto every single immunocomplex, could further catalyze the ascorbic acid 2-phosphate (AA-p) reaction to amplify the electrochemical signal. Transmission electron microscopy (TEM) images of the AuNPs/IL/Gr nanocomposites revealed the formation of a functionalized surface network structure. The resulting immunosensor exhibited a linear response to APE1 in the concentration range of 0.1-80 pg mL(-1) with a detection limit of 0.04 pg mL(-1), indicating potential applications in clinical diagnostics.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/sangre , Técnicas Electroquímicas/instrumentación , Oro/química , Grafito/química , Inmunoensayo/instrumentación , Nanopartículas del Metal/química , Fosfatasa Alcalina/química , Anticuerpos Inmovilizados/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , Pruebas de Enzimas/instrumentación , Enzimas Inmovilizadas/química , Diseño de Equipo , Humanos , Límite de DetecciónRESUMEN
Apurinic/apyrimidinic endonuclease 1 (APE-1), a kind of multifunctional protein widely-distributed in the body, plays an essential role in the DNA base excision repair and serves as multiple possible roles in the response of human cancer to radiotherapy and chemotherapy. In this work, an ultrasensitive solid-state electrochemiluminescence (ECL) immunosensor is designed to determine APE-1 based on the new Ru(bpy)3(2+)/bi-arginine system. The bi-arginine (bi-Arg) is decorated on the Au nanoparticles functionalized magnetic Fe3O4/reduced graphene oxide (bi-Arg/Au@Fe3O4-rGO) according to the self-assembling and covalent cross-linking interaction to obtain the functionalized nanocomposite of bi-Arg/Au@Fe3O4-rGO. Herein, the bi-Arg/Au@Fe3O4-rGO plays not only an amplification label to enhance the ECL signal of Ru(bpy)3(2+) due to the coreactant of bi-Arg but also an ideal nanocarrier to load numerous secondary antibody. Based on sandwich-type immunoassay format, this proposed method offers a linear range of 1.0fgmL(-1)-5.0pgmL(-1) and an estimated detection limit of 0.3fgmL(-1) for the APE-1. Moreover, the reagentless ECL immunosensor also exhibits high sensitivity, excellent selectivity and good stability, which has greatly potential development and application in clinical diagnostics, immunology and biomedical research.
Asunto(s)
2,2'-Dipiridil/análogos & derivados , Arginina/química , Técnicas Biosensibles/métodos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , Técnicas Electroquímicas/métodos , Inmunoensayo/métodos , 2,2'-Dipiridil/química , Complejos de Coordinación , Oro/química , Grafito/química , Humanos , Límite de Detección , Mediciones Luminiscentes/métodos , Nanopartículas del Metal/química , Modelos MolecularesRESUMEN
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 1α, 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.