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Journal of Peking University(Health Sciences) ›› 2019, Vol. 51 ›› Issue (3): 487-492. doi: 10.19723/j.issn.1671-167X.2019.03.016

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Fluorescence assay for the detection of apurinic/apyrimidinic endonuclease 1 (APE1) activity in human blood samples

Jia-yu WANG,Mei-ping ZHAO△()   

  1. Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
  • Received:2019-03-13 Online:2019-05-22 Published:2019-06-26
  • Supported by:
    Supported by the National Natural Science Foundation of China (81571130100), the Fundamental Research Funds for the Central Universities: Peking University Medicine Seed Fund for Interdisciplinary Research (BMU2017MC008), Innovation Fund of Sunan Institute for Molecular Engineering, Peking University (SIM2018002848)

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Abstract: 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.

Key words: Spectrometry, fluorescence, Apurinic/apyrimidinic endonuclease 1, DNA probes, Serum, Peripheral blood mononuclear cells

CLC Number: 

  • R446.1

Figure 1

A, Fluorescence responses of the APE1 probe (200 nmol/L) to APE1 (2.0 U/mL) in different reaction buffers. NEB buffer 4+0.04% Triton X-100 is the new reaction buffer developed in this work; B, Fluorescence responses of the APE1 probe (300 nmol/L) to APE1 at different concentrations in the range from 0.01 to 2.0 U/mL in the new reaction buffer; C, Fluorescence responses of the APE1 probe (300 nmol/L) to APE1 at different concentrations in the range from 0.005 to 0.1 U/mL in the new reaction buffer; D, Linear working range of the APE1 probe in the new reaction buffer"

Figure 2

A, Isolation of PBMCs by density gradient centrifugation; B, Comparison of the APE1 concentration in human serum determined by different dilution folds PBMC, peripheral blood mononuclear cells."

[1] Li M , Wilson DM 3rd. Human apurinic/apyrimidinic endo-nuclease 1[J]. Antioxid Redox Signal, 2014,20(4):678-707.
doi: 10.1089/ars.2013.5492
[2] Whitaker AM, Freudenthal BD . APE1: A skilled nucleic acid surgeon[J]. DNA Repair (Amst), 2018,71:93-100.
doi: 10.1016/j.dnarep.2018.08.012
[3] Traversi D, Cervella P, Gilli G . Evaluating the genotoxicity of urban PM2.5 using PCR-based methods in human lung cells and the Salmonella TA98 reverse test[J]. Environ Sci Pollut Res, 2015,22(2):1279-1289.
doi: 10.1007/s11356-014-3435-1
[4] Simeonov A, Kulkarni A, Dorjsuren D , et al. Identification and characterization of inhibitors of human apurinic/apyrimidinic endonuclease APE1[J]. PLoS One, 2009,4(6):e5740.
doi: 10.1371/journal.pone.0005740
[5] Han J, Zhuo Y, Chai Y , et al. Ultrasensitive electrochemical strategy for trace detection of APE-1 via triple signal amplification strategy[J]. Biosens Bioelectron, 2013,41:116-122.
doi: 10.1016/j.bios.2012.07.082
[6] Kreklau EL, Limp-Foster M, Liu N , et al. A novel fluorometric oligonucleotide assay to measure O 6-methylguanine DNA methyltransferase, methylpurine DNA glycosylase, 8-oxoguanine DNA glycosylase and abasic endonuclease activities: DNA repair status in human breast carcinoma cells overexpressing methylpurine DNA glycosylase [J]. Nucleic Acids Res, 2001,29(12):2558-2566.
doi: 10.1093/nar/29.12.2558
[7] Zhang S, He L, Dai N , et al. Serum APE1 as a predictive marker for platinum-based chemotherapy of non-small cell lung cancer patients[J]. Oncotarget, 2016,7(47):77482-77494.
[8] Kirkali G, Jaruga P, Reddy P . Identification and quantification of DNA repair protein apurinic/apyrimidinic endonuclease 1 (APE1) in human cells by liquid chromatography/isotope-dilution tandem mass spectrometry[J]. PLoS One, 2013,8(7):e69894.
doi: 10.1371/journal.pone.0069894
[9] Coskun E, Jaruga P, Reddy P . Extreme expression of DNA repair protein apurinic/apyrimidinic endonuclease 1 (APE1) in human breast cancer as measured by liquid chromatography and isotope dilution tandem mass spectrometry[J]. Biochemistry, 2015,54(38):5787-5790.
doi: 10.1021/acs.biochem.5b00928
[10] Fang S, Chen L, Zhao M . Unimolecular chemically modified DNA fluorescent probe for one-step quantitative measurement of the activity of human apurinic/apyrimidinic endonuclease 1 in biological samples[J]. Anal Chem, 2015,87(24):11952-11956.
doi: 10.1021/acs.analchem.5b03939
[11] Greening DW, Simpson RJ . Characterization of the low-molecular-weight human plasma peptidome[J]. Methods Mol Biol, 2017,1619:63-79.
doi: 10.1007/978-1-4939-7057-5
[12] Wang D, Zhuo JQ, Zhao MP . A simple and rapid competitive enzyme-linked immunosorbent assay (cELISA) for high-throughput measurement of secretory immunoglobulin A (sIgA) in saliva[J]. Talanta, 2010,82(1):432-436.
doi: 10.1016/j.talanta.2010.04.040
[13] Li XJ, Mei X, Huang YF , et al. Simple label-free fluorescence detection of apurinic/apyrimidinic endonuclease 1 activity and its inhibitor using the abasic site-binding fluorophore[J]. Anal Methods, 2019,11(6):739-743.
doi: 10.1039/C8AY02633E
[14] Evans AR, Limp-Foster M, Kelley MR . Going APE over ref-1[J]. Mutat Res, 2000,461(2):83-108.
doi: 10.1016/S0921-8777(00)00046-X
[15] Antoniali G, Malfatti MC, Tell G . Unveiling the non-repair face of the base excision repair pathway in RNA processing: A missing link between DNA repair and gene expression?[J]. DNA Repair, 2017,56:65-74.
doi: 10.1016/j.dnarep.2017.06.008
[16] Kelley MR, Cheng L, Foster R , et al. Elevated and altered expression of the multifunctional DNA base excision repair and redox enzyme Ape1/ref-1 in prostate cancer[J]. Clin Cancer Res, 2001,7(4):824-830.
[17] Wang D, Xiang DB, Yang XQ , et al. APE1 overexpression is associated with cisplatin resistance in non-small cell lung cancer and targeted inhibition of APE1 enhances the activity of cisplatin in A549 cells[J]. Lung Cancer, 2009,66(3):298-304.
doi: 10.1016/j.lungcan.2009.02.019
[18] Moore DH, Michael H, Tritt R , et al. Alterations in the expression of the DNA repair/redox enzyme APE/ref-1 in epithelial ovarian cancers[J]. Clin Cancer Res, 2000,6(2):602-609.
[19] Shin JH, Choi S, Lee YR , et al. APE1/Ref-1 as a serological biomarker for the detection of bladder cancer[J]. Cancer Res Treat, 2015,47(4):823-833.
doi: 10.4143/crt.2014.074
[20] Dai N, Cao XJ, Li MX , et al. Serum APE1 autoantibodies: a novel potential tumor marker and predictor of chemotherapeutic efficacy in non-small cell lung cancer[J]. PLoS One, 2013,8(3):e58001.
doi: 10.1371/journal.pone.0058001
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