A robust luminescent assay for screening alkyladenine DNA glycosylase inhibitors to overcome DNA repair and temozolomide drug resistance / 药物分析学报

Temozolomide(TMZ)is an anticancer agent used to treat glioblastoma,typically following radiation therapy and/or surgical resection.However,despite its effectiveness,at least 50％of patients do not respond to TMZ,which is associated with repair and/or tolerance of TMZ-induced DNA lesions.Studies have demonstrated that alkyladenine DNA glycosylase(AAG),an enzyme that triggers the base excision repair(BER)pathway by excising TMZ-induced N3-methyladenine(3meA)and N7-methylguanine le-sions,is overexpressed in glioblastoma tissues compared to normal tissues.Therefore,it is essential to develop a rapid and efficient screening method for AAG inhibitors to overcome TMZ resistance in glio-blastomas.Herein,we report a robust time-resolved photoluminescence platform for identifying AAG inhibitors with improved sensitivity compared to conventional steady-state spectroscopic methods.As a proof-of-concept,this assay was used to screen 1440 food and drug administration-approved drugs against AAG,resulting in the repurposing of sunitinib as a potential AAG inhibitor.Sunitinib restored glioblastoma(GBM)cancer cell sensitivity to TMZ,inhibited GBM cell proliferation and stem cell char-acteristics,and induced GBM cell cycle arrest.Overall,this strategy offers a new method for the rapid identification of small-molecule inhibitors of BER enzyme activities that can prevent false negatives due to a fluorescent background.

Use of uracil-DNA glycosylase enzyme to reduce DNA-related artifacts from formalin-fixed and paraffin-embedded tissues in diagnostic routine

Background: Detection of somatic mutations is a mandatory practice for therapeutic definition in precision oncology. However, somatic mutation detection protocols use DNA from formalin-fixed and paraffin-embedded (FFPE) tumor tissues, which can result in detection of nonreproducible sequence artifacts, especially C:G > T:A transitions, in DNA. In recent studies, DNA pretreatment with uracil DNA glycosylase (UDG), an enzyme involved in base excision repair, significantly reduced the number of DNA artifacts after mutation detection by next-generation sequencing (NGS) and other methods, without affecting the capacity to detect real mutations. This study aimed to evaluate the effects of UDG enzymatic pretreatment in reducing the number of DNA sequencing artifacts from FFPE tumor samples, to improve the accuracy of genetic testing in the molecular diagnostic routine. Methods: We selected 12 FFPE tumor samples (10 melanoma, 1 lung, and 1 colorectal tumor sample) with different storage times. We compared sequencing results of a 16-hotspot gene panel of NGS libraries prepared with UDG-treated and untreated samples. Results: All UDG-treated samples showed large reductions in the total number of transitions (medium reduction of 80%) and the transition/transversion ratio (medium reduction of 75%). In addition, most sequence artifacts presented a low variant allele frequency (VAF < 10%) which are eliminated with UDG treatment. Conclusion: Including UDG enzymatic treatment before multiplex amplification in the NGS workflow significantly decreased the number of artifactual variants detected in FFPE samples. Thus, including this additional step in the current methodology should improve the rate of true mutation detection in the molecular diagnostic routine.

Effect of sumoylation on the structure and activity of human thymine DNA glycosylase / 上海交通大学学报（医学版）

Objective: To study the effect of sumoylation on the structure, stability and activity of human thymine DNA glycosylase (TDG). Methods: Expression and purification systems were established for obtaining SUMO-1-TDG protein with high purity which can be used for crystal screening and activity detection. Structure of SUMO-1-TDG was solved after crystal screening, diffraction data collection and structure analysis. The change of TDG stability led by sumoylation was detected through a protein thermal shift assay. In addition, an activity assay was applied to investigate the effect of sumoylation on the activity of TDG. Results: A high-resolution structure of SUMO-1-TDG which could clearly describe the interaction between TDG and SUMO-1 was solved. The melting temperature (Tm) value of SUMO-1-TDG increased by about 16℃ and the catalytic activity increased by 9.70%, comparing with TDG protein. Conclusion: SUMO-1 binds to TDG to modify the intermolecular interaction of amino acids near the binding site, and further participates in the regulation of the stability and catalytic activity of TDG protein.

Effect of sumoylation on the structure and activity of human thymine DNA glycosylase / 上海交通大学学报（医学版）

Objective·To study the effect of sumoylation on the structure,stability and activity of human thymine DNA glycosylase (TDG).Methods·Expression and purification systems were established for obtaining SUMO-1-TDG protein with high purity which can be used for crystal screening and activity detection.Structure of SUMO-1-TDG was solved after crystal screening,diffraction data collection and structure analysis.The change of TDG stability led by sumoylation was detected through a protein thermal shift assay.In addition,an activity assay was applied to investigate the effect of sumoylation on the activity of TDG.Results·A high-resolution structure of SUMO-1-TDG which could clearly describe the interaction between TDG and SUMO-1 was solved.The melting temperature ? value of SUMO-1-TDG increased by about 16 ℃ and the catalytic activity increased by 9.70％,comparing with TDG protein.Conclusion·SUMO-1 binds to TDG to modify the intermolecular interaction of amino acids near the binding site,and further participates in the regulation of the stability and catalytic activity of TDG protein.

Genetic polymorphisms of hOGG1 in the 5'-flank region in population of radon exposure area in Guangdong Province / 中国职业医学

OBJECTIVE: To screen genetic polymorphisms in the 5'-flank region of 8-hydroxyguanine DNA glycosylase( hOGG1) gene and analyze the characteristics of their genetic distribution in Han population of radon exposure area in Guangdong Province. METHODS: A simple random sampling method was used to select 60 subjects as radon exposure population. The genomic DNA samples were extracted from peripheral blood. The single nucleotide at-1721 nt-+ 164 nt locus of hOGG1 were screened using polymerase chain reaction( PCR) amplification,purification and direct sequencing for polymorphisms. Genetic characteristics of single nucleotide polymorphisms( SNP) in the study population were analyzed and compared with different populations reported in Hap Map data. RESULTS: The 5'-flank region of hOGG1 were amplified and sequenced in these 60 individuals( 120 chromosomes) of healthy Han Chinese in radon exposure area. Eight SNPs were identified by sequence alignment in the study population. Among them,there was 3 known polymorphisms and their minor allele frequencies( MAF) were-1493 G > A( 4. 2%),-834 G > C( 0. 8%) and-18 G > T( 3. 3%),respectively. The MAF of other 5 novel variations were-1455 G > A( 0. 8%),-1293 A > T( 23. 3%),-1187 C > A( 7. 5%),-337 C > A( 36. 7%) and-323 G > A( 0. 8%),respectively. The differences in the MAF distribution of-1493 G > A between the study population and the Hap Map-CEU were statistically significant( P < 0. 05). CONCLUSION: Eight SNPs and their genetic characteristics were screened and identified in the 5'-flank region of hOGG1 of Han Chinese population in radon exposure area. This result provides a basis for construction of polymorphism haplotypes and functional analysis for the target population.

Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination

Here, we describe a uracil-DNA glycosylase (UNG)-treated reverse transcription loop-mediated isothermal amplification (uRT-LAMP) for the visual detection of all subtypes of avian influenza A virus (AIV). The uRT-LAMP assay can prevent unwanted amplification by carryover contamination of the previously amplified DNA, although the detection limit of the uRT-LAMP assay is 10-fold lower than that of the RT-LAMP without a UNG treatment. To the best of our knowledge, this is the first successful application of deoxyuridine triphosphate/UNG strategy in RT-LAMP for AIV detection, and the assay can be applied for the rapid, and reliable diagnosis of AIVs, even in contaminated samples.

DNA damage and repair gene N-methylpurine DNA glycosylase expression in human osteosarcoma / 第三军医大学学报

Objective To determine N-methylpurine DNA glycosylase (MPG) expression in human osteosarcoma and analyze its association with clinicopathology and prognosis, to construct adenoviral vector of MPG and study its ability to sensitize human osteosarcoma cell HOS to DNA damage agents. Methods The MPG expression was determined by immunohistochemical staining in 60 specimens and 3 cell lines (HOS, SAOS-2, U2OS) of human osteosarcoma. High expression group and low expression group were divided according to their staining intensity, and their association with clinicopathological data and prognosis was analyzed statistically with SAS software. The adenoviral infection and MPG expression, as well as enzyme activity were determined by flow cytometry, Western blotting, and HEX labeled oligonucleotide-based assay respectively. The cell survival/proliferation was measured using MTS, SRB, and thymidine incorporation assay. Apoptotic cells were assayed by flow cytometry after treated with phycoerythin (PE)-conjugated Annexin V and 7-amino-actinomycin (7-AAD). Results The high expression of MPG was found in 40 cases (67%), while low expression in three cell lines of osteosarcoma. MPG expression was significantly associated with WHO histological classification and prognosis by single factor regression analysis. The recombinant adenovirus of 10 MOI was found to infect more than 90% of HOS cells within 24 h by EGFP fluorescence, in which the MPG overexpression and MPG enzyme activity were also detected. The HOS cells with MPG overexpression were more sensitive to the DNA damage agents including MMS, MNNG, and TMZ, and the IC_(50) of three DNA damage agents decreased by 6.0, 4.5, and 2.5 fold respectively. Conclusion Transient MPG overexpression is a potential therapeutic approach for increasing HOS cellular sensitivity to DNA damage agent in chemotherapy.

Expression of the DNA Repair Gene, N-Methylpurine-DNA Glycosylase in Astrocytic Tumors

OBJECTIVE: This study is designed to investigate the association of tumorigenesis with DNA repair gene, N-methylpurine-DNA-glycosylase(MPG) in astrocytic tumors. METHODS: MPG mRNA expression and localization in the 30 astrocytic tumors and 7 tumor-adjacent brain tissues was examined by reverse transcriptase-polymerase chain reaction(RT-PCR) and RNA in situ hybridization. Expression and intracellular localization of MPG protein was determined by immunohistochemistry. Statistical analysis was performed by ANOVA with a p value<0.05 considered statistically significant. RESULTS: MPG mRNA expression in RT-PCR was significantly higher in grade IV tumor tissues than in brain tissues adjacent to tumor or in grade II-III astrocytic tumor tissues(p<0.05). MPG mRNA in in situ hybridization was detected both in brain tissues adjacent to tumor and in astrocytic tumor tissues, regardless of the tumor grades. However, MPG protein localization in immunohistochemical study was detected only in the nucleus of all tumor tissues. In brain tissues adjacent to tumor, immunohistochemical study for MPG was not stained both in the nucleus and in cytoplasm. CONCLUSION: These results suggest MPG's role in human astrocytic tumors and raise the possibility that the increased mRNA level and intracellular localization could be associated with astrocytic tumorigenesis. Further studies about control of MPG gene expression in astrocytic tumors are warranted.

Study on the Effect of Residual Uracil DNA Glycosylase upon HCV cDNA / 中国医师杂志

To explore the destructive effect of residual uracil DNA glycosylase on dUTP incorporation to the cDNA PCR products,PAGE and DNA-EIA hybridization technique were applied to detect the efficiency of anti-contamination and the rate of DNA hybridization.In this study,it was found that:⑴Good effect could be achieved with the reaction of 2u of uracil DNA glycosylase(UDG)for 20min at 37℃.⑵UDG could be inactivated at 94℃ for 10min.The hybridization rate of control group of PCR products was 94 18% at room temperature for 65 hours.The hybridization rates of test groups at-20℃ for 65 hours were 87 69%,77 24%,76 83%,respectively.And the inactivation times of anti-contamination groups at 94℃ were“0”min,“10”min and “20”min,respectively;at room temperature for 65 hours,these hybridization rates were 74 77%,72 50%,70 29%,respectively.There were significant differences between control and rest groups(P0 05).Our study suggested that the hybridization rate of test groups kept at-20℃ decreased 19 41%,after 65 hours,the rate decreased 27 45%,but in control group only decreased 5 82% for 65h at room temperature.So that,we conclude that the residual UDP can cause above results.