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1.
Cell Mol Life Sci ; 76(15): 2957-2966, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31143960

RESUMO

DNA modifications are a major form of epigenetic regulation that eukaryotic cells utilize in concert with histone modifications. While much work has been done elucidating the role of 5-methylcytosine over the past several decades, only recently has it been recognized that N(6)-methyladenine (N6-mA) is present in quantifiable and biologically active levels in the DNA of eukaryotic cells. Unlike prokaryotes which utilize N6-mA to recognize "self" from "foreign" DNA, eukaryotes have been found to use N6-mA in varying ways, from regulating transposable elements to gene regulation in response to hypoxia and stress. In this review, we examine the current state of the N6-mA in research field, and the current understanding of the biochemical mechanisms which deposit and remove N6-mA from the eukaryotic genome.


Assuntos
Adenina/análogos & derivados , Eucariotos/metabolismo , Adenina/metabolismo , Animais , Metilação de DNA , Enzimas Reparadoras do DNA/metabolismo , Epigenômica , Eucariotos/genética , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Oxirredutases N-Desmetilantes/metabolismo , Estresse Fisiológico
2.
Nat Commun ; 10(1): 2045, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053733

RESUMO

Long noncoding RNAs (lncRNAs) have emerged as new regulatory molecules implicated in diverse biological processes, including therapeutic resistance. However, the mechanisms underlying lncRNA-mediated temozolomide (TMZ) resistance in glioblastoma (GBM) remain largely unknown. To illustrate the role of lncRNA in TMZ resistance, we induce TMZ-resistant GBM cells, perform a lncRNA microarray of the parental and TMZ-resistant cells, and find an unreported lncRNA in GBM, lnc-TALC (temozolomide-associated lncRNA in glioblastoma recurrence), correlated with TMZ resistance via competitively binding miR-20b-3p to facilitate c-Met expression. A phosphorylated AKT/FOXO3 axis regulated lnc-TALC expression in TMZ-resistant GBM cells. Furthermore, lnc-TALC increased MGMT expression by mediating the acetylation of H3K9, H3K27 and H3K36 in MGMT promoter regions through the c-Met/Stat3/p300 axis. In clinical patients, lnc-TALC is required for TMZ resistance and GBM recurrence. Our results reveal that lnc-TALC in GBM could serve as a therapeutic target to overcome TMZ resistance, enhancing the clinical benefits of TMZ chemotherapy.


Assuntos
Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Glioblastoma/tratamento farmacológico , MicroRNAs/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , RNA Longo não Codificante/metabolismo , Temozolomida/farmacologia , Adulto , Animais , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Glioblastoma/genética , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Análise de Sobrevida , Temozolomida/uso terapêutico , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Eur J Med Chem ; 175: 107-113, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31077996

RESUMO

The DNA-repair enzyme MutT homolog 1 (MTH1) is a potential target for a broad range of tumors. Its substrate binding site features a non-catalytical pair of aspartic acids which resembles the catalytic dyad of aspartic proteases. We hypothesized that inhibitors of the latter might be re-targeted for MTH1 despite the two enzyme classes having different substrates and catalyze different reactions. We selected from the crystal structures of holo aspartic proteases a library of nearly 350 inhibitors for in silico screening. Three fragment hits were identified by docking and scoring according to a force field-based energy with continuum dielectric solvation. These fragments showed good ligand efficiency in a colorimetric assay (MW <300 Da and IC50<50µM). Molecular dynamics simulations were carried out for determining the most favorable interaction patterns. On the basis of the simulation results we evaluated in vitro seven commercially available compounds, two of which showed submicromolar potency for MTH1. To obtain definitive evidence of the predicted binding modes we solved the crystal structures of five of the 10 inhibitors predicted in silico. The final step of hit optimization was guided by protein crystallography and involved the synthesis of a single compound, the lead 11, which shows nanomolar affinity for MTH1 in two orthogonal binding assays, and selectivity higher than 2000-fold against its original target (BACE1). The high rate of fragment-hit identification and the fast optimization suggest that ligand retargeting by binding site analogy is an efficient strategy for drug design.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Enzimas Reparadoras do DNA/antagonistas & inibidores , Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Enzimas Reparadoras do DNA/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular , Monoéster Fosfórico Hidrolases/metabolismo , Reprodutibilidade dos Testes
4.
Science ; 364(6437): 286-289, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-31000663

RESUMO

CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Edição de Genes/métodos , Proteína Homóloga a MRE11/metabolismo , Análise de Sequência de DNA/métodos , Adenoviridae , Animais , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , DNA/química , DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas , Células K562 , Proteína Homóloga a MRE11/genética , RNA Guia
5.
Anticancer Res ; 39(4): 1839-1847, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30952724

RESUMO

BACKGROUND/AIM: Casticin shows anti-cancer effects in many types of cancer. However, there is no information regarding its role in DNA damage in human bladder cancer. The aim of this study was to investigate the effects of casticin on TSGH-8301 cells in vitro. MATERIALS AND METHODS: Viability of cells was assayed by flow cytometry. DNA damage was assayed by DAPI staining, comet assay, and gel electrophoresis. Protein levels were examined by western blotting and confocal laser microscopy. RESULTS: Casticin decreased viability of cells and induced DNA damage. Furthermore, casticin decreased expression of p-ATM, p-ATR, MDC1 and MGMT levels after 48 h of treatment, however, it increased p-ATR and MGMT levels after 12 h. In contrast, casticin increased the levels of p-p53, p-H2A.X, and PARP after 48 h of treatment. As shown by confocal microscopy, casticin affected the translocation of DNA-PKcs and p-p53 to the nucleus of TSGH-8301 cells. CONCLUSION: Casticin decreased viability of human bladder cancer cells through DNA damage.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Flavonoides/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Transporte Ativo do Núcleo Celular , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Histonas/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Fosforilação , Poli(ADP-Ribose) Polimerases/metabolismo , Transativadores/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia
6.
Int J Mol Sci ; 20(8)2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30999567

RESUMO

Ubiquitin-like/ubiquitin-associated proteins (UbL-UbA) are a well-studied family of non-proteasomal ubiquitin receptors that are evolutionarily conserved across species. Members of this non-homogenous family facilitate and support proteasomal activity by promoting different effects on proteostasis but exhibit diverse extra-proteasomal activities. Dysfunctional UbL-UbA proteins render cells, particularly neurons, more susceptible to stressors or aging and may cause earlier neurodegeneration. In this review, we summarized the properties and functions of UbL-UbA family members identified to date, with an emphasis on new findings obtained using Drosophila models showing a direct or indirect role in some neurodegenerative diseases.


Assuntos
Doenças Neurodegenerativas/metabolismo , Neurônios/patologia , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Drosophila , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteostase , Fatores de Transcrição/metabolismo
7.
Neuropathology ; 39(2): 78-84, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30937985

RESUMO

Adult thalamic glioblastomas (GBM) are uncommon tumors with limited available molecular data. One of the reported molecular alterations in these tumors is the H3K27M mutation. It has been documented that H3K27M mutation is found in a high proportion of pediatric thalamic gliomas. In this study, we have analyzed the molecular alterations exclusive to adult thalamic GBM. This is a 6 years retrospective study of adult thalamic GBM patients who underwent surgical decompression of the tumor. Clinical data were obtained from the case records. Immunohistochemistry (IHC) was performed on the tumors using antibodies directed against the gene products of R132H mutant isocitrate dehydrogenase 1 (IDH1), alpha-thalassemia/mental retardation X-linked (ATRX), p53, H3K27M, H3K27me3, and V600E mutant BRAF. Molecular analyses were carried out to detect other IDH1 and IDH2 mutations, O6 -methylguanine-DNA-methyltransferase gene (MGMT) promoter methylation, and epidermal growth factor gene (EGFR) and telomerase reverse transcriptase gene (TERT) promoter mutations. A total of 42 cases of adult thalamic GBM were studied. The mean age of presentation was 42 years with age range of 19-58 years. Male predominance was noted. All the tumors were IDH wild-type, BRAF (V600E)-immunonegative and unmethylated for MGMT promoter. H3K27M immunopositivity was noted in 60% of tumors. Of these 33.3% were from older adults above the age of 50 years. Of the H3K27M-immunopositive cases, ATRX loss of expression was seen in 32%, p53 immunopositivity in 24% and EGFR amplification in 12%. Higher frequency of TERT promoter mutations was noted in H3K27M-immunonegative cases (58.8%) compared to immunopositive cases (20%). Ours is one of the few studies elucidating the molecular alterations exclusive to adult thalamic GBM. We show a high frequency of H3K27M immunopositivity, suggestive of its mutational status in these tumors, including in older adults.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Histonas/metabolismo , Tálamo/metabolismo , Adulto , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Receptores ErbB/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Isocitrato Desidrogenase/metabolismo , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Telomerase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína Nuclear Ligada ao X/metabolismo , Adulto Jovem
8.
Anal Chim Acta ; 1060: 30-44, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-30902329

RESUMO

DNA repair pathways are closely associated with the maintenance of genomic integrity, disease outbreak, and the development of therapeutics. Owing to these significances, novel analytical methods for enzymes that are involved in the DNA repair pathways have been actively investigated. This review focuses on discussions on nucleic acid-based methods, especially those based on the fluorescence, for the determination of DNA repair enzymes. Furthermore, this review not only provides meaningful insights in creating ingenious fluorescent detection methods but it also suggests future directions that will be followed in developing new analytical technologies for the DNA repair enzymes.


Assuntos
Enzimas Reparadoras do DNA/análise , DNA/química , Fluorescência , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo
9.
Zhonghua Bing Li Xue Za Zhi ; 48(3): 186-191, 2019 Mar 08.
Artigo em Chinês | MEDLINE | ID: mdl-30831643

RESUMO

Objective: To investigate the prognostic impact of alterations of epidermal growth factor receptor(EGFR) and MGMT in glioblastoma. Methods: The retrospective study included 161 supratentorial glioblastomas diagnosed in the Department of Pathology, Xuanwu Hospital, Capital Medical University from 2009 to 2015. EGFR and EGFRvⅢ protein expression was detected by immunohistochemistry; EGFR amplification was detected by fluorescence in situ hybridization; MGMT promoter methylation was detected by pyrosequencing. The change of molecular genetics EGFR and MGMT and outcome were assessed statistically. Results: There were 161 patients, including 85 (52.8%) males and 76 (47.2%) females. The mean age was 53 years, and the median overall survival was 13 months. The integrated classification of glioblastoma included 16 IDH-mutant, 134 wild type, and 11 NOS. The rate of overexpression of EGFR protein was 32.9%(53/161), and that of EGFR amplification was 37.5%(18/48). There was high concordance between immunohistochemistry and FISH(85.4%, Kappa=0.475, P<0.01) and between the level of EGFR protein and EGFR amplification (P<0.01). Twelve cases showed EGFRvⅢ expression, and all also showed EGFR protein overexpression; 149 cases were EGFRv Ⅲ wild type, and EGFR protein overexpression was seen in 27.5%(41/149) of cases. There was no correlation between EGFR and EGFRv Ⅲ expression. Of all cases, 70.2%(106/151) showed MGMT promoter methylation by pyrosequencing. The changes of molecular genetics of EGFR and MGMT were not related. EGFR amplification and protein overexpression had no significant relationship with prognosis. Patients with EGFRv Ⅲ-mutant had shorter survival time than the EGFRv Ⅲ-wild type(P=0.014); patients with MGMT promoter methylation had better prognosis than without (PFS:P=0.002,OS:P=0.006),and MGMT promoter methylation was an independent predictor for overall survival (HR=0.269, 95%CI 0.124-0.583, P=0.001). Conclusions: EGFR protein expression by immunohistochemistry correlates with the status of EGFR amplification. Patients with EGFRv Ⅲ-mutant tumors have poorer prognosis than that with EGFRv Ⅲ-wild type tumors. MGMT promoter methylation is closely associated with prognosis and an independent predictor for overall survival.


Assuntos
Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Neoplasias Supratentoriais/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adulto , Biomarcadores Tumorais/metabolismo , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Receptores ErbB/genética , Feminino , Amplificação de Genes , Glioblastoma/genética , Humanos , Hibridização in Situ Fluorescente , Masculino , Metilação , Pessoa de Meia-Idade , Prognóstico , Regiões Promotoras Genéticas , Estudos Retrospectivos , Neoplasias Supratentoriais/genética , Proteínas Supressoras de Tumor/genética
10.
Nat Commun ; 10(1): 1242, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30886146

RESUMO

The ataxia-telangiectasia mutated (ATM) kinase, an upstream kinase of the DNA damage response (DDR), is rapidly activated following DNA damage, and phosphorylates its downstream targets to launch DDR signaling. However, the mechanism of ATM activation is still not completely understood. Here we report that UFM1 specific ligase 1 (UFL1), an ufmylation E3 ligase, is important for ATM activation. UFL1 is recruited to double strand breaks by the MRE11/RAD50/NBS1 complex, and monoufmylates histone H4 following DNA damage. Monoufmylated histone H4 is important for Suv39h1 and Tip60 recruitment. Furthermore, ATM phosphorylates UFL1 at serine 462, enhancing UFL1 E3 ligase activity and promoting ATM activation in a positive feedback loop. These findings reveal that ufmylation of histone H4 by UFL1 is an important step for amplification of ATM activation and maintenance of genomic integrity.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reparo do DNA , Histonas/metabolismo , Processamento de Proteína Pós-Traducional/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Lisina Acetiltransferase 5/metabolismo , Proteína Homóloga a MRE11/metabolismo , Metiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismo , Serina/metabolismo , Ubiquitina-Proteína Ligases/genética
11.
Chromosoma ; 128(1): 41-52, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30612150

RESUMO

Aurora-A is a conserved mitotic kinase overexpressed in many types of cancer. Growing evidence shows that Aurora-A plays a crucial role in DNA damage response (DDR) although this aspect has been less characterized. We isolated a new aur-A mutation, named aur-A949, in Drosophila, and we showed that it causes chromosome aberrations (CABs). In addition, aur-A949 mutants were sensitive to X-ray treatment and showed impaired γ-H2Av foci dissolution kinetics. To identify the pathway in which Aur-A works, we conducted an epistasis analysis by evaluating CAB frequencies in double mutants carrying aur-A949 mutation combined to mutations in genes related to DNA damage response (DDR). We found that mutations in tefu (ATM) and in the histone variant H2Av were epistatic over aur-A949 indicating that Aur-A works in DDR and that it is required for γ-H2Av foci dissolution. More interestingly, we found that a mutation in lig4, a gene belonging to the non-homologous end joining (NHEJ) repair pathway, was epistatic over aur-A949. Based on studies in other systems, which show that phosphorylation is important to target Lig4 for degradation, we hypothesized that in aur-A949 mutant cells, there is a persistence of Lig4 that could be, in the end, responsible for CABs. Finally, we observed a synergistic interaction between Aur-A and the homologous recombination (HR) repair system component Rad 51 in the process that converts chromatid deletions into isochromatid deletions. Altogether, these data indicate that Aur-A depletion can elicit chromosome damage. This conclusion should be taken into consideration, since some anticancer therapies are aimed at reducing Aurora-A expression.


Assuntos
Aurora Quinase A/genética , Cromossomos de Insetos/química , Reparo do DNA por Junção de Extremidades , Enzimas Reparadoras do DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Epistasia Genética , Animais , Aurora Quinase A/deficiência , Aberrações Cromossômicas/efeitos da radiação , Cromossomos de Insetos/efeitos da radiação , Dano ao DNA , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/efeitos da radiação , Feminino , Instabilidade Genômica , Histonas/genética , Histonas/metabolismo , Masculino , Mutação , Fosforilação/efeitos da radiação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise/efeitos da radiação , Raios X
12.
BMC Bioinformatics ; 20(1): 30, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30646838

RESUMO

BACKGROUND: Single-molecule localization microscopy is a super-resolution microscopy technique that allows for nanoscale determination of the localization and organization of proteins in biological samples. For biological interpretation of the data it is essential to extract quantitative information from the super-resolution data sets. Due to the complexity and size of these data sets flexible and user-friendly software is required. RESULTS: We developed SMoLR (Single Molecule Localization in R): a flexible framework that enables exploration and analysis of single-molecule localization data within the R programming environment. SMoLR is a package aimed at extracting, visualizing and analyzing quantitative information from localization data obtained by single-molecule microscopy. SMoLR is a platform not only to visualize nanoscale subcellular structures but additionally provides means to obtain statistical information about the distribution and localization of molecules within them. This can be done for individual images or SMoLR can be used to analyze a large set of super-resolution images at once. Additionally, we describe a method using SMoLR for image feature-based particle averaging, resulting in identification of common features among nanoscale structures. CONCLUSIONS: Embedded in the extensive R programming environment, SMoLR allows scientists to study the nanoscale organization of biomolecules in cells by extracting and visualizing quantitative information and hence provides insight in a wide-variety of different biological processes at the single-molecule level.


Assuntos
Gráficos por Computador , Enzimas Reparadoras do DNA/metabolismo , Microscopia de Fluorescência/métodos , Análise de Célula Única/métodos , Software , Algoritmos , Interpretação Estatística de Dados , Humanos
13.
Oncol Rep ; 41(3): 1851-1862, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30569141

RESUMO

Human MutT homolog 1 (MTH1) hydrolyses oxidised nucleotide triphosphates, thereby preventing them from being incorporated into DNA; MTH1 has been found to be elevated in many types of cancers, including lung, stomach cancer, melanoma and breast cancer. Thus, tumour­targeted hMTH1 may be valuable for developing novel anticancer therapies. In the present study, we prepared human MTH1 protein and its monoclonal antibody (mAb). The hMTH1 gene was cloned into the prokaryotic expression vector pET28a and optimally expressed in the E. coli Transetta (DE3) strain. Using an Ni­NTA column and a G­50 gel filtration column, 20.1 mg of active hMTH1 was obtained from 1,000 ml of bacterial culture, and the purity was over 98%, as detected by high­performance liquid chromatography (HPLC). The half maximal inhibitory concentration (IC50) of TH287 (hMTH1 inhibitor) was determined to be 3.53±0.47 nM using the recombinant hMTH1 protein (rhMTH1). The enzyme activity assay showed the Michaelis constant (Km) and the catalytic constant (kcat) of the protein were 106.13±48.83 µM and 3.64±0.58 sec­1, respectively. The anti­hMTH1 mAb was obtained via the hybridoma technique and validated by western blot analysis. In addition, an immunofluorescence assay (IFA) and ELISA determined that the mAb could efficiently bind to natural hMTH1 expressed on the human breast cancer cell line MCF­7. Taken together, the results showed the rhMTH1 is an active protein and has practical applications for inhibitor selection, and our prepared hMTH1 mAb will provide a valuable tool for the further characterisation of hMTH1 and antitumour medicinal development in future.


Assuntos
Anticorpos Monoclonais/imunologia , Enzimas Reparadoras do DNA/imunologia , Enzimas Reparadoras do DNA/metabolismo , Desenvolvimento de Medicamentos/métodos , Monoéster Fosfórico Hidrolases/imunologia , Monoéster Fosfórico Hidrolases/metabolismo , Anticorpos Monoclonais/isolamento & purificação , Linhagem Celular Tumoral , Clonagem Molecular , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/isolamento & purificação , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios Enzimáticos/métodos , Humanos , Oxirredução , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/isolamento & purificação , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
14.
Int J Oncol ; 54(1): 229-238, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30387839

RESUMO

Radiotherapy resistance in patient with non­small cell lung cancer (NSCLC) reduces patient survival and remains a significant challenge for the treatment of NSCLC. Radiation resistance has been demonstrated to be affected by secreted factors, yet it remains unclear how autocrine secretions affect the radioresistance of NSCLC cells. In the present study, the NSCLC cell line, NCI­H460, was irradiated with γ­rays (4 Gy) and then cultured in medium from H460 cells or normal medium to examine the potential influence of cell secretions on the radiation resistance of H460 cells. Cell viability, accumulation of reactive oxygen species and DNA repair capacity were all markedly improved in the irradiated H460 cells that were cultured in conditioned medium (CM), compared with those cells cultured in normal medium. In addition, G2/M cell cycle arrest and upregulation of homologous recombination repair proteins were observed in the CM­treated cells, while exosomes secreted by H460 cells had no influence on the radiation resistance of H460 cells. Taken together, these results indicate that autocrine secretions enhance the radiation resistance of γ­irradiated H460 cells and that these secretions mainly affect the DNA repair process.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Exossomos/metabolismo , Neoplasias Pulmonares/metabolismo , Tolerância a Radiação , Comunicação Autócrina , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Sobrevivência Celular , Meios de Cultivo Condicionados/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Neoplasias Pulmonares/radioterapia , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima
15.
Acta Biochim Biophys Sin (Shanghai) ; 51(2): 131-138, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30576408

RESUMO

Translin/TRAX complex, also named as C3PO, is evolutionarily conserved and participates in diverse cellular processes in different organisms from yeast to human. C3PO plays a critical role in the activation of RNA-induced silencing complexes by promoting the unwinding and degradation of passenger strand of exogenous siRNAs (exo-siRNAs) in Drosophila and human. Moreover, human C3PO (hC3PO) has been found to broadly repress miRNAs by degrading miRNA precursors. However, the effect of Drosophila melanogaster C3PO (dmC3PO) on endogenous siRNA (endo-siRNA) and miRNA pathways remains unknown. Here, we found that the loss of dmC3PO promoted the accumulation of the passenger strand of esi-2.1 (hp-CG4068B), and resulted in the de-repression of the DNA-damage-response gene mutagensensitive 308 (mus308), which is an endogenous slicer target of esi-2.1 in Drosophila. Moreover, we also found that depletion of dmC3PO increased the accumulation of miR-bantam. Taken together, our findings indicated that dmC3PO not only involves in siRNA pathway triggered by dsRNA, but also regulates the abundance of certain endogenous small RNAs in Drosophila.


Assuntos
Proteínas de Transporte/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Inativação Gênica , MicroRNAs/genética , RNA/genética , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Humanos
16.
Biochemistry ; 58(6): 561-574, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30570250

RESUMO

The nonbulky 5',8-cyclopurine DNA lesions (cP) and the bulky, benzo[ a]pyrene diol epoxide-derived stereoisomeric cis- and trans- N2-guanine adducts (BPDE-dG) are good substrates of the human nucleotide excision repair (NER) mechanism. These DNA lesions were embedded at the In or Out rotational settings near the dyad axis in nucleosome core particles reconstituted either with native histones extracted from HeLa cells (HeLa-NCP) or with recombinant histones (Rec-NCP). The cP lesions are completely resistant to NER in human HeLa cell extracts. The BPDE-dG adducts are also NER-resistant in Rec-NCPs but are good substrates of NER in HeLa-NCPs. The four BPDE-dG adduct samples are excised with different efficiencies in free DNA, but in HeLa-NCPs, the efficiencies are reduced by a common factor of 2.2 ± 0.2 relative to the NER efficiencies in free DNA. The NER response of the BPDE-dG adducts in HeLa-NCPs is not directly correlated with the observed differences in the thermodynamic destabilization of HeLa-NCPs, the Förster resonance energy transfer values, or hydroxyl radical footprint patterns and is weakly dependent on the rotational settings. These and other observations suggest that NER is initiated by the binding of the DNA damage-sensing NER factor XPC-RAD23B to a transiently opened BPDE-modified DNA sequence that corresponds to the known footprint of XPC-DNA-RAD23B complexes (≥30 bp). These observations are consistent with the hypothesis that post-translational modifications and the dimensions and properties of the DNA lesions are the major factors that have an impact on the dynamics and initiation of NER in nucleosomes.


Assuntos
Adutos de DNA/química , Dano ao DNA , Reparo do DNA , DNA/química , Nucleossomos/química , Purinas/química , Adutos de DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Nucleossomos/genética
17.
J Biochem ; 165(4): 317-322, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30535026

RESUMO

Radiotherapy is the major treatment modality for malignant glioma. However, the treatment response of radiotherapy is suboptimal due to resistance. Here we aimed to explore the effect and mechanism of Mothers against decapentaplegic homologue (SMAD3) silencing in sensitizing malignant glioma to radiotherapy. Clonogenic assay was used to evaluate the sensitivity of glioma cells to increasing doses of radiation. Glioma cells were transfected with small-interfering RNAs (siRNAs) specific to SMAD3. Overexpression of SMAD3 was achieved by transfecting expression plasmid encoding SMAD3 cDNA. Changes in MRE11-RAD50-NBS1 mRNA and protein levels were assessed through qPCR analysis and western blot analysis, respectively. Chromatin immunoprecipitation (ChIP) was used to confirm the interaction between SMAD3 and MRE11-RAD50-NBS1 (MRN) complex. Silencing of SMAD3 increased sensitivity of glioma cells to radiotherapy. MRE11, RAD50 and NBS1 were overexpressed in response to radiotherapy, which was attenuated by SMAD3 silencing while boosted by SMAD3 overexpression. ChIP analysis confirmed the interaction of SMAD3 with MRE11, RAD50 and NBS1 under radiotherapy, which was inhibited by SMAD3 silencing. SMAD3 silencing is an effective strategy for sensitizing glioma to radiotherapy, which is mediated by the interaction of SMAD3 with the MRN complex.


Assuntos
Neoplasias Encefálicas , Proteínas de Ciclo Celular , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA , Inativação Gênica , Glioblastoma , Proteína Homóloga a MRE11 , Complexos Multiproteicos , Proteínas de Neoplasias , Proteínas Nucleares , Proteína Smad3 , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/radioterapia , Humanos , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo
18.
Oncol Rep ; 41(2): 908-916, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30535433

RESUMO

Glioma originates from the glial cells of the spine or brain, and promoter methylation of O6­methylguanine­DNA methyltransferase (MGMT) can promote the chemosensitivity of glioma. The present study aimed to reveal the key genes implicated in MGMT promoter methylation in patients with glioma. RNA­sequencing data and methylation data for glioma were extracted from The Cancer Genome Atlas database. Following expression characteristic analysis and differential expression analysis using unsupervised hierarchical clustering and a rank sum test, the feature genes were identified between high and low methylation groups. Furthermore, multivariate survival analysis for the feature genes was performed using the survival package in R. Additionally, the independent glioma RNA expression datasets GSE7696 and GSE42669 were used to validate the prognostic efficiency of the gene combination. The results indicated that the prognosis of the low methylation group was significantly worse than that of the high methylation group. The ten genes corresponding to the cut­off value of 0.56 (Rho GTPase­activating protein 21, CECR2, histone acetyl­lysine reader, endosulfine α, G­patch domain­containing 8, KIAA1109, MGMT, protocadherin ß 13, selenoprotein M, sperm­associated antigen 9 and WD repeat domain 6) were able to significantly predict prognosis and were differentially expressed between the two groups. Multivariate survival analysis suggested that the ten genes were effective for sample classification and prognostic prediction. Furthermore, the validation datasets confirmed the correlation of the ten genes with prognosis. In conclusion, these 10 genes may be mediated by MGMT promoter methylation in glioma. In addition, the ten­gene combination may be associated with the prognosis of patients with glioma.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Glioma/genética , Regiões Promotoras Genéticas/genética , Proteínas Supressoras de Tumor/genética , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Biologia Computacional , Metilação de DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Conjuntos de Dados como Assunto , Perfilação da Expressão Gênica , Glioma/mortalidade , Glioma/patologia , Humanos , Análise de Sequência de RNA , Análise de Sobrevida , Proteínas Supressoras de Tumor/metabolismo
19.
Med Hypotheses ; 122: 22-25, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30593415

RESUMO

Aerobic exercise can delay aging and extend lifespan, but its specific mechanism still remains unclear. One popular theory is that with age and the cell division times increasing, DNA damage will inevitably accumulate, leading to dysfunction and failure of various tissues and organs, which will eventually lead to aging. Thus, repairing damaged DNA is a key strategy to extend lifespan. Excision repair cross-complementary gene 1 (ERCC1) is a DNA repair enzyme that recognizes, excises and repairs damaged DNA. Defects or reduced activity of the enzyme can lead to DNA damage accumulation. This study provides that aerobic exercise can significantly extend rats' lifespan and increase the expression of ERCC1 in heart, brain, liver and kidney. Therefore, based on our experiments, we propose the following scientific hypothesis: aerobic exercise can up-regulate the expression of ERCC1 and then may reduce DNA damage accumulation to maintain genomic integrity and stability, thereby delaying aging and prolonging lifespan in humans.


Assuntos
Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Exercício , Envelhecimento , Animais , Encéfalo/metabolismo , DNA/análise , Dano ao DNA , Reparo do DNA , Homeostase , Humanos , Rim/metabolismo , Fígado/metabolismo , Longevidade , Modelos Teóricos , Miocárdio/metabolismo , Condicionamento Físico Animal , RNA Mensageiro/metabolismo , Ratos , Distribuição Tecidual
20.
Int J Cancer ; 144(12): 3023-3030, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-30536544

RESUMO

Hypermutagenesis refers to marked increase in the number of mutations due to continuous mutagenic process. Hypermutated tumors, have being found in several tumor types, are associated with inherited or acquired alterations in the DNA repair pathways. Hypermutation has been observed in a subset of adult glioma patients as a direct result of temozolomide(TMZ)-induced mutagenesis. In our study, we have identified a rare subset of treatment-naïve adult gliomas with de novo hypermutator phenotype and explored the evolution of spontaneous and treatment-induced hypermutagenesis. We conducted Whole-Exome Sequencing (WES), Whole-Transcriptome Sequencing (WTS), and Single-Cell Sequencing (SCS) of TMZ-naïve and post-TMZ-treated hypermutated tumors to identify distinct clinical or genomic manifestations that contribute to the development of hypermutation in untreated adult gliomas. TMZ-naïve hypermutated tumors were marked by absence of IDH1 somatic mutation and MGMT promoter (pMGMT) methylation, two genomic traits that were significantly associated with the TMZ-induced hypermutagenic event in glioblastoma, and harbored inherited alterations in the mismatch repair (MMR) machinery. The immediate family members of the TMZ-naive hypermutated glioma patients were also previous diagnosed with cancer development history, suggesting that germline dysfunction of the MMR pathway could potentially pose hereditary risk to genetic predisposition of carcinogenesis in gliomas. Lastly, both TMZ-naïve and post-TMZ-treated hypermutated tumors exhibited a significant accumulation of neoantigen loads, suggesting immunotherapeutic alternatives. Our results present new and unique understanding of hypermutagenic process in adult gliomas and an important step towards clinical implication of immunotherapy in glioma treatment.


Assuntos
Neoplasias do Sistema Nervoso Central/genética , Reparo de Erro de Pareamento de DNA , Mutação em Linhagem Germinativa , Glioblastoma/genética , Adulto , Idoso , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Transformação Celular Neoplásica/genética , Neoplasias do Sistema Nervoso Central/metabolismo , Metilação de DNA , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Temozolomida/uso terapêutico , Transcriptoma , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Sequenciamento Completo do Exoma , Adulto Jovem
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