RESUMO
DNA glycosylases initiate the base excision repair (BER) pathway by catalyzing the removal of damaged or mismatched bases from DNA. The Arabidopsis DNA glycosylase methyl-CpG-binding domain protein 4 like (MBD4L) is a nuclear enzyme triggering BER in response to the genotoxic agents 5-fluorouracil and 5-bromouracil. To date, the involvement of MBD4L in plant physiological processes has not been analyzed. To address this, we studied the enzyme functions in seeds. We found that imbibition induced the MBD4L gene expression by generating two alternative transcripts, MBD4L.3 and MBD4L.4. Gene activation was stronger in aged than in non-aged seeds. Seeds from mbd4l-1 mutants displayed germination failures when maintained under control or ageing conditions, while 35S:MBD4L.3/mbd4l-1 and 35S:MBD4L.4/mbd4l-1 seeds reversed these phenotypes. Seed nuclear DNA repair, assessed by comet assays, was exacerbated in an MBD4L-dependent manner at 24 h post-imbibition. Under this condition, the BER genes ARP, APE1L, and LIG1 showed higher expression in 35S:MBD4L.3/mbd4l-1 and 35S:MBD4L.4/mbd4l-1 than in mbd4l-1 seeds, suggesting that these components could coordinate with MBD4L to repair damaged DNA bases in seeds. Interestingly, the ATM, ATR, BRCA1, RAD51, and WEE1 genes associated with the DNA damage response (DDR) pathway were activated in mbd4l-1, but not in 35S:MBD4L.3/mbd4l-1 or 35S:MBD4L.4/mbd4l-1 seeds. These results indicate that MBD4L is a key enzyme of a BER cascade that operates during seed imbibition, whose deficiency would cause genomic damage detected by DDR, generating a delay or reduction in germination.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , DNA Glicosilases , Reparo do DNA , Germinação , Sementes , Sementes/genética , Sementes/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , DNA Glicosilases/metabolismo , DNA Glicosilases/genética , Regulação da Expressão Gênica de Plantas , Dano ao DNARESUMO
Photobiomodulation (PBM) induced by non-ionizing radiations emitted from low-power lasers and light-emitting diodes (LEDs) has been used for various therapeutic purposes due to its molecular, cellular, and systemic effects. At the molecular level, experimental data have suggested that PBM modulates base excision repair (BER), which is responsible for restoring DNA damage. There is a relationship between the misfunction of the BER DNA repair pathway and the development of tumors, including breast cancer. However, the effects of PBM on cancer cells have been controversial. Breast cancer (BC) is the main public health problem in the world and is the most diagnosed type of cancer among women worldwide. Therefore, the evaluation of new strategies, such as PBM, could increase knowledge about BC and improve therapies against BC. Thus, this work aims to evaluate the effects of low-power red laser (658 nm) and blue LED (470 nm) on the mRNA levels from BER genes in human breast cancer cells. MCF-7 and MDA-MB-231 cells were irradiated with a low-power red laser (69 J cm-2, 0.77 W cm-2) and blue LED (482 J cm-2, 5.35 W cm-2), alone or in combination, and the relative mRNA levels of the APTX, PolB, and PCNA genes were assessed by reverse transcription-quantitative polymerase chain reaction. The results suggested that exposure to low-power red laser and blue LED decreased the mRNA levels from APTX, PolB, and PCNA genes in human breast cancer cells. Our research shows that photobiomodulation induced by low-power red laser and blue LED decreases the mRNA levels of repair genes from the base excision repair pathway in MCF-7 and MDA-MB-231 cells.
Assuntos
Neoplasias da Mama , Terapia com Luz de Baixa Intensidade , Humanos , Feminino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/radioterapia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Lasers , Reparo do DNA/genética , Terapia com Luz de Baixa Intensidade/métodosRESUMO
The functions of annexin A1 (ANXA1), which is expressed on membranes and in cytoplasmic granules, have been fully described. Nonetheless, the role of this protein in protecting against DNA damage in the nucleus is still emerging and requires further investigation. Here, we investigated the involvement of ANXA1 in the DNA damage response in placental cells. Placenta was collected from ANXA1 knockout mice (AnxA1-/-) and pregnant women with gestational diabetes mellitus (GDM). The placental morphology and ANXA1 expression, which are related to the modulation of cellular response markers in the presence of DNA damage, were analyzed. The total area of AnxA1-/- placenta was smaller due to a reduced labyrinth zone, enhanced DNA damage, and impaired base excision repair (BER) enzymes, which resulted in the induction of apoptosis in the labyrinthine and junctional layers. The placentas of pregnant women with GDM showed reduced expression of AnxA1 in the villous compartment, increased DNA damage, apoptosis, and a reduction of enzymes involved in the BER pathway. Our translational data provide valuable insights into the possible involvement of ANXA1 in the response of placental cells to oxidative DNA damage and represent an advancement in investigations into the mechanisms involved in placental biology.
Assuntos
Anexina A1 , Diabetes Gestacional , Camundongos , Animais , Gravidez , Humanos , Feminino , Placenta/metabolismo , Diabetes Gestacional/genética , Diabetes Gestacional/metabolismo , Anexina A1/metabolismo , Processamento de Proteína Pós-Traducional , Dano ao DNARESUMO
DNA glycosylases remove mispaired or modified bases from DNA initiating the base excision repair (BER) pathway. The DNA glycosylase MBD4 (methyl-CpG-binding domain protein 4) has been functionally characterized in mammals, but not yet in plants, where it is called MBD4-like (MBD4L). Mammalian MBD4 and Arabidopsis recombinant MBD4L excise U and T mispaired with G, as well as 5-fluorouracil (5-FU) and 5-bromouracil (5-BrU) in vitro. Here, we investigate the ability of Arabidopsis MBD4L to remove some of these substrates from the nuclear genome in vivo in coordination with uracil DNA glycosylase (AtUNG). We found that mbd4l mutants are hypersensitive to 5-FU and 5-BrU, as they displayed smaller size, less root growth, and higher cell death than control plants in both media. Using comet assays, we determined BER-associated DNA fragmentation in isolated nuclei and observed reduced DNA breaks in mbd4l plants under both conditions, but particularly with 5-BrU. The use of ung and ung x mbd4l mutants in these assays indicated that both MBD4L and AtUNG trigger nuclear DNA fragmentation in response to 5-FU. Consistently, we here report the nuclear localization of AtUNG based on the expression of AtUNG-GFP/RFP constructs in transgenic plants. Interestingly, MBD4L and AtUNG are transcriptionally coordinated but display not completely overlapping functions. MBD4L-deficient plants showed reduced expression of BER genes and enhanced expression of DNA damage response (DDR) gene markers. Overall, our findings indicate that Arabidopsis MBD4L is critical for maintaining nuclear genome integrity and preventing cell death under genotoxic stress conditions.
Assuntos
Arabidopsis , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , DNA/metabolismo , Dano ao DNA , Reparo do DNA/genética , Fluoruracila/metabolismo , Mamíferos/genética , Mamíferos/metabolismo , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismoRESUMO
The aim of this study was to evaluate photobiomodulation effects on mRNA relative levels from genes of base excision repair and genomic stabilization in heart tissue from an experimental model of acute lung injury by sepsis. For experimental procedure, animals were randomly assigned to six main groups: (1) control group was animals treated with intraperitoneal saline solution; (2) LASER-10 was animals treated with intraperitoneal saline solution and exposed to an infrared laser at 10 J cm-2; (3) LASER-20 was animals treated with intraperitoneal saline solution and exposed to an infrared laser at 20 J cm-2; (4) acute lung injury (ALI) was animals treated with intraperitoneal LPS (10 mg kg-1); (5) ALI-LASER10 was animals treated with intraperitoneal LPS (10 mg kg-1) and, after 4 h, exposed to an infrared laser at 10 J cm-2 and (6) ALI-LASER20 was animals treated with intraperitoneal LPS (10 mg kg-1) and, after 4 h, exposed to an infrared laser at 20 J cm-2. Irradiation was performed only once and animal euthanasias for analysis of mRNA relative levels by RT-qPCR. Our results showed that there was a reduction of mRNA relative levels from ATM gene and an increase of mRNA relative levels from P53 gene in the heart of animals with ALI when compared to the control group. In addition, there was an increase of mRNA relative levels from OGG1 and APE1 gene in hearts from animals with ALI when compared to the control group. After irradiation, an increase of mRNA relative levels from ATM and OGG1 gene was observed at 20 J cm-2. In conclusion, low-power laser modulates the mRNA relative levels from genes of base excision repair and genomic stabilization in the experimental model of acute lung injury evaluated.
Assuntos
Lesão Pulmonar Aguda , Lipopolissacarídeos , Lesão Pulmonar Aguda/genética , Animais , Reparo do DNA , Genômica , Lasers , Lipopolissacarídeos/farmacologia , Pulmão/efeitos da radiação , Modelos Teóricos , RNA Mensageiro/genética , Solução SalinaRESUMO
Pathogenic microbes are exposed to a number of potential DNA-damaging stimuli during interaction with the host immune system. Microbial survival in this situation depends on a fine balance between the maintenance of DNA integrity and the adaptability provided by mutations. In this study, we investigated the association of the DNA repair response with the virulence of Cryptococcus neoformans, a basidiomycete that causes life-threatening meningoencephalitis in immunocompromised individuals. We focused on the characterization of C. neoformansAPN1 and APN2 putative genes, aiming to evaluate a possible role of the predicted Apurinic/apyrimidinic (AP) endonucleases 1 and 2 of the base excision repair (BER) pathway on C. neoformans response to stress conditions and virulence. Our results demonstrated the involvement of the putative AP-endonucleases Apn1 and Apn2 in the cellular response to DNA damage induced by alkylation and by UV radiation, in melanin production, in tolerance to drugs and in virulence of C. neoformans in vivo. We also pointed out the potential use of DNA repair inhibitor methoxy-amine in combination with conventional antifungal drugs, for the development of new therapeutic approaches against this human fungal pathogen. This work provides new information about the DNA damage response of the highly important pathogenic fungus C. neoformans.
RESUMO
Epithelial ovarian cancer is a serious public health problem worldwide with the highest mortality rate of all gynecologic cancers. The current standard-of-care for the treatment of ovarian cancer is based on chemotherapy based on adjuvant cisplatin/carboplatin and taxane regimens that represent the first-line agents for patients with advanced disease. The DNA repair activity of cancer cells determines the efficacy of anticancer drugs. These features make DNA repair mechanisms a promising target for novel cancer treatments. In this context a better understanding of the DNA damage response caused by antitumor agents has provided the basis for the use of DNA repair inhibitors to improve the therapeutic use of DNA-damaging drugs. In this review, we will discuss the functions of DNA repair proteins and the advances in targeting DNA repair pathways with special emphasis in the inhibition of HRR and BER in ovarian cancer. We focused in the actual efforts in the development and clinical use of poly (ADPribose) polymerase (PARP) inhibitors for the intervention of BRCA1/BRCA2-deficient ovarian tumors. The clinical development of PARP inhibitors in ovarian cancer patients with germline BRCA1/2 mutations and sporadic high-grade serous ovarian cancer is ongoing. Some phase II and phase III trials have been completed with promising results for ovarian cancer patients.
Assuntos
Antineoplásicos/farmacologia , Reparo do DNA/efeitos dos fármacos , Terapia de Alvo Molecular , Neoplasias Ovarianas , Antineoplásicos/uso terapêutico , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêuticoRESUMO
Accumulation of oxidative mitochondrial DNA (mtDNA) damage and impaired base excision repair (BER) in brains have been associated with Alzheimer's disease (AD). However, it is still not clear how these affect mtDNA stability, as reported levels of mtDNA mutations in AD are conflicting. Thus, we investigated whether alterations in BER correlate with mtDNA instability in AD using postmortem brain samples from cognitively normal AD subjects and individuals who show neuropathological features of AD, but remained cognitively normal (high-pathology control). To date, no data on DNA repair and mtDNA stability are available for these individuals. BER activities, mtDNA mutations, and mtDNA copy number were measured in the nuclear and mitochondrial extracts. Significantly lower uracil DNA glycosylase activity was detected in nuclear and mitochondrial extracts from AD subjects, while apurinic/apyrimidinic endonuclease activity was similar in all groups. Although mtDNA mutation frequency was similar in all groups, mtDNA copy number was significantly decreased in the temporal cortex of AD brains but not of high-pathology control subjects. Our results show that lower mitochondrial uracil DNA glycosylase activity does not result in increased mutagenesis, but rather in depletion of mtDNA in early-affected brain regions during AD development.
Assuntos
Doença de Alzheimer/genética , Encéfalo/metabolismo , Reparo do DNA/genética , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Feminino , Dosagem de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Estresse Oxidativo/genética , Lobo Temporal/metabolismo , Uracila-DNA Glicosidase/metabolismoRESUMO
Trypanosoma cruzi, the causative agent of Chagas' disease survives to DNA damage generated by ROS/RNS inside to their different hosts. In recent eukaryotes, oxidative DNA damage is repaired mainly by the Base Excision Repair (BER) pathway, being essential the apurinic/apyrimidinic endonuclease activity. Using a pTREX-gfp vector, the nucleotide sequence that encodes T. cruzi AP endonuclease TcAP1 (orthologue of human APE1) and a putative TcAP1 dominant negative (TcAP1DN), were transfectedand expressed in T. cruzi epimastigotes. TcAP1-GFP and TcAP1DN-GFP were expressed in those modified epimastigotes and found in the parasite nucleus. The endonucleases were purified under native conditions and the AP endonuclease activity was evaluated. While TcAP1 presents the expected AP endonuclease activity TcAP1DN does not. Moreover, TcAP1DN partially inhibits in vitro TcAP1 enzymatic activity. Transfected epimastigotes expressing TcAP1-GFP and TcAP1DN-GFP were differentiated to infective trypomastigotes. The infective parasites maintained both proteins (TcAP1-GFP and TcAP1DN-GFP) in the nucleus. The overexpression of TcAP1-GFP in epimastigotes and trypomastigotes increases the viability of both parasite forms when exposed to oxidative stress while the expression of TcAP1DN-GFP did not show any in vivo inhibitory effect, suggesting that endogenous TcAP1 constitutive expression overcomes the TcAP1DN inhibitory activity. Our results show that TcAP1 is important for trypomastigote survival under oxidative conditions similar to those found in infected mammalian cells, then increasing its permanence in the infected cells and the possibility of development of Chagas disease.
Assuntos
Doença de Chagas/patologia , Dano ao DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Estresse Oxidativo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismo , Sequência de Aminoácidos , Doença de Chagas/genética , Doença de Chagas/parasitologia , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Humanos , Estágios do Ciclo de Vida , Mutação , Oxirredução , Proteínas de Protozoários/genética , Homologia de Sequência , Trypanosoma cruzi/genéticaRESUMO
Plant mitochondrial and chloroplast genomes encode essential proteins for oxidative phosphorylation and photosynthesis. For proper cellular function, plant organelles must ensure genome integrity. Although plant organelles repair damaged DNA using the multi-enzyme Base Excision Repair (BER) pathway, the details of this pathway in plant organelles are largely unknown. The initial enzymatic steps in BER produce a 5'-deoxyribose phosphate (5'-dRP) moiety that must be removed to allow DNA ligation and in plant organelles, the enzymes responsible for the removal of a 5'-dRP group are unknown. In metazoans, DNA polymerases (DNAPs) remove the 5'-dRP moiety using their intrinsic lyase and/or strand-displacement activities during short or long-patch BER sub-pathways, respectively. The plant model Arabidopsis thaliana encodes two family-A DNAPs paralogs, AtPolIA and AtPolIB, which are the sole DNAPs in plant organelles identified to date. Herein we demonstrate that both AtPolIs present 5'-dRP lyase activities. AtPolIB performs efficient strand-displacement on a BER-associated 1-nt gap DNA substrate, whereas AtPolIA exhibits only moderate strand-displacement activity. Both lyase and strand-displacement activities are dependent on an amino acid insertion that is exclusively present in plant organellar DNAPs. Within this insertion, we identified that residue AtPollB-Lys593 acts as nucleophile for lyase activity. Our results demonstrate that AtPolIs are functionally equipped to play a role in short-patch BER and suggest a major role of AtPolIB in a predicted long-patch BER sub-pathway. We propose that the acquisition of insertion 1 in the polymerization domain of AtPolIs was a key component in their evolution as BER associated and replicative DNAPs.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Domínio Catalítico , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Dano ao DNA , DNA de Cloroplastos/metabolismo , DNA Mitocondrial/metabolismo , DNA de Plantas/metabolismo , DNA Polimerase Dirigida por DNA/química , Fósforo-Oxigênio Liases/metabolismo , Alinhamento de SequênciaRESUMO
Autophagy and DNA repair are biological processes vital for cellular homeostasis maintenance and when dysfunctional, they lead to several human disorders including premature aging, neurodegenerative diseases, and cancer. The interchange between these pathways is complex and it may occur in both directions. Autophagy is activated in response to several DNA lesions types and it can regulate different mechanisms and molecules involved in DNA damage response (DDR), such as cell cycle checkpoints, cell death, and DNA repair. Thus, autophagy may modulate DNA repair pathways, the main focus of this review. In addition to the already well-documented autophagy positive effects on homologous recombination (HR), autophagy has also been implicated with other DNA repair mechanisms, such as base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). Given the relevance of these cellular processes, the clinical applications of drugs targeting this autophagy-DNA repair interface emerge as potential therapeutic strategies for many diseases, especially cancer.
Assuntos
Autofagia/fisiologia , Reparo do DNA/fisiologia , Animais , Autofagia/genética , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA por Junção de Extremidades/fisiologia , Reparo do DNA/genética , Recombinação Homóloga/genética , Recombinação Homóloga/fisiologia , HumanosRESUMO
Colorectal cancer (CRC) is prevalent worldwide, and treatment often involves surgery and genotoxic chemotherapy. DNA repair mechanisms, such as base excision repair (BER) and mismatch repair (MMR), may not only influence tumour characteristics and prognosis but also dictate chemotherapy response. Defective MMR contributes to chemoresistance in colorectal cancer. Moreover, BER affects cellular survival by repairing genotoxic base damage in a process that itself can disrupt metabolism. In this study, we characterized BER and MMR gene expression in colorectal tumours and the association between this repair profile with patients' clinical and pathological features. In addition, we exploited the possible mechanisms underlying the association between altered DNA repair, metabolism and response to chemotherapy. Seventy pairs of sporadic colorectal tumour samples and adjacent non-tumour mucosal specimens were assessed for BER and MMR gene and protein expression and their association with pathological and clinical features. MMR-deficient colon cancer cells (HCT116) transiently overexpressing MPG or XRCC1 were treated with 5-FU or TMZ and evaluated for viability and metabolic intermediate levels. Increase in BER gene and protein expression is associated with more aggressive tumour features and poor pathological outcomes in CRC. However, tumours with reduced MMR gene expression also displayed low MPG, OGG1 and PARP1 expression. Imbalancing BER by overexpression of MPG, but not XRCC1, sensitises MMR-deficient colon cancer cells to 5-FU and TMZ and leads to ATP depletion and lactate accumulation. MPG overexpression alters DNA repair and metabolism and is a potential strategy to overcome 5-FU chemotherapeutic resistance in MMR-deficient CRC.
RESUMO
Reactive species play an important role in physiological functions. Overproduction of reactive species, notably reactive oxygen (ROS) and nitrogen (RNS) species along with the failure of balance by the body's antioxidant enzyme systems results in destruction of cellular structures, lipids, proteins, and genetic materials such as DNA and RNA. Moreover, the effects of reactive species on mitochondria and their metabolic processes eventually cause a rise in ROS/RNS levels, leading to oxidation of mitochondrial proteins, lipids, and DNA. Oxidative stress has been considered to be linked to the etiology of many diseases, including neurodegenerative diseases (NDDs) such as Alzheimer diseases, Amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Multiple sclerosis, and Parkinson's diseases. In addition, oxidative stress causing protein misfold may turn to other NDDs include Creutzfeldt-Jakob disease, Bovine Spongiform Encephalopathy, Kuru, Gerstmann-Straussler-Scheinker syndrome, and Fatal Familial Insomnia. An overview of the oxidative stress and mitochondrial dysfunction-linked NDDs has been summarized in this review.
Assuntos
Doenças Mitocondriais/etiologia , Doenças Neurodegenerativas/complicações , Estresse Oxidativo/fisiologia , Animais , HumanosRESUMO
PURPOSE: We examined the influence of OGG1 c.977C>G (rs1052133), APEX1 c.444T>G (rs1130409), XRCC1 c.-77T>C (rs3213245), c.580C>T (rs1799782), c.839G>A (rs25489) and c.1196G>A (rs25487) single-nucleotide polymorphisms (SNPs), involved in base-excision repair (BER) pathway, on oropharyngeal squamous cell carcinoma (OPSCC) risk and prognosis. METHODS: Aiming to identify the genotypes, DNA from 200 consecutive OPSCC patients and 200 controls was analyzed by PCR-RFLP. The prognostic impact of genotypes of SNPs on progression-free survival (PFS) and overall survival of OPSCC patients was examined using the Kaplan-Meier estimates and Cox regression analyses. RESULTS: XRCC1 c.580CT or TT genotypes (19.5 vs. 11.0 %, P = 0.04) and XRCC1 TTGG haplotype from c.-77T>C, c.580C>T, c.839G>A and c.1196G>A SNPs (17.5 vs. 10.0 %, P = 0.04) were more common in patients with OPSCC than in controls. Carriers of combined genotypes of c.580C>T and TTGG haplotype of XRCC1 gene were under 3.35- and 3.22-fold increased risk of OPSCC than others. For survival analysis, we selected only patients with tumor at stage IV. The median follow-up time was 24.5 months. At 24 months of follow-up, PFS was shorter in patients with OGG1 c.977CC genotype when compared with others genotypes (35.5 vs. 52.1 %, log-rank test, P = 0.03). After multivariate Cox analysis, patients with OGG1 c.977CC genotype had more chance to present tumor progression when compared with others (HR 1.68, P = 0.02). CONCLUSIONS: Our data present, for the first time, evidence that inherited OGG1 c.977C>G; XRCC1 c.-77T>C, c.580C>T, c.839G>A and c.1196G>A abnormalities of DNA BER pathway are important determinants of OPSCC and predictors of patient outcomes.
Assuntos
Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/genética , DNA Glicosilases/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Neoplasias Orofaríngeas/diagnóstico , Neoplasias Orofaríngeas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Prognóstico , Fatores de Risco , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.
Assuntos
Dano ao DNA/genética , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Escherichia coli/genética , Leishmania braziliensis/genética , Mutação/genética , Sequência de Aminoácidos , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Dados de Sequência MolecularRESUMO
Temozolomide (TMZ) is widely used for patients with glioblastoma (GBM); however, tumor cells frequently exhibit drug-resistance. Base excision repair (BER) has been identified as a possible mediator of TMZ resistance, and an attractive approach to sensitizing cells to chemotherapy. Human apurinic/apyrimidinic endonuclease/redox factor-1 (APE1) is an essential enzyme with a role in the BER pathway by repairing abasic sites, and it also acts as a reduction factor, maintaining transcription factors in an active reduced state. Thus, we aimed to investigate whether the down-regulation of APE1 expression by siRNA can interfere with the resistance of GBM to TMZ, being evaluated by several cellular and molecular parameters. We demonstrated that APE1 knockdown associated with TMZ treatment efficiently reduced cell proliferation and clonogenic survival of resistant cells (T98G), which appears to be a consequence of increased DNA damage, S-phase arrest, and H2AX phosphorylation, resulting in apoptosis induction. On the contrary, for those assays, the sensitization effects of APE1 silencing plus TMZ treatment did not occur in the TMZ-sensitive cell line (U87MG). Interestingly, TMZ-treatment and APE1 knockdown significantly reduced cell invasion in both cell lines, but TMZ alone did not reduce the invasion capacity of U87MG cells, as observed for T98G. We also found that VEGF expression was down-regulated by TMZ treatment in T98G cells, regardless of APE1 knockdown, but U87MG showed a different response, since APE1 silencing counteracted VEGF induction promoted by TMZ, suggesting that the APE1-redox function may play an indirect role, depending on the cell line. The present results support the contribution of BER in the GBM resistance to TMZ, with a greater effect in TMZ-resistant, compared with TMZ-sensitive cells, emphasizing that APE1 can be a promising target for modifying TMZ tolerance. Furthermore, genetic characteristics of tumor cells should be considered as critical information to select an appropriate therapeutic strategy.
Assuntos
Antineoplásicos Alquilantes/farmacologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Dacarbazina/análogos & derivados , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/antagonistas & inibidores , Dacarbazina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Glioblastoma/tratamento farmacológico , Humanos , RNA Interferente Pequeno/metabolismo , TemozolomidaRESUMO
Most human tissues used in research are of post mortem origin. This is the case for all brain samples, and due to the difficulty in obtaining a good number of samples, especially in the case of neurodegenerative diseases, male and female samples are often included in the same experimental group. However, the effects of post mortem interval (PMI) and gender differences in the endpoints being analyzed are not always fully understood, as is the case for DNA repair activities. To investigate these effects, in a controlled genetic background, base excision repair (BER) activities were measured in protein extracts obtained from Wistar rat brains from different genders and defined PMI up to 24 hours, using a novel fluorescent-based in vitro incision assay. Uracil and AP-site incision activity in nuclear and mitochondrial extracts were similar in all groups included in this study. Our results show that gender and PMI up to 24 hours have no influence in the activities of the BER proteins UDG and APE1 in rat brains. These findings demonstrate that these variables do not interfere on the BER activities included in these study, and provide a security window to work with UDG and APE1 proteins in samples of post mortem origin.
Assuntos
Encéfalo/metabolismo , Reparo do DNA , Mudanças Depois da Morte , Caracteres Sexuais , Animais , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Feminino , Humanos , Masculino , Ratos , Fatores de TempoRESUMO
Tobacco smoke and air pollutants contain carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and tobacco specific nitrosamines (TSNA), that are substrates of metabolizing enzymes generating reactive metabolites that can bind to DNA. Variation in the activity of these enzymes may modify the extent to which these metabolites can interact with DNA. We compared the levels of bulky DNA adducts in blood leukocytes from 93 volunteers living in Mexico City with the presence of 13 single nucleotide polymorphisms (SNPs) in genes related to PAH and TSNA metabolism (AhR rs2044853, CYP1A1 rs1048943, CYP1A1 rs1048943, CYP1A1 rs1799814, EPHX1 rs1051740, EPHX1 rs2234922, GSTM1 null, GSTT1 null and GSTP1 rs947894), DNA repair (XRCC1 rs25487, ERCC2 rs13181 and MGMT rs12917) and cell cycle (TP53 rs1042522). (32)P-postlabeling analysis was used to quantify bulky DNA adduct formation. Genotyping was performed using PCR-RFLP. The mean levels of bulky DNA adducts were 8.51±3.66 adducts/10(8) nucleotides (nt) in smokers and 8.38±3.59 adducts/10(8) nt in non-smokers, being the difference not statistically significant. Without taking into account the smoking status, GSTM1 null individuals had a marginally significant lower adduct levels compared with GSTM1 volunteers (p=0.0433) and individuals heterozygous for MGMT Leu/Phe had a higher level of bulky adducts than those who were homozygous wild type (p=0.0170). A multiple regression analysis model showed a significant association between the GSTM1 (deletion) and MGMT rs12917 (Phe/Phe) haplotype and the formation of DNA adducts in smokers (R(2)=0.2401, p=0.0215). The presence of these variants conferred a greater risk for higher adduct levels in this Mexican population.
Assuntos
Adutos de DNA/sangue , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Glutationa Transferase/genética , Haplótipos , Leucócitos/química , Proteínas Supressoras de Tumor/genética , Feminino , Humanos , Masculino , Polimorfismo de Nucleotídeo ÚnicoRESUMO
O cloreto estanoso (SnCI2) e a radiação ultravioleta A (UVA) são agentes que lesam diversas estruturas celulares, inclusive o DNA, principalmente pela geração de espécies reativas de oxigênio. O objetivo deste trabalho foi estudar a mutagênese e o reparo das lesões produzidas pela combinação do UVA, na condição de pré-iluminação, com o SnCI2. Avaliou-se a ação de enzimas do reparo por excisão de bases (BER), em Escherichia coli (E. coli), por eletroforese em gel alcalino de agarose e sobrevivência bacteriana. Também se estudou a indução do sistema SoxRS pelo cromoteste, e a mutagênese pelo teste de Ames. De acordo com os resultados: i)o UVA induziu quebras no DNA das cepas testadas e os mutantes fpg-nfo e fpg apresentaram maior retardo no reparo das lesões; ii) o SnCI2 induziu mais quebras que o UVA e os mutantes nfo e fpg mostraram maior dificuldade em reparar as lesões; iii) o UVA+SnCI2 provocou mais quebras que o SnCI2 e os mutantes nfo e fpg também apresentaram maior lentidão no reparo das lesões; iv) o UVA não inativou as cepas testadas; v) as cepas nfo e fpg foram as mais sensíveis ao SnCI2; vi) o UVA+SnCI2 provocou maior letalidade em todas as cepas testadas, em relação ao SnCI2, e os mutantes nfo e fpg também foram os mais sensíveis ao tratamento com ambos os agentes; vii) a transformação dos mutantes nfo com o plasmídio pBW21 (nfo+) e dos mutantes fpg com o plasmídio pFPG (fpg+) aumentou a sobrevivência das cepas aos tratamentos com SnCI2 e UVA+SnCI2; viii) o SnCI2 induziu o sistema SoxRS; ix) o SnCI2, UVA e UVA+SnCI2 não induziram mutagênese; x) o reparo das lesões parece ser preferencialmente realizado pelas proteínas Fpg e Nfo.
Stannous chloride (SnCI2) and ultraviolet radiation A (UVA) are able to induce lesions in different cellular structures, including DNA, manly through ROS generation. The aim of this work was to study the mutagenesis and repair of lesions induced by the association of UVA (pre treatment) with SnCI2. It was evaluated the action of base excision repair (BER) enzymes in Escherichia coli (E. coli) by alkaline gel electrophoresis and bacterial survival. It was also evaluated the SoxRS system induction by chromotest and mutagenesis through the Ames test. According to the results: i) UVA induced DNA strand breaks in all strains and fpg-nfo and fpg mutants showed greater delay in the repair of lesions; ii) SnCI2 induced more breaks than UVA and nfo and fpg mutants showed more difficult to repair the damage; iii) UVA + SnCI2 caused more breaks than the SnCI2 and nfo and fpg mutants also showed a slowest repair of injuries; iv) UVA did not inactivate any bacterial strains tested; v) nfo and fpg strains were more sensitive to SnCI2; vi) UVA + SnCI2 caused higher mortality in all strains tested, when compared to SnCI2, and, again, nfo and fpg mutants were the most sensitives to the treatment with both agents; vii) the transformation of nfo mutant with the plasmid pBW21 (nfo+) and fpg mutants with plasmid pFPG (fpg+) increased the survival of the strains to SnCI2 and UVA + SnCI2 treatments; viii) SnCI2 was able to induce SoxRS system; ix) SnCI2, UVA + SnCI2 and UVA did not induce mutagenesis; x) damage repair seems to be preferentially performed by Fpg and Nfo proteins.
Assuntos
Humanos , Masculino , Feminino , Compostos de Estanho/farmacologia , Compostos de Estanho/toxicidade , Dano ao DNA/genética , Enzimas Reparadoras do DNA/genética , Escherichia coli , Escherichia coli/efeitos da radiação , Escherichia coli/genética , Reparo do DNA/genética , Testes de Mutagenicidade/métodos , Raios Ultravioleta , Recombinação GenéticaRESUMO
Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl2) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl2 in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl2 was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms.
Espécies reativas de oxigênio (ERO) podem induzir lesões em diferentes alvos celulares, incluindo o DNA. O cloreto estanoso (SnCl2) é um gerador de ERO que induz letalidade em E. coli, sendo o reparo por excisão de bases (BER) um mecanismo importante neste processo. Técnicas como o ensaio cometa (em eucariotos) e a eletroforese de DNA plasmidial em gel de agarose têm sido utilizadas para detectar genotoxicidade. No presente estudo, uma adaptação do método de eletroforese em gel alcalino de agarose foi usada para verificar a indução de quebras, pelo SnCl2, no DNA de E. coli, bem como a participação de enzimas do BER na restauração das lesões. Os resultados mostraram que o SnCl2 induziu quebras no DNA de todas as cepas testadas. Além disso, endonuclease IV e exonuclease III estão envolvidas na reparação dos danos. Em resumo, os dados obtidos indicam que a metodologia de eletroforese em gel alcalino de agarose pode ser empregada tanto para o estudo de quebras no DNA, quanto para avaliação dos mecanismos de reparação associados.