RESUMO
BACKGROUND/OBJECTIVES: 8-Hydroxydeoxyguanosine (8-OHdG) is an indicator of oxidative stress and causes transversion mutations and carcinogenesis. 8-OHdG is excision repaired by 8-OHdG DNA glycosylase 1 (OGG1), which is classified as nuclear and mitochondrial subtypes. We aimed to clarify the role of OGG1 in pancreatic ductal adenocarcinoma (PDAC). METHODS: Ninety-two patients with PDAC who had undergone surgical resection at multiple institutions were immunohistochemically analyzed. The OGG1 and 8-OHdG expression levels were scored using the Germann Immunoreactive Score. The cutoff values of OGG1, as well as that of 8-OHdG, were determined. RESULTS: The low nuclear OGG1 expression group (n = 41) showed significantly higher carbohydrate antigen (CA)19-9 (p = 0.026), and higher s-pancreas antigen (SPAN)-1 (p = 0.017) than the high expression group (n = 51). Nuclear OGG1 expression has no effect on the prognosis. The low mitochondrial OGG1 expression group (n = 40) showed higher CA19-9 (p = 0.041), higher SPAN-1 (p = 0.032), and more histological perineural invasion (p = 0.037) than the high expression group (n = 52). The low mitochondrial OGG1 expression group had a significantly shorter recurrence-free survival (p = 0.0080) and overall survival (p = 0.0073) rates. The Cox proportional hazards model revealed that low mitochondrial OGG1 expression is an independent risk factor of the PDAC prognosis. OGG1 expression was negatively correlated with 8-OHdG expression (p = 0.0004), and high 8-OHdG expression shortened the recurrence-free survival of patients with PDAC. CONCLUSIONS: Low mitochondrial OGG1 expression might aggravate the PDAC prognosis.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , DNA Glicosilases/biossíntese , Mitocôndrias/metabolismo , Neoplasias Pancreáticas/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Adulto , Antígeno CA-19-9 , Carcinoma Ductal Pancreático/cirurgia , Núcleo Celular/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/cirurgia , Prognóstico , Estudos Retrospectivos , Fatores de Risco , Análise de SobrevidaRESUMO
Human Mpv17-like protein (M-LPH) has been suggested to participate in prevention of mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) damage. To clarify the molecular mechanism of M-LPH function, we knocked out M-LPH in human hepatoma HepG2 using CRISPR-Cas9 technology. An increase in mtDNA damage in M-LPH-KO HepG2 cells was demonstrated by PCR-based quantitation and 8-hydroxy-2'-deoxyguanosine (8-OHdG) measurement. Furthermore, confocal immunofluorescence analysis and Western blot analysis of mitochondrial extracts demonstrated that M-LPH-KO caused reductions in the protein levels of mitochondrial transcription factor A (TFAM), an essential factor for transcription and maintenance of mtDNA, and two DNA repair enzymes, 8-oxoguanine DNA glycosylase (OGG1) and DNA ligase 3 (LIG3), both involved in mitochondrial base excision repair (BER). Accordingly, it was suggested that the increase in mtDNA damage was due to a cumulative effect of mtDNA instability resulting from deficiencies of TFAM and diminished ability for BER arising from deficiencies in BER-related enzymes. These findings suggest that M-LPH could be involved in the maintenance of mtDNA, and therefore mitochondrial function, by protecting proteins essential for mtDNA stability and maintenance, in an integrated manner.
Assuntos
Dano ao DNA/fisiologia , DNA Glicosilases/biossíntese , DNA Ligase Dependente de ATP/biossíntese , DNA Mitocondrial , Proteínas de Ligação a DNA/biossíntese , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/biossíntese , Proteínas de Ligação a Poli-ADP-Ribose/biossíntese , Fatores de Transcrição/biossíntese , Técnicas de Inativação de Genes , Células Hep G2 , HumanosRESUMO
DNA lesions caused by UV damage are thought to be repaired solely by the nucleotide excision repair (NER) pathway in human cells. Patients carrying mutations within genes functioning in this pathway display a range of pathologies, including an increased susceptibility to cancer, premature aging, and neurological defects. There are currently no curative therapies available. Here we performed a high-throughput chemical screen for agents that could alleviate the cellular sensitivity of NER-deficient cells to UV-induced DNA damage. This led to the identification of the clinically approved anti-diabetic drug acetohexamide, which promoted clearance of UV-induced DNA damage without the accumulation of chromosomal aberrations, hence promoting cellular survival. Acetohexamide exerted this protective function by antagonizing expression of the DNA glycosylase, MUTYH. Together, our data reveal the existence of an NER-independent mechanism to remove UV-induced DNA damage and prevent cell death.
Assuntos
Dano ao DNA , DNA Glicosilases/metabolismo , Reparo do DNA/efeitos da radiação , Raios Ultravioleta , Acetoexamida/farmacologia , Linhagem Celular Tumoral , DNA Glicosilases/biossíntese , DNA Glicosilases/genética , Reparo do DNA/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Humanos , MasculinoRESUMO
It is unknown whether DNA damage accumulation is an upstream instigator or secondary effect of the cell death process in different populations of adult postmitotic neurons in the central nervous system. In two different mouse models of injury-induced neurodegeneration characterized by relatively synchronous accumulation of mitochondria, oxidative stress, and DNA damage prior to neuronal apoptosis, we enforced the expression of human 8-oxoguanine DNA glycosylase (hOGG1) and human apurinic-apyrimidinic endonuclease-1/Ref1 (hAPE) using recombinant adenoviruses (Ad). Thalamic lateral geniculate neurons and lumbar spinal cord motor neurons were transduced by Ad-hOGG1 and Ad-hAPE injections into the occipital cortex and skeletal muscle, respectively, prior to their target deprivation- and axotomy-induced retrograde apoptosis. Enforced expression of hOGG1 and hAPE in thalamus and spinal cord was confirmed by western blotting and immunohistochemistry. In injured populations of neurons in thalamus and spinal cord, a DNA damage response (DDR) was registered, as shown by localization of phospho-activated p53, Rad17, and replication protein A-32 immunoreactivities, and this DDR was attenuated more effectively by enforced hAPE expression than by hOGG1 expression. Enforced expression of hOGG1 and hAPE significantly protected thalamic neurons and motor neurons from retrograde apoptosis induced by target deprivation and axotomy. We conclude that a DDR response is engaged pre-apoptotically in different types of injured mature CNS neurons and that DNA repair enzymes can regulate the survival of retrogradely dying neurons, suggesting that DNA damage and activation of DDR are upstream mechanisms for this form of adult neurodegeneration in vivo, thus identifying DNA repair as a therapeutic target for neuroprotection.
Assuntos
Adenoviridae , Apoptose , DNA Glicosilases/biossíntese , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/biossíntese , Neurônios Motores/metabolismo , Doenças Neurodegenerativas/metabolismo , Transdução Genética , Animais , Axotomia , DNA Glicosilases/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/terapia , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
A large percentage of redox-responsive gene promoters contain evolutionarily conserved guanine-rich clusters; guanines are the bases most susceptible to oxidative modification(s). Consequently, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most abundant base lesions in promoters and is primarily repaired via the 8-oxoguanine DNA glycosylase-1 (OOG1)-initiated base excision repair pathway. In view of a prompt cellular response to oxidative challenge, we hypothesized that the 8-oxoG lesion and the cognate repair protein OGG1 are utilized in transcriptional gene activation. Here, we document TNFα-induced enrichment of both 8-oxoG and OGG1 in promoters of pro-inflammatory genes, which precedes interaction of NF-κB with its DNA-binding motif. OGG1 bound to 8-oxoG upstream from the NF-κB motif increased its DNA occupancy by promoting an on-rate of both homodimeric and heterodimeric forms of NF-κB. OGG1 depletion decreased both NF-κB binding and gene expression, whereas Nei-like glycosylase-1 and -2 had a marginal effect. These results are the first to document a novel paradigm wherein the DNA repair protein OGG1 bound to its substrate is coupled to DNA occupancy of NF-κB and functions in epigenetic regulation of gene expression.
Assuntos
DNA Glicosilases/biossíntese , Epigênese Genética , Regulação Enzimológica da Expressão Gênica , Guanina/análogos & derivados , NF-kappa B/metabolismo , Elementos de Resposta , Animais , DNA Glicosilases/genética , Reparo do DNA , Guanina/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , NF-kappa B/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
China is one of the countries with the highest incidence of gastric cancer, and accounts for over 40% of all new gastric cancer cases in the world. Genetic factors as well as environmental factors play a role in development of gastric cancer. To investigate the independent roles of single nucleotide polymorphisms (SNPs) in base excision repair (BER) genes (APE1 and NEIL2), carcinogen metabolism gene (CYP2E1) and tumor suppressor pathway gene (MDM2) for gastric cancer susceptibility in a Chinese population, we conducted a hospital based case-control study to evaluate the potential association between these polymorphisms and susceptibility to gastric cancer in a Northern Jiangsu population. We also associated the NEIL-2 mRNA expression with the studied NEIL2 SNP genotypes to assess whether the genotypes have influence on the NEIL2 mRNA (hence protein) expression. Five SNPs, APE 1 (rs2275008), NEIL 2 (rs804270), MDM2 (rs2279744), and CYP 2E1 (rs2480256 and rs2031920), were genotyped by TaqMan assays in 105 gastric cancer cases and 118 controls. Genotype frequency distribution showed that the APE 1 SNP (rs2275008), NEIL 2 SNP (rs804270), MDM2 SNP (rs2279744), and CYP 2E1 SNP (rs2031920) had more mutant alleles in gastric cancer cases than controls (76.19, 68.57, 54.29, and 43.81%, respectively), while CYP 2E1 SNP (rs2480256) had large percentage of both alleles (43.81%). Risk analysis revealed that there was increased risk for gastric cancer in subjects with mutant alleles in APE 1 (rs2275008: OR 5.49, 95% CI = 2.6-5.7, p <.0001), NEIL 2 (rs804270: OR 2.3, 95% CI = 1.22-4.3, p=0.01), MDM2 (rs2279744: OR 14.65, 95% CI = 5.63-8.15, p < .0001), and CYP 2E1 (rs2031920: OR 8.385, 95% CI = 3.2-5.3, p < .0001) SNPs. Moreover, the NEIL2 mRNA expression analysis showed that there was significant differential expression of NEIL2 mRNA among the randomly tested NEIL2 genotypes (p = 0.005), with low expression seen in variant genotypes than in other genotypes. In conclusion, variant alleles in the NEIL2 (rs804270), APE1 (rs2275008), CYP2E1 (rs2031920) and MDM2 (rs2279744) SNPs may independently influence susceptibility to gastric cancer in a Northern Jiangsu Chinese population. The genotypes may also independently influence their respective gene mRNA expression, as seen in our study, where there was differential expression of the NEIL2 mRNA among the genotypes, with low NEIL2 mRNA expression seen in the variant genotype.
Assuntos
Citocromo P-450 CYP2E1/genética , DNA Glicosilases/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-mdm2/genética , Neoplasias Gástricas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Povo Asiático , Estudos de Casos e Controles , China/epidemiologia , Citocromo P-450 CYP2E1/biossíntese , DNA Glicosilases/biossíntese , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/biossíntese , Feminino , Regulação da Expressão Gênica , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-mdm2/biossíntese , Neoplasias Gástricas/epidemiologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
BACKGROUND: Atmospheric pressure cold plasma (APCP) might be considered a novel tool for tissue disinfection in medicine since the active chemical species produced by low plasma doses, generated by ionizing helium gas in air, induces reactive oxygen species (ROS) that kill microorganisms without substantially affecting human cells. OBJECTIVES: In this study, we evaluated morphological and functional changes in human corneas exposed for 2 minutes (min) to APCP and tested if the antioxidant n-acetyl l-cysteine (NAC) was able to inhibit or prevent damage and cell death. RESULTS: Immunohistochemistry and western blotting analyses of corneal tissues collected at 6 hours (h) post-APCP treatment demonstrated no morphological tissue changes, but a transient increased expression of OGG1 glycosylase that returned to control levels in 24 h. Transcriptome sequencing and quantitative real time PCR performed on different corneas revealed in the treated corneas many differentially expressed genes: namely, 256 and 304 genes showing expression changes greater than ± 2 folds in the absence and presence of NAC, respectively. At 6 h post-treatment, the most over-expressed gene categories suggested an active or enhanced cell functioning, with only a minority of genes specifically concerning oxidative DNA damage and repair showing slight over-expression values (<2 folds). Moreover, time-related expression analysis of eight genes up-regulated in the APCP-treated corneas overall demonstrated the return to control expression levels after 24 h. CONCLUSIONS: These findings of transient oxidative stress accompanied by wide-range transcriptome adjustments support the further development of APCP as an ocular disinfectant.
Assuntos
Córnea/efeitos dos fármacos , Desinfecção/métodos , Gases em Plasma/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Acetilcisteína/farmacologia , Idoso , Ar , Antioxidantes/farmacologia , Pressão Atmosférica , Temperatura Baixa , Córnea/metabolismo , Dano ao DNA , DNA Glicosilases/biossíntese , DNA Glicosilases/genética , Indução Enzimática/efeitos dos fármacos , Desenho de Equipamento , Proteínas do Olho/genética , Perfilação da Expressão Gênica , Hélio , Humanos , Técnicas In Vitro , Pessoa de Meia-Idade , Estresse Oxidativo/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fatores de TempoRESUMO
Ochratoxin A (OTA) is a nephrotoxic mycotoxin produced by Aspergillus and Penicillium fungi. It contaminates human and animal food products, and chronic exposure is associated with renal fibrosis in humans (Balkan endemic nephropathy). Resveratrol, a phytoalexin, possesses anti-cancer and antioxidant properties. We investigated the mechanism of cellular oxidative stress induced by OTA, and the effect of resveratrol in human embryonic kidney (HEK293) cells over 24 and 48 h. Cells were exposed to OTA [IC50 = 1.5 µM (24 h) and 9.4 µM (48 h) determined using MTT assay] and 25 µM resveratrol. Glutathione was quantified by luminometry and gene expression of Nrf2 and OGG1 was determined by qPCR. Protein expression of Nrf2, LonP1, SIRT3, and pSIRT1 was assessed by Western blot, DNA damage (comet assay), and intracellular reactive oxygen species (flow cytometry). At 24 h, resveratrol increased mRNA expression of the DNA repair enzyme, OGG1 (P < 0.05), whereas OTA and OTA+resveratrol significantly decreased OGG1 expression (P < 0.05). OGG1 expression increased during 48-h exposure to resveratrol and OTA+resveratrol (P < 0.05). Comet tail lengths doubled in 48-h OTA-treated cells, whereas at both time periods, OTA+resveratrol yielded shorter comet tails (P < 0.0001). During 24- and 48-h exposure, OTA, resveratrol, and OTA+resveratrol significantly decreased mRNA expression of Nrf2 (P < 0.05). Luminometry analysis of GSH revealed an increase by OTA+resveratrol for 24 and 48 h (P < 0.05 and P < 0.001, respectively). Western blot analysis showed decreased Nrf2 protein expression during 24-h exposure, but increased Nrf2 expression during 48 h. LonP1 protein expression increased during 24-h exposure to OTA (P < 0.05) and OTA+resveratrol (P < 0.0011) and during 48-h exposure to resveratrol (P < 0.0005).
Assuntos
Nefropatia dos Bálcãs/tratamento farmacológico , Ocratoxinas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Estilbenos/administração & dosagem , Proteases Dependentes de ATP/biossíntese , Apoptose/efeitos dos fármacos , Aspergillus/patogenicidade , Nefropatia dos Bálcãs/patologia , Dano ao DNA , DNA Glicosilases/biossíntese , Microbiologia de Alimentos , Células HEK293 , Humanos , Proteínas Mitocondriais/biossíntese , Penicillium/patogenicidade , Espécies Reativas de Oxigênio/metabolismo , Resveratrol , Sesquiterpenos/administração & dosagem , FitoalexinasRESUMO
RAD9 participates in DNA damage-induced cell cycle checkpoints and DNA repair. As a member of the RAD9-HUS1-RAD1 (9-1-1) complex, it can sense DNA damage and recruit ATR to damage sites. RAD9 binding can enhance activities of members of different DNA repair pathways, including NEIL1 DNA glycosylase, which initiates base excision repair (BER) by removing damaged DNA bases. Moreover, RAD9 can act independently of 9-1-1 as a gene-specific transcription factor. Herein, we show that mouse Rad9(-/-) relative to Rad9(+/+) embryonic stem (ES) cells have reduced levels of Neil1 protein. Also, human prostate cancer cells, DU145 and PC-3, knocked down for RAD9 demonstrate reduced NEIL1 abundance relative to controls. We found that Rad9 is required for Neil1 protein stability in mouse ES cells, whereas it regulates NEIL1 transcription in the human cells. RAD9 depletion enhances sensitivity to UV, gamma rays and menadione, but ectopic expression of RAD9 or NEIL1 restores resistance. Glycosylase/apurinic lyase activity was reduced in Rad9(-/-) mouse ES and RAD9 knocked-down human prostate cancer whole cell extracts, relative to controls. Neil1 or Rad9 addition restored this incision activity. Thus, we demonstrate that RAD9 regulates BER by controlling NEIL1 protein levels, albeit by different mechanisms in human prostate cancer versus mouse ES cells.
Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA Glicosilases/metabolismo , Reparo do DNA , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , DNA Glicosilases/biossíntese , DNA Glicosilases/genética , Células-Tronco Embrionárias/metabolismo , Masculino , Camundongos , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/metabolismoRESUMO
Glial cells are very important for normal brain function and alterations in their activity due to hyperglycemia, could contribute to diabetes-related cognitive dysfunction. Oxidative insults often cause rapid changes in almost all cells including glial cells. However, pathophysiologic mechanisms that lead to diabetic complications are not completely elucidated. Therefore, we examined whether elevated glucose levels directly or indirectly disrupt antioxidant defense mechanisms causing alterations in signaling pathways, cell cycle dysregulation, and reactive oxygen/nitrogen species-mediated apoptosis in glial cells. Findings of this study demonstrated that exposure of glial cells to high glucose markedly induces cellular and molecular injuries, as evidenced by elevated levels of reactive oxygen/nitrogen species, biomolecules damage, cell cycle dysregulation, decrease in antioxidant enzymes, and decrease in cell viability. Pretreatment of cells with N-acetyl-L-cysteine reduced high glucose-induced cytotoxicity by increasing the levels of antioxidant enzymes, and decreasing the number of apoptotic cells. Further, at molecular level high glucose treatment resulted in a significant increase in phosphorylation of Akt, MAPKs, tuberin, down regulation of 8-oxoG-DNA glycosylase and increase in 8-hydroxydeoxyguanosine accumulations. Pretreatment of cells with N-acetyl-L-cysteine, phosphatidylinositol3-kinase/Akt and ERK1/2 inhibitors completely abolished the apoptotic effects of high glucose. Moreover, N-acetyl-L-cysteine significantly inhibited reactive oxygen/nitrogen species generation, elevated antioxidants levels, inhibited Akt, ERK1/2, tuberin phosphorylation, decreased 8-hydroxydeoxyguanosine accumulation and upregulated 8-oxoG-DNA glycosylase expression. Our results demonstrate that high glucose induces apoptosis and inhibits proliferation of glial cells, which may be mediated by the phosphorylation of tuberin, down regulation of 8-oxoG-DNA glycosylase and 8-hydroxydeoxyguanosine accumulation via activation of Akt and ERK1/2MAPK pathways.
Assuntos
Apoptose/genética , DNA Glicosilases/biossíntese , Neuropatias Diabéticas/genética , Neuroglia/efeitos dos fármacos , Estresse Oxidativo , Animais , Apoptose/efeitos dos fármacos , Cisteína/metabolismo , Neuropatias Diabéticas/etiologia , Neuropatias Diabéticas/patologia , Glucose/administração & dosagem , Humanos , Hiperglicemia/induzido quimicamente , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Peroxidação de Lipídeos/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neuroglia/metabolismo , Neuroglia/patologia , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
The common Ser326Cys polymorphism in the base excision repair protein 8-oxoguanine glycosylase 1 is associated with a reduced capacity to repair oxidative DNA damage particularly under conditions of intracellular oxidative stress and there is evidence that Cys326-OGG1 homozygous individuals have increased susceptibility to specific cancer types. Indirect biochemical studies have shown that reduced repair capacity is related to OGG1 redox modification and also possibly OGG1 dimer formation. In the current study we have used bimolecular fluorescence complementation to study for the first time a component of the base excision repair pathway and applied it to visualise accumulation of Cys326-OGG1 protein complexes in the native cellular environment. Fluorescence was observed both within and around the cell nucleus, was shown to be specific to cells expressing Cys326-OGG1 and only occurred in cells under conditions of cellular oxidative stress following depletion of intracellular glutathione levels by treatment with buthionine sulphoximine. Furthermore, OGG1 complex formation was inhibited by incubation of cells with the thiol reducing agents ß-mercaptoethanol and dithiothreitol and the antioxidant dimethylsulfoxide indicating a causative role for oxidative stress in the formation of OGG1 cellular complexes. In conclusion, this study has provided for the first time evidence of redox sensitive Cys326-OGG1 protein accumulation in cells under conditions of intracellular oxidative stress that may be related to the previously reported reduced repair capacity of Cys326-OGG1 specifically under conditions of oxidative stress.
Assuntos
DNA Glicosilases/biossíntese , Reparo do DNA/efeitos dos fármacos , Butionina Sulfoximina/farmacologia , Linhagem Celular Tumoral , DNA Glicosilases/genética , Dimetil Sulfóxido/farmacologia , Humanos , Estresse Oxidativo/genética , Espectrometria de FluorescênciaRESUMO
PROBLEM: The most common DNA lesion generated by oxidative stress (OS) is 7, 8-dihydro-8-oxoguanine (8-oxoG) whose excision repair is performed by 8-oxoguanine glycosylase (OGG1). We investigated OGG1 expression changes in fetal membranes from spontaneous preterm birth (PTB) and preterm premature rupture of the membranes (pPROM) and its changes in vitro in normal fetal membranes exposed to OS inducer water-soluble cigarette smoke extract (CSE). METHOD OF STUDY: DNA damage was determined in amnion cells treated with CSE by comet and FLARE assays. OGG1 mRNA expression and localization in fetal membranes from clinical specimens and in normal term membranes exposed to CSE were examined by QRT-PCR and by immunohistochemistry. RESULTS: DNA strand and base damage was seen in amnion cells exposed to CSE. OGG1 expression was 2.5-fold higher in PTB samples compared with pPROM (P = 0.045). No significant difference was seen between term and pPROM or PTB and term. CSE treatment showed a nonsignificant decrease in OGG1. OGG1 was localized to both amnion and chorion with less intense staining in pPROM and CSE-treated membranes. CONCLUSION: Increased OS-induced DNA damage predominated by 8-oxoG is likely to persist in fetal cells due to reduced availability of base excision repair enzyme OGG1. This can likely lead to fetal cell senescence associated with some adverse pregnancy outcome.
Assuntos
Dano ao DNA/fisiologia , DNA Glicosilases/biossíntese , Membranas Extraembrionárias/enzimologia , Oxidantes/toxicidade , Adulto , Células Cultivadas , Ensaio Cometa , Feminino , Ruptura Prematura de Membranas Fetais/enzimologia , Feto , Humanos , Imuno-Histoquímica , Estresse Oxidativo/fisiologia , Gravidez , Nascimento Prematuro/enzimologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fumaça/efeitos adversos , Nicotiana/toxicidadeRESUMO
The modified guanine base 8-oxoguanine (8-oxoG) is abundantly produced by oxidative stress, can contribute to carcinogenesis, and can be removed from DNA by 8-oxoguanine DNA glycosylase-1 (OGG1), which acts as an 8-oxoG glycosylase and endonuclease. This study investigated the mechanism by which 7,8-dihydroxyflavone (DHF) inhibits oxidative stress-induced 8-oxoG formation in hamster lung fibroblasts (V79-4). DHF significantly reduced the amount of 8-oxoG induced by hydrogen peroxide (H2O2) and elevated the levels of OGG1 mRNA and protein. DHF increased the binding of nuclear factor erythroid 2-related factor 2 (Nrf2) to antioxidant response element sequences in the upstream promoter region of OGG1. Moreover, DHF increased the nuclear levels of Nrf2, small Maf proteins, and the Nrf2/small Maf complex, all of which are decreased by H2O2 treatment. Likewise, the level of phosphorylated Akt, which activates Nrf2, was decreased by H2O2 treatment but restored by DHF treatment. The levels of OGG1 and nuclear translocation of Nrf2 protein were decreased upon treatment with PI3K inhibitor or Akt inhibitor, and DHF treatment did not restore OGG1 and nuclear Nrf2 levels in these inhibitor-treated cells. Furthermore, PI3K and Akt inhibitors abolished the protective effects of DHF in cells undergoing oxidative stress. These data indicate that DHF induces OGG1 expression via the PI3K-Akt pathway and protects cells against oxidative DNA base damage by activating DNA repair systems.
Assuntos
DNA Glicosilases/genética , Flavonas/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Cricetinae , DNA Glicosilases/biossíntese , DNA Glicosilases/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Pulmão/citologia , Pulmão/efeitos dos fármacos , Estresse Oxidativo/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Damage to DNA can lead to many different acute and chronic pathophysiological conditions, ranging from cancer to endothelial damage. The current study has been initiated to determine whether the flavonoid wogonin can attenuate etoposide-induced oxidative DNA damage and apoptosis in mouse bone marrow cells. We found that oral administration of wogonin before etoposide injection significantly attenuates etoposide-induced oxidative DNA damage and apoptosis in a dose dependent manner. Etoposide induced a significant down-regulation of mRNA expression of the OGG1 repair gene and marked biochemical alterations characteristic of oxidative DNA stress, including increased 8-OHdG, enhanced lipid peroxidation and reduction in reduced glutathione. Prior administration of wogonin ahead of etoposide challenge restored these altered parameters. Importantly, wogonin had no antagonizing effect on etoposide-induce topoisomerase-II inhibition. Conclusively, our study indicates that wogonin has a protective role in the abatement of etoposide-induced oxidative DNA damage and apoptosis in the bone marrow cells of mice via suppression of oxidative DNA stress and enhancing DNA repair through modulation of OGG1 repair gene expression. Therefore, wogonin can be a promising chemoprotective agent and might be useful to avert secondary leukemia and other drug-related cancers in cured cancer patients and medical personnel exposing to the potent carcinogen etoposide.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , DNA Glicosilases/metabolismo , Flavanonas/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Leucemia/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Animais , Anticarcinógenos/administração & dosagem , Anticarcinógenos/efeitos adversos , Anticarcinógenos/uso terapêutico , Antioxidantes/administração & dosagem , Antioxidantes/efeitos adversos , Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , DNA Glicosilases/biossíntese , DNA Glicosilases/genética , Relação Dose-Resposta a Droga , Etoposídeo/efeitos adversos , Etoposídeo/antagonistas & inibidores , Flavanonas/administração & dosagem , Flavanonas/efeitos adversos , Leucemia/induzido quimicamente , Leucemia/metabolismo , Leucemia/patologia , Masculino , Camundongos , Testes para Micronúcleos , Testes de Mutagenicidade , RNA Mensageiro/metabolismo , Distribuição Aleatória , Inibidores da Topoisomerase II/efeitos adversos , Inibidores da Topoisomerase II/químicaRESUMO
PURPOSE: Human 8-oxoguanine DNA glycosylase (hOGG1) is an ubiquitous protein. It initiates the DNA base excision repair (BER) pathway to repair the 8-oxoguanine lesion. This may be associated with chemotherapeutics. In this article, the effect of hOGG1 over-expression on cisplatin resistance in esophageal squamous cell carcinoma (ESCC) EC9706 and ET13 cells was investigated. METHODS: Recombinant adenovirus pAd/CMV/V5-DEST-hogg1 and control adenovirus pAd/CMV/5-GW/lacZ were constructed and transferred into EC9706 and ET13 cells, respectively. The protein expression and localization were determined by Western blot and by immunofluorescence assay. The cell growth viability was determined by 3-(4,5-dimethylthiazol-2yl)-2,5 diphe-nyltetrazolium bromide (MTT) assay and clonogenic survival assay. The apoptotic cells were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining and flow cytometry. The oxidative DNA damage (8-Hydroxyguanine [8-oxoG] DNA level) was semi-quantified by immunohistochemistry assay. RESULTS: The over-expression of hOGG1 protein was mainly in the nucleus in hOGG1 cells. After exposure to a common chemotherapeutic agent cisplatin, hOGG1 over-expression cells exhibited longer survival ability, lower cell apoptosis, and less 8-oxoG oxidative damage, compared with vector-treated cells and no-treated cells (p<0.05). CONCLUSION: BER pathway to repair 8-oxoG lesion may be associated with ESCC sensitivity to cisplatin, and over-expression of hOGG1 in the nucleus can repair more 8-oxoG oxidative damage. The findings implied that over-expression of hOGG1 can protect ESCC cells from cisplatin-induced apoptosis and prolong cancer cell survival time. Modulation of DNA damage repair activity in the nucleus or in the mitochondria may lead to a different approach regarding cisplatin-induced resistance to chemotherapy.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/enzimologia , Cisplatino/farmacologia , DNA Glicosilases/metabolismo , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/enzimologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , DNA Glicosilases/biossíntese , DNA Glicosilases/genética , Resistencia a Medicamentos Antineoplásicos , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Humanos , TransfecçãoRESUMO
BACKGROUND: Estrogen metabolism-mediated oxidative stress is suggested to play an important role in estrogen-induced breast carcinogenesis. We have earlier demonstrated that antioxidants, vitamin C (Vit C) and butylated hydroxyanisole (BHA) inhibit 17ß-estradiol (E2)-mediated oxidative stress and oxidative DNA damage, and breast carcinogenesis in female August Copenhagen Irish (ACI) rats. The objective of the present study was to characterize the mechanism by which above antioxidants prevent DNA damage during breast carcinogenesis. METHODS: Female ACI rats were treated with E2; Vit C; Vit C + E2; BHA; and BHA + E2 for up to 240 days. mRNA and protein levels of a DNA repair enzyme 8-Oxoguanine DNA glycosylase (OGG1) and a transcription factor NRF2 were quantified in the mammary and mammary tumor tissues of rats after treatment with E2 and compared with that of rats treated with antioxidants either alone or in combination with E2. RESULTS: The expression of OGG1 was suppressed in mammary tissues and in mammary tumors of rats treated with E2. Expression of NRF2 was also significantly suppressed in E2-treated mammary tissues and in mammary tumors. Vitamin C or BHA treatment prevented E2-mediated decrease in OGG1 and NRF2 levels in the mammary tissues. Chromatin immunoprecipitation analysis confirmed that antioxidant-mediated induction of OGG1 was through increased direct binding of NRF2 to the promoter region of OGG1. Studies using silencer RNA confirmed the role of OGG1 in inhibition of oxidative DNA damage. CONCLUSIONS: Our studies suggest that antioxidants Vit C and BHA provide protection against oxidative DNA damage and E2-induced mammary carcinogenesis, at least in part, through NRF2-mediated induction of OGG1.
Assuntos
Antioxidantes/farmacologia , Dano ao DNA , DNA Glicosilases/biossíntese , Estrogênios/toxicidade , Neoplasias Mamárias Experimentais/metabolismo , Fator 2 Relacionado a NF-E2/biossíntese , 8-Hidroxi-2'-Desoxiguanosina , Animais , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Dano ao DNA/fisiologia , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análise , Desoxiguanosina/biossíntese , Feminino , Humanos , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/patologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Interferência de RNA , Ratos , Ratos Endogâmicos ACI , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Regulação para CimaRESUMO
MUTYH variants are differently distributed in geographical areas of the world. In MUTYH-associated polyposis (MAP) patients from North-Eastern Italy, c.933+3A>C (IVS10+3A>C), a transversion causing an aberrant splicing process, accounts for nearly 1/5 of all mutations. The aim of this study was to verify whether its high frequency in North-Eastern Italy is due to a founder effect and to clarify its impact on MUTYH transcripts and protein. Haplotype analysis and age estimate performed on members of eleven Italian MAP families and cancer-free controls provided evidence that c.933+3A>C is a founder mutation originated about 83 generations ago. In addition, the Italian haplotype associated with the c.933+3A>C was also found in German families segregating the same mutation, indicating it had a common origin in Western Europe. Altogether c.933+3A>C and the two common Caucasian mutations p.Tyr179Cys and p.Gly396Asp represent about 60% of MUTYH alterations in MAP patients from North-Eastern Italy, suggesting the opportunity to perform targeted molecular screening for these variants in the diagnostic setting. Expression analyses performed on lymphoblastoid cell lines supported the notion that MUTYH c.933+3A>C alters splicing causing the synthesis of a non functional protein. However, some primary transcripts escape aberrant splicing, producing traces of full-length transcript and wild-type protein in a homozygote; this is in agreement with clinical findings that suggest a relatively mild phenotypic effect for this mutation. Overall, these data, that demonstrate a founder effect and further elucidate the splicing alterations caused by the MUTYH c.933+3A>C mutation, have important implications for genetic counseling and molecular diagnosis of MAP.
Assuntos
Polipose Adenomatosa do Colo/genética , DNA Glicosilases/genética , Mutação , População Branca/genética , Polipose Adenomatosa do Colo/metabolismo , Estudos de Casos e Controles , Linhagem Celular , DNA Glicosilases/biossíntese , Expressão Gênica , Predisposição Genética para Doença , Haplótipos , Humanos , ItáliaRESUMO
AIM: To examine the expression of N-methylpurine-DNA glycosylase (MPG) gene and protein in glioma samples with different WHO grades and its association with patients' survival. METHODS: Immunohistochemistry assay, quantitative real-time PCR and Western blot analysis were carried out to investigate the expression of MPG gene and protein in 128 glioma and 10 non-neoplastic brain tissues. RESULTS: MPG gene expression level in glioma tissues was significantly higher than that in non-neoplastic brain tissues (P < 0.001). Immunohistochemistry also showed that MPG protein was over-expressed in glioma tissues, which was consistent with the resutls of Western blot analysis. Additionally, the expression levels of MPG gene and protein both increase from grade I to grade IV glioma according to the results of real-time PCR, immunohistochemistry and western blot analysis. Moreover, the survival rate of MPG-positive patients was significantly lower than that of MPG-negative patients (P < 0.001). We further confirmed that the over-expression of MPG was a significant and independent prognostic indicator in glioma by multivariate analysis (P < 0.001). CONCLUSIONS: Our data showed the over-expression of MPG gene and protein in human gliomas, and also suggested for the first time that MPG be an unfavorable independent prognostic indicator for glioma patients.
Assuntos
Neoplasias Encefálicas/enzimologia , DNA Glicosilases/biossíntese , Glioma/enzimologia , Adolescente , Adulto , Idoso , Western Blotting , Neoplasias Encefálicas/química , Estudos de Casos e Controles , Criança , DNA Glicosilases/genética , Feminino , Glioma/química , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase em Tempo RealRESUMO
AIM: The present study aimed to identify genes that influence the susceptibility of cancer cells to radiation. MATERIALS AND METHODS: The sensitivities of eight colorectal cancer cell lines to gamma radiation were tested. Microarray data and cells with stable overexpression were used to identify candidate genes. Candidate genes correlating with radioresistance were validated with the use of 22 clinical specimens obtained before preoperative radiotherapy from patients with rectal cancer. RESULTS: Regenerating islet-derived protein 4 (REG4) gene expression was 12-fold higher in radioresistant cells. REG4-overexpressing cells had higher survival rates and fewer DNA strand breaks after gamma irradiation. Expression of the antiapoptotic gene baculoviral inhibitor of apoptosis repeat containing 5 (BIRC5) and base excision-repair pathway gene nei endonuclease VIII-like 2 (NEIL2) in REG4-overexpressing cells, was also three to four times higher than that of the parental cell lines. REG4, BIRC5 and NEIL2 expression levels were significantly higher in non-responding patients (n=14) than in responders (n=8). CONCLUSION: The REG4, BIRC5 and NEIL2 genes might be useful predictors of the sensitivity of cancer patients to radiotherapy.
Assuntos
DNA Glicosilases/biossíntese , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/biossíntese , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas Inibidoras de Apoptose/biossíntese , Lectinas Tipo C/biossíntese , Neoplasias Retais/metabolismo , Neoplasias Retais/radioterapia , Apoptose , Linhagem Celular Tumoral , Ensaio Cometa , Desoxirribonuclease (Dímero de Pirimidina)/genética , Raios gama , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Associadas a Pancreatite , Tolerância a Radiação/genética , Radioterapia/métodos , SurvivinaRESUMO
The aim of the study was to determine the extent of oxidative DNA damage (levels of 8-oxo2dG) and expression of OGG1 and p53 and TNF-α proteins in lymphocytes of Alzheimer's disease (AD) patients and a control group. The studies were conducted on 41 patients with AD, including 25 women and 16 men aged 34-84 years. The control group included 51 individuals, 20 women and 31 men aged 22-83 years. The level of 8-oxo2dG was determined using HPLC/EC/UV, and the level of OGG1 and p53 and TNF-α proteins was determined with the Western blot method. The results showed that both proteins participating in DNA repair (OGG1, p53) and the inflammatory protein TNF-α are involved in pathogenesis of neurodegenerative diseases. It also seems that a specific system for DNA repair (OGG1) may contribute to downregulation of the inflammatory factor (TNF-α) level, especially in the early stages of dementia. Moreover, the results showed that p53 protein can fulfil its function in DNA damage repair only in early stages of dementia. It is possible that OGG1 and p53 and TNF-α proteins together or separately may be involved in pathogenesis of AD by repair of oxidative DNA damage and/or apoptosis.