Análise da imunoexpressão de proteínas de reparo do DNA envolvidas nas vias BER e NER em lesões odontogênicas epiteliais benignas / Immunoexpression analysis of DNA repair proteins involved in BER and NER pathways in benign epithelial odontogenic lesions

As lesões odontogênicas epiteliais benignas apresentam comportamento biológico heterogêneo e patogênese ainda não totalmente esclarecida. As vias de reparo do ácido desoxirribonucleico (DNA) atuam em tipos específicos de danos ao material genético, realizando o reparo e regulando diversos processos celulares. Dentre as principais vias de reparo do DNA, destacamse o reparo por excisão de bases (BER) e o reparo por excisão de nucleotídeos (NER). Investigações têm demonstrado que as proteínas envolvidas nessas vias se encontram desreguladas e, por vezes, altamente expressas em algumas neoplasias malignas, contribuindo para a progressão tumoral. Levando em consideração a heterogeneidade do comportamento biológico das lesões odontogênicas epiteliais benignas e a escassez de estudos que tenham avaliado a expressão de proteínas de reparo do DNA nestas lesões, este trabalho avaliou a imunoexpressão de proteínas da via BER (APE-1 e XRCC-1) e NER (XPF) em ameloblastomas (AMEs) sólidos (n = 30), ceratocistos odontogênicos não sindrômicos (CONS) (n = 30), ceratocistos odontogênicos sindrômicos (COS) (associados à Síndrome de Gorlin) (n = 29), cistos dentígeros (CDs) (n = 30) e folículos dentários (FDs) (n = 20). A análise da expressão imunoistoquímica de APE-1, XRCC-1 e XPF foi realizada de forma quantitativa por um avaliador previamente calibrado e sem acesso aos dados clínicos dos casos. Em cinco campos de maior imunorreatividade, foram quantificadas as células positivas e negativas para as proteínas no componente epitelial de todos os casos, sendo estabelecido o percentual de células positivas em relação ao número total de células contadas para cada anticorpo. As marcações nucleares e citoplasmáticas foram analisadas separadamente para APE-1 e XPF, enquanto apenas a imunoexpressão nuclear foi considerada para XRCC-1. As comparações das medianas dos percentuais de imunorreatividade em relação aos grupos estudados foram realizadas por meio dos testes não paramétricos de Kruskal-Wallis e Mann-Whitney. Possíveis correlações entre a expressão de APE-1, XRCC-1 e XPF foram avaliadas por meio do teste de correlação de Spearman. O nível de significância foi estabelecido em 5% (p < 0,05). Foi verificada uma maior imunoexpressão nuclear de APE-1 nos CONSs, COSs e AMEs sólidos, em comparação com os CDs (p < 0,001). Dentre todos os grupos avaliados, a expressão citoplasmática de APE1 só foi encontrada em 4 CONSs e 6 COSs. A expressão nuclear de XRCC-1 foi estatisticamente maior nos CONSs e COSs em relação aos CDs (p < 0,05). Em nível nuclear, a expressão de XPF foi significativamente maior nos CONSs e COSs em relação aos CDs e AMEs (p < 0,05) e, embora sem significância estatística, foi observada uma maior expressão nuclear dessa proteína nos AMEs quando comparado aos CDs. Em relação à expressão citoplasmática de XPF, foi observada uma maior expressão nos COSs em relação aos CDs (p = 0,04). Nenhuma diferença estatisticamente significativa foi encontrada entre as expressões nucleares de APE-1, XRCC-1 e XPF entre CONSs e COSs (p > 0,05). Além disso, todas as lesões odontogênicas estudadas revelaram uma maior expressão estatisticamente significativa de APE-1 (nuclear), XRCC-1 (nuclear) e XPF (nuclear e citoplasmática) quando comparados aos FDs (p < 0,05). Para todas as lesões, o teste de correlação de Spearman mostrou uma correlação positiva entre a expressão nuclear de APE-1 e XRCC-1 ou XPF, em nível nuclear (p < 0,05). Os resultados deste estudo sugerem um potencial envolvimento das proteínas APE-1, XRCC-1 e XPF na patogênese das lesões odontogênicas epiteliais benignas, com destaque para aquelas com comportamento biológico mais agressivo (AU).

The benign epithelial odontogenic lesions present a heterogeneous biological behavior and their pathogenesis are not fully understood. The deoxyribonucleic acid (DNA) repair pathways act on specific types of damage to the genetic material, performing the repair and regulating several cellular processes. Among the main DNA repair pathways, the most notable are the base excision repair (BER) and the nucleotide excision repair (NER). Investigations have shown that the proteins involved in these pathways are deregulated and sometimes highly expressed in some malignancies, contributing to tumor progression. Taking into account the heterogeneity of the biological behavior of benign epithelial odontogenic lesions and the scarcity of studies that have evaluated the expression of DNA repair proteins in these lesions, this study evaluated the immunoexpression of BER (APE-1 and XRCC-1) proteins and NER (XPF) in solid ameloblastomas (AMEs) (n = 30), non-syndromic odontogenic keratocysts (NSOKCs) (n = 30), syndromic odontogenic keratocysts (SKOCs) (associated with Gorlin's Syndrome) (n = 29), dentigerous cysts (DCs) (n = 30) and dental follicles (DFs) (n = 20). The immunohistochemical analysis of APE-1, XRCC-1 and XPF was performed quantitatively by a previously calibrated evaluator and without access to the clinical data of the cases. In five fields of higher immunoreactivity, positive and negative cells were quantified for the proteins in the epithelial component of all cases, and the percentage of positive cells was established in relation to the total number of cells counted for each antibody. Nuclear and cytoplasmic markers were analyzed separately for APE-1 and XPF, while only nuclear immunoexpression was considered for XRCC-1. The comparisons of the median percentages of immunoreactivity in relation to the studied groups were performed using the non-parametric Kruskal-Wallis and MannWhitney tests. Possible correlations between the expression of APE-1, XRCC-1 and XPF were assessed by Spearman's correlation test. The level of significance was set at 5% (p < 0.05). A higher nuclear immunoexpression of APE-1 in the NSOKCs, SOKCs and solid AMEs was verified in comparison with the DCs (p < 0.001). Among all the evaluated groups, the cytoplasmic expression of APE-1 was only found in 4 NSOKCs and 6 SOKCs. Nuclear expression of XRCC-1 was statistically higher in NSOKCs and SOKCs than in DCs (p < 0.05). At the nuclear level, XPF expression was significantly higher in NSOKCs and SOKCs than in DCs and AMEs (p < 0.05) and, although without statistical significance, a higher nuclear expression of this protein was observed in AMEs when compared to CDs. Regarding the cytoplasmic expression of XPF, a greater expression was observed in the SOKCs in relation to the DCs (p = 0.04). No statistically significant difference was found between the nuclear expressions of APE-1, XRCC-1 and XPF between NSOKCs and SOKCs (p > 0.05). In addition, all the odontogenic lesions studied revealed a statistically significant expression of APE-1 (nuclear), XRCC-1 (nuclear) and XPF (nuclear and cytoplasmic) when compared to DFs (p < 0.05). For all lesions, Spearman's correlation test showed a positive correlation between nuclear expression of APE-1 and XRCC-1 or XPF at the nuclear level (p < 0.05). The results of this study suggest a potential involvement of APE-1, XRCC-1 and XPF proteins in the pathogenesis of benign epithelial odontogenic lesions. The role played by these proteins may be more important in odontogenic lesions with more aggressive biological behavior (AU).

A pilot study of the ERCC1 and XPF genes in forensic age estimation / 中国法医学杂志

Objective The aim of this study is to detect the mRNA and protein expression levels of ERCC1 and XPF genes among different age groups of healthy Chinese Han individuals,and to analyze the correlation between the mRNA and protein expression levels andthe age of individuals in order to find new molecular markers for forensic age estimation.Methods Peripheral blood samples were obtained from 150 unrelated healthy Chinese Han individuals.The plasma was centrifuged from the whole blood by gradient centrifugation,and the totalRNA was extractedwithTrizol fromperipheral blood mononuclear cells(PBMCs).Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to quantitatively analyze the mRNA relative expression levels of ERCC1and XPF in PBMCs.Enzyme linked immunosorbent assay (ELISA) was used to quantitatively analyze the protein expression levels of ERCC1and XPF in plasma.Results There were no significant differences in the mRNA relative expression levels of ERCC1 and XPF in PBMCs between males and females(P＞0.05).Significant differences were found in the mRNA relative expression levels of ERCC1 and XPF between different age groups (P＜0.05).Regression analysis showed thatthe mRNA relative expression levels of ERCC1 and XPF were both negatively correlated with age.The correlation coefficients(r) were-0.578 and-0.844,respectively.When the age was used as independent variable(x) and the mRNA expression relative level as dependent variable (y),the fitting curveswere Y=3.3E-5X2-0.0261X+1.9175 (R2=0.3244,P＜0.01),Y=0.0003X2-0.0459X+2.0439 R2=0.729,P＜0.01),respectively.There were no significant differences inthe protein expression levels of ERCC1 and XPF in plasma between different age groups or genders (P＞0.05).Conclusion The mRNA relative expression levels of ERCC1 and XPF in PBMCsdeclined with the increase of age,however,the protein expression levels in plasma were unrelated to age.ERCC 1 and XPF genes can be used asnew molecular markers for forensic age estimation,so as to providetheoretical basis for establishing the mathematical model of ERCC1/XPF genesin concern ofindividual ages.

Associação da imunoexpressão das proteínas XRCC1, TFIIH E XPF com características clinicopatológicas e sobrevida em carcinoma epidermoide de língua oral / Association of immunoexpression of XRCC1, TFIIH and XPF proteins with clinicopathological characteristics and survival in oral tongue squamous cell carcinoma

Sistemas de reparo do DNA, genes e proteínas, são essenciais para manutenção da integridade do genoma, evitando graves doenças como o câncer. Desrregulação na expressão destas proteínas vem sendo associado tanto ao risco do desenvolvimento, como na evolução de variados cânceres humanos com destaque para o carcinoma epidermoide oral. O objetivo deste trabalho foi analisar a imunoexpressão das proteínas de reparo do DNA, XRCC1, THIIF e XPF em carcinoma epidermoide de língua oral (CELO) e investigar associação com parâmetros clínicos, histopatológicos, de desfecho e sobrevida em cinco anos. Setenta e quatro casos de CELO foram analisados por meio da técnica da imuno-histoquímica de forma semiquantitativa. Observou-se alta expressão das proteínas pesquisadas nas células parenquimatosas, identificando associação significativa da elevada expressão de XRCC1 com melhor estadiamento clínico (p=0,02). A regressão de Cox revelou tamanho do tumor (p<0,01), comprometimento linfonodal (p=0,04), estágio do tumor (p=0,02) e profundidade de invasão >4mm (p=0,05) como fatores prognósticos para CELO. Os resultados deste experimento sugerem que as proteínas XRCC1, TFIIH e XPF participam do processo de tumorigênese, entretanto a imunoexpressão das mesmas não pode ser utilizada como indicador independente de prognóstico para CELO (AU).

DNA repair systems, genes and proteins are essential for genome integrity maintenance, avoiding serious diseases such as cancer. Deregulation in the expression of those proteins has been associated with both the risk of development and evolution of various human cancers, including oral squamous cell carcinoma. The purpose of this study was to analyze the immunoreactivity of the DNA repair proteins XRCC1, THIIF and XPF in oral tongue squamous cell carcinoma (OTSCC) and to investigate its association with clinical and histopathological parameters, outcome and 5-year survival rate. Seventy-four cases of OTSCC were analyzed semi-quantitatively through immunohistochemistry. We observed that DNA repair proteins were highly expressed in parenchymal cells; however, we only observed a significant association between XRCC1 high expression and better clinical staging (p=0,02). Cox regression showed that tumor size (p<0,01), lymph node involvement (p=0,04), tumor stage (p=0,02) and depth of invasion> 4mm (p=0,05) were prognostic factors. The results of this experiment suggest that XRCC1, TFIIH and XPF participate in the tumorigenic process, however, their immunoexpression may not be used as an independent prognostic indicator for OTSCC (AU).

Relationship between Helicobacter pylori L-form infection and expression of XPF and p53 in gastric carcinoma / 临床与实验病理学杂志

Purpose To investigate the relationship between Helicobacter pylori L-form ( Hp-L) infection and expression of XPF and p53 in gastric carcinoma. Methods XPF, p53 and Hp-L were examined in 239 cases of gastric carcinoma and healthy individuals by means of Giemsa stain and immunohistochemical staining with ABC method. Results XPF and p53 expression in gastric carcinoma was significantly higher than that of the surrounding tissue (P<0. 01). There were positive correlation between the expression of XPF and p53 (r=0. 196, P<0. 01). The expression of XPF was related to the Laurén classification (P<0. 05). The expression of p53 was associated to the depth of invasion (P<0. 05). (2) 175 (73. 22%) cases were Hp-L positive by both Giemsa and immunohisto-chemical staining. Hp-L expression in gastric carcinoma were significantly higher than that of the surrounding tissue (P<0. 01). (3) In the Hp-L positive group, XPF and p53 were significantly higher than in that of the Hp-L negative group (P<0. 01). Positive corre-lation existed between positive Hp-L staining and the expression of XPF and p53 (r=0. 32, r=0. 296, P<0. 01). (4) Univariate a-nalysis showed that depth of invasion, distant metastasis, TNM stage, XPF and p53 were related to the prognosis of gastric carcinoma ( P<0. 05). Multivariate Cox model analysis showed that XPF was an independent prognostic factor for the patients (P<0. 05). The pa-tients with positive expression of XPF had a shorter survival time. When combined with the depth of invasion and distant metastasis, the correction risk increased by 3. 791 times. Conclusion XPF, p53 and Hp-L may play very important roles in the development of gastric carcinoma. The high expression of XPF implies poorer prognosis. Hp-L infection may be related to the up-regulated expression of XPF and p53.

Avaliação de polimorfismos funcionais nos genes de reparo XRCC1, APEX1, XPD e XPF em carcinomas de células escamosas orais / Evaluation of functional polymorphisms in the repair genes XRCC1, APEX1, XPD and XPF in oral squamous cell carcinomas

As vias de reparo por excisão de base (BER) e por excisão de nucleotídeo (NER) desempenham um papel crucial na manutenção da integridade genômica. Polimorfismos em genes das vias BER e NER, que modulam a capacidade de reparo do DNA, podem estar relacionados ao risco de desenvolvimento e prognóstico do câncer oral. O presente trabalho teve como objetivo investigar a frequência de polimorfismos de nucleotídeos simples, em dois genes da via de reparo do DNA por excisão de base (XRCC1 ­ rs25487 e APEX1 ­ rs1130409) e dois genes da via de reparo por excisão de nucleotídeo (XPD ­ rs13181 e XPF ­ rs1799797), em pacientes com carcinoma de células escamosas oral (CCEO), buscando associações com o risco de desenvolver esta neoplasia maligna e o seu prognóstico. Um total de 92 amostras de DNA de pacientes com CCEO e 130 controles foram genotipadas utilizando o método da reação em cadeia da polimerase em tempo real. O software estatístico GraphPad Prism version 6.0.1. foi utilizado para a aplicação dos testes apropriados. Odds ratio (OR) e hazard ratio (HR), e seus intervalos de confiança (IC) de 95%, foram calculados pela regressão logística. A avaliação do prognóstico foi realizada por meio da curva de Kaplan-Meier e análise multivariada de Cox. A presença das variantes polimórficas nos genes XRCC1, APEX1, XPD, e XPF não foram associadas ao risco de desenvolver CCEO. A interação da presença da variante polimórfica com o hábito de fumar não foi significativa para nenhum dos polimorfismos analisados. Já a presença do polimorfismo em XPD, somada ao hábito de beber, aumentou o risco de desenvolver CCEO (OR 1,86, 95% IC: 0,86 ­ 4,01, p=0,03). Apenas o SNP do APEX1 (rs1130409) esteve associado a uma diminuição da sobrevida específica (HR 3,94, 95% IC: 1,31 ­ 11,88, p=0,01). O presente estudo sugere uma interação entre o consumo de álcool e a presença do polimorfismo estudado no gene XPD. Além disso, indica um pior prognóstico para pacientes que possuem o polimorfismo estudado em APEX1 (AU).

Base excision repair (BER) and nucleotide excision repair (NER) pathways play critical role in maintaining genome integrity. Polymorphisms in BER and NER genes which modulate the DNA repair capacity may affect the susceptibility and prognosis of oral cancer. This study was conducted with genomic DNA from 92 patients with oral squamous cell carcinomas (OSCC) and 130 controls. The cases were followed up to explore the associations between BER and NER genes polymorphisms and the risk and prognosis of OSCC. Four single-nucleotide polymorphisms (SNPs) in XRCC1 (rs25487), APEX1 (rs1130409), XPD (rs13181) and XPF (rs1799797) genes were tested by polymerase chain reaction ­ quantitative real time method. The GraphPad Prism version 6.0.1 statistical software was applied for statistical analysis of association. Odds ratio (OR), hazard ratio (HR), and their 95 % confidence intervals (CIs) were calculated by logistic regression. Kaplan-Meier curve and Cox proportional hazard model were used for prognostic analysis. The presence of polymorphic variants in XRCC1, APEX1, XPD and XPF genes were not associated with an increased risk of OSCC. Gene-environment interactions with smoking were not significant for any polymorphism. The presence of polymorphic variants of the XPD gene in association with alcohol consumption conferred an increased risk of 1.86 (95% CI: 0.86 ­ 4.01, p=0.03) for OSCC. Only APEX1 was associated with decreased specific survival (HR 3.94, 95% CI: 1.31 ­ 11.88, p=0.01). These results suggest an interaction between polymorphic variants of the XPF gene and alcohol consumption. Additionally APEX1 may represent a prognostic marker for OSCC (AU).

Effects of melatonin on DNA damage induced by cyclophosphamide in rats

The antioxidant and free radical scavenger properties of melatonin have been well described in the literature. In this study, our objective was to determine the protective effect of the pineal gland hormone against the DNA damage induced by cyclophosphamide (CP), an anti-tumor agent that is widely applied in clinical practice. DNA damage was induced in rats by a single intraperitoneal injection of CP (20 or 50 mg/kg). Animals received melatonin during the dark period for 15 days (1 mg/kg in the drinking water). Rat bone marrow cells were used for the determination of chromosomal aberrations and of formamidopyrimidine DNA glycosylase enzyme (Fpg)-sensitive sites by the comet technique and of Xpf mRNA expression by qRT-PCR. The number (mean ± SE) of chromosomal aberrations in pinealectomized (PINX) animals treated with melatonin and CP (2.50 ± 0.50/100 cells) was lower than that obtained for PINX animals injected with CP (12 ± 1.8/100 cells), thus showing a reduction of 85.8% in the number of chromosomal aberrations. This melatonin-mediated protection was also observed when oxidative lesions were analyzed by the Fpg-sensitive assay, both 24 and 48 h after CP administration. The expression of Xpf mRNA, which is involved in the DNA nucleotide excision repair machinery, was up-regulated by melatonin. The results indicate that melatonin is able to protect bone marrow cells by completely blocking CP-induced chromosome aberrations. Therefore, melatonin administration could be an alternative and effective treatment during chemotherapy.