Análise comparativa da imunoexpressão das proteínas hmlh1 e hmsh2 em carcinomas epidermóides de lábio inferior e queilites actínicas com graus variados de displasia

Lip squamous cell carcinoma (SCC) may develop from a premalignant condition, actinic cheilitis (AC) in 95% of the cases. Both premalignant and neoplastic lip diseases are caused mainly by chronic exposure to the ultraviolet component of solar radiation, especially UVB. This exposure causes disruption of the cell cycle and damage to DNA repair systems, like mismatch repair, altering proteins repair as hMLH1 and hMSH2. This research aimed to investigate the immunohistochemical expression of hMLH1 and hMSH2 proteins in lower lip SCCs and ACs, providing additional information about carcinogenesis of the lower lip. The sample consisted 40 cases of ACs and 40 cases of lower lip SCCs. Histological sections of 3 μm were submitted to immunoperoxidase method, for immunohistochemical analysis of lesions were counted in 1000 cells (positive and negative), data were evaluated both in absolute numbers and percentage of immunostained cells, the latter by assigning scores. Associations of the variables and comparative analysis of biomarker expression were performed by Fisher s exact and Pearson s chi-square, "t" student, one-way ANOVA, Mann- Whitney e Kruskal-Wallis tests. The level of significance was 5%. It was found that, in lower lip SCC, the mean of the proteins was higher in female patients (hMLH1= 369,80 + 223,98; hMHS2 = 534,80 + 343,62), less than 50 years old (hMLH1 = 285,50 + 190,65; hMHS2 = 540,00 + 274,79) and classified as low-grade malignancy (hMLH1 = 264,59 + 179,21; hMHS2 = 519,32 + 302,58), in these data only to sex, for hMLH1 protein, was statistically significant (p=0.034). Comparing the different lesions, we observed that for both hMLH1 and hMSH2 protein, the average of positive epithelial cells decreased as the lesion was graded at later stages. The ACs classified without dysplasia or mild dysplasia had the highest average of immunostained cells (hMLH1 = 721.23 + 88.116; hMHS2 = 781.50 + 156.93). The ACs classified as moderate or severe dysplasia had intermediate values (hMLH1 = 532,86 + 197,72; hMHS2 = 611,14 + 172,48) and SSCs of the lower lip had the lowest averages (hMLH1 = 255,03 + 199,47; hMHS2 = 518,38 + 265,68). There was a statistically significant difference between groups (p<0.001). In conclusion, our data support the hypothesis that changes in immunoexpression of these proteins is related to the process of carcinogenesis of the lower lip

Caracterização de dois cDNAs homológos e uma AP endonuclease em cana-de-açúcar

The genome of all organisms is subject to injuries that can be caused by endogenous and environmental factors. If these lesions are not corrected, it can be fixed generating a mutation which can be lethal to the organisms. In order to prevent this, there are different DNA repair mechanisms. These mechanisms are well known in bacteria, yeast, human, but not in plants. Two plant models Oriza sativa and Arabidopsis thaliana had the genome sequenced and due to this some DNA repair genes have been characterized. The aim of this work is to characterized two sugarcane cDNAs that had homology to AP endonuclease: scARP1 and scARP3. In silico has been done with these two sequences and other from plants. It has been observed domain conservation on these sequences, but the cystein at 65 position that is a characteristic from the redox domain in APE1 protein was not so conservated in plants. Phylogenetic relationship showed two branches, one branch with dicots and monocots sequence and the other branch with only monocots sequences. Another approach in order to characterized these two cDNAs was to construct overexpression cassettes (sense and antisense orientation) using the 35S promoter. After that, these cassettes were transferred to the binary vector pPZP211. Furthermore, previously in the laboratory was obtained a plant from nicotiana tabacum containing the overexpression cassette in anti-sense orientation. It has been observed that this plant had a slow development and problems in setting seeds. After some manual crossing, some seeds were obtained (T2) and it was analyzed the T2 segregation. The third approach used in this work was to clone the promoter region from these two cDNAs by PCR walking. The sequences obtained were analyzed using the program PLANTCARE. It was observed in these sequences some motives that may be related to oxidative stress response

Os efeitos das queimadas na Amazônia em nível celular e molecular

The Amazon holds over half of the planet's remaining tropical forests and comprises the largest biodiversity in the world, accounting for approximately 60 % of the Brazilian territory. However, deforestation fires in the region causes serious problems to exposed human. The aim of this study was to evaluate the chemical compounds as well as the cellular and molecular effects after exposure to organic material extracted from particulate matter less than 10 µm (PM10) in the Amazon region. As for the chemical composition, n-alkanes analysis showed a prevalence of anthropogenic influence during the fires in the region. In addition, there was a predominance of monosaccharides from biomass burning markers. Also, the Polycyclic Aromatic Hydrocarbons (PAH) and their derivatives have also been identified in samples collected in the Amazon. By using the PAH concentrations was possible to calculate the BaP-equivalent and it was found that the dibenz(a) anthracene contributes with 83% to potential carcinogenic risk. As for the potential mutagenic risk, the benzo (a) pyrene is the HPA that has a major contribution in this analysis. It may be noted that the retene was the most abundant PAH. This compound was genotoxic and cause death by necrosis in the human lung cells. In biological tests, the data showed that organic PM10 is capable of causing genetic damage in both plant cells and in human lung cells. This damage cause an arrest in the G1 phase of the cell cycle exposed, increasing the expression of p53 and p21. Additionally, the PM10 caused cell death by apoptosis, increasing the foci of histone - H2AX. Given these results, it is important to emphasize the reduction and better control of biomass burning in the Amazon region thus improving the quality of health of the population being exposed. As clearly stated recently by the World Health Organization, the reduction of air pollution could save millions of lives annually.

Avaliação de polimorfismos funcionais nos genes de reparo XRCC1, APEX1, XPD e XPF em carcinomas de células escamosas orais

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.

Avaliação do efeito da inibição do reparo de sítios abásicos na resposta inflamatória celular

Base excision repair (BER) proteins has been associated with functions beyond DNA repair. Apurynic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein involved in a plethora of cellular activities, such as redox activation of transcription factors, RNA processing and DNA repair. Some studies have described the action of the protein 8-oxoguanine (OGG1) in correcting oxidized lesions in promoters as a step in the transcription of pro-inflammatory cytokines. Despite being especially important in redox activation of transcription factors such as nuclear factor κB (NF-κB) and AP- 1, the repair activity of APE1 has not yet been associated with the inflammatory response. In this study, experimental and bioinformatic analysis approaches have been used to investigate the relationship between inhibition of the repair of abasic sites in DNA by MX, a synthetic molecule designed to inhibt the repair activity of APE1, and the modulation of the inflammatory response. The results showed that treatment of monocytes with lipopolysaccharide (LPS) and MX reduced the expression of cytokines, chemokines and toll-like receptors, and negatively regulated biological immune processes, as macrophages activation, and NF-κB and tumor necrosis factor (TNF-α) and interferon pathways, without inducing cell death. The transcriptomic analysis suggests that LPS/MX treatment induces mitochondrial dysfunction, endoplasmic reticulum stress and activation of autophagy pathways, probably activated by impairment of cellular energy and/or the accumulation of nuclear and mitochondria DNA damage. Additionally, it is proposed that the repair activity of APE1 is required for transcription of inflammatory genes by interaction with abasic sites at specific promoters and recruitment of transcriptional complexes during inflammatory signaling. This work presents a new perspective on the interactions between the BER activity and the modulation of inflammatory response, and suggests a new activity for APE1 protein as modulator of the immune response in a redox-independent manner.

Imunoexpressão das proteínas APE-1 e XRCC-1 em carcinoma epidermoide de língua oral

DNA repair systems play a critical role in protecting the human genome from damage caused by carcinogens present in the environment. Mutations in DNA repair genes may be responsible for tumor development and resistance of malignant cells to chemotherapeutic agents. The major pathway for oxidative DNA damage repair is the base excision repair pathway. The objective of this study was to investigate the immunoexpression of APE-1 and XRCC-1, which are proteins involved in DNA base excision repair and its association with clinical and histopathological parameters in oral tongue squamous cell carcinoma (OTSCC), in order to investigate a possible prognostic value for those proteins. The expression of APE-1 and XRCC-1 was evaluated semi-quantitatively by immunohistochemistry in 50 OTSCC cases. Clinical data was collected from patients’ medical charts and histopathological grading was performed for each case. Statistical analysis (Chi-square and Fisher’s exact tests; significance of 5%) was performed to determine the association between protein expressions and clinico-pathological characteristics. APE-1 was highly expressed in nucleus and cytoplasm in 56% of cases. XRCC-1 showed overexpression only in nucleus in 60% of cases. High expression of XRCC-1 was significantly associated to clinical stages I and II (P=0.02). Both proteins were not associated to other clinical parameters or histopathological grading. Our findings demonstrate that DNA base excision repair proteins APE-1 and XRCC-1 are upregulated in OTSCC, however, they are not related to clinical and histologic parameters, except for XRCC-1 association to better clinical staging. Our results indicate that the immunohistochemical expression of these proteins has no association with prognostic parameters in this tumor.

Imunoexpressão das proteínas APE-1 e XRCC-1 em carcinoma epidermoide de língua oral

DNA repair systems play a critical role in protecting the human genome from damage caused by carcinogens present in the environment. Mutations in DNA repair genes may be responsible for tumor development and resistance of malignant cells to chemotherapeutic agents. The major pathway for oxidative DNA damage repair is the base excision repair pathway. The objective of this study was to investigate the immunoexpression of APE-1 and XRCC-1, which are proteins involved in DNA base excision repair and its association with clinical and histopathological parameters in oral tongue squamous cell carcinoma (OTSCC), in order to investigate a possible prognostic value for those proteins. The expression of APE-1 and XRCC-1 was evaluated semi-quantitatively by immunohistochemistry in 50 OTSCC cases. Clinical data was collected from patients’ medical charts and histopathological grading was performed for each case. Statistical analysis (Chi-square and Fisher’s exact tests; significance of 5%) was performed to determine the association between protein expressions and clinico-pathological characteristics. APE-1 was highly expressed in nucleus and cytoplasm in 56% of cases. XRCC-1 showed overexpression only in nucleus in 60% of cases. High expression of XRCC-1 was significantly associated to clinical stages I and II (P=0.02). Both proteins were not associated to other clinical parameters or histopathological grading. Our findings demonstrate that DNA base excision repair proteins APE-1 and XRCC-1 are upregulated in OTSCC, however, they are not related to clinical and histologic parameters, except for XRCC-1 association to better clinical staging. Our results indicate that the immunohistochemical expression of these proteins has no association with prognostic parameters in this tumor.

Association of the XPD and XRCC3 gene polymorphisms with oral squamous cell carcinoma in a Northeastern Brazilian population: A pilot study

Objective to evaluate the association between XPD and XRCC3 polymorphisms and oral squamous cell carcinoma (OSCC). Design the sample consisted of 54 cases of OSCC and 40 cases of inflammatory fibrous hyperplasia (IFH). Genotypes were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Results XPD-Lys/Gln was more common in IFH (n = 28; 70%) than in OSCC (n = 24; 44.4%) (OR: 0.3; p < 0.05). XPD-Gln was more frequent in high-grade lesions (0.48) than in low-grade lesions (0.21) (OR: 3.4; p < 0.05). The Gln/Gln genotype was associated with III and IV clinical stages (OR: 0.07; p < 0.05). XRCC3-Met was more frequent in OSCC (0.49) than in IFH (0.35) (OR: 2.6; p < 0.05). The Met/Met genotype was associated with the presence of metastases (OR: 8.1; p < 0.05) and with III and IV clinical stages (OR: 0.07; p < 0.05). Conclusions in this sample, the frequency of XPD-Gln in IFH suggests that this variant may protect against OSCC. The presence of the XRCC3-Met allele seems to contribute to the development of OSCC, metastases and more advanced stages in these lesions.

Association of the XPD and XRCC3 gene polymorphisms with oral squamous cell carcinoma in a Northeastern Brazilian population: A pilot study

Objective to evaluate the association between XPD and XRCC3 polymorphisms and oral squamous cell carcinoma (OSCC). Design the sample consisted of 54 cases of OSCC and 40 cases of inflammatory fibrous hyperplasia (IFH). Genotypes were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Results XPD-Lys/Gln was more common in IFH (n = 28; 70%) than in OSCC (n = 24; 44.4%) (OR: 0.3; p < 0.05). XPD-Gln was more frequent in high-grade lesions (0.48) than in low-grade lesions (0.21) (OR: 3.4; p < 0.05). The Gln/Gln genotype was associated with III and IV clinical stages (OR: 0.07; p < 0.05). XRCC3-Met was more frequent in OSCC (0.49) than in IFH (0.35) (OR: 2.6; p < 0.05). The Met/Met genotype was associated with the presence of metastases (OR: 8.1; p < 0.05) and with III and IV clinical stages (OR: 0.07; p < 0.05). Conclusions in this sample, the frequency of XPD-Gln in IFH suggests that this variant may protect against OSCC. The presence of the XRCC3-Met allele seems to contribute to the development of OSCC, metastases and more advanced stages in these lesions.

Associação da imunoexpressão das proteínas XRCC1, TFIIH E XPF com características clinicopatológicas e sobrevida em carcinoma epidermoide de língua oral

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.

Associação da imunoexpressão das proteínas XRCC1, TFIIH E XPF com características clinicopatológicas e sobrevida em carcinoma epidermoide de língua oral

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.

Análise filogenética e funcional de dois genes de reparo homólogos a AP endonuclease em cana-de-açúcar: ScARP1 e ScARP3

The genome of all organisms constantly suffers the influence of mutagenic factors from endogenous and/or exogenous origin, which may result in damage for the genome. In order to keep the genome integrity there are different DNA repair pathway to detect and correct these lesions. In relation to the plants as being sessile organisms, they are exposed to this damage frequently. The Base Excision DNA Repair (BER) is responsible to detect and repair oxidative lesions. Previous work in sugarcane identified two sequences that were homologous to Arabidopsis thaliana: ScARP1 ScARP3. These two sequences were homologous to AP endonuclease from BER pathway. Then, the aim of this work was to characterize these two sequence using different approaches: phylogenetic analysis, in silico protein organelle localization and by Nicotiana tabacum transgenic plants with overexpression cassette. The in silico data obtained showed a duplication of this sequence in sugarcane and Poaceae probably by a WGD event. Furthermore, in silico analysis showed a new localization in nuclei for ScARP1 protein. The data obtained with transgenic plants showed a change in development and morphology. Transgenic plants had slow development when compared to plants not transformed. Then, these results allowed us to understand better the potential role of this sequence in sugarcane and in plants in general. More work is important to be done in order to confirm the protein localization and protein characterization for ScARP1 and ScARP3

Identificação de genes em Chromobacterium violaceum relacionados à resposta ao estresse

The sequencing of the genome of Chromobacterium violaceum identified one single circular chromosome of 4.8 Mb, in which approximately 40% of the founded ORFs are classified as hypothetical conserved or hypothetical. Some genic regions of biotechnological and biological interest had been characterized, e. g., environmental detoxification and DNA repair genes, respectively. Given this fact, the aim of this work was to identify genes of C. violaceum related to stress response, as the ones involved with mechanisms of DNA repair and/or genomic integrity maintenance. For this, a genomic library of C. violaceum was built in Escherichia coli strain DH10B (RecA-), in which clones were tested to UVC resistance, resulting in five candidates clones. In the PLH6A clone were identified four ORFs (CV_3721 to 3724). Two ORFs, CV_3722 and CV_3724, were subcloned and a synergic complementation activity was observed. The occurrence of an operon was confirmed using cDNA from C. violaceum in a RT-PCR assay. Further, it was observed the induction of the operon after the treatment with UVC. Thus, this operon was related to the stress response in C. violaceum. The mutagenesis assay with rifampicin after the treatment with UVC light showed high frequency of mutagenicity for the ORF CV_3722 (Pol III δ subunit). In this way, we propose that the C. violaceum δ subunit can act in DH10B in the translesion synthesis using Pol IV in a RecA independent-manner pathway. In growth curve assays other four clones (PLE1G, PLE7B, PLE10B and PLE12H) were able to complement the function at the dose 5 J/m2 and in mutagenicity assays PLE7B, PLE10B and PLE12H showed frequencies of mutation with significant differences upon the control (DH10B), demonstrating that in some way they are involved with the stress response in C. violaceum. These clones appear to be interrelated, probably regulated by a messenger molecule (eg., nucleotide c-di-GMP) and/or global regulatory molecule (eg., σS subunit of RNA polymerase).The results obtained contribute for a better genetic knowledge of this specie and its response mechanisms to environmental stress.

Análise da estabilidade genética de células-tronco mesenquimais humanas

Human multipotent mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have become an important and attractive therapeutic tool since they are easily isolated and cultured, have in vitro expansion potential, substantial plasticity and secrete bioactive molecules that exert trophic effects. The human umbilical cord as a cell source for cell therapy will help to avoid several ethical, political, religious and technical issues. One of the main issues with SC lines from different sources, mainly those of embryonic origin, is the possibility of chromosomal alterations and genomic instability during in vitro expansion. Cells isolated from one umbilical cord exhibited a rare balanced paracentric inversion, likely a cytogenetic constitutional alteration, karyotype: 46,XY,inv(3)(p13p25~26). Important genes related to cancer predisposition and others involved in DNA repair are located in 3p25~26. Titanium is an excellent biomaterial for bone-implant integration; however, the use can result in the generation of particulate debris that can accumulate in the tissues adjacent to the prosthesis, in the local bone marrow, in the lymph nodes, liver and spleen. Subsequently may elicit important biological responses that aren´t well studied. In this work, we have studied the genetic stability of MSC isolated from the umbilical cord vein during in vitro expansion, after the cryopreservation, and under different concentrations and time of exposition to titanium microparticles. Cells were isolated, in vitro expanded, demonstrated capacity for osteogenic, adipogenic and chondrogenic differentiation and were evaluated using flow cytometry, so they met the minimum requirements for characterization as MSCs. The cells were expanded under different concentrations and time of exposition to titanium microparticles. The genetic stability of MSCs was assessed by cytogenetic analysis, fluorescence in situ hybridization (FISH) and analysis of micronucleus and other nuclear alterations (CBMN). The cells were able to internalize the titanium microparticles, but MSCs preserve their morphology, differentiation capacity and surface marker expression profiles. Furthermore, there was an increase in the genomic instability after long time of in vitro expansion, and this instability was greater when cells were exposed to high doses of titanium microparticles that induced oxidative stress. It is necessary always assess the risks/ benefits of using titanium in tissue therapy involving MSCs, considering the biosafety of the use of bone regeneration using titanium and MSCs. Even without using titanium, it is important that the therapeutic use of such cells is based on analyzes that ensure quality, security and cellular stability, with the standardization of quality control programs appropriate. In conclusion, it is suggested that cytogenetic analysis, FISH analysis and the micronucleus and other nuclear alterations are carried out in CTMH before implanting in a patient

Estudo do papel da proteína multifuncional APE1/Ref-1 sobre a resposta inflamatória na meningite bacteriana

Despite advances in antibiotic therapy, bacterial meningitis (BM) remains with high mortality and morbidity rates in worldwide. One important mechanism associated to sequels during disease is the intense inflammatory response which promotes an oxidative burst and release of reactive oxygen species, consequently leading to cell death. Activation of DNA repair enzymes during oxidative stress has been demonstrated in several neurological disorders. APE1/Ref-1 is a multifunctional protein involved in DNA repair and plays a redox function on transcription factors such as NFkB and AP-1.The aim of this study was assess the role of APE1/Ref-1 on inflammatory response and the possibility of its modulation to reduce the sequels of the disease. Firstly it was performed an assay to measure cytokine in cerebrospinal fluid of patients with BM due to Streptococcus pneumoniae and Neisseriae meningitides. Further, a cellular model of inflammation was used to observe the effect of the inhibition of the endonuclease and redox activity of APE1/Ref-1 on cytokine levels. Additionally, APE1/Ref-1 expression in cortex and hippocampus of rat with MB after vitamin B6 treatment was evaluated. Altogether, results showed a similar profile of cytokines in the cerebrospinal fluid of patients from both pathogens, although IFNy showed higher expression in patients with BM caused by S. pneumoniae. On the other hand, inhibitors of APE1/Ref-1 reduced cytokine levels, mainly TNF-α. Reduction of oxidative stress markers was also observed after introduction of inhibitors in the LPS-stimulated cell. In the animal model, BM increased the expression of the protein APE1/Ref-1, while vitamin B6 promoted reduction. Thereby, this data rise important factors to be considered in pathogenesis of BM, e.g., IFNy can be used as prognostic factor during corticosteroid therapy, APE1/Ref-1 can be an important target to modulate the level of inflammation and VIII oxidative stress, and vitamin B6 seems modulates several proteins related to cell death. So, this study highlights a new understanding on the role of APE1/Ref-1 on the inflammation and the oxidative stress during inflammation condition