Eleonidas Moura Lima

hTERT methylation and expression in gastric cancer

Gastric cancer is the second most prevalent cause of cancer death worldwide. DNA methylation is a common event in gastric carcinogenesis. hTERT seems to be the rate-limiting determinant of telomerase activation, which is responsible for stability and life span. hTERT hypermethylation has been associated with telomerase expression. In the present study, we investigated the promoter methylation status and hTERT protein expression in gastric cancer and normal mucosa samples. One hundred and nine gastric cancer and 53 normal mucosa samples were investigated through methylation-specific PCR. Immunohistochemistry was analysed using peroxidase in 55 gastric cancer and 18 normal gastric mucosa samples. This is the first study evaluating hTERT methylation status in gastric carcinogenesis. We did not observe hTERT protein expression in normal gastric mucosa. Moreover, hTERT expression was observed in 80% of tumours and was associated with gastric cancer (p < 0.0001). Partial methylation was the most frequent pattern in gastric samples, even in normal mucosa. The frequency of specimens presenting hypermethylation was significantly higher in tumours than in normal mucosa samples (p = 0.0002), although the presence of hypermethylated promoter was not associated with a higher frequency of hTERT expression. A low correlation between hTERT protein expression and methylation was verified in gastric cancer samples. There was a clear difference in the frequency of hTERT expression and methylation within tumoral and non-tumoral tissues. Methylation status and telomerase expression may be useful for the diagnosis of gastric cancer and may have an impact on the anti-telomerase strategy for cancer therapy.

Interrelationship between TP53 gene deletion, protein expression and chromosome 17 aneusomy in gastric adenocarcinoma

BackgroundThis study evaluates the existence of numerical alterations of chromosome 17 and TP53 gene deletion in gastric adenocarcinoma. The p53 protein expression was also evaluated, as well as, possible associations with clinicopathological characteristics.MethodsDual-color fluorescence in situ hybridization and immunostaining were performed in twenty gastric cancer samples of individuals from Northern Brazil.ResultsDeletion of TP53 was found in all samples. TP53 was inactivated mainly by single allelic deletion, varying to 7–39% of cells/case. Aneusomy of chromosome 17 was observed in 85% of cases. Chromosome 17 monosomy and gain were both observed in about half of cases. Cells with gain of chromosome 17 frequently presented TP53 deletion. The frequency of cells with two chr17 and one TP53 signals observed was higher in diffuse than in intestinal-type GC. Immunoreactivity of p53 was found only in intestinal-type samples. The frequency of cells with two chr17 and two TP53 signals found was higher in samples with positive p53 expression than in negative cases in intestinal-type GC.ConclusionWe suggest that TP53 deletion and chromosome 17 aneusomy is a common event in GC and other TP53 alterations, as mutation, may be implicated in the distinct carcinogenesis process of diffuse and intestinal types.

SMARCA5 Methylation and Expression in Gastric Cancer

Here, we first evaluated SMARCA5 expression and promoter DNA methylation in gastric carcinogenesis. Immunohistochemistry and methylation-specific PCR were analyzed in 19 and 48 normal mucosa and in 52 and 92 gastric cancer samples, respectively. We observed higher immunoreactivity of SMARCA5 in gastric cancer samples than in normal mucosa. Moreover, SMARCA5 promoter methylation was associated with the absence of protein expression. Our findings suggest that SMARCA5 has a potential role in proliferation and malignancy in gastric carcinogenesis.

Insulin-like growth factor binding protein-3 gene methylation and protein expression in gastric adenocarcinoma.

OBJECTIVE The aim of this study was to evaluate IGFBP-3 protein expression, its correlation with gene promoter methylation pattern in gastric carcinogenesis and with clinicopathological characteristics. DESIGN Forty-three normal gastric mucosa and 94 adenocarcinoma samples were investigated through methylation specific PCR, after bisulfite modification. Immunohistochemistry was analyzed using peroxidase in 54 gastric cancer and 20 normal gastric mucosa samples. RESULTS IGFBP-3 expression was higher in tumor samples than in normal mucosa (p<0.0001). Intestinal type presented a higher frequency of protein expression than diffuse type (p=0.0412). Methylation frequency of IGFBP-3 promoter in gastric samples revealed, respectively, 95.7% and 97.7% in neoplastic and non-neoplastic samples. The frequency of IGFBP-3 methylation did not differ between tumor and normal samples (95.7% versus 97.7%, p=0.7810). We did not observe a significant correlation between IGFBP-3 promoter methylation and protein expression. CONCLUSION In summary, our study did not observe any influence of IGFBP-3 promoter methylation on protein expression. Moreover we propose that IGFBP-3 immunostaining in gastric tissue may be a useful marker for malignancy.

Association study of SNPs of genes IFNGR1 (rs137854905), GSTT1 (rs71748309), and GSTP1 (rs1695) in gastric cancer development in samples of patient in the northern and northeastern Brazil

Cancer is a multifactorial disease with a high mortality rate in Brazil and worldwide. Gastric cancer (GC) is considered the fourth type of malignancy more frequent in the population worldwide and the second leading cause of death. This work aimed to evaluate single nucleotide polymorphisms (SNPs) of IFNGR1, GSTT1, and GSTP1 genes samples in gastric cancer. We analyzed 60 samples of gastric cancer, 26 diffuse and 34 intestinal types, totaling 120 alleles for each SNP. The results were obtained by PCR and allele-specific PCR. Statistical analyzes performed using BioEstat 5.0 software, applying the Fisher’s exact test and chi-square. Only the SNP gene GSTP1 (rs1695) were significantly associated with gastric cancer in the samples analyzed (χ2 = 8.73, P < 0.05). Our results suggest that the GSTP1 gene SNP (rs1695) can be considered a risk factor associated with gastric carcinogenesis.

Identification of IL11RA and MELK amplification in gastric cancer by comprehensive genomic profiling of gastric cancer cell lines

AIM To identify common copy number alterations on gastric cancer cell lines. METHODS Four gastric cancer cell lines (ACP02, ACP03, AGP01 and PG100) underwent chromosomal comparative genome hybridization and array comparative genome hybridization. We also confirmed the results by fluorescence in situ hybridization analysis using the bacterial artificial chromosome clone and quantitative real time PCR analysis. RESULTS The amplification of 9p13.3 was detected in all cell lines by both methodologies. An increase in the copy number of 9p13.3 was also confirmed by fluorescence in situ hybridization analysis. Moreover, the interleukin 11 receptor alpha (IL11RA) and maternal embryonic leucine zipper kinase (MELK) genes, which are present in the 9p13.3 amplicon, revealed gains of the MELK gene in all the cell lines studied. Additionally, a gain in the copy number of IL11RA and MELK was observed in 19.1% (13/68) and 55.9% (38/68) of primary gastric adenocarcinoma samples, respectively. CONCLUSION The characterization of a small gain region at 9p13.3 in gastric cancer cell lines and primary gastric adenocarcinoma samples has revealed MELK as a candidate target gene that is possibly related to the development of gastric cancer.

Study of Methylation Pattern of de Novo DNA Methyltransferase Genes and its Correlation with DNA Methylation Pattern of RUNX3 in Individuals with Gastric Cancer from Northern Region of Brazil

El cancer gastrico es la cuarta patologia mas frecuente en el mundo. En el norte del Brasil, es la segunda neoplasia mas frecuente en hombres y la tercera en mujeres. Alteraciones geneticas y epigeneticas relacionadas con la carcinogenesis gastrica y la metilacion del DNA son las alteraciones epigeneticas mejor estudiadas. En este trabajo, analizamos el estado de novo de metilacion de genes DNA metiltransferases y su asociacion con el estado de metilacion del gen RUNX3 en muestras de individuos brasilenos con cancer gastrico de los tipos intestinal y difuso. Fue usada la Reaccion en Cadena de la Polimerasa (PCR), metilacion especifica, para analizar el estado de metilacion del DNA. Fueron estudiados 66 tejidos tumorales. Solamente el gen RUNX3 presento un estado de metilacion alterado, estuvo metilado en 38,5% de las muestras de cancer gastrico tipo intestinal y en 70% de muestras de cancer gastrico tipo difuso, lo que sugiere que estaria relacionado con la genesis de esta neoplasia. Hubo una diferencia estadistica significativa entre muestras de los tipos difuso e intestinal (p=0.0418) y entre tejidos normal y tumoral (p<0.0001)parael gen RUNX3. Esta asociacion no fue encontrada para los genes DNMT3A, DNMT3B y DNMT3 en las islas CpG analizadas. Alteraciones del estado de metilacion de RUNX3 no estan asociadas con alteraciones de novo de genes DNA metiltransferases. De esta forma se hace necesaria una mejor comprension de este fenomeno en la carcinogenesis gastrica

Dideoxy single allele-specific PCR - DSASP new method to discrimination allelic

Gastric cancer (GC) is a multifactorial disease with a high mortality rate in Brazil and worldwide. This work aimed to evaluate single nucleotide polymorphisms (SNP) rs1695, in the Glutathione S-Transferase Pi (GSTP1) gene in GC samples by comparative analysis Specific PCR - ASP and Dideoxy Single Allele-Specific PCR - DSASP methods. The DSASP is the proposed new method for allelic discrimination. This work analyzed 60 GC samples, 26 diffuse and 34 intestinal types. The SNP rs1695 of the GSTP1 gene was significantly associated with GC analyzed by DSASP method (χ2 = 9.7, P 0.05). These results suggest that the SNP rs1695 of the GSTP1 gene was a risk factor associated with gastric carcinogens is and the DSASP method was a new successfully low-cost strategy to study allelic discrimination.

Prevalência de hemoglobinas variantes em comunidades quilombolas no estado do Piauí, Brasil

Resumo As hemoglobinas variantes (Hb) decorrem de mutacoes nos genes da globina. As variantes estruturais mais frequentes sao HbS, HbC, HbD e HbE. O gene da hemoglobina S tem frequencia elevada na America, enquanto que no Brasil e maior no Sudeste e Nordeste. O presente artigo tem por objetivo investigar a presenca de hemoglobinas variantes em 15 comunidades quilombolas do estado do Piaui. Foram analisadas 1.239 amostras, nas quais as hemoglobinas foram triadas pela cromatografia liquida de alta eficiencia (HPLC). Aplicou-se questionario referente a genero, etnia e consanguinidade das populacoes. Das 1.239 amostras, 5,4% apresentaram o traco falciforme AS, as doencas falciformes SS e SC apareceram em 0,8% do total, nas hemoglobinas AC, AD e DD. Das 1.069 pessoas negras, 84 apresentaram alteracao das hemoglobinas; destas, 34 eram do sexo masculino e 53 do feminino. Ocorreu a presenca de 13 casamentos consanguineos dentre as 84 alteracoes das hemoglobinas. O estudo das hemoglobinas variantes em 15 comunidades remanescentes de quilombos do Piaui contribui para sua educacao em saude frente aos aspectos da heranca genetica destas proteinas, relevante questao de saude publica, proporcionando subsidios para a implantacao do Programa Estadual da Doenca Falciforme do Piaui.Hemoglobin variants (Hb) result from mutations in globin genes, with amino acid substitution in the polypeptide chain. Among the most common structural variants are HbS, HbC, HbD and HbE. The S hemoglobin gene is a high frequency gene across America and Brazil, where it is more frequent in the Southeast and Northeast. The scope of this article is to investigate the presence of hemoglobin variants in 15 quilombos (fugitive slave communities) of Piaui. The sample was of 1,239 people and hemoglobin was screened by high-performance liquid chromatography (HPLC). A questionnaire was applied related to gender, ethnicity and consanguinity. Of the samples analyzed, 5.4% had AS sickle cell trait, while SS and SC sickle cell anemia showed a rate of 0.8%, with AC, AD and DD hemoglobin. Of the 1,069 Afro-descendants, 84 revealed hemoglobin abnormalities, 34 being male 53 being female. There were 13 consanguineous marriages among the 84 hemoglobin alterations. The study of hemoglobin variants in 15 former quilombo communities in the state of Piaui contributes to their education in health in the aspects of genetic inheritance of hemoglobin, a relevant public health issue, providing input for the implementation of the State Program of Sickle Cell Disease of Piaui.