RESUMO
Diffuse type gastric carcinoma is the most aggressive type of gastric cancer. This type of tumor is not preceded by precancerous changes and is associated with early-onset and hereditary syndromes. To test the hypothesis that DNA methylation profile would be useful for molecular classification of the diffuse type gastric carcinoma, DNA methylation patterns of the CpG Island of 17 genes were studied in 104 cases and 47 normal adjacent gastric mucosa by Methylation-specific PCR, Immunohistochemistry and Hierarchicalclustering analysis. The most frequent methylated genes were FHIT, E-cadherin, BRCA1 and APC (>50%),followed by p14, p16, p15, p73, MGMT and SEMA3B (20-49%). Hierarchical clustering analysis reveals four groups with different clinical features. The first was characterized by hypermethylation of BRCA1 and younger age (<45 years old), and the second by hypermethylation of p14 and p16 genes, male predominance and Epstein-Barr virus infection. The third group was characterized by hypermethylation of FHIT and antrum located tumors and the fourth was not associated with any clinical variables. In normal adjacent mucosa only the p73 gene was significantly less methylated in comparison to tumor mucosa. DNA methylation identified subgroups of diffuse type gastric cancer. Hypermethylation of BRCA1 associated with young age suggests a role in early-onset gastric carcinoma.
Assuntos
Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Metilação de DNA/genética , DNA de Neoplasias/genética , Genes BRCA1 , Neoplasias Gástricas/genética , Análise por Conglomerados , Ilhas de CpG/genética , Diagnóstico Precoce , Mucosa Gástrica/patologia , Imuno-Histoquímica , Reação em Cadeia da Polimerase , Lesões Pré-Cancerosas/genética , Regiões Promotoras Genéticas/genética , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/patologiaRESUMO
BRCA1 gene mutations account for nearly all families with multiple cases of both early onset breast and/or ovarian cancer and about 30% of hereditary breast cancer. Although to date more than 1,237 distinct mutations, polymorphisms, and variants have been described, several mutations have been found to be recurrent in this gene. We have analyzed 63 Chilean breast/ovarian cancer families for eighteen frequent BRCA1 mutations. The analysis of the five exons and two introns in which these mutations are located was made using mismatch PCR assay, ASO hybridization assay, restriction fragment analysis, allele specific PCR assay and direct sequentiation techniques. Two BRCA1 mutations (185delAG and C61G) and one variant of unknown significance (E1250K) were found in four of these families. Also, a new mutation (4185delCAAG) and one previously described polymorphism (E1038G) were found in two other families. The 185delAG was found in a 3.17 % of the families and the others were present only in one of the families of this cohort. Therefore these mutations are not prominent in the Chilean population. The variant of unknown significance and the polymorphism detected could represent a founder effect of Spanish origin.
Assuntos
Humanos , Masculino , Feminino , Neoplasias da Mama/genética , Genes BRCA1 , Mutação/genética , Neoplasias Ovarianas/genética , Neoplasias da Mama Masculina/genética , Chile , Análise Mutacional de DNA , DNA de Neoplasias , População Branca , Predisposição Genética para Doença , Mutação em Linhagem Germinativa/genética , Linhagem , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Fatores de RiscoRESUMO
Attacin, a bactericidal small protein is produced by the giant silk moth Hyalophora cecropia. This paper deals with our efforts to clone the attacin cDNA in a bacterial vector to express it in Escherichia coli and produce the protein in sufficient amount, for further studies. We chose two inducible expression vector/bacterial cell systems: pPL-lambda/N99cI+ cells which is able to be induced by nalidixic acid, and pET3d/BL21(DE3) cells carrying a T7 RNA polymerase gene which is IPTG-inducible. After cloning in the pPL-lambda system and under no addition of the inducer, isolated transformants carried this plasmid with at least 2 concurrent deletions that drastically affected attacin expression, even though attacin gene seems to be intact as deduced by its PCR amplification. It was concluded that basal attacin expression occurred in this system and bacterial growth was limited. Plasmid deletions may have emerged by selection pressure as a way to avoid bactericidal expression and allow bacteria survival. The second cloning attempt was done in pET3d vector/BL21 cells, that should not express the cloned sequence (they lack T7 RNA polymerase gene). Transformed BL21 cells gave 3 recombinant plasmids, 2 of them presented a C deletion that generated an early stop signal in the attacin coding region. The third clone, pET-ATT18, carrying an intact gene, was transferred to BL21(DE3)-IPTG inducible cells in order to be expressed. Attacin was undetectable in stained gels or by Western blot analysis. However, expression was visualized in grown cells after 30 min of IPTG induction and 5 min of [35S]-methionine labeling, as a 22.5 kDa protein band by using gel electrophoresis and fluorography. This low level of expression drastically affected bacterial growth. Considering that attacin has no lytic activity, these results suggest that this molecule should block bacterial growth directly at the cytoplasm by an unknown mechanism, since no signal peptide coding sequence was incorporated in this gene construction, precluding periplasmic or external destination of this protein