RESUMO
The ADAM23 gene is frequently silenced in different types of tumors, and, in breast tumors, silencing is correlated with tumor progression, suggesting that it might be associated with the acquisition of a metastatic phenotype. ADAM23 exerts its function mainly through the disintegrin domain, because its metalloprotease domain is inactive. Analysis of ADAM23 binding to integrins has revealed a specific interaction with alpha(v)beta(3) integrin mediated by the disintegrin domain. Altered expression of alpha(v)beta(3) integrin has been observed in different types of tumors, and expression of this integrin in the activated form has been shown to promote metastasis formation. Here, we investigated the possibility that interaction between ADAM23 and alpha(v)beta(3) integrin might negatively modulate alpha(v)beta(3) activation during metastatic progression. ADAM23 expression was knocked down using short hairpin RNA in the MDA-MB-435 cell line, which has been extensively used as a model for alpha(v)beta(3) integrin activation. Ablation of ADAM23 enhanced alpha(v)beta(3) integrin activation by at least 2- to 4-fold and ADAM23 knockdown cells showed enhanced migration and adhesion to classic alpha(v)beta(3) integrin ligands. Ablation of ADAM23 expression also enhanced pulmonary tumor cell arrest in immunodeficient mice. To complement our findings with clinical evidence, we showed that silencing of ADAM23 gene by DNA promoter hypermethylation in a collection of 94 primary breast tumors was significantly associated with lower distant metastases-free and disease-specific survivals and was an independent prognostic factor for poor disease outcome. Our results strongly support a functional role of ADAM23 during metastatic progression by negatively modulating alpha(v)beta(3) integrin activation.
Assuntos
Proteínas ADAM/genética , Integrina alfaVbeta3/genética , Metástase Neoplásica/genética , Proteínas ADAM/deficiência , Proteínas ADAM/fisiologia , Animais , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Metilação de DNA , DNA de Neoplasias/genética , Feminino , Humanos , Integrina alfaVbeta3/fisiologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos SCID , Metástase Neoplásica/patologia , Reação em Cadeia da Polimerase , RNA Catalítico/genéticaRESUMO
BACKGROUND: Aberrant methylation of gene promoter region is responsible for inappropriate gene silencing, and it has been associated to initiation and progression of cancer. Aberrant promoter methylation is frequently observed in adult patients with myelodysplastic syndrome (MDS), but in pediatric patients it has been poorly investigated. METHODS: We examined the promoter methylation status of 13 genes in bone marrow cells collected at diagnosis of 21 pediatric patients with MDS (subtype RAEB or RAEB-t). For this analysis, we performed sodium bisulfite treatment of genomic DNA, followed by methylation specific PCR (MSP). RESULTS: In pediatric MDS samples, we observed two genes frequently methylated: CALCA was methylated in 85.7% (18/21) of the analyzed samples and CDKN2B in 50% (6/12). CONCLUSIONS: Our findings indicate that CALCA and CDKN2B are frequently methylated in pediatric MDS. It suggests that aberrant methylation in pediatric MDS seems to be similar to adult MDS, thus pediatric patients could be also benefited with treatment using demethylating agents.
Assuntos
Calcitonina/genética , Inibidor de Quinase Dependente de Ciclina p15/genética , Metilação de DNA , Síndromes Mielodisplásicas/genética , Precursores de Proteínas/genética , Adolescente , Peptídeo Relacionado com Gene de Calcitonina , Criança , Pré-Escolar , Aberrações Cromossômicas , DNA/análise , Perfilação da Expressão Gênica , Humanos , Lactente , Síndromes Mielodisplásicas/diagnóstico , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA/métodosRESUMO
Promoter hypermethylation occurs early in leukemogenesis and seems to be associated with poor prognosis in acute lymphoblastic leukemia (ALL). The methylation status of the promoter region of six genes was analyzed in 71 children with ALL using methylation specific PCR (MSP). Calcitonin (CALCA) and E-cadherin (CDH1) were the most frequently methylated genes in this group of patients. Considering the patients with central nervous system (CNS) infiltration, the estimated 2-year overall survival (OS) was 20% for those with methylation in CALCA promoter and 85% for those without (p=0.001). Our results suggest that the hypermethylation of CALCA promoter is a promising prognostic marker and may predict a higher risk for ALL patients with CNS infiltration.
Assuntos
Calcitonina/genética , Neoplasias do Sistema Nervoso Central/secundário , Ilhas de CpG , Metilação de DNA , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Regiões Promotoras Genéticas/genética , Neoplasias do Sistema Nervoso Central/diagnóstico , Criança , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Reação em Cadeia da Polimerase/métodos , Prognóstico , Fatores de Risco , Sensibilidade e Especificidade , Taxa de SobrevidaRESUMO
Genome stability and normal gene expression are maintained by a fixed and predetermined DNA methylation pattern, which becomes abnormal in malignant cells. Hypomethylation of satellite DNA sequences is frequently found in tumors and has been associated with an increased frequency of DNA rearrangements and chromosome instability. In this work, we used methylation-sensitive arbitrarily primed polymerase chain reaction (MSAP-PCR) to identify differentially methylated DNA fragments in normal and tumor breast samples. We identified a novel differentially methylated fragment located on chromosome 5 with high similarity to a SATR-1 satellite sequence. This fragment was found to be hypomethylated in 63% of breast tumor cell lines and in 86% of breast tumors relative to normal breast tissue. We found that normal tissue adjacent to breast tumors displayed a variable decrease in methylation and that the decrease observed for most of these adjacent samples was higher than observed for normal breast tissue derived from reduction mammoplasty. The methylation decrease was, however, significantly higher in tumor samples than in adjacent tissue (chi2= 154, 1 df, P < 10(-4)), suggesting that SATR-1 hypomethylation frequently occurs in the early stages of tumor development. Our results highlight the importance of global DNA hypomethylation as a contributing factor in breast tumorigenesis.
Assuntos
Neoplasias da Mama/genética , Metilação de DNA , Repetições de Microssatélites/genética , Sequência de Bases , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Primers do DNA , Feminino , Humanos , Reação em Cadeia da PolimeraseRESUMO
Cancer patients receiving chemotherapy are exposed to high doses of cytotoxic and genotoxic drugs which, in some cases, can lead to treatment related leukemia. Since this only occurs in a minority of patients, however, it is possible some individuals are predisposed due to genetic polymorphisms in genes for enzymes that mediate drug metabolism. To address this possibility we measured the genotoxicity of chemotherapeutic agents in patients receiving treatment for ALL by the frequency of the Vgamma/Jbeta trans-rearrangement in their peripheral blood leukocytes and compared this with CYP3A4 genotype. CYP3A4 is the most abundant of the cytochrome P450 (CYP) enzyme in the liver and intestine which contains a common -392A>G substitution in the promoter region (CYP3A4*1B allele). We found a significant increase in the frequency of rearrangements during chemotherapy only in patients homozygous for the wild type CYP3A4*1A allele. This provides a direct link between CYP3A4 genotype and susceptibility to drug genotoxicity thus strengthening the possibility that predisposition to treatment related leukemia may be measurable by simple genetic testing.
