Identificação de regiões diferencialmente metiladas em câncer de mama através da técnica AP-PCR sensível à metilação (MSAP-PCR) / Identification of differentially methylated regions in breast tumors by methylation sensitive AP-PCR (MSAP-PCR)

RESUMO

A estabilidade do genoma e o perfil normal de expressão gênica são mantidos por um padrão fixo e pré-determinado de metilação do DNA. Esse padrão pode, no entanto, ser alterado nas células tumorais e contribuir na tumorigênese. Neste trabalho, nós utilizamos a técnica de AP-PCR sensível à metilação (MSAP-PCR) com o objetivo de identificar regiões diferencialmente metiladas em tumores de mama. Através desta metodologia, fomos capazes de identificar duas regiões diferencialmente metiladas. O primeiro fragmento de DNA isolado foi mapeado no cromossomo 2q33-34, nas proximidades do gene ADAM23... O tratamento das linhagens celulares MCF-7 e SKBR-3 com o agente desmetilante 5?-aza-2?-deoxycytidina levou a uma reexpressão do gene ADAM23 e a um decréscimo significativo nos níveis de metilação. Uma maior porcentagem de metilação foi verificada em tumores de mama com um estádio mais avançado e a presença de metilação parece estar associada com a presença de linfonodos axilares comprometidos e de metástases, assim como, com a ocorrência de recidivas da doença... Nossos resultados sugerem que a diminuição nos níveis de metilação da região SATR1 é um evento comum em tumores de mama, o qual pode facilitar a ocorrência de rearranjos cromossômicos e contribuir no processo de progressão tumoral. Nossos dados também reforçam a importância da hipometilação global do genoma na tumorigênese e devem ser considerados no desenvolvimento de novos protocolos de tratamento contra o câncer que utilizam agentes desmetilantes...(AU)

ABSTRACT

Genome stability and normal gene expression are maintained by a fixed and predetermined DNA methylation pattern. However, this pattern may be altered in tumor cells, contributing to tumorigenesis. In this work, we used the Methylation Sensitive Arbitrarilly Primed - PCR (MSAP-PCR) in arder to identify differentially methylated regions in breast tumors. Using this methodology, we were able to identify two differentially methylated regions. The first isolated DNA fragment was mapped to chromosome 2q33-34, in the proximity of the ADAM23 gene. The members of the ADAM family are cell surface proteins, which present two characteristic domains the desintegrin domain and the metalloprotease doma in. The ADAM23 protein presents an inactive metalloprotease domain and it is thought to be involved exclusively in cell-cell adhesion. We verified that the promoter region of the ADAM23 gene is hypermethyllated in 66,7°/o of the tumor celllines and in 51,4- 69,2°/o of the analyzed primary breast tumors. The presence of methylation is strongly associated with reductions in both mRNA and protein expression, suggesting that the silencing of ADAM23 gene may be related to alterations in cell adhesion properties and to tumor progression. A threshold of 40-60°/o of methylated dinucleotides within the promoter region is necessary for the complete silencing of the gene. Treatment of MCF-7 and SKBR-3 cell lines with 5'-Aza 2'-deoxycytidine led to a reactivation of ADAM23 gene expression and a marked decrease in the methylation levei. A higher percentage of methylation was observed in breast tumors in advanced stages and the presence of methylation seems to be associated with the presence of positive axillary lymph nades and metastases as well as with disease recurrence. In ali, these results suggest that the methylation of the AM23 gene may be used as a molecular marker in breast tumors.The second DNA fragment isolated was mapped to chromosome 5 and presented a high similarity with a satellite sequence named SA TR 1. Satellite sequences correspond to blocks of tandemly repeated sequences usually located in regions of pericentromeric and/or telomeric heterochromatin. The loss of methylation in satellite sequences is frequently found in tumors and has been associated with an increased frequency of DNA rearrangements and chromosome instability. The loss of methylation in the STAR1 sequence was observed in 63°/o of the breast tumor cell !ines and in 63-87°/o of primary breast tumors. Patients with a ecrease in the leveis of methylation in the SA TR 1 region presented a shorter disease-free survival in relation to patients with normal leveis of methylation (log-rank, p=0.1963). Our results suggest that a decrease in the leveis of methylation in the SATR1 region is common in breast tumors and may facilitate the occurrence of chromosome rearrangements and contribute to tumor progression. Our results also reinforce the importance of global genome hypomethylation in tumorigenesis and must be considered in the development of new treatment protocols, which use demethylating agents in the treatment of cancer (AU)