Restriction landmark genomic scanning to identify novel methylated and amplified DNA sequences in human lung cancer.

摘要

The introductory chapter briefly reviews our current knowledge on lung cancer. Both loss-of-function of tumor suppressor genes by promoter methylation and gain-of-function of oncogenes by DNA amplification are involved in lung cancer development. Restriction Landmark Genomic Scanning (RLGS) is a two-dimensional gel electrophoresis technique that has been successfully applied to scan cancer genomes for aberrant DNA methylation and DNA amplification. Based on current knowledge, we hypothesize that utilizing this genome-wide approach to identify methylation changes, as well as DNA amplifications, can lead to the discovery of aberrant DNA methylation patterns, and the identification of novel genes that are silenced or activated in lung cancer.In chapter 2, we successfully established Asc I RLGS, an AscI-EcoRV boundary plasmid library, and library mixing RLGS gels to facility the identification of methylated and amplified genes in cancer development. In chapter 3, we selected 1184 CpG islands for RLGS analysis and determined their methylation status in 16 primary non-small cell lung cancers. The global methylation pattern of lung cancer is heterogeneous. We demonstrated that methylation is part of the silencing process of BMP3B in primary tumors and lung cancer cell lines. Chapter 4 describes the detailed characterization of BMP3B as a cancer related gene. In a large set of NSCLC, we found that BMP3B showed increased methylation in approximately 45% of primary lung cancers. Among the markers that we used, the closest marker to BMP3B, D10S196, showed 35% LOH. Restoration of gene expression suppresses in vitro and in vivo cell growth of human lung cancer cell line A549. In chapter 5, the amplified RLGS fragments were summarized and four amplicons in primary lung cancer have been cloned. The 11q22 amplicon was further characterized by Southern hybridizations to define the amplified region.In conclusion, the identification of novel methylated genes with loss-of-function and the amplified genes with gain-of-function during lung cancer development will improve the understanding of lung cancer tumorigenesis. Our study should provide better knowledge of the mechanism of lung cancer for better diagnosis and treatment.