MOLECULAR STUDIES OF ESOPHAGEAL ADENOCARCINOMA

这一持续应用的目标是在食管腺癌中识别重要的、可能是新的扩增的癌症相关基因。这个项目涉及在癌症和分子遗传学方面拥有丰富专业知识的研究人员的合作，并将重点放在食管腺癌上，这是一种发病率正在上升的致命癌症。识别新的基因组扩增，以及由此产生的在这些肿瘤中过度表达的重要基因，将为深入了解与这种疾病的发展和进展相关的分子事件提供重要的见解。这项工作可能会为早期发现提供新的基因工具，并有可能为治疗干预寻找新的途径。我们已经使用高灵敏度的定量二维基因组扫描(2D GELL)方法在食管腺癌中确定了一些潜在的新的基因组扩增事件。这项技术涉及对数千个来自基因组功能成分的基因组限制片段进行比较分析，这些片段来自单个患者的正常组织和肿瘤组织。我们将使用我们在食管腺癌中成功展示的一种新的扩增子的程序来鉴定和表征扩增和过度表达的基因。二维凝胶特别适合于DNA扩增区域的鉴定。扩增片段将使用我们已经成功实施的程序进行克隆，并使用荧光原位杂交和/或数据库搜索绘制其染色体位置。利用一种创新的定量聚合酶链式反应分析方法和迄今收集到的200多例腺癌，将确定“最小的共同扩增区域”。这将有效地定义寻找重要的扩增和过度表达的癌症相关基因的区域。潜在的候选基因可能包括已知基因、未确定特征的表达序列标签或从映射到最小区域内的基因组克隆中分离出来的基因。新基因与癌症的相关性将通过功能分析来检验，包括对永生化细胞的转基因，以及对细胞生长、侵袭性、锚定独立性和小鼠肿瘤形成的影响的分析。这些研究将确定在食管腺癌中扩增的特定基因的特性和频率，并提供对这一重要癌症发生和/或进展的潜在机制的更深入的了解。

The goals of this continuation application are to identify important, and potentially novel amplified cancer-related genes in esophageal adenocarcinomas. This project involves the collaboration of investigators with substantial expertise in cancer and molecular genetics and focuses upon esophageal adenocarcinoma, a deadly cancer which is increasing in incidence. The identification of novel genomic amplifications, and the resulting important genes overexpressed in these tumors, will provide significant insight into the molecular events associated with the development and progression of this disease. This work may provide new genetic tools for early detection, and potentially for identifying new avenues for therapeutic intervention. We have identified a number of potentially novel genomic amplification events in esophageal adenocarcinomas using the highly sensitive method of quantitative two-dimensional genomic scanning (2D gels). This technology involves the comparative analysis of several thousand genomic restriction fragments derived from functional components of the genome between the normal and tumor tissue from an individual patient. The amplified and overexpressed genes encompassed in the amplicons will be identified and characterized, using the procedures we have successfully demonstrated for a novel amplicon in esophageal adenocarcinomas. 2D gels are particularly well suited for the identification of amplified regions of DNA. Amplified fragments will be cloned using procedures that we have successfully implemented and their chromosomal locations mapped using fluorescence in situ hybridization and/or database searches. Utilizing an innovative quantitative PCR assay and over 200 adenocarcinomas collected to date, the "minimal region of common amplification" will be determined. This will effectively define the region to search for important amplified and overexpressed cancer-related genes. Potential candidates may include known genes, uncharacterized expressed sequence tags, or genes isolated from genomic clones that map within the minimal region. The cancer-relatedness of novel genes will be examined using functional assays involving transfection into immortalized cells and analysis of the effects upon cell growth, invasiveness, anchorage-independence and tumor formation in mice. These studies will determine both the identity and frequency of specific genes amplified in esophageal adenocarcinomas and also provide a greater insight into the mechanisms underlying the development and/or progression of this important cancer.