MOLECULAR STUDIES OF ESOPHAGEAL ADENOCARCINOMA

这项持续应用的目标是识别食管癌中重要的、潜在的新型扩增癌症相关基因。该项目涉及在癌症和分子遗传学方面具有丰富专业知识的研究人员的合作，重点研究食管癌，这是一种发病率不断上升的致命癌症。鉴定新的基因组扩增，以及由此产生的重要基因在这些肿瘤中过表达，将为与该疾病的发生和进展相关的分子事件提供重要的见解。这项工作可能为早期检测提供新的遗传工具，并有可能为治疗干预确定新的途径。我们利用高灵敏度的定量二维基因组扫描（2D凝胶）方法，在食管腺癌中发现了许多潜在的新的基因组扩增事件。该技术涉及对来自个体患者正常和肿瘤组织之间的基因组功能成分的数千个基因组限制性片段进行比较分析。扩增子中包含的扩增和过表达基因将被识别和表征，使用我们已经成功演示的食管腺癌新扩增子的程序。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。利用创新的定量PCR检测和迄今收集的200多个腺癌，将确定“共同扩增的最小区域”。这将有效地定义该区域，以寻找重要的扩增和过表达的癌症相关基因。潜在的候选基因可能包括已知基因，未表征的表达序列标签，或从最小区域内的基因组克隆中分离的基因。新基因的癌症相关性将通过功能分析进行检验，包括转染到永生化细胞中，并分析其对小鼠细胞生长、侵袭性、锚定独立性和肿瘤形成的影响。这些研究将确定在食管腺癌中扩增的特定基因的身份和频率，并对这种重要癌症的发生和/或进展的机制提供更深入的了解。

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.