The goals of this renewal application are to identify important and potentially novel amplified cancer-related genes in esophageal adenocarcinomas, a deadly cancer that is increasing in incidence. The identification of genomic amplifications, and the resulting important genes over-expressed 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) and using gene expression profiling with oligonucleotide arrays. The 2D-gel 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 over-expressed genes encompassed in the amplicons are identified and characterized as we have successfully demonstrated. Utilizing well-defined chromosomal markers, a quantitative PCR assay and the DNA from large numbers of normal tissue-adenocarcinoma paired samples, we have identified the """"""""minimal region of common amplification"""""""" for a number of specific amplicons for immediate analysis and other amplicons will be determined. The important amplified and over-expressed cancer-related genes and will be defined. 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 the analysis of the effects upon cell growth, invasiveness, anchorage-independence, apoptosis and in selected cases tumor formation in mice. In vitro studies using adenocarcinoma cells containing amplified genes of interest of containing overexpressed genes as the result of transfection will be examined for potential targeted therapy and thus providing clinical relevance to identification of specific amplification events. 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.
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