The molecular events underlying colonic neoplastic progression potentially involve at least three classes of genetic elements: oncogenes, tumor suppressor genes and DNA repair genes. Although alterations in specific loci have been identified in different colonic lesions, the consequences of activation or inactivation and the interplay between these altered elements in the colonic neoplastic process remain little understood. The long term goal of the work outlined in this proposal is to link genotypic change with the in vivo phenotype generated and to gain an understanding of the individual and cumulative role of genetic changes underlying colon carcinogenesis. Toward this end we have developed a panel of immortalized rat colon epithelial cell lines which act as a vehicle for analysis of the action of both individual and combination genes. The desirability of a human counterpart of these cell lines is self evident and we propose to use culture techniques we have established, to achieve this goal. Immortalized cell lines will be used to isolate novel phosphotyrosyl proteins associated with early events in neoplastic progression, to which monoclonal antibodies will be raised and assessed as potential markers of change in colon mucosa. Analysis of individual genetic events implicated in colon carcinogenesis will be directed not only toward gaining an understanding of their role in progression involving interaction with other altered genes, but also the cellular events accompanying activation or inactivation. These studies will focus on cellular events associated with c-src activation in colon epithelium including the identification and isolation of cellular substrates and protein interactions involved in src signaling pathways. From the use of a heterotopic colon model, with the ability to localize implanted cells, we propose to establish genotypic/phenotypic links associated with altered genetic loci. Alternative use of the heterotopic model is proposed to evaluate possible complementation between APC and K-ras gene mutations in early stages of colonic neoplastic progression. Early and late stage events associated with mutation of the MSH2 locus in colonic tissue will be performed in a mouse model currently under construction.
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