The primary goal of this revised proposal is to examine the relationship of the expression of protein kinase C isoforms with tight junction permeability and colon tumors. The hypothesis to be tested is that an increase in tight junction leakiness (and therefore in the paracellular permeability across an epithelial barrier) plays a major role in epithelial carcinogenesis through mechanisms regulated by protein kinase C isoforms. Specifically, to test this hypothesis, changes in PKC-induced tight junction permeability will be studied with regard to the different stages of development of epithelial cancers, namely normal mucosa, polyps and carcinomas of a rat model of colon carcinogenesis.
Four specific aims are proposed: (a) Measurement of tight junction permeability via transepithelial electrical resistance, paracellular flux of solutes and by electron microscopy with electron- dense dyes; (b) Expression and distribution of PKC isoforms; (c) Expression and distribution of tight junction-associated proteins; and quantitative subcellular distribution of epithelial growth factor receptors by immunocytochemistry and autoradiography.
