The long-term objectives are to determine cellular origins and lineage pathways in hepatoma formation in experimental hepatocarcinogenesis rat models. The discovery of new cell types within bile ductules (basal blast-like cells, transitional cells) in rat livers during chemical carcinogenesis suggested that other stem-like cells may exist in carcinogen-treated livers besides the previously reported oval/bile ductule cells (BD) cells.
The specific aims are to determine: l) the specific cells within bile ductules that give rise to preneoplastic nodules (PN) and that repopulate the liver; 2) the mechanisms by which BD cells migrate and invade liver cords to form PN and whether these mechanisms are similar to those utilized by malignant cells undergoing metastasis; and 3) functional properties of ductule transitional cells that have integrated into the hepatic cord. Livers from carcinogen-partial hepatectomy (PH) protocols (Solt-Farber) will be studied for the following: l) expression of proteins during migration of BD cells into sinusoids (e.g.cytoskeleton), invasion of BD cells into liver parenchyma (e.g. extracellular matrix, adhesion receptors, endothelium, matrix metalloproteinases and inhibitors) and acquisition by transitional cells of either hepatocyte or nodular differentiation markers (e.g. transporter, secretory, cell communication) and 2) the presence of point mutations in the pS3 tumor suppressor gene in BD cells and nodules. Livers will also be injected with retroviral marker gene (E.coli-beta gal lacZ nls gene) after PH step of the protocols to label replicating basal cells to determine cell lineage pathways in PN development and in liver restoration. BD cell types and their interrelations to each other and with extracellular components will be analyzed by microscopic methods (light, confocal, electron, immunocytochemistry). Laser capture microdissection microscopy will be used to isolate specific hepatic cells from liver sections for analysis of p53 gene mutations by PCR/single strand conformation polymorphism/DNA sequencing procedures.
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