Hepatocarcinogenesis is a complex process requiring cell-to-cell interactions within the three-dimensional tissue architecture that constitutes the mammalian liver. We seek to enhance our abilities to investigate hepatocarcinogenesis by extending the process to the culture dish as much as possible. We have developed an organotypic rat liver model in which viable tissue has been maintained in culture for longer than 90 days. The overall objective for this application is to further develop this organotypic culture model for rat liver (and in the future, for human liver tumor tissue, as well), and to demonstrate its usefulness for investigating choline-deficient hepatocarcinogenesis in a rat model. Toward this end, we propose three Specific Aims: (1) For our organotypic rat liver culture model, we will analyze and test all medium components until we arrive at a defined medium that provides long periods of viability as judged by dual vital staining, by gene expression patterns revealed by DNA arrays, by metabolic capabilities revealed by enzyme amounts and activities. (2) We will further characterize CWSV-1 cells, which form islands of tissue in culture that approximate normal liver in pattern of organization, especially as to their ability to give rise to more than one liver cell type and their progression to tumorigenic cells in choline-deficient medium. (3) We will compare and contrast these two culture models in Aims (1) and (2) with respect to the ability of PBN and 4-OH-PBN to increase apoptosis and inhibit tumor development. Ultimately, we seek an organotypic culture model in which the full process of carcinogenesis from tumor initiation through progression to full metastatic cancer can be manipulated and cures tested.
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