MANIPULATING HEPATOCYTE PROLIFERATION BY GENE TARGETING.

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The aim of this RO3 proposal is to examine the ability of cyclin D1 and other cell cycle regulatory proteins to promote hepatocyte proliferation in culture and in vivo. Mature hepatocytes retain the ability to function as stem cells and can repopulate the live after hepatic injury. Previous studies have suggested that up-regulation of cyclin D1 plays an important role in governing progression of hepatocytes through G1 phase of the cell cycle. To test whether cyclin D1 is capable of promoting mitogen-independent hepatocyte proliferation, we have constructed a replication-defective adenovirus encoding this protein (ADV-D1). Our preliminary studies indicate that ADV-D1 but not a control adenovirus, promotes cell cycle progression in primary hepatocytes in the absence of growth factor, and induces hepatocyte proliferation on extracellular matrices that are normally growth-inhibitory. Pilot in vivo studies indicate that ADV-D1 effectively transfects hepatocytes in the intact animal. These results suggest that transfection of hepatocytes with ADV-D1 can promote hepatocyte proliferation independent of extracellular signals, and warrants testing as an agent to promote liver regeneration. The three Specific Aims of this study will examine the mechanisms by which cyclin D1 promotes hepatocyte growth, and will examine the ability of other G1 phase cyclins and E2F transcription factors to promote hepatocyte cell cycle progression. These studies are intended to provide unique mechanistic insight in the regulation of hepatocyte growth, and to identify candidate proteins for gene targeting strategies to therapeutically enhance liver regeneration.