Targeting p27kip1 to Improve Hepatocyte Transplantation, Bench Research

In characterizing the cell-cycle regulatory role of the cyclin-dependent kinase inhibitor, p27kip1, it was discovered that p27 knockout mice exhibit pronounced multi-organomegaly with no increased spontaneous carcinogenesis in the liver. This suggested that p27 manipulation could improve the success of hepatocyte transplantation. It was then shown that donor hepatocytes from p27 knockout mice more actively proliferate and rescue liver failure in the FAH mouse model. These results were limited by the fact that p27 was suppressed throughout the donor animal from early embryogenesis and increase the risk of carcinogenesis due to long-term p27 absence. In this proposal we develop three new techniques for targeting p27 in the mouse liver. Mx1-Cre transgenic mice will allow us to knockout p27 in the liver alone at controlled timepoints of hepatogenesis. The tetracycline-controlled overexpression of the F-box protein Skp2 will allow us to lower p27 protein abundance in the hepatocytes of Alb-rTA transgenic mice for controlled periods of time. Finally, the tetracycline-controlled expression short hairpin RNA structures (shRNAs) will allow us to target p27 for RNA interference (RNAi) in the hepatocytes of Alb-rTA transgenic mice for controlled periods of time. Tet-controlled Skp2 and p27shRNA expression will also be delivered to wild type isolated primary hepatocytes in culture by an advance lentivirus vector, giving us p27 targeting strategies which are directly relevant to human therapeutics. In vivo and in vitro comparisons of our various delivery systems will give us detailed information of the role of p27 in hepatocyte proliferation and transformation, and will be complimented by a model of induced hepatocellular carcinoma. Finally, we will perform a controlled series of hepatocyte transplantation studies in which various p27 targeting strategies are employed in conjunction with variations of the FAH mouse liver failure model. The results will give us a clear assessment of the potential benefits of p27 suppression in the treatment of human liver failure. I submitted a K08 application in November of 2003 with score of 130 will result in funding by the NIDDK in the winter of 2004.