The mammalian liver is one of the few adult organs capable of completely regenerating itself in response to injury or two-thirds partial hepatectomy in which terminally differentiated hepatocytes are induced to reenter the cell cycle and undergo DNA replication and mitosis. The proliferative response of differentiated hepatocytes is initiated by the release of growth factors and cytokines that stimulate expression and nuclear translocation of immediate early transcription factors. However, the transcriptional mechanisms mediating hepatocyte entry into S-phase and mitosis are not completely understood. In the previous funding period, we developed transgenic mice in which the Transthyretin promoter functioned to prematurely express the Forkhead Box (Fox) M1B (previously called HFH-11B) transcription factor. Liver regeneration studies with these TG mice demonstrated that premature FoxM1B expression accelerates the onset of hepatocyte proliferation through earlier induction of cell cycle regulatory genes. Furthermore, albumin enhancer/promoter driven Cre recombinase (AIb-Cre) mediated deletion of the FoxM1b fl/fl (LoxP targeted) allele in postnatal hepatocytes resulted in significant reduction in regenerating hepatocyte DNA replication and mitosis. This was associated with increased levels of the cyclin dependent kinase (Cdk) inhibitor p21Cip1 protein and undetectable expression of Cdc25B phosphatase, which is required for Cdk1 activity and entry into mitosis. Our broad, long-term goals are to determine the role of Foxm1b in mediating regenerating hepatocyte proliferation and liver morphogenesis and examine whether Foxm1b is required for development of hepatocellular carcinoma. We propose the following three Specific Aims: (1) To test the hypothesis that deleting the cdk inhibitor p21Cip1 gene and restoring Cdc25B expression will increase hepatocyte proliferation in regenerating AIb-Cre Foxm1b -/- liver. We have shown that deletion of the Foxm1b fl/fl allele in the embryonic liver causes abnormal liver development and embryonic lethality. (2) To further characterize the Foxm1b embryonic liver defect and use of inducible hepatic Cre recombinase to delete the Foxm1b fl/fl allele at various times during liver development to determine the stages of liver development at which Foxm1b function is required. (3) Because Foxm1b is essential for regenerating hepatocyte proliferation, we will test the hypothesis that AIb-Cre Foxm1b -/- hepatocytes are refractory to developing hepatocellular carcinoma in response to a Diethylnitrosamine/Phenobarbital liver tumor induction protocol. ? ? ? ?
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