During liver regeneration after partial hepatectomy (PH), normally quiescent hepatocytes enter the cell cycle and replicate. Although-more than 95% of hepatocytes of young adult rats and mice divide during regeneration, the process is strictly regulated and terminates when the liver regains its original weight. In contrast, hepatic carcinogenesis requires sustained hepatocyte replication and gradual loss of proliferative control. Our general goals are: a) to understand the molecular mechanisms that control the initiation of liver regeneration and the sequential steps that lead to DNA synthesis and b) to determine the critical steps required for the development of tumors from hepatocytes which acquire the capacity to proliferate. Our work has centered on studies of protooncogenes and growth factors during liver regeneration and on the analysis of hepatocarcinogenesis in transgenic mice which overexpress transforming growth factor alpha (TGFalpha). During the last period of grant support, we showed that the activation of the transcription factor NF-kappaB occurs almost immediately after PH and have proposed that DNA binding of NF-kappaB is one of the events that make hepatocytes competent to enter the cell cycle. Using partially hepatectomized rats, transgenic and knockout mice and newly developed lines of replicating differentiated hepatocytes, we propose to investigate: a) the molecular mechanisms of NF-kappaB activation after PH and in hepatocyte cultures, b) the role of reactive oxygen intermediates (ROI) in NF-kappaB activation after PH; c) the role of tumor necrosis factor alpha (TNF) as an activator of NF-kappaB and initiator of liver regeneration; d) c-myc transactivation by NF-kappaB in differentiated hepatocytes using transient transfection assays to determine whether c-myc is a NF-kappaB target gene. The major goals of the proposed experiments are to determine whether NF-kappaB activation after PH is required for DNA synthesis and to identify the inducers of NFkappaB as well as its potential target genes in the regenerating liver. We will also investigate whether the increased DNA binding of the transcription factors AP1 and STAT3, which occur during the first hours after PH, may be induced by cytokines and growth factors and be modulated by ROI. The development of neoplasia involves not only genes which promote and suppress growth but also genes which regulate programmed cell death. The balance between proliferation and apoptosis is a major factor in determining the persistence and expansion of early neoplastic foci. In our studies of TGFalpha transgenic mice we showed that TGFalpha causes liver hyperplasia in young animals, that hyperplasia is associated with high hepatocyte turnover as the animals age and that more than 75% of 12-15 month mice develop hepatocellular tumors. We hypothesize that TGFalpha mediated hyperplasia may be compensated by apoptosis but that constitutive hepatocyte proliferation eventually causes genomic instability leading to tumor formation. We will test this hypothesis by: a) measuring the expression of apoptosis regulating genes in TGFalpha transgenic mouse livers with high hepatocyte proliferation and turnover and by studying tumor development in TGFalpha/Bd-2 double transgenic mice; b) using flow cytometry to analyze the ploidy distribution of proliferating hepatocytes and the proportion of apoptotic cells in each ploidy class and c) determining whether proliferating hepatocytes and tumor cells are mostly diploid cells which have microsatellite instability.
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