These studies are devoted to increasing our understanding of the mechanisms of hepatocellular transformation. In primary hepatocytes and HepG2 hepatoma cells, prolonged Mitogen Activated Protein (MAP) kinase activation increased expression of cyclin dependent kinase inhibitor (cdki) proteins and reduced DNA synthesis. However, the amount of MAP kinase activity required to increase cdki expression and inhibit DNA synthesis had increased at least 6-fold in the hepatoma cells. Thus one mechanism by which loss of growth control and hepatocellular transformation may take place is by a reduced ability of MAP kinase signaling to cause increased cdki protein levels. i.e. the threshold MAP kinase activity required to elevate p2l Cip-1 expression has increased in the hepatoma cells. The likely candidate molecules for this alteration in the threshold are downstream of MAP kinase signaling and upstream of the cdki proteins. The tumor suppresser p53 is unlikely to be a candidate because both primary hepatocytes and HepG2 cells express functional p53.
The aim of this work is to understand in primary hepatocytes the mechanisms downstream of the MAP kinase cascade by which this pathway signals to increase the expression of the cdki proteins p2l Cip-1 and p16 INK4a. We will determine using knock-out mice and anti-sense technologies whether chronic MAP kinase signaling can (a) force primary hepatocytes into S phase in the absence of either p2l Cip-1/WAF1 or p16 INK4a expression, and (b) increase p2l Cip-1 and p16 INK4a protein levels by increasing the rates of transcription and/or translation, or by increasing the stabilities of p2l Cip-1/p16 INK4a mRNA and/or protein. We will determine whether the increased protein levels of p2l Cip-1 due to chronic activation of the MAP kinase cascade in primary hepatocytes is/are mediated by the transcription factors C/EBPalpha, C/EBPbeta, Sp1, and Sp3. It is only by understanding the signaling mechanisms which regulate growth arrest in primary hepatocytes that we may begin to understand what has become defective within these mechanisms in hepatoma cells.
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