SIGNALING PATHWAYS REGULATING EPITHELIAL CELL GROWTH

这些研究致力于增加我们对肝细胞转化机制的理解。 在原代肝细胞和HepG 2肝癌细胞中，长时间的丝裂原活化蛋白（MAP）激酶激活增加了细胞周期蛋白依赖性激酶抑制剂（cdki）蛋白的表达，并减少了DNA合成。 然而，在肝癌细胞中，增加cdki表达和抑制DNA合成所需的MAP激酶活性的量增加了至少6倍。 因此，可能发生生长控制丧失和肝细胞转化的一种机制是MAP激酶信号传导导致cdki蛋白水平增加的能力降低。即在肝癌细胞中提高p21 Cip-1表达所需的阈值MAP激酶活性增加。 这种阈值改变的可能候选分子是MAP激酶信号传导的下游和cdki蛋白的上游。 肿瘤抑制因子p53不太可能是候选者，因为原代肝细胞和HepG 2细胞都表达功能性p53。 这项工作的目的是了解在原代肝细胞中MAP激酶级联的下游机制，该通路通过该机制发出信号以增加cdki蛋白p21 Cip-1和p16 INK 4a的表达。 我们将使用基因敲除小鼠和反义技术来确定慢性MAP激酶信号传导是否能够（a）在p21 Cip-1/WAF 1或p16 INK 4a表达缺失的情况下迫使原代肝细胞进入S期，和（B）通过增加转录和/或翻译速率来增加p21 Cip-1和p16 INK 4a蛋白水平，或通过增加p21 Cip-1/p16 INK 4a mRNA和/或蛋白的稳定性。 我们将确定由于原代肝细胞中MAP激酶级联反应的慢性激活而导致的p2 l Cip-1蛋白水平增加是否是由转录因子C/EBPalpha、C/EBPbeta、Sp1和Sp3介导的。只有通过了解原代肝细胞中调节生长停滞的信号传导机制，我们才能开始了解肝癌细胞中这些机制中的缺陷。

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.