SIGNALING PATHWAYS REGULATING EPITHELIAL CELL GROWTH

这些研究致力于加深我们对肝细胞转化机制的了解。在原代肝细胞和HepG2肝癌细胞中，延长丝裂原活化蛋白(MAP)激酶的激活时间可增加细胞周期蛋白依赖性激酶抑制物(CDKI)蛋白的表达，减少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MAPK活性至少增加了6倍。因此，失去生长控制和肝细胞转化的一个机制可能是由于MAP激酶信号转导能力降低而导致CDKI蛋白水平升高。也就是说，在肝癌细胞中，上调p21-Cip-1表达所需的MAPK活性阈值已经增加。这种阈值变化的可能候选分子是MAP激酶信号的下游和CDKI蛋白的上游。肿瘤抑制基因P53不太可能是候选基因，因为原代肝细胞和HepG2细胞都表达功能性P53。这项工作的目的是在原代肝细胞中了解MAP激酶级联下游的机制，该途径通过信号传递增加CDKI蛋白p21、Cip-1和p16INK4a的表达。我们将利用基因敲除小鼠和反义技术来确定慢性MAP激酶信号是否可以(A)在p2lCip-1/WAF1或p16INK4a缺失的情况下迫使原代肝细胞进入S期，以及(B)通过提高转录和/或翻译速率或通过增加p2lCip-1/p16INK4a mRNA和/或蛋白的稳定性来增加p2lCip-1和p16INK4a的蛋白水平。我们将确定原代肝细胞中由于MAP激酶级联的慢性激活而导致的p21-Cip-1蛋白水平的增加是否是由转录因子C/EBPalpha、C/EBPbeta、Sp1和Sp3介导的。只有通过了解调节原代肝细胞生长停滞的信号机制，我们才能开始了解肝癌细胞中这些机制中的缺陷是什么。

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.