ROLE OF C/EBP ALPHA IN GROWTH INHIBITION

这项提案的主要目标是界定核能的作用 转录因子C/EBPalpha与生长抑制和 永垂不朽。我们已经证明C/EBPalpha抑制细胞 包括人在内的几种转化细胞系的体外增殖 肝癌细胞和缺少P53和Rb蛋白的细胞系Saos2。我们 进一步表明诱导C/EBPalpha在稳定的 转化的人纤维肉瘤细胞抑制细胞生长和 同时上调p21/SDI-1mRNA和蛋白表达。因为p21 成纤维细胞中的蛋白质与细胞衰老有关 推测C/EBPalpha可能至少在一定程度上对 体内正常肝细胞的静息或静止状态。P21/SDI-1 在肝脏中，mrna的表达水平很容易被 北方印迹。 我们将在三个实验中研究C/EBPalpha在生长抑制中的作用 不同的方式。首先，我们将克隆C/EBPalpha的靶基因 参与了我们开发的一种新的细胞系统的生长停滞 实验室。我们将使用酶降解消减杂交来 识别作为C/EBPalpha靶标的基因以及 对增长停滞负责。第二，我们将测定生化 P21/SDI-1在生长细胞中表达增加的基础 被C/EBPalpha逮捕。我们将确定p21/SDI-1是否表达 对于通过阻断p21/SDI-1来阻止生长是必要的和/或充分的 使用反义策略进行表达。初步数据显示，P53和 C/EBPalpha不需要RB来阻止增长；我们将建立 这两个抑癌基因中的任何一个是否参与了 C/EBPalpha的生长抑制作用。最后，我们将利用C/EBP敲门- 我们在实验室里制作的小鼠模型。源自的单元格 这些动物将在体外培养，以检验这一假设 失去C/EBPalpha将改变生长特性或 细胞永生化的频率。

The major goal of this proposal is to define the role of the nuclear transcription factor, C/EBPalpha in growth inhibition and immortalization. We have shown that C/EBPalpha inhibits cell proliferation in several transformed cell lines in vitro including human hepatoma cells and Saos2, a cell line that lacks p53 and Rb protein. We have further shown that induction of C/EBPalpha expression in stably transformed human fibrosarcoma cells inhibits cell growth and simultaneously elevates p21/SDI-1 mRNA and protein. Because the p21 protein is associated with cellular senescence in fibroblasts, we speculate that C/EBPalpha may be responsible, at least in part, for the resting or quiescent state of the normal hepatocyte in vivo. p21/SDI-1 mRNA is expressed in liver at levels that are easily detectable by Northern blotting. We will examine the role of C/EBPalpha in growth inhibition in three different ways. First, we will clone the target genes of C/EBPalpha that are involved in growth arrest in a novel cell system developed in our laboratory. We will use enzyme degrading subtractive hybridization to identify the genes that are targets of C/EBPalpha and that are responsible for growth arrest. Second, we will determine the biochemical basis for increased expression of p21/SDI-1 in cells that are growth arrested by C/EBPalpha. We will determine whether p21/SDI-1 expression is necessary and/or sufficient for growth arrest by blocking p21/SDI-1 expression using antisense strategies. Preliminary data suggest p53 and Rb are not required for growth arrest by C/EBPalpha; we will establish whether or not either of these tumor suppressor genes is involved in growth inhibition by C/EBPalpha. Finally, we will exploit a C/EBP knock- out mouse model that we have made in our laboratory. Cells derived from these animals will be cultured in vitro in order to test the hypothesis that the loss of C/EBPalpha will alter the growth properties or immortalization frequency of the cells.