ROLE OF C/EBP ALPHA IN GROWTH INHIBITION

该提案的主要目标是界定核武器的作用， 转录因子，C/EBPalpha在生长抑制和 永生 我们已经证明，C/EBPalpha抑制细胞生长， 在包括人在内的几种体外转化细胞系中增殖 肝癌细胞和Saos 2，缺乏p53和Rb蛋白的细胞系。 我们 进一步表明，在稳定的细胞中诱导C/EBPalpha表达， 转化的人纤维肉瘤细胞抑制细胞生长， 同时上调p21/SDI-1 mRNA和蛋白表达。 因为p21 蛋白质与成纤维细胞的细胞衰老有关，我们 推测C/EBPalpha可能负责，至少部分负责， 体内正常肝细胞的静息或静止状态。 p21/SDI-1 mRNA在肝脏中的表达水平很容易通过 北方印迹法。 我们将研究C/EBPalpha在三种生长抑制中的作用。 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型细胞系统的生长停滞。 实验室 我们将使用酶降解消减杂交， 确定C/EBPalpha的靶基因， 负责生长停滞。 其次，我们将确定生物化学 p21/SDI-1在生长的细胞中表达增加的基础 被C/EBPalpha逮捕。 我们将确定p21/SDI-1表达是否 是通过阻断p21/SDI-1生长停滞所必需和/或充分的 使用反义策略表达。 初步数据表明p53和 Rb不需要C/EBPalpha的生长停滞;我们将建立 这些肿瘤抑制基因是否参与了 通过C/EBPalpha的生长抑制。 最后，我们将利用C/EBP爆震- 这是我们在实验室制作的小鼠模型。 衍生的细胞 这些动物将在体外培养，以测试假设， C/EBPalpha的丧失将改变生长特性，或者 细胞的永生化频率。

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.