ROLE OF E2F IN REGULATION OF CELL CYCLE AND TUMOR DEVELOPMENT

视网膜母细胞瘤的发育和肿瘤抑制特性 蛋白质（pRB）似乎至少部分依赖于其 调节转录活性的能力， E2 F转录因子 动物模型将用于研究 pRB特异性E2 F的发育作用，并确定它们如何 调节有助于视网膜母细胞瘤蛋白的性质。 在E2 F-2和E2 F-3基因中具有种系突变的新型小鼠品系 将使用标准的基因定位技术。 分析 这些小鼠将确定单个或多个pRB的丢失是如何发生的， 特异性E2 Fs影响胚胎和成年小鼠的发育。 然后将使用杂交来确定这些突变是否 基因足以阻断肿瘤形成， Rb+/-小鼠或Rb-/-胚胎中发生的广泛凋亡。 这将有助于确定E2 F监管如何有助于 pRB的发育和肿瘤抑制特性。 的影响 这些突变也将在分子水平上进行检查， 单一和复合突变小鼠品系以产生原代细胞系。 在每种情况下，E2 F复合物的光谱，E2 F复合物的程度和时间， E2 F应答基因的激活和这些突变体的生长特性 将对细胞进行分析。 这些研究将有助于将pRB特异性 E2 Fs对特定靶基因的激活，并确定这些基因如何被激活。 个别事件有助于生长刺激特性的 内源性E2 F。 最后，我们将继续进行体外开发 转录检测可以让我们研究 pRB与其靶E2 Fs之间的相互作用。 这尤其 系统将进行优化，以研究pRB消除 与其结合的启动子的基础转录。 这些目标 体外研究是为了分离E2 F特异性和全局抑制， pRB的性质，以建立这些分子机制， 影响是介导的，并确定如何每一个有助于 这种蛋白质的生长抑制特性。

The developmental and tumor-suppressive properties of the retinoblastoma protein (pRB) both appear to be at least partially dependent upon its ability to regulate the transcriptional activity of a discrete subset of the E2F transcription factors. Animal models will be used to study the developmental role of the pRB-specific E2Fs and determine how their regulation contributes to the properties of the retinoblastoma protein. Novel mouse strains with germline mutations in the E2F-2 and E2F-3 genes will be created using standard gene targeting technology. Analysis of these mice will establish how loss of either single or multiple pRB- specific E2Fs affects the development of embryonic and adult mice. Crossbreeding will then be used to determine whether mutation of the these genes is sufficient to block either the tumor formation that occurs in the Rb+/- mice or the widespread apoptosis that occurs in the Rb-/- embryos. This will help to establish how E2F-regulation contributes to the developmental and tumor-suppressive properties of pRB. The effect of these mutations will also be examined at the molecular level using the single and compound mutant mouse strains to generate primary cells lines. In each case, the spectrum of E2F complexes, the degree and timing of activation of E2F responsive genes and growth properties of these mutant cells will be analyzed. These studies will help to link the pRB-specific E2Fs to the activation of specific target genes and determine how these individual events contribute to growth stimulatory properties of the endogenous E2F. Finally, we will continue to develop in vitro transcription assays that will allow us to study the direct consequences of the interaction between pRB and its target E2Fs. In particular, this system will be optimized to study the mechanism by which pRB abolishes the basal transcription of promoters to which it is bound. The goal of these in vitro studies is to separate the E2F-specific and global repression properties of pRB, to establish the molecular mechanisms by which these effects are mediated, and to determine how each one contributes to the growth suppressive properties of this protein.