Signal Transduction Events and the Regulation of Cell Growth

该项目旨在增加我们对癌细胞生物学的理解，并通过研究生长调节信号转导事件来开发癌症治疗的新方法。这项工作目前集中在（1）视网膜母细胞瘤易感基因家族调控癌细胞生长的新方面和（2）环AMP负生长调控的分子机制。视网膜母细胞瘤蛋白目前被认为仅在细胞核中起作用，并且仅在细胞周期的G1期起作用。相比之下，使用共聚焦显微镜和免疫共沉淀技术，我们发现，Rb是与细胞骨架，这种定位作为一个功能的细胞周期的调节。这些数据得到了支持，在体外转录-翻译实验中使用野生型和突变型GST-Rb结构，并通过瞬时转染使用血凝素标记的野生型和突变型Rb结构。这些数据提示了一种新的肿瘤抑制基因功能模型，其中肿瘤抑制基因产物对细胞-细胞或细胞-基质相互作用有反应。在研究环AMP的抗癌作用时，我们确定细胞内环AMP的升高导致细胞周期蛋白A表达的丧失和细胞周期蛋白A调节的细胞周期蛋白依赖性激酶活性的下调。我们最近的研究表明，环腺苷酸诱导前列腺癌细胞G1期阻滞的机制表明，环腺苷酸诱导转录的细胞周期蛋白依赖性激酶抑制剂p21。我们正在绘制p21启动子上的环AMP响应元件，并已开始表征响应环AMP激活近端启动子的因子。 这些研究表明，cAMP通过一种新的非CRE介导的机制激活p21启动子。 此外，数据表明组蛋白乙酰化酶（HAT）在p21启动子激活响应cAMP的作用。

This project is designed to increase our understanding of cancer cell biology and to develop a new approach to cancer treatment through the study of growth-regulatory signal transduction events. This work is currently focused on (1) novel aspects of the regulation of cancer cell growth by the retinoblastoma susceptibility gene family and (2) the molecular mechanism of negative growth regulation by cyclic AMP. The retinoblastoma protein is currently thought to function only in the cell nucleus and only in the G1 phase of the cell cycle. In contrast, using confocal microscopy and co-immunoprecipitation techniques we found that Rb is associated with the cell cytoskeleton and that this localization is regulated as a function of the cell cycle. These data were supported by in vitro transcription-translation experiments using wild type and mutant GST-Rb constructs, and by transient transfections using hemagglutinin-tagged wild type and mutant Rb constructs. These data suggest a new model of tumor suppressor gene function, in which the tumor suppressor gene product is responsive to cell-cell or cell-substratum interactions. While studying the anticancer action of cyclic AMP we determined that elevation of intracellular cyclic AMP causes loss of cyclin A expression and down-regulation of cyclin A-regulated cyclin-dependent kinase activity. Our recent studies on the mechanism of cyclic AMP-induced G1 arrest in prostate cancer cells demonstrate that cyclic AMP induces transcription of the cyclin-dependent kinase inhibitor p21. We are mapping the cyclic AMP-responsive element on the p21 promoter and have begun characterizing the factors activating the proximal promoter in response to cyclic AMP. These studies demonstrate that cAMP activates the p21 promoter through a novel, non-CRE-mediated mechanism. Furthermore, the data demonstrate the role of histone acetylases (HATs) in p21 promoter activation in response to cAMP.