Control of topoisomerose II through the cell cycle

通过细胞周期控制拓扑异构糖 II

• 批准号: 6634084
• 负责人: DENNIS W STACEY
• 金额: $ 22.22万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6634084
• 关键词: 3T3 cells; DNA topoisomerases; bladder neoplasm; breast neoplasms; cell cycle; enzyme activity; enzyme induction /repression; gene mutation; genetic promoter element; guanine nucleotide binding protein; in situ hybridization; messenger RNA; oncoproteins; protein protein interaction

DESCRIPTION (provided by applicant): The levels of topoisomerase Ha (topo Ha) are elevated in many tumors, contributing to increased sensitivity to a number of clinical anti-cancer drugs. Clearly, the regulation of this protein is of critical importance. We have studied the regulation of topo Ha expression with the use of time lapse and fluorescence imaging techniques which allow cell cycle analyses without cell synchronization. A portion of the topo Ha promoter was identified which is remarkably dependent upon and responsive to Ras activity. Nevertheless, topo IIalpha protein levels in cycling cells were not altered by this oncogene. Only a small population of slowly cycling NJH3T3 cells increased topo Ha levels following injection of Ras protein. Interestingly, these slowly cycling cells containing Ras activity were highly sensitive to the anti-topo II drug etoposide. This proposal is designed to extend our studies of topo IIalpha regulation by testing models to explain these results. First, we will determine why topo IIalpha protein levels are not altered by Ras activity in cycling cells, despite a ras responsive region in the promoter. Is the difference between promoter activity and protein expression due to alterations in topo Ha protein or mRNA stability? Is it possible that other regions of the topo IIalpha promoter suppress the ras responsive region in cycling cells? These experiments will utilize traditional biochemical procedures, as well as our new imaging techniques. Next, we will analyze slowly cycling cells. These cells behave similarly to many tumor cells. While they retain proliferative capacity, they are temporarily located in a prolonged Gi-phase. Experiments involving time lapse analysis, studies of signaling molecules, and analysis of promoter activity will be performed in these cells to determine how topo Ha regulation differs in these cells compared to cycling NIH3T3 cells. Finally, we will perform experiments to demonstrate that these slowly cycling NlH3T3 cells serve as a culture model for tumor cells. It should be emphasized that most cells of a tumor are not cycling at any give time, but must, nevertheless, be sensitive to an anti-tumor treatment. Perhaps our studies of these slowly cycling cells will yield information of value in understanding these temporarily non-cycling tumor cells and their sensitivity to drug treatment.

描述(申请人提供)：拓扑异构酶Ha(Topo Ha)水平 在许多肿瘤中升高，有助于增加对一些数字的敏感性 临床抗癌药物。显然，这种蛋白质的调节是 至关重要。我们已经研究了Topo Ha表达的调节 使用延时和荧光成像技术使细胞 没有细胞同步的周期分析。Topo Ha启动子的一部分 被确定为显著依赖于RAS并对其做出响应的 活动。然而，周期细胞中的topo IIpha蛋白水平并没有 被这种致癌基因改变了。只有一小部分骑行缓慢的NJH3T3 注射RAS蛋白后，细胞Topo Ha水平升高。 有趣的是，这些缓慢循环的细胞含有RAS活性， 对抗TOPO II类药物依托泊苷敏感。这项提议旨在 通过测试模型来解释我们对Topo IIpha调控的研究 这些结果。首先，我们将确定为什么topo IIpha蛋白水平不是 被循环细胞中的RAS活性改变，尽管在 发起人。启动子活性和蛋白质之间的区别 Topo Ha蛋白或mRNA稳定性改变导致的表达？是吗 可能是topo IIpha启动子的其他区域抑制了ras 细胞周期中的响应区？这些实验将利用传统的 生化程序，以及我们的新成像技术。接下来，我们将 分析缓慢循环的细胞。这些细胞的行为与许多肿瘤细胞相似。 虽然它们保留了增殖能力，但它们暂时位于 胃动期延长。涉及时间推移分析的实验，研究 信号分子和启动子活性的分析将在 以确定Topo Ha调控在这些细胞中的不同之处 到NIH3T3细胞的循环。最后，我们将进行实验来演示 这些缓慢循环的NlH3T3细胞可以作为肿瘤的培养模型 细胞。应该强调的是，肿瘤的大多数细胞不是在 任何给予时间，但必须，但必须对抗肿瘤治疗敏感。 也许我们对这些缓慢循环的细胞的研究将会得到 对了解这些暂时非周期肿瘤细胞及其相互关系的价值 对药物治疗敏感。