Control of topoisomerose II through the cell cycle

描述（由申请人提供）：拓扑异构酶Ha（topo Ha）的水平 在许多肿瘤中升高，导致对一些 临床抗癌药物的应用。显然，这种蛋白质的调节是 至关重要。我们研究了topo Ha表达的调控， 使用时间推移和荧光成像技术， 没有细胞同步的循环分析。拓扑异构酶Ha启动子的一部分 这是显着依赖和响应Ras 活动尽管如此，在周期细胞中的拓扑异构酶II α蛋白水平并不 被这个致癌基因改变了只有一小部分缓慢循环的NJH3T3 细胞在注射Ras蛋白后增加topo Ha水平。 有趣的是，这些含有Ras活性的缓慢循环细胞高度表达Ras。 对抗拓扑异构酶II药物依托泊苷敏感这项建议旨在 通过测试模型来扩展我们对拓扑II α调节的研究， 这些结果。首先，我们将确定为什么拓扑异构酶II α蛋白水平不是 尽管在细胞周期中存在ras反应区， 发起人。是启动子活性和蛋白质 表达由于改变拓扑异构酶Ha蛋白或mRNA的稳定性？吗 topo II α启动子的其他区域可能抑制ras基因的表达， 循环细胞的反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。接下来我们就 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 GI期延长。涉及时间推移分析的实验， 信号分子和启动子活性分析将在 这些细胞，以确定如何topo Ha调节不同，在这些细胞相比， 循环NIH3T3细胞。最后，我们将进行实验，以证明 这些缓慢循环的N1H3T3细胞用作肿瘤的培养模型， 细胞应该强调的是，肿瘤的大多数细胞在24小时内不循环。 任何给定的时间，但必须，尽管如此，敏感的抗肿瘤治疗。 也许我们对这些缓慢循环的细胞的研究将产生以下信息： 了解这些暂时不循环的肿瘤细胞及其 对药物治疗的敏感性。

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.