INTRACELLULAR CA2+ HOMEOSTASIS AND CELLULAR RADIOSENSITIVITY

描述：(申请者的描述)目的是测试 电离辐射(IR)诱导的钙瞬变对于 细胞周期检查点的激活；以及如果对钙离子的遗传操作 动态平衡将钝化IR诱导的钙瞬变，短路IR 诱导细胞周期检查点，改变放射敏感性。Ca2+ 两种乳腺癌细胞株MCF-7和MDA-MB-231的动态平衡 通过稳定的B淋巴细胞转染法进行基因操作 钙通道，CD20，在可诱导启动子的控制下。首字母A 研究表明，CD20的表达增加了细胞内的游离 [Ca2+]，缩短细胞周期的G1期，并阻断IR诱导的G1期 逮捕。提出了三个目标：(1)继续刻画 CD20表达克隆包括胞浆游离[Ca~(2+)]， 生长和细胞周期分析。代表性克隆和零点 开发了一种测定细胞外[Ca~(2+)]的方法。 亲本和非亲本的生物学和细胞周期调控特性 诱导后的CD20细胞系在CD20的表达上有所恢复 对IR的敏感反应；(2)检测CD20的表达是否改变 红外线诱导的钙振荡和内钙储备的大小和 建立IR诱导的钙瞬变与细胞之间的联系 周期检查点；(3)定义钙敏感IR诱导的细胞周期 检查站。首先，将检查MCF7细胞的G1/S检查点 因为这在CD20Rb的磷酸化检查点中被废除到了钙离子- 敏感事件。研究将集中在合成，稳定性和 关键调节蛋白的活性，例如细胞周期蛋白依赖蛋白和 它们的内源性抑制物，目标是定义最直接的， 检查点激活所需的依赖于CA2+的事件。以结果为基础 从AIM#1开始，对钙离子敏感的S或G2检查点将使用 在G1/S检查站分析指导下的总体做法。结果 从这项实验中可以看出治疗的可能靶点 操纵肿瘤细胞的放射敏感性。

DESCRIPTION: (Applicant's Description) The objective is to test whether ionizing radiation (IR) induced Ca2+ transients are critical for activation of cell cycle checkpoints; and if genetic manipulation of Ca2+ homeostasis will blunt IR-induced Ca2+ transients, short circuit IR induced cell cycle checkpoints and alter radio-sensitivity. Ca2+ homeostasis of two breast carcinoma cell lines, MCF-7 and MDA-MB-231, has been genetically manipulated by stable transfection with the B-lymphocyte Ca2+ channel, CD20, under control of an inducible promoter. An initial characterization reveals that CD20 expression increases cytosolic free [Ca2+], shortens G1 of the cell cycle, and blocks the IR-induced G1 arrest. Three aims are proposed: (1) Continue characterization of the CD20-expressing clones to include measurements of cytosolic free [Ca2+], growth and cell cycle analysis. Representative clones and a null point assay developed to determine at what extracellular [Ca2+], the radio- biological and cell-cycle control properties of the parental and non- induced CD20 transfected cell lines are recovered in the CD20 expressing sensitive responses to IR; (2) Test whether expression of CD20 alters the IR-induced Ca2+ oscillations and the size of internal Ca2+ stores and establish the linkage between the IR-induced Ca2+ transients and cell cycle checkpoints; (3) Define Ca2+-sensitive IR-induced cell cycle checkpoints. Initially, the G1/S checkpoint of MCF7 cells will be examined since this is abrogated in CD20 Rb phosphorylation checkpoint to the Ca2+- sensitive event. Studies will focus on the synthesis, stabilities, and activities of key regulatory proteins, e.g. cyclin dependent kinases and their endogenous inhibitors, the goal being to define the most immediate, Ca2+-dependent event necessary for checkpoint activation. Based on results from Aim #1, Ca2+-sensitive S-or G2-checkpoints will be examined with the overall approach guided by the analysis of the G1/S checkpoint. Results from this experimentation may indicate possible targets for therapeutic manipulation of tumor cell radio-sensitivity.