Converting an Oncogene to an Apoptotic Factor by Manipulating Signal Sequences

描述（由申请人提供）：改变参与疾病的信号转导蛋白的亚细胞定位是一种新的治疗干预方法。一些蛋白质的亚细胞定位在疾病的病因学中起着关键作用。Bcr-Abl蛋白就是一个很好的例子，它是慢性粒细胞白血病（CML）的病原体.当Bcr-Abl在细胞质中时，它表现为癌基因，但如果被迫进入细胞核，它就成为凋亡因子。CML是一种骨髓增生性疾病，其特征在于粒细胞及其未成熟前体细胞的增殖增加;中位生存时间为4至6年。本研究的目的是利用我们的配体反应蛋白开关结构来控制Bcr-Abl的亚细胞定位，并将Bcr-Abl从癌基因转化为凋亡因子。已经表明，通过Bcr-Abl的核捕获从细胞质中耗尽Bcr-Abl可导致细胞凋亡。如果Bcr-Abl能被导向细胞核，它就能从癌基因转化为凋亡因子。由于Bcr-Abl与自身寡聚形成四聚体，因此通过引入外源性定位可控的Bcr-Abl可以实现核捕获，在配体诱导下，定位可控的Bcr-Abl将与wt Bcr-Abl寡聚并转运至核，随后发生细胞凋亡。在目的1中，我们将亚克隆具有荧光标签的Bcr-Abl（Bcr-Abl蛋白开关，PS）的定位可控版本，并显示与野生型（wt）Bcr-Abl的寡聚化，易位到细胞核，并引起Bcr-Abl阳性K562细胞的凋亡。通过荧光显微镜监测Bcr-Abl PS的定位，并使用标准细胞死亡测定法检测细胞凋亡。将使用wt Bcr-Abl和Bcr-Abl蛋白开关之间的体内寡聚化和哺乳动物双杂交测定来确定wt Bcr-Abl与Bcr-Abl PS的相互作用。目标2将测试格列卫中的Bcr-Abl PS。耐药白血病细胞相似。目的3将测试和使用允许仅在白血病细胞中优先表达Bcr-Abl PS的特异性启动子。目的4将使用注射人白血病细胞的Balb/C裸小鼠测试Bcr-Abl PS是否将在人异种移植物模型中根除/减少白血病。我们的目标是使用配体反应蛋白开关结构来控制Bcr-Abl的亚细胞定位，并将Bcr-Abl从癌基因转化为凋亡因子。我们的长期、最终目标是使用Bcr-Abl（作为基因治疗）的定位可控版本来治疗CML。

DESCRIPTION (provided by applicant): Changing the subcellular localization of a signal transducing protein involved in disease is a novel approach for therapeutic intervention. The subcellular location of some proteins plays a critical role in the etiology of disease. A precise example of this is Bcr- Abl protein, the causative agent of chronic myelogenous leukemia (CML). When Bcr-Abl is in the cytoplasm of cells, it behaves as an oncogene, but if forced to the nucleus, it becomes an apoptotic factor. CML is a myeloproliferative disorder characterized by increased proliferation of granulocytes and their immature precursors; with a median survival time of 4 to 6 years. The goal of this study is to use our ligand responsive protein switch constructs to control the subcellular location of Bcr-Abl, and convert Bcr- Abl from an oncogene to an apoptotic factor. It has been shown that depletion of Bcr- Abl from the cytoplasm by nuclear trapping of Bcr-Abl can result in apoptosis. If Bcr-Abl can be directed to the nucleus, it can be converted from an oncogene to an apoptotic factor. Since Bcr-Abl oligomerizes with itself to form tetramers, nuclear trapping could be achieved by introducing exogenously localization-controllable Bcr-Abl. Upon ligand induction, localization controllable Bcr-Abl will oligomerize with wt Bcr-Abl and will undergo transport to the nucleus, followed by cellular apoptosis. In Aim 1 we will subclone localization controllable versions of Bcr-Abl (Bcr-Abl protein switch, PS) with a fluorescent tag and show oligomerization with wild-type (wt) Bcr-Abl, translocate to the nucleus, and cause apoptosis of Bcr-Abl positive K562 cells. Localization of Bcr-Abl PS will be monitored by fluorescence microscopy, and apoptosis will be tested using standard cell death assays. Interaction of wt Bcr-Abl with Bcr-Abl PS will be determined using an in vivo oligomerization between wt Bcr-Abl and Bcr-Abl protein switch and mammalian two-hybrid assay. Aim 2 will test the Bcr-Abl PS in Gleevec.-resistant leukemic cells similarly. Aim 3 will test and use specific promoters that allow preferential expression of Bcr-Abl PS in leukemia cells only. Aim 4 will test if Bcr-Abl PS will eradicate/diminish leukemia in a human xenograft model using Balb/C nude mice injected with human leukemia cells. Our goal is to use ligand responsive protein switch constructs to control the subcellular location of Bcr-Abl, and convert Bcr-Abl from an oncogene to an apoptotic factor. Our long-term, ultimate goal is to use localization controllable versions of Bcr-Abl (as gene therapy) for treatment of CML.