Converting an Oncogene to an Apoptotic Factor by Manipulating Signal Sequences

通过操纵信号序列将癌基因转化为凋亡因子

基本信息

批准号：
7758311
负责人：
Carol S. Lim
金额：
$ 28.37万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-01 至 2013-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7758311
关键词：
Accounting Address Adverse effects Amino Acids Animal Model Animals Apoptosis Apoptosis Inhibitor Apoptotic Back Binding Binding Sites Biological Assay Breast Cancer Cell Caspase Cell Cycle Cell Death Cell Line Cell Nucleus Cell Proliferation Cell Survival Cells Cellular Morphology Chronic Myeloid Leukemia Chronic Phase Chronic Phase of Disease Clinical Cloning Coiled-Coil Domain Complex Cytoplasm Dexamethasone Disease Dose Drug Delivery Systems Etiology Family Female Fluorescence Fluorescence Microscopy Genetic Transcription Gleevec Goals Human Imagery Imatinib mesylate Immunoblotting Immunocompromised Host Immunoprecipitation K-562 K562 Cells Length Leukemic Cell Ligand Binding Domain Ligands Location Malignant Neoplasms Measures Messenger RNA Metabolic Diseases Methodology Mifepristone Modality Modeling Monitor Mus Mutate Mutation Myeloid Cells Myeloproliferative disease Normal Cell Nuclear Nuclear Export Nuclear Localization Signal Nude Mice Oncogenes Oncogenic Output Paper Patients Peptide Signal Sequences Plasmids Play Point Mutation Polymers Protein Tyrosine Kinase Proteins Resistance Resistance development Reverse Transcriptase Polymerase Chain Reaction Role S100A8 gene Signal Transduction Site-Directed Mutagenesis Specificity Staining method Stains Techniques Telomerase Testing Therapeutic Therapeutic Intervention Time Transfection Tumor Suppressor Proteins Two-Hybrid System Techniques Tyrosine Kinase Inhibitor Viral Work Xenograft Model abl Oncogene annexin A5 clinically relevant gene therapy granulocyte immortalized cell in vivo leukemia member neoplastic cell non-oncogenic novel strategies nucleocytoplasmic transport promoter protein function standard measure survivin tumor tumor eradication tumorigenesis

项目摘要

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.

描述（由申请人提供）：改变与疾病相关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至关重要的作用。一个精确的例子是 Bcr-Abl 蛋白，它是慢性粒细胞白血病 (CML) 的病原体。当Bcr-Abl位于细胞的细胞质中时，它表现为癌基因，但如果被迫进入细胞核，它就会成为凋亡因子。 CML 是一种骨髓增殖性疾病，其特征是粒细胞及其未成熟前体细胞增殖增加；中位生存时间为 4 至 6 年。本研究的目标是使用我们的配体响应蛋白开关结构来控制 Bcr-Abl 的亚细胞位置，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可以导致细胞凋亡。如果Bcr-Abl可以定向到细胞核，它就可以从癌基因转化为凋亡因子。由于Bcr-Abl自身寡聚形成四聚体，因此可以通过引入外源定位可控的Bcr-Abl来实现核捕获。配体诱导后，定位可控的 Bcr-Abl 将与 wt Bcr-Abl 寡聚，并转运至细胞核，随后发生细胞凋亡。在目标 1 中，我们将亚克隆带有荧光标签的 Bcr-Abl 定位可控版本（Bcr-Abl 蛋白开关，PS），并显示与野生型 (wt) Bcr-Abl 的寡聚化，易位到细胞核，并导致 Bcr 凋亡-Abl阳性K562细胞。 Bcr-Abl PS 的定位将通过荧光显微镜监测，并且细胞凋亡将使用标准细胞死亡测定法进行测试。 wt Bcr-Abl 与 Bcr-Abl PS 的相互作用将使用 wt Bcr-Abl 和 Bcr-Abl 蛋白质开关之间的体内寡聚化以及哺乳动物双杂交测定来确定。目标 2 将类似地测试格列卫耐药白血病细胞中的 Bcr-Abl PS。目标 3 将测试和使用仅允许 Bcr-Abl PS 在白血病细胞中优先表达的特定启动子。目标 4 将使用注射人类白血病细胞的 Balb/C 裸鼠来测试 Bcr-Abl PS 是否会在人类异种移植模型中根除/减少白血病。我们的目标是使用配体响应蛋白开关结构来控制 Bcr-Abl 的亚细胞位置，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 的本地化可控版本（作为基因疗法）来治疗 CML。