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）：改变参与疾病的信号转导蛋白的亚细胞定位是治疗干预的一种新方法。某些蛋白质的亚细胞位置在疾病病因中起着至关重要的作用。一个确切的例子是bcr- abl蛋白，这是慢性粒细胞性白血病（CML）的病因。当BCR-ABL处于细胞的细胞质中时，它表现为癌基因，但是如果被迫进入细胞核，它将成为凋亡因素。 CML是一种髓增生性疾病，其特征是粒细胞增殖及其未成熟的前体的增殖。中位生存时间为4至6年。这项研究的目的是使用我们的配体响应蛋白开关构建体来控制BCR-ABL的亚细胞位置，并将BCR- ABL从癌基因转换为凋亡因子。已经表明，通过BCR-ABL的核捕获从细胞质中耗尽了BCR-ABL会导致细胞凋亡。如果可以将BCR-ABL定向到核，则可以将其从癌基因转换为凋亡因子。由于BCR-ABL与自身的寡聚形成四聚体，因此可以通过引入外源定位可控制的BCR-ABL来实现核捕获。配体诱导后，可控制的BCR-ABL将与WT BCR-ABL寡聚，并将经历到细胞核，然后进行细胞凋亡。在AIM 1中，我们将带有荧光标签的BCR-ABL（BCR-ABL蛋白开关，PS）的可控制版本，并用野生型BCR-ABL显示寡聚，转化为核，并导致BCR-ABL阳性K562细胞的凋亡。 BCR-ABL PS的定位将通过荧光显微镜监测，并将使用标准细胞死亡测定法测试凋亡。 WT BCR-ABL与BCR-ABL PS的相互作用将使用WT BCR-ABL和BCR-ABL蛋白开关和哺乳动物两杂化测定之间的体内寡聚确定。 AIM 2也将在Gleevec中测试BCR-ABL PS。同样，抗性白血病细胞。 AIM 3将测试和使用特定的启动子，仅在白血病细胞中允许BCR-ABL PS的优先表达。 AIM 4将使用BALB/C裸小鼠注入人类白血病细胞的BALB/C裸小鼠在人异种移植模型中是否会消除/减少白血病。我们的目标是使用配体响应蛋白开关构建体来控制BCR-ABL的亚细胞位置，并将BCR-ABL从癌基因转换为凋亡因子。我们的长期，最终的目标是使用可控制的BCR-ABL（作为基因疗法）的可控版本来治疗CML。