Cutaneous Biology KIT Ligand

皮肤生物学 KIT 配体

基本信息

批准号：
7261475
负责人：
B Jack Longley
金额：
$ 29.4万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-01-01 至 2009-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7261475
关键词：
Acetylation Affect Apoptosis Base Sequence Binding Biological Assay Biology Cell Line Cells Cholesterol Clinical Cutaneous Cutaneous Mastocytosis Data Development Digestion Exonuclease Gene Activation Gene Expression Gene Targeting Genes Genetic Transcription Goals Growth Health Homologous Protein Human Human Cell Line Immunoblot Analysis Immunoblotting Immunohistochemistry Immunologics Immunoprecipitation In Vitro Injection of therapeutic agent Lesion Ligands MDM2 gene MDM2 gene Malignant Neoplasms Mast Cell Neoplasm Messenger RNA Metabolism Methylation Mus Mutate Nevus Nuclear Pathway interactions Patients Peritoneal Phosphorylation Physiologic pulse Plasmids Polymerase Chain Reaction Polyubiquitination Promoter Regions Protein Binding Proteins Public Health Pulse taking RNA Interference Receptor Protein-Tyrosine Kinases Regression Analysis Regulation Research Personnel Resistance Reverse Transcription Role Running Signal Pathway Site-Directed Mutagenesis Skin Mastocytoma Small Interfering RNA Stem Cell Factor Stem cells Subfamily lentivirinae TP53 gene Testing Tetracycline Tetracyclines Therapeutic Uses Time Two-Hybrid System Techniques Ubiquitin X Chromosome Xeno Xenograft procedure base citrate carrier concept gene function human disease improved in vivo inhibitor/antagonist knock-down mast cell mastocytosis melanocyte melanoma multicatalytic endopeptidase complex mung bean novel prevent programs promoter protein expression research study sperm cell tumor tumor growth yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Stem Cell Factor (SCF) is the ligand for KIT, a receptor tyrosine kinase. We hypothesize that KIT activation can affect the expression of MAGE genes, which are usually only expressed in developing sperm and in tumors. We also hypothesize that MAGE proteins promote tumor survival by suppressing p53 dependant apoptosis. The goals of this proposal are to determine the relationship between KIT activation and MAGE induced apoptosis and to develop means of treating MAGE positive malignancies by interfering with MAGE protein expression in vivo. These goals will be achieved by the following specific aims: Specific Aim 1: will test the hypothesis that KIT activation directly affects MAGE gene expression by determining whether KIT controls MAGE gene promoter methylation, and will determine whether MAGE protein expression is common in mastocytosis by immunohistochemistry and reverse transcription-PCR. Specific Aim 2: will define mechanisms by which MAGE proteins regulate apoptosis by critically testing the requirement for p53 using inducible lentivirus based suppression of p53 and by determining the effect of MAGE gene expression on p53 metabolism including: p53 phosphorylation and acetylation by immunoblot analysis, p53 transcription by real time PCR and nuclear run on analysis; p53 function by p53 target gene activation assays; and p53 degradation by ubiquitin immunoblot and pulse chase analysis. We will then test the hypothesis that multiple MAGE proteins regulate apoptosis via Kap1 binding to the MAGE common homology domain using tagged MAGE and Kap1 proteins expressed from nested MAGE cDNAs in immunoprecipitation and mammalian two hybrid assays. Specific Aim 3. will test the hypothesis that mastocytosis and other tumors may be treated by inhibiting MAGE expression in vivo using human cell lines xenografted onto nu/nu mice and treated systemically with MAGE siRNA. Public Health Relevance: These studies will improve health by causing a major shift of the current clinical paradigm that envisions the therapeutic use of tumor specific MAGE proteins as targets for immunologic attack, rather than as targets for functional manipulation. The results will impact multiple aspects of health by developing novel therapies for malignant tumors and increasing our understanding of the development of sperm from spermatogonial stem cells.

描述（由申请人提供）：干细胞因子（SCF）是受体酪氨酸激酶试剂盒的配体。我们假设套件激活会影响法术基因的表达，法术基因通常仅在发育中的精子和肿瘤中表达。我们还假设法师蛋白通过抑制依赖p53的凋亡来促进肿瘤存活。该提案的目标是确定试剂盒激活与法师诱导的凋亡之间的关系，并通过干扰体内法师蛋白表达来开发治疗法师阳性恶性肿瘤的方法。这些目标将通过以下特定目的来实现：具体目标1：将通过确定KIT控制MAGE基因启动子甲基化直接影响MAGE基因表达的假设，并将确定MAGE蛋白表达是否通过免疫组织化学和反向转录PCR在乳腺癌中常见。具体目的2：将定义机制，通过批判性地测试基于诱导的慢病毒对p53的抑制以及MAGE GENE表达对p53代谢的影响，通过对p53代谢的影响，包括：通过以下方式通过以下方式确定：p53的磷酸化和乙基分析，通过IMMUNOBLOT ANTINSRANTION，PR -53 TIRE ANTINE，PIRE STRANTION p53 TIRE ANTIME，通过p53代谢对p53的抑制进行批判性测试对p53进行调节的机制，并通过p53代谢进行了影响。 p53靶基因激活测定的p53功能；和p53通过泛素免疫印迹和脉冲追逐分析降解。然后，我们将检验以下假设：多种法师蛋白通过KAP1使用标记的MAGE和KAP1蛋白在免疫沉淀和哺乳动物两种杂交分析中以嵌套法MAGE cDNA表达的标记MAGE和KAP1蛋白来调节凋亡。具体目标3。将检验以下假设：肥大细胞增多症和其他肿瘤可以通过使用异种细胞系在NU/NU小鼠上抑制MAGE表达，并用Mage siRNA进行系统的治疗。公共卫生相关性：这些研究将通过引起当前临床范式的重大转移来改善健康，该临床范式设想使用特异性法MAGE蛋白作为免疫攻击的靶标，而不是作为功能操纵的靶标。结果将通过开发新的恶性肿瘤疗法来影响健康的多个方面，并增加我们对精子干细胞精子发展的理解。