Cutaneous Biology KIT Ligand

描述（由申请人提供）：干细胞因子（SCF）是KIT（一种受体酪氨酸激酶）的配体。我们假设KIT激活可以影响法师基因的表达，而这些基因通常只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p53依赖性凋亡促进肿瘤存活。本发明的目的是确定KIT活化和法师诱导的细胞凋亡之间的关系，并开发通过在体内干扰法师蛋白表达来治疗法师阳性恶性肿瘤的方法。这些目标将通过以下具体目标实现：具体目标1：将通过确定KIT是否控制法师基因启动子甲基化来检验KIT激活直接影响法师基因表达的假设，并将通过免疫组织化学和逆转录-PCR来确定法师蛋白表达是否在肥大细胞增多症中常见。具体目标二：将定义法师蛋白调节细胞凋亡的机制，通过使用基于诱导型慢病毒的p53抑制来严格测试对p53的需要，并通过确定法师基因表达对p53代谢的影响，包括：通过免疫印迹分析的p53磷酸化和乙酰化，通过真实的时间PCR和核运行分析的p53转录;通过p53靶基因活化测定的p53功能;通过泛素免疫印迹和脉冲追踪分析检测p53降解。然后，我们将使用标记的法师和Kap1蛋白质在免疫沉淀和哺乳动物双杂交试验中从巢式法师cDNA表达来检验多个法师蛋白质通过Kap1结合到法师共同同源结构域来调节细胞凋亡的假设。具体目标3。将检验肥大细胞增多症和其它肿瘤可以通过使用异种移植到nu/nu小鼠上并用法师siRNA全身治疗的人细胞系在体内抑制法师表达来治疗的假设。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变来改善健康，所述临床范式设想肿瘤特异性法师蛋白作为免疫攻击的靶标而不是作为功能操纵的靶标的治疗用途。这些结果将通过开发恶性肿瘤的新疗法和增加我们对精原干细胞精子发育的理解来影响健康的多个方面。

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.