Cutaneous Biology KIT Ligand

描述（由申请人提供）：干细胞因子（SCF）是酪氨酸激酶受体KIT的配体。我们假设KIT激活可以影响MAGE基因的表达，而MAGE基因通常只在发育中的精子和肿瘤中表达。我们还假设MAGE蛋白通过抑制p53依赖的细胞凋亡来促进肿瘤存活。本研究的目的是确定KIT激活与MAGE诱导的细胞凋亡之间的关系，并通过在体内干扰MAGE蛋白表达来治疗MAGE阳性恶性肿瘤。具体目的1：通过确定KIT是否控制MAGE基因启动子甲基化来检验KIT激活直接影响MAGE基因表达的假设，并通过免疫组织化学和逆转录- pcr确定MAGE蛋白表达是否在肥大细胞增多症中常见。具体目标2：将通过使用可诱导的慢病毒抑制p53来严格测试对p53的需求，并通过确定MAGE基因表达对p53代谢的影响，确定MAGE蛋白调节细胞凋亡的机制，包括：通过免疫印迹分析p53磷酸化和乙酰化，通过实时PCR和核运行分析p53转录；通过P53靶基因激活检测P53功能；用泛素免疫印迹和脉冲追踪法检测p53的降解情况。然后，我们将在免疫沉淀和哺乳动物双杂交实验中使用嵌套MAGE cdna表达的标记MAGE和Kap1蛋白，测试多个MAGE蛋白通过Kap1结合到MAGE共同同源结构域来调节凋亡的假设。具体目标3。将通过将人细胞系移植到nu/nu小鼠身上，并全身使用MAGE siRNA处理，来验证肥大细胞增多症和其他肿瘤可能通过抑制体内MAGE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模式的重大转变来改善健康，目前的临床模式设想使用肿瘤特异性MAGE蛋白作为免疫攻击的靶标，而不是作为功能操纵的靶标。这一结果将通过开发恶性肿瘤的新疗法和增加我们对精子从精原干细胞发育的理解，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.