Regulation of Class I Gene Expressions in Tumor Cells

描述：（改编自申请人摘要）大多数恶性肿瘤 肿瘤目前无法治愈，除非在早期发现;免疫学 这些方法通常有望改善治疗。本报告的总体目标 拟议的项目是获得新的理解的基本机制， 肿瘤细胞中的人主要组织相容性抗原基因表达是 由干扰素调节。预计这些知识最终将 促进尝试开发更有效的治疗目前无法治愈的 通过细胞免疫疗法和疫苗策略治疗癌症。中央 这一建议的假设是干扰素刺激了 人白细胞抗原（HLA）Ia类基因、HLA-A2和HLA Ib类基因， HLA-E，通过不同的和新的机制。由于这两个HLA I类 基因在细胞毒性T细胞和自然杀伤细胞中起关键作用 淋巴细胞介导的肿瘤细胞杀伤，了解其选择性 调节应增加肿瘤免疫学的基础知识。主要 该提案的具体目标是：（1）确定 干扰素-γ介导HLA-A2基因的转录后刺激 通过3'干扰素应答元件，和（2）表征不同的干扰素应答元件， 在HLA-E基因启动子中的干扰素-γ转录应答元件。 为了实现这些特定的目标，一组连续的分子生物学基因 将进行转移和体外诱变试验，以确定 这两种基因的不同调节所需的确切DNA和RNA序列 肿瘤细胞系和新鲜白血病细胞中的基因。细胞和分子 生物测定，包括共聚焦显微镜和体外RNA剪接 分析，将进行定义的新机制， HLA-A2基因的转录后调节。生物化学方法， 将用于鉴定、表征和纯化推定的蛋白质 介导HLA-A2和HLA-E基因的干扰素应答的因子。 在鉴定了顺式作用DNA序列和反式作用DNA序列之后， 介导HLA-E基因转录反应的因子， 将进行生物化学和遗传学研究来定义蛋白质-蛋白质 相互作用和介导干扰素的信号转导事件 反应从这个项目中获得的知识将增加基本的 了解HLA Ia类和Ib类基因的差异 在肿瘤细胞中调节，并可能最终导致新的治疗方法 优化癌症的免疫治疗以及同种异体骨髓 通过组织相容性抗原屏障进行移植。

DESCRIPTION: (Adapted from applicant's abstract) The majority of malignant tumors are currently incurable unless detected at an early stage; immunologic approaches often promise in improving therapy. The overall objective of this proposed project is to obtain new understanding of the basic mechanisms by which human major histocompatibility antigen gene expression in tumor cells is regulated by interferon. It is anticipated that such knowledge will ultimately facilitate attempts to develop more effective treatment of currently incurable cancers through cellular immune therapy and vaccine strategies. The central hypothesis of this proposal is that interferon stimulates expression of the human leukocyte antigen (HLA) Class Ia gene, HLA-A2, and the HLA Class lb gene, HLA-E, through differing and novel mechanisms. Since these two HLA Class I genes play critical roles in both cytotoxic T-cell and natural killer cell lymphocyte mediated tumor cell killing, understanding their selective regulation should increase basic knowledge about tumor immunology. The major specific aims of this proposal are (1) to determine the mechanism by which interferon-gamma mediates post-transcriptional stimulation of the HLA-A2 gene through a 3' interferon response element, and (2) to characterize the distinct interferon-gamma transcriptional response elements in the HLA-E gene promoter. To achieve these specific aims, a sequential set of molecular biological gene transfer and in vitro mutagenesis assays will be carried out to define the exact DNA and RNA sequences required for the differing regulation of these two genes in tumor cell lines and fresh leukemia cells. Cell and molecular biological assays, including confocal microscopy and in vitro RNA splicing analysis, will be carried out to define the novel mechanism of post-transcriptional regulation of the HLA-A2 gene. Biochemical methods then will be utilized to identify, characterize and purify the putative protein factors that mediate the interferon response of the HLA-A2 and HLA-E genes. Following the identification of the cis-acting DNA sequences and trans-acting factors that mediate the transcriptional response of the HLA-E gene, biochemical and genetic studies will be carried out to define protein-protein interactions and the signal transduction events that mediate the interferon response. The knowledge gained from this project will increase the basic understanding of how the HLA Class Ia and Class lb genes are differentially regulated in tumor cells, and may ultimately lead to new therapeutic approaches to optimize immunotherapy of cancer as well as allogeneic bone marrow transplantation across histocompatibility antigen barriers.