CHARACTERIZATION OF NOVEL BUTADIENE-INDUCED PUTATIVE p53 TARGET GENES

新型丁二烯诱导的推定 p53 靶基因的表征

• 批准号: 7981635
• 负责人: PERPETUA M MUGANDA
• 金额: $ 41.9万
• 依托单位: NORTH CAROLINA AGRI & TECH ST UNIV
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-09 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7981635
• 关键词: Address; Air Pollutants; Apoptosis; Apoptotic; Arts; Atrophic; Biological Assay; Bone Marrow; Butadiene; CCL1 gene; CEACAM1; CXCL9 gene; Carcinogens; Cell Line; Cells; Cellular biology; Chemicals; Complement component C4a; Data; Development; Environment; Future; Gene Activation; Gene Targeting; Genes; Goals; Hematopoietic; Human; Intervention; Investigation; Knowledge; Laboratories; Lead; Lymphocyte; Mediating; Molecular; Mutagens; Neurologic; Ovarian; Polymerase Chain Reaction; Process; Production; Protein p53; Proteins; Protocols documentation; Reagent; Regulation; Reporter Genes; Reporting; Research; Risk Assessment; Role; Scientist; Signal Transduction; Small Interfering RNA; Specific qualifier value; Spleen; Stress; System; TP53 gene; Testing; Therapeutic Intervention; Thymus Gland; Time; Toxic Environmental Substances; Toxic effect; Work; carcinogenesis; cell type; chromatin immunoprecipitation; design; environmental chemical; erythritol anhydride; expectation; experience; innovation; novel; p53 Signaling Pathway; promoter; public health relevance; reproductive; response; stem; success; toxicant; transcription factor

DESCRIPTION (provided by applicant): Butadiene (BD) is a high production volume chemical that is currently regulated as a hazardous air pollutant. This environmental chemical is a known mutagen and human carcinogen, and possesses a wide range of hematopoietic, reproductive, and neurological toxicity, which includes bone marrow depletion, ovarian, spleen and thymus atrophy. Although the toxic effects of BD are known to be mediated through its active metabolites, the cellular and molecular mechanisms responsible for BD's toxic effects are not completely understood. Elucidation of the molecular mechanisms responsible for BD toxicity is a necessary pre-requisite to the development of therapeutic and intervention protocols for the management of BD exposure, and this is the long-term goal of this application. Butadiene's most potent active metabolite, diepoxybutane (DEB), has previously been reported to induce p53-regulated apoptosis in human lymphocytes. Although the tumor suppressor p53 protein is a multifunctional transcription factor that mediates apoptosis in response to various forms of stress, the mechanisms of p53 action and p53 signaling are known to be cell type and damage specific. Efforts towards the understanding of the mechanisms of p53 action in DEB-exposed lymphocytes have led to the discovery of novel DEB-induced putative p53-target genes. The objective of this proposal, therefore, is to determine how p53 regulates the expression of these novel DEB-induced putative p53-target genes, and determine the functional aspects of this regulation in DEB-exposed human lymphocytes. The central hypothesis to be tested is that the novel DEB-induced putative p53-target genes are regulated by p53 at the transcriptional and/or post-transcriptional level to signal and/or execute DEB-induced p53-dependent apoptosis in exposed lymphocytes. This project will utilize high-throughput approaches involving small interfering RNA, chromatin immunoprecipitation-quantitative PCR assays, reporter gene assays, polymerase chain reaction arrays, primary lymphocytes, as well as genetically-paired p53-deficient and proficient cell lines to test the central hypothesis through two specific aims: (1). Determine how p53 regulates the expression of the novel DEB-induced putative p53-target genes in DEB-exposed human lymphocytes. (2). Determine the role of the novel DEB-induced putative p53-target genes in the cellular and molecular toxicity of DEB within exposed human lymphocytes. At the completion of these studies, our expectation is that novel DEB-induced direct and/or indirect p53 targets will be identified, and their functions in DEB-exposed cells will be deduced. Collectively, this information is important because it will lead to a better understanding, of not only the toxicity of butadiene at the cellular and molecular level, but also the role of p53 in that process. This knowledge will have an important vertical impact on butadiene risk assessment as well as better means of therapeutic intervention after butadiene exposure and carcinogenesis. PUBLIC HEALTH RELEVANCE: The research proposed in this application is important because it will lead to a better understanding on novel mechanisms of p53 signaling and action as they occur in lymphocytes that have been exposed to the environmental toxicant butadiene, a mutagen and human carcinogen. This knowledge will have an important vertical impact on butadiene risk assessment, as well as on the design of better means of therapeutic intervention after butadiene exposure.

描述（由申请人提供）：丁二烯 (BD) 是一种高产量化学品，目前被作为有害空气污染物进行监管。这种环境化学物质是一种已知的诱变剂和人类致癌物，具有广泛的造血、生殖和神经毒性，包括骨髓耗竭、卵巢、脾脏和胸腺萎缩。尽管已知 BD 的毒性作用是通过其活性代谢物介导的，但造成 BD 毒性作用的细胞和分子机制尚不完全清楚。阐明BD毒性的分子机制是制定BD暴露管理的治疗和干预方案的必要先决条件，也是本申请的长期目标。丁二烯最有效的活性代谢物二环氧丁烷 (DEB) 先前已被报道可诱导人类淋巴细胞中 p53 调节的细胞凋亡。尽管肿瘤抑制蛋白 p53 蛋白是一种多功能转录因子，可介导细胞凋亡以响应各种形式的应激，但 p53 作用和 p53 信号传导的机制已知是细胞类型和损伤特异性的。为了了解 p53 在暴露于 DEB 的淋巴细胞中的作用机制，我们发现了新的 DEB 诱导的推定 p53 靶基因。因此，本提案的目的是确定 p53 如何调节这些新的 DEB 诱导的推定 p53 靶基因的表达，并确定这种调节在暴露于 DEB 的人淋巴细胞中的功能方面。要测试的中心假设是，新的 DEB 诱导的假定 p53 靶基因在转录和/或转录后水平上受 p53 调节，以在暴露的淋巴细胞中发出信号和/或执行 DEB 诱导的 p53 依赖性细胞凋亡。该项目将利用高通量方法，包括小干扰RNA、染色质免疫沉淀定量PCR检测、报告基因检测、聚合酶链式反应阵列、原代淋巴细胞以及基因配对的p53缺陷和熟练细胞系，通过两个具体目标来检验中心假设：(1)。确定 p53 如何调节暴露于 DEB 的人淋巴细胞中新型 DEB 诱导的推定 p53 靶基因的表达。 （2）。确定新型 DEB 诱导的假定 p53 靶基因在暴露的人淋巴细胞内 DEB 的细胞和分子毒性中的作用。这些研究完成后，我们期望将鉴定新的 DEB 诱导的直接和/或间接 p53 靶标，并推断它们在 DEB 暴露细胞中的功能。总的来说，这些信息很重要，因为它不仅可以帮助人们更好地了解丁二烯在细胞和分子水平上的毒性，还可以了解 p53 在此过程中的作用。这些知识将对丁二烯风险评估以及丁二烯暴露和致癌后更好的治疗干预手段产生重要的纵向影响。 公共健康相关性：本申请中提出的研究很重要，因为它将导致人们更好地了解 p53 信号传导和作用的新机制，因为它们发生在暴露于环境毒物丁二烯（一种诱变剂和人类致癌物）的淋巴细胞中。这些知识将对丁二烯风险评估以及丁二烯暴露后更好的治疗干预手段的设计产生重要的纵向影响。