Protein kinase C-delta regulates chromatin remodeling and DNA repair

蛋白激酶 C-delta 调节染色质重塑和 DNA 修复

• 批准号: 10393176
• 负责人: Trisiani Affandi
• 金额: $ 0.25万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-21 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10393176
• 关键词: ABL1 gene; Address; Affect; Apoptosis; Apoptotic; Binding; Biological Assay; Cancer Biology; Cell Nucleus; Chromatin; Chromatin Structure; Chronic; Comet Assay; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; Data; Development; Diagnosis; Double Strand Break Repair; Enzymes; Exposure to; Fellowship; Goals; Head and Neck Cancer; Histone Deacetylase; Histones; Immunoprecipitation; In Vitro; Lead; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methods; Modification; Molecular; Molecular Conformation; Nonhomologous DNA End Joining; Normal tissue morphology; Nuclear; Nuclear Import; Nuclear Protein; Operative Surgical Procedures; Oral cavity; Oral mucous membrane structure; Pathway interactions; Patients; Phosphorylation; Phosphotransferases; Play; Production; Protein Inhibition; Protein Tyrosine Kinase; Proteins; Quality of life; Radiation Protection; Regulation; Repair Complex; Reporter; Research; Research Personnel; Research Training; Role; SRC gene; Saliva; Salivary Glands; Site; Therapeutic Intervention; Training; Tyrosine Kinase Inhibitor; Xerostomia; alpha Karyopherins; cancer cell; chemotherapy; chromatin remodeling; experience; experimental study; head and neck cancer patient; histone acetyltransferase; histone modification; homologous recombination; in vivo; inhibitor/antagonist; irradiation; novel; oral biology; oral infection; oral tissue; protein activation; protein complex; protein function; protein kinase C-delta; repaired; response; salivary acinar cell; side effect; skills; small hairpin RNA

PROJECT ABSTRACT Head and neck cancer (HNC) accounts for 4% of all cancers in the US, and HNC patients are mostly treated with irradiation (IR) therapy or combination of treatments. Unfortunately, besides killing cancer cells, IR treatment also kills non-tumor cells in the oral cavity and damages the salivary glands, causing side effects such as chronic oral infection, decreased saliva production, and xerostomia. IR-induced damage to the salivary glands is thought to occur in part due from loss of salivary acinar cells through apoptosis. Our lab has identified protein kinase C delta (PKCδ) as a key regulator of IR-induced apoptosis in the salivary acinar cells in vitro and in the salivary gland in vivo. We have shown that upon IR damage PKCδ is activated and imported into the nucleus. Although we know that nuclear import of PKCδ is necessary for apoptosis, the mechanism of how nuclear PKCδ regulates DNA damage-induced apoptosis is unknown. Previous studies from our lab have suggested that PKCδ does not activate the apoptotic pathway directly but may instead regulate upstream pathways that contribute to the DNA damage response and DNA repair. My preliminary studies suggest a novel mechanism of PKCδ-mediated regulation of apoptosis through chromatin remodeling and DNA repair. My preliminary data shows that depletion of PKCδ increases IR-induced DNA damage repair, and that PKCδ regulates DNA repair through both non- homologous end joining (NHEJ) and homologous recombination (HR) pathways. Furthermore, my preliminary data suggests that PKCδ may regulate chromatin structure through altering histone modifications. Thus, my overall goal is to understand mechanistically how nuclear PKCδ regulates chromatin accessibility and DNA repair in response to IR damage. In Aim 1 I will characterize the role of nuclear PKCδ in NHEJ and HR pathways and analyze whether PKCδ is biased toward NHEJ or HR using in vivo fluorescent reporter assay. I will also determine if PKCδ affects the formation of NHEJ and HR repair complexes at sites of double-stranded breaks (DSBs) and perform rapid immunoprecipitation mass spectrometry to identify chromatin interacting proteins influenced by PKCδ. In Aim 2 I will ask how PKCδ regulates histone modifications and chromatin structure, and how this relates to DNA repair regulation. I will explore the role of PKCδ in histone modifications before and after IR, and the nuclear function of PKCδ in chromatin remodeling. To further understand the mechanism of this regulation, we will analyze if PKCδ regulates upstream histone-modifying enzymes. Overall, my proposed studies should enhance our understanding of the mechanism by which PKCδ regulates DNA damage-induced apoptosis, and may lead to new safe therapeutic interventions to protect salivary gland and oral tissues of patients undergoing IR for HNC. This F32 fellowship will also provide me with specific scientific and research training, including the development of new research skills, and exposure to basic and translational cancer and oral biology. Together with a wide variety of professional development opportunities, this training will prepare me for my long-term goal to become an independent cancer researcher.

项目摘要 头颈癌（HNC）占美国所有癌症的4%，HNC患者大多接受治疗 用辐射（IR）疗法或治疗的组合。不幸的是，除了杀死癌细胞，红外治疗 也会杀死口腔中的非肿瘤细胞，损害唾液腺，引起慢性副作用， 口腔感染、唾液分泌减少和口腔干燥。人们认为，放射线对唾液腺的损伤 部分原因是由于唾液腺泡细胞通过凋亡而丧失。我们的实验室已经鉴定出蛋白激酶C 在体外和唾液腺中，PKCδ作为IR诱导的腺泡细胞凋亡的关键调节因子， 体内腺体。我们已经表明，在IR损伤时，PKCδ被激活并输入到细胞核中。虽然 我们知道PKCδ的核输入是细胞凋亡所必需的，核PKCδ如何调节细胞凋亡的机制是， DNA损伤诱导的细胞凋亡是未知的。我们实验室以前的研究表明，PKCδ不 直接激活凋亡途径，但可能会调节上游途径，有助于DNA 损伤反应和DNA修复。我的初步研究表明，PKCδ介导的一种新的机制， 通过染色质重塑和DNA修复调节细胞凋亡。我的初步数据显示 PKCδ增加IR诱导的DNA损伤修复，并且PKCδ通过两种非- 同源末端连接（NHEJ）和同源重组（HR）途径。此外，初步 数据表明PKCδ可能通过改变组蛋白修饰来调节染色质结构。所以我的 总体目标是从机制上了解核PKCδ如何调节染色质可及性， DNA修复反应IR损伤。目的1：研究PKCδ在NHEJ和HR中的作用 并使用体内荧光报告基因测定分析PKCδ是否偏向NHEJ或HR。我 还将确定PKCδ是否影响NHEJ和HR修复复合物在双链 断裂（DSB），并进行快速免疫沉淀质谱法，以确定染色质相互作用 PKCδ影响的蛋白质。在目标2中，我将探讨PKCδ如何调节组蛋白修饰和染色质 结构，以及这与DNA修复调节的关系。我将探讨PKCδ在组蛋白修饰中的作用 以及PKCδ在染色质重构中的核功能。进一步了解 为了阐明这种调节的机制，我们将分析PKCδ是否调节上游组蛋白修饰酶。总的来说， 我所提出的研究应该能增强我们对PKCδ调节DNA的机制的理解 损伤诱导的细胞凋亡，并可能导致新的安全的治疗干预，以保护唾液腺和 对HNC进行IR的患者的口腔组织。这个F32奖学金也将为我提供具体的科学 和研究培训，包括新的研究技能的发展，并接触基础和翻译 癌症和口腔生物学。加上各种各样的专业发展机会，这项培训将 为我成为一名独立的癌症研究人员的长期目标做好准备。