Protein kinase C-delta regulates chromatin remodeling and DNA repair - Supplement

蛋白激酶 C-delta 调节染色质重塑和 DNA 修复 - 补充

• 批准号: 10401150
• 负责人: Trisiani Affandi
• 金额: $ 2.43万
• 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: 2021-07-01 至 2021-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10401150
• 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 不幸的是，除了杀死癌细胞之外，IR 治疗还需要进行放射治疗或联合治疗。 还会杀死口腔中的非肿瘤细胞并损害唾液腺，引起慢性等副作用 口腔感染、唾液分泌减少和红外线引起的唾液腺损伤。 发生的部分原因是唾液腺泡细胞因细胞凋亡而损失。我们的实验室已鉴定出蛋白激酶 C。 δ (PKCδ) 作为体外和唾液中 IR 诱导的唾液腺泡细胞凋亡的关键调节因子 我们已经证明，在 IR 损伤时，PKCδ 会被激活并输入细胞核。 我们知道 PKCδ 的入核对于细胞凋亡是必需的，核 PKCδ 的调节机制 DNA 损伤诱导的细胞凋亡尚不清楚，我们实验室之前的研究表明 PKCδ 不会。 直接激活细胞凋亡途径，但可能调节有助于 DNA 的上游途径 我的初步研究表明 PKCδ 介导的新机制。 通过染色质重塑和 DNA 修复来调节细胞凋亡 我的初步数据表明耗竭。 PKCδ 增加了 IR 诱导的 DNA 损伤修复，并且 PKCδ 通过非 同源末端连接（NHEJ）和同源重组（HR）途径。 数据表明 PKCδ 可能通过改变组蛋白修饰来调节染色质结构。 总体目标是从机制上理解核 PKCδ 如何调节染色质可及性和 IR 损伤后的 DNA 修复 在目标 1 中，我将描述核 PKCδ 在 NHEJ 和 HR 中的作用。 途径并使用体内荧光报告分析 I 分析 PKCδ 是否偏向 NHEJ 或 HR。 还将确定 PKCδ 是否影响双链位点 NHEJ 和 HR 修复复合物的形成 断裂 (DSB) 并进行快速免疫沉淀质谱分析以识别染色质相互作用 受 PKCδ 影响的蛋白质 在目标 2 中，我将询问 PKCδ 如何调节组蛋白修饰和染色质。 我将探讨 PKCδ 在组蛋白修饰中的作用。 IR前后，以及PKCδ在染色质重塑中的核功能。 为了了解这种调节的机制，我们将分析 PKCδ 是否调节上游组蛋白修饰酶。 我提出的研究应该增强我们对 PKCδ 调节 DNA 机制的理解 -诱导细胞凋亡，并可能导致新的安全治疗干预措施的损害，以保护唾液腺和 接受 IR 治疗 HNC 的患者的口腔组织。这项 F32 奖学金还将为我提供具体的科学知识。 和研究培训，包括发展新的研究技能，以及接触基础和转化 癌症和口腔生物学以及各种专业发展机会，该培训将 为我成为一名独立癌症研究员的长期目标做好准备。