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 通过辐照（IR）疗法或治疗的组合。不幸的是，除了杀死癌细胞，红外治疗 还会杀死口腔中的非肿瘤细胞并损害唾液腺，从而导致副作用，例如慢性 口腔感染，改善唾液产生和静态。 IR引起的唾液腺损害被认为 部分原因是由于细胞凋亡失去了唾液腺泡细胞。我们的实验室已经鉴定了蛋白激酶C Delta（PKCδ）作为IR诱导的唾液腺泡细胞中IR诱导凋亡的关键调节剂，并在体外和唾液中 体内腺体。我们已经表明，在IR损伤后，PKCδ被激活并导入到核中。虽然 我们知道，PKCδ的核进口对于凋亡是必要的，这是核PKCδ如何调节的机制 DNA损伤诱导的凋亡尚不清楚。我们实验室的先前研究表明，PKCδ不得 直接激活凋亡途径，但可以调节有助于DNA的上游途径 损伤响应和DNA修复。我的初步研究表明了PKCδ介导的一种新型机制 通过染色质重塑和DNA修复来调节凋亡。我的初步数据表明部署 PKCδ增加了IR诱导的DNA损伤修复，PKCδ通过这两个非 - 调节DNA修复 同源末端连接（NHEJ）和同源重组（HR）途径。此外，我的初步 数据表明，PKCδ可以通过改变组蛋白的修饰来调节染色质结构。那，我的 总体目标是机械地了解核PKCδ如何调节染色质的可及性和 DNA修复响应IR损伤。在AIM 1中，我将表征核PKCδ在NHEJ和HR中的作用 途径并分析使用体内荧光报告测定法分析PKCδ是否偏向NHEJ或HR。我 还将确定PKCδ是否影响双链位点的NHEJ和HR修复复合物的形成 断裂（DSB）并执行快速免疫沉淀质谱法以鉴定染色质相互作用 受PKCδ影响的蛋白质。在AIM 2中，我将询问PKCδ如何调节组蛋白修饰和染色质 结构以及与DNA修复调节的关系。我将探讨PKCδ在组蛋白修饰中的作用 IR之前和之后，PKCδ在染色质重塑中的核功能。进一步了解 该调节的机制，我们将分析PKCδ是否调节上游组蛋白改性酶。全面的， 我提出的研究应增强我们对PKCδ调节DNA的机制的理解 损伤引起的凋亡，并可能导致新的安全治疗干预措施，以保护唾液腺和 患有HNC的IR的患者的口腔组织。 F32奖学金还将为我提供特定的科学 和研究培训，包括发展新的研究技能以及接触基本和翻译 癌症和口服生物学。加上各种各样的专业发展机会，这种培训将 我为我的长期目标做好准备，成为一名独立的癌症研究员。