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Investigating a novel role for Top2a in R-loop resolution and in YM155 mechanism of action

研究 Top2a 在 R 环解析和 YM155 作用机制中的新作用

基本信息

批准号：
10684809
负责人：
Ryan Patrick Mackay
金额：
$ 4.88万
依托单位：
LOUISIANA STATE UNIV HSC SHREVEPORT
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-30 至 2025-01-09
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10684809
关键词：
ATP phosphohydrolase Amino Acids Basic Science Benign Binding Binding Proteins Biological Assay Biology Biometry Bromodeoxyuridine Cancer Patient Cancer cell line Cell Death Cell physiology Cells Chromatin Loop Clinical Clinical Research Comet Assay Complementary DNA Complex Conserved Sequence Core Facility DNA DNA Damage DNA Double Strand Break DNA Structure DNA replication fork DNA-Directed RNA Polymerase Data Data Analyses Deamination Dedications Development Discipline Disease Educational process of instructing Educational workshop Enzymes Evaluation Exonuclease Experimental Designs Faculty Family Fiber Focus Groups Functional disorder Gamma-H2AX Gene Expression Genetic Transcription Genomic Instability Goals Grant Health Sciences Healthcare Human Hybrids Immunofluorescence Immunologic Immunoglobulin Class Switching Immunoglobulin Switch Recombination Institution Instruction Knowledge Label Laboratories Link Louisiana Malignant Neoplasms Measures Mentors Messenger RNA Methods Mitochondria Normal Cell Papillary thyroid carcinoma Pathologic Pharmaceutical Preparations Physiologic pulse Physiological Processes Play Prevention Process Protein Overexpression Proteins Publications Publishing RNA RNA Binding RNA Interference RNA Processing RNA Splicing RNA annealing Recombinants Replication Initiation Research Research Design Research Personnel Research Training Residencies Resolution Resources Review Literature Ribonucleases Ribonucleoproteins Role Sampling Single-Stranded DNA Site-Directed Mutagenesis Small Nuclear Ribonucleoproteins Statistical Data Interpretation Structure Students Superhelical DNA TOP2A gene Techniques Technology Testing Therapeutic Thyroid Gland Tissues Topoisomerase Training Transcriptional Regulation Universities Western Blotting Writing anaplastic thyroid cancer cancer cell clinical training data modeling design doctoral student experience graduate school helicase human disease improved in silico interest knock-down laboratory development malignant neurologic neoplasms mammalian genome medical schools medical specialties meetings mutant nanomolar nervous system disorder novel nuclease overexpression prevent programs skills small molecule statistics

项目摘要

R-loops are DNA:RNA hybrids formed when newly transcribed RNA binds to its complementary DNA template, preventing reannealing of the two DNA strands. R-loops occur naturally and play roles in normal physiological processes including immunoglobulin class switch, transcription regulation, and mitochondrial replication initiation. Pathologic R-loop accumulation has also been demonstrated in disease states and has been linked to DNA damage and genome instability. Various factors are involved in resolving R-loops as part of normal cellular physiology including mRNA binding proteins, helicases, and RNaseH as well as topoisomerases 1 and 3b. Based on preliminary data investigating YM155, a small molecule, in the treatment of anaplastic thyroid cancer, this project will investigate two hypotheses - Topoisomerase 2α (Top2α) plays a novel role in the resolution of R-loops, and Top2α inhibition and R-loop accumulation are important for YM155 mechanism of action which will be tested in two aims: 1) to assess the role of Top2α in R-loop resolution and 2) to investigate the role of Top2α and R-loop accumulation in YM155 mechanism of action. R-loop formation (immunofluorescence), DNA damage (COMET assay and p-H2AX foci), and replication fork stalling (DNA fiber assay) will be measured after Top2α knockdown. The interaction between Top2α and YM155 will be characterized via site-directed mutagenesis. RNaseH is specific to RNA in DNA:RNA hybrids and is often used to confirm R-loop accumulation. We will see if RNaseH1 overexpression rescues cells from Top2α knockdown and YM155 treatment. Currently, there is no known role for Top2α in R-loop resolution. While R-loops have been implicated in cancer, little is known about whether increased R-loop accumulation can be turned into a therapeutic strategy. This project will further understanding of R-loop biology as well as cancer pathophysiology. This proposal also describes an integrated research and clinical training plan for an MD-PhD student. Activities under this proposal include development of lab methods, first author publication, and presentation skills including institutional seminars and national scientific meetings, and teaching students in graduate healthcare programs. The student will also gain valuable experience in research design, statistics, and data interpretation and evaluation by participating in literature review clubs and graduate coursework. A clinical training sponsor is identified in the student’s specialty of interest with opportunities for clinical instruction and observation, for clinical research, and for mentored guidance to facilitate transition into a research-oriented residency program. Research and clinical training will be performed at Louisiana State University Health Sciences Center – Shreveport (LSUHSC-S). LSUHSC-S has a medical school and graduate school with faculty performing research in basic science and clinical disciplines. LSUHSC-S has various resources for investigators including dedicated lab space and research core facilities to assist with the latest technologies as well as seminars, focus groups, and professional development workshops in grant writing, conduct of research, biostatistics and data analysis.

R 环是新转录的 RNA 与其互补 DNA 模板结合时形成的 DNA:RNA 杂交体，防止两条 DNA 链重新退火。 R 环自然发生并在正常生理中发挥作用过程包括免疫球蛋白类别转换、转录调节和线粒体复制引发。病理性 R 环积累也已在疾病状态中得到证实，并与 DNA 损伤和基因组不稳定。作为正常细胞一部分的 R 环的解析涉及多种因素生理学，包括 mRNA 结合蛋白、解旋酶、RNaseH 以及拓扑异构酶 1 和 3b。基于研究小分子 YM155 治疗未分化甲状腺癌的初步数据，该项目将研究两个假设 - 拓扑异构酶 2α (Top2α) 在分辨率中发挥新作用 R 环的数量、Top2α 抑制和 R 环积累对于 YM155 的作用机制很重要将测试两个目标：1) 评估 Top2α 在 R 环分辨率中的作用，2) 研究该作用 YM155 作用机制中 Top2α 和 R 环积累的研究。 R 环形成（免疫荧光），将测量 DNA 损伤（COMET 测定和 p-H2AX 焦点）和复制叉停滞（DNA 纤维测定） Top2α 敲除后。 Top2α 和 YM155 之间的相互作用将通过定点表征诱变。 RNaseH 对 DNA:RNA 杂交体中的 RNA 具有特异性，通常用于确认 R 环积累。我们将看看 RNaseH1 过度表达是否可以将细胞从 Top2α 敲低和 YM155 治疗中拯救出来。现在， Top2α 在 R 环解析中的作用尚不清楚。虽然 R 环与癌症有关，但鲜有报道了解增加的 R 环积累是否可以转化为治疗策略。该项目将进一步了解 R 环生物学以及癌症病理生理学。该提案还描述了针对医学博士生的综合研究和临床培训计划。活动该提案包括实验室方法的开发、第一作者的出版物和演讲技巧，包括机构研讨会和国家科学会议，以及在研究生医疗保健项目中教授学生。学生还将获得研究设计、统计和数据解释方面的宝贵经验通过参加文献综述俱乐部和研究生课程进行评估。临床培训赞助商是根据学生的兴趣专业确定临床指导和观察的机会，以进行临床研究，并提供指导指导，以促进向以研究为导向的住院医师计划的过渡。研究和临床培训将在路易斯安那州立大学健康科学中心进行 – 什里夫波特 (LSUHSC-S)。 LSUHSC-S 设有医学院和研究生院，并配备从事研究的教师在基础科学和临床学科。 LSUHSC-S 为研究人员提供各种资源，包括专门的资源实验室空间和研究核心设施，以协助最新技术以及研讨会、焦点小组、以及拨款写作、研究开展、生物统计学和数据分析方面的专业发展研讨会。