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DNA：新转录RNA与其完整DNA模板结合时形成的RNA杂交。防止两条DNA链的重新进行。 R环自然发生，并在正常生理中发挥作用包括免疫球蛋白类开关，转录调节和线粒体复制的过程引发。病理R环的积累也已在疾病状态中得到证明，并与 DNA损伤和基因组不稳定性。作为正常细胞的一部分，解决R-loops的各种因素涉及生理学，包括mRNA结合蛋白，解旋酶和RNASEH以及拓扑异构酶1和3b。基于研究YM155的初步数据（一种小分子），用于治疗甲状腺甲状腺癌的治疗该项目将研究两个假设 - 拓扑异构酶2α（TOP2α）在分辨率中起新作用 R环，TOP2α抑制和R环的积累对于YM155作用机理很重要这将在两个目标中进行测试：1）评估TOP2α在R-loop分辨率中的作用和2）研究角色 YM155作用机理中TOP2α和R环的积累。 R环形成（免疫荧光），将测量DNA损伤（彗星测定和P-H2AX焦点），并测量复制叉失速（DNA纤维测定） TOP2α敲低之后。 TOP2α和YM155之间的相互作用将通过位置定向来表征诱变。 RNASEH是DNA中的RNA特异性：RNA杂交中的RNA，通常用于确认R环的积累。我们将看到RNASEH1过表达是否会使细胞免于TOP2α敲低和YM155处理。现在， TOP2α在R环分辨率中没有已知的作用。尽管癌症中隐含了R环，但几乎没有知道是否增加了R环的积累可以将其变成一种治疗策略。这个项目将进一步了解R-loop生物学以及癌症的病理生理学。该建议还描述了MD-PHD学生的综合研究和临床培训计划。活动根据该建议，包括开发实验室方法，第一作者出版物和演示技巧，包括机构半货和国家科学会议，以及教学生研究生医疗计划的教学。该学生还将在研究设计，统计和数据解释方面获得宝贵的经验，并且通过参加文学审查俱乐部和研究生课程来评估。临床培训赞助商是临床的临床教学和观察机会，以学生感兴趣的专业识别研究，并为指导指导，以促进过渡到以研究为导向的居住计划。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 - Shreveport（LSUHSC-S）。 LSUHSC-S有一所医学院和研究生院在基础科学和临床学科中。 LSUHSC-S为调查人员提供了各种资源，包括专用实验室空间和研究核心设施，以协助采用最新技术以及焦点小组，以及赠款写作，研究，生物统计学和数据分析的专业发展研讨会。