Telomerase-Mediated Healing of Double-Strand Breaks in Human Cells

端粒酶介导的人体细胞双链断裂修复

基本信息

批准号：
10560471
负责人：
Charles Gunnar Kinzig
金额：
$ 5.27万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
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：
2021-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10560471
关键词：
Aneuploidy Apoptosis Award Base Pairing Binding Biological Assay Cell Aging Cell Survival Cell division Cells Centromere Chromosomal Breaks Chromosome 16 Chromosome Deletion Chromosomes Clinical Complex DNA Damage DNA Double Strand Break DNA Primase Data Development Dicentric chromosome Distal Doctor of Philosophy Double Strand Break Repair Doxycycline Ectopic Expression Environment Event Frequencies Genes Genome Genome Stability Genomic Instability Genomics Germ Cells Goals Gonadal structure Haploidy Hela Cells Human In Vitro Institution Length Link Malignant Neoplasms Mediating Medical Methods Molecular Neoplasms Pathologic Pathway interactions Patient Care Patients Physicians Physiological Polymerase Process Proliferating Regulation Reporter Repression Role Running Rupture Scientist Signal Transduction Somatic Cell TP53 gene Telomerase Telomerase Inhibitor Telomerase RNA Component Telomerase inhibition Telomere Maintenance Telomere Shortening Testing Trees alpha-Thalassemia cancer cell cancer diagnosis cancer prevention carcinogenesis career cell transformation chromosome fusion chromothripsis design detection assay expectation experimental study genetic approach healing improved mortality neoplastic cell novel strategies p53-binding protein 1 prevent programs reconstitution recruit repaired response senescence stem cells telomere tumor tumorigenesis

项目摘要

PROJECT SUMMARY/ABSTRACT Telomeres—which define and protect the ends of humans’ linear chromosomes—serve as a natural check on carcinogenesis. Genome stability requires cells to differentiate telomeres from perilous DNA double-strand breaks (DSBs) to block inappropriate DSB repair and DNA damage response (DDR) signaling, which humans accomplish with the shelterin complex. Telomerase maintains telomere length in the gonads and some stem cells, but telomeres in somatic cells shorten with each cell division due to developmental silencing of telomerase. Unfettered cell division in early neoplasms eventually leads to a few telomeres becoming critically short and activating persistent DDR signaling, which causes cells with functional p53 and Rb pathways to undergo senescence or apoptosis. Cells defective in these pathways continue to divide until multiple telomeres become de-protected and then enter telomere crisis, defined by poor cell viability due to intolerable genomic instability, as chromosomes repeatedly fuse at their ends and break. Clinical tumors emerge from crisis with rearranged, aneuploid genomes and a telomere maintenance mechanism. To escape from telomere crisis, I predict that malignant cells must reconstitute their telomeres and that telomerase may accomplish this by directly repairing non-telomeric chromosome ends with neotelomeres. The objective of the proposed project is to identify and mechanistically characterize telomerase- mediated DSB repair in human cells. In vitro, telomerase can add TTAGGG repeats to a non-telomeric breakpoint sequence derived from a patient with α-thalassemia due to a terminal chromosomal truncation. Using this sequence, I have designed a PCR-based reporter assay to detect neotelomere formation in cells at an inducible DSB and have gathered evidence that suggests that telomere healing occurs in human cells in a telomerase-dependent manner. I will improve this assay with TaqMan probes on a qPCR platform to rigorously quantify telomere healing events and will perform further experiments to demonstrate that telomerase is responsible for TTAGGG repeat addition. Because telomerase-mediated repair threatens to convert DSBs into terminal chromosome deletions, I hypothesize that human cells have evolved mechanisms to block telomerase activity at DSBs. I will implement a genetic approach with my telomere healing assay to identify the physiologic repressors of this aberrant mode of DSB repair. Ultimately, I aim to unveil a new role for telomerase in enabling incipient cancers to traverse the bottleneck of telomere crisis. This leap in our understanding of genomic instability in early tumorigenesis may lead to unexpected ways to detect and prevent cancer in patients. With the aid of this award and the stimulating environment of the Tri-Institutional MD/PhD Program, I will grow scientifically, medically, and professionally in ways that will enable me to advance toward my long-term career goal of leading a cancer-centric lab while providing patient care as a transformative physician-scientist.

项目概要/摘要端粒——定义和保护人类线性染色体的末端——作为一种自然检查致癌作用需要细胞将端粒与危险的 DNA 双链区分开来。断裂（DSB）以阻止不适当的 DSB 修复和 DNA 损伤反应（DDR）信号传导，人类通过端粒酶复合物来维持性腺和一些茎中的端粒长度。细胞，但体细胞中的端粒随着每次细胞分裂而缩短，这是由于早期肿瘤中不受限制的细胞分裂最终导致一些端粒变得严重。短且激活持续的 DDR 信号传导，导致具有功能性 p53 和 Rb 途径的细胞这些途径有缺陷的细胞会继续分裂，直到出现多个端粒。失去保护，然后进入端粒危机，其定义是由于无法耐受的基因组导致细胞活力较差不稳定，因为染色体在其末端反复融合并断裂。重新排列的非整倍体基因组和端粒维持机制为了摆脱端粒危机，我。预测恶性细胞必须重建它们的端粒，而端粒酶可以通过以下方式完成这一任务：用新端粒直接修复非端粒染色体末端。拟议项目的目标是识别端粒酶并对其进行机械表征在人体细胞中介导的 DSB 修复在体外，端粒酶可以将 TTAGGG 重复序列添加到非端粒上。断点序列源自因染色体末端截断而患有 α 地中海贫血的患者。利用该序列，我设计了一种基于 PCR 的报告基因检测方法来检测细胞中新端粒的形成一种诱导型 DSB，并收集了证据表明端粒愈合发生在人类细胞中我将在 qPCR 平台上使用 TaqMan 探针改进这种依赖于端粒酶的方式。量化端粒愈合事件并将进行进一步的实验来证明端粒酶因为端粒酶介导的修复威胁将 DSB 转化为 TTAGGG 重复序列。末端染色体缺失，我凯文认为人类细胞已经进化出阻断端粒酶的机制我将通过我的端粒愈合分析实施遗传学方法来识别生理学。最终，我的目标是揭示端粒酶在实现这一异常模式方面的新作用。早期癌症跨越了端粒危机的瓶颈，这是我们对基因组理解的飞跃。早期肿瘤发生的不稳定性可能会导致以意想不到的方式检测和预防患者的癌症。在这个奖项的帮助和三机构医学博士/博士项目的激励环境下，我会成长以科学、医学和专业的方式使我能够朝着我的长期职业生涯前进目标是领导一个以癌症为中心的实验室，同时作为变革性的医生科学家为患者提供护理。