Elucidating Mechanisms of Loss of Heterozygosity in Diploid Cells

阐明二倍体细胞杂合性丧失的机制

• 批准号: 10579177
• 负责人: Samantha Brooke Regan
• 金额: $ 4.77万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-22 至 2025-06-21
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10579177
• 关键词: Affect; Aneuploidy; Biological Assay; Breast; CRISPR/Cas technology; Cell Line; Cells; Chromatids; Chromosomal Breaks; Chromosomal Instability; Chromosome Arm; Chromosomes; Colon Carcinoma; Complex; Cruciform DNA; DNA Damage; DNA Double Strand Break; DNA Repair Gene; Dangerousness; Data; Diploid Cells; Double Effect; Double Strand Break Repair; Event; Flow Cytometry; Fluorescent in Situ Hybridization; Frequencies; Gene Conversion; Genes; Genetic Crossing Over; Genetic Nondisjunction; Genetic Recombination; Genome; Genome Stability; Heterozygote; Homologous Gene; Individual; Location; Loss of Heterozygosity; Malignant Neoplasms; Mammalian Cell; Modeling; Mutation; Nonhomologous DNA End Joining; Normal Cell; Outcome; Ovarian; Pathway interactions; Patients; Pattern; Premalignant Cell; Proteins; Research; Retinoblastoma; Role; Sister Chromatid Exchange; Site; Structure; Syndrome; System; Topoisomerase III; Tumor Suppressor Genes; Work; Yeasts; anticancer research; cancer initiation; daughter cell; early screening; endonuclease; functional loss; helicase; high risk; homologous recombination; knock-down; mutant; nuclease; overexpression; prevent; segregation; tumor; tumor progression

Project Summary Abstract Loss of heterozygosity (LOH) is the loss of the functional copy of a tumor suppressor gene in heterozygous individuals, leaving only the mutant copy, thereby contributing to cancer initiation and progression. Understanding how LOH arises in healthy cells is a vital component of cancer research, as it could lead to opportunities to prevent cancer in patients with increased early screening for individuals with mutations putting them at high risk for LOH, or with identification and targeting of cells that have undergone dangerous LOH. LOH can develop through deletions of whole chromosome arms or smaller regions of the genome, nondisjunction incidents, or homologous recombination events between homologous chromosomes (i.e., interhomolog homologous recombination, IH-HR). Despite decades of research, a critical need remains to identify the causes of LOH in normal or precancerous cells. Detecting these mechanisms in already established tumors is often difficult due to high levels of aneuploidy, ongoing chromosome instability, and DNA damage. We have recently developed a high throughput flow cytometry-based system which is sensitive for detecting LOH in normal diploid cells. Our objective is to exploit this system to identify and quantify mechanisms of LOH that arise from DNA double-strand breaks (DSBs), and the factors that modulate these events. We hypothesize that mechanisms of LOH which occur as a result of DSBs include copy number neutral LOH, as from IH-HR or non-disjunction associated with chromosome duplication and furthermore, that these events are modulated by the location of the DSB and DNA repair proteins, including proteins that act on recombination intermediates. We will address this hypothesis through the following specific aims: In Aim 1, we will investigate how proteins that act on recombination intermediates impact LOH arising from IH-HR, utilizing our flow-cytometry based system. We will assay the frequency of LOH that arises from IH-HR in the presence or absence of proteins that both prevent and contribute to crossover events, which can lead to long-range LOH. In Aim 2, we will take an unbiased approach to explore additional mechanisms of LOH arising from a DSB. We will determine how LOH mechanism is affected by the location of a DSB along a chromosome and by loss of different DSB repair pathways. This work will allow us to identify the factors that can contribute to or even prevent LOH, and give a better understanding of how these initiating events in cancer occur.

项目摘要 杂合性缺失（洛）是指肿瘤细胞中肿瘤抑制基因功能拷贝的缺失， 杂合个体，只留下突变拷贝，从而有助于癌症的发生 和进步。了解洛缺失如何在健康细胞中产生是癌症的重要组成部分 研究，因为它可能会导致机会，以预防癌症患者增加早期 筛查具有使其处于洛缺失高风险的突变的个体，或 并靶向已经经历危险洛缺失的细胞。洛缺失可通过缺失发生 整个染色体臂或基因组的较小区域，不分离事件，或 同源染色体之间的同源重组事件（即，同源物间 同源重组，IH-HR）。尽管进行了数十年的研究，但仍然迫切需要 确定正常或癌前细胞中洛缺失的原因。检测这些机制， 由于高水平的非整倍体，已经建立的肿瘤通常是困难的， 染色体不稳定和DNA损伤。我们最近开发了一种高通量流程， 该系统对正常二倍体细胞中的洛检测敏感。我们 目的是利用该系统来识别和量化由以下原因引起的洛缺失机制： DNA双链断裂（DSB），以及调节这些事件的因素。我们假设 DSB导致的洛缺失机制包括拷贝数中性的洛缺失， 从IH-HR或与染色体复制相关的不分离，此外， 这些事件由DSB和DNA修复蛋白的位置调节，包括 作用于重组中间体的蛋白质。我们将通过以下方式来解决这个假设： 以下具体目标：在目标1中，我们将研究蛋白质如何作用于重组 中间体影响由IH-HR引起的洛缺失，利用我们的基于流式细胞术的系统。我们 将分析在存在或不存在蛋白质的情况下由IH-HR引起的洛缺失的频率 既能防止又能促进交叉事件，而交叉事件可导致长距离洛缺失。在Aim中 2，我们将采取无偏见的方法来探讨洛缺失的其他机制， DSB。我们将确定洛机制是如何影响的DSB沿着 染色体和不同的DSB修复途径的损失。这项工作将使我们能够确定 可能导致甚至预防洛缺失的因素，并更好地了解如何 这些癌症的起始事件发生。