Elucidating Mechanisms of Loss of Heterozygosity in Diploid Cells

项目摘要摘要 杂合性丢失(LOH)是指肿瘤抑制基因功能拷贝的丢失。 杂合子个体，只留下突变副本，从而导致癌症的发生 和进步。了解LOH是如何在健康细胞中产生的是癌症的重要组成部分 研究，因为它可能带来机会，以预防癌症患者的早期增加 筛查具有突变的个体，使他们处于杂合性缺失的高危状态，或具有身份识别 并以经历危险杂合性缺失的细胞为靶点。杂合性血友病可通过缺失而发展 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(即同源间 同源重组，IH-HR)。尽管进行了几十年的研究，但仍有一个迫切需要 确定正常或癌前细胞中杂合性缺失的原因。检测中的这些机制 已经建立的肿瘤通常很困难，因为高水平的非整倍体，正在进行 染色体不稳定和DNA损伤。我们最近开发了一种高吞吐量的流量 检测正常二倍体细胞杂合性缺失的细胞学检测系统。我们的 目的是利用这个系统来识别和量化由以下原因引起的LOH的机制 DNA双链断裂(DSB)以及调节这些事件的因素。我们假设 作为DSB结果发生的LOH机制包括拷贝数中性的LOH，AS 来自IH-HR或与染色体复制相关的非分离，而且， 这些事件受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解决这一假设 以下是具体目标：在目标1中，我们将研究蛋白质如何作用于重组 利用我们的基于流式细胞术的系统，中间体影响IH-HR产生的LOH。我们 将分析在蛋白质存在或不存在的情况下由IH-HR引起的杂合性缺失的频率 这既防止了交叉事件，也导致了交叉事件，从而可能导致远程LOH。在AIM 2，我们会以不偏不倚的态度，探讨因 DSB。我们将确定DSB沿线的位置如何影响LOH机制 染色体和不同的DSB修复途径的丢失。这项工作将使我们能够识别 可能导致或甚至预防LOH的因素，并让我们更好地了解 这些启动癌症的事件就会发生。

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.