苯通过影响DNA修复引起造血干祖细胞基因组突变的机制研究

Leukemia is a heterogenous group of diseases originated from uncontrolled clonal expansion of hematopoietic stem cells and progenitors. Although tremendous progress has been made in the understanding of disease pathogenesis, little is known regrading the etiology of leukemia development. Benzene is an important industrial chemical and major components in automobile exhaust and cigarette smoke. Benzene exposure has been associated with genetic mutation and chromosomal aberration, and induce acute leukemia in human population. Earlier studies have focused on the fact that benzene can directly induce genetic lesions as a harmful environmental chemical. Based on the finding that benzene causes increased levels of error-prone repair of DNA damage, we propose a model in which benzene contributes to mutagenesis through aberrant regulation of DNA repair. Our preliminary results have confirmed disregulated expression of key repair genes in benzene exposure population, as well as cellular reporter gene system of DNA repair. Using an embryonic stem cell differentiation model, we plan to examine the impact of benzene on mutagenesis in different lineages of hematopoietic progenitors. We will also determine the specific repair pathway involved upon benzene exposure, and use functional analysis to identify key repair genes critical for benzene-mediated aberrant repair. Finally, we plan to set up reporter gene DNA repair systems for gene mutation and chromosomal translocation, as well as a cellular model of topoisomerase II inhibitor etoposide-induced MLL translocation. We will explore how benzene regulates DNA repair and exert its effect on the formation of point mutation, small insertion/deletion and chromosomal translocation. This study will shed light on the molecular mechanisms in benzene-induced leukemia, and provide the basis for the development of novel prevention and prognostic tools in acute leukemia.

白血病是一类造血干祖细胞来源的恶性克隆性疾病。尽管白血病发病的机理研究近年来取得很大的进展，但对其病因仍然知之甚少。作为重要的工业原料和香烟烟雾及汽车尾气的主要成分，苯可引起基因突变和染色体畸变并诱发急性白血病。以往研究多侧重于苯对DNA的直接损伤，但我们根据接苯后DNA修复能力紊乱和错误增加，提出苯通过干扰DNA修复引起基因组突变。课题组已经在接苯人群中证实修复基因表达紊乱，将利用干细胞分化系统确定苯对不同谱系造血干祖细胞基因组突变的影响，运用损伤修复报告基因系统确定苯调控的特定修复通路，用基因敲低等功能实验寻找苯影响DNA修复的关键基因。进一步在报告基因体系及白血病MLL基因易位模型中，利用PCR克隆后测序等手段探讨苯通过调控DNA修复对点突变、小片段插入缺失和染色体不平衡易位等基因组突变的影响。本研究有助于阐明苯引起白血病的分子机理，为预防苯致白血病的发生及发展新治疗手段提供理论基础

白血病发病的机理研究近年来取得很大的进展，但对其病因仍然知之甚少。作为重要的工业原料和香烟烟雾及汽车尾气的主要成分，苯可引起基因突变和染色体畸变并诱发急性白血病。以往研究多侧重于苯对DNA的直接损伤，本项目中，我们通过损伤修复报告基因系统发现苯的重要代谢产物氢醌可增加DNA双链断裂同源重组修复（HR）及单链复性修复(SSA)。在此基础上，我们重点研究了苯早期损伤对修复能力的影响。我们发现，苯的早期损伤修复后DNA双链断裂修复蛋白BRCA1向断裂处募集的增加，同源重组修复基因RAD51、BRCA1、BRCA2、RAD50、RAD51C、NBN的mRNA表达水平显著增加，单链复性修复基因RAD52及重要抑癌基因TP53的mRNA表达水平也呈增加趋势。通过RNA测序及生物信息学分析进一步发现两条重要通路-活性氧通路及NF-kB通路被激活。NF-kB蛋白编码基因（NF-kB1,NF-kB2,RelA及RelB）及其靶基因（BIC3, CCL2, CCL20及 CD83）mRNA在修复后表达显著升高，同时，免疫印迹分析更进一步证实NF-kB 家族重要蛋白p65(RelA)在修复后高表达。利用MLL白血病模型发现，氢醌对MLL白血病前体造血干祖细胞的修复能力有促进作用，同时能显著维持干细胞的自我更新。综上所述，我们的结果显示，苯的代谢物氢醌可促进DNA双链断裂同源重组修复及单链复性修复，活性氧通路及NF-kB通路可能在苯所引起的早期双链断裂修复中起重要作用。这些研究成果为苯通过异常修复引发突变的机制研究提供了新的理论依据。