MECHANISMS OF MUTATIONS IN HUMAN TUMOR CELLS

描述：肿瘤的发生和发展是一个循序渐进的过程。 涉及多种基因和表型的积累 改装。这些改变包括所有类型的基因组 重排和点突变激活显性癌基因或 消除肿瘤抑制因子。虽然已经取得了相当大的进展 在识别肿瘤中改变的基因方面，人们对 影响它们的突变机制。有人争辩说，这一速度 肿瘤细胞中的点突变太低，无法解释所有突变 参与了肿瘤的发展。这里提出的工作目标是 目的：阐明人类肿瘤细胞过度突变的机制并探讨其机制。 确定这些因素如何导致肿瘤的遗传不稳定性 细胞。具体地说，生产多个 突变(其中单个低频启动事件显然 在目标序列中产生多个改变的结果) 人结直肠癌细胞系微卫星不稳定性的研究 将会被调查。莫特博士提出，这些形式的 过度突变是容易出错的DNA合成或改变的结果 错配修复，并可能显著促进积累 肿瘤细胞中的点突变。具体目标是：I)审查 这些突变的细胞和基因位点特异性。如果这些事件 导致基因组不稳定，那么它们应该在很大范围内发生 肿瘤细胞和整个基因组。的性质和速度 显示多个突变的品系中的突变将是一个特别的焦点。 Ii)确定多重突变的结构和来源。这个 多个突变相对于单点突变的频率和 这些事件的复杂性(突变发生的距离 下降和这一区域突变的频率)将被确定。 这些数据为容易出错的合成提供了推定的“补丁”大小 以及在体内测量该贴片中复制的保真度。 DNA损伤剂触发这些突变的能力将是 检查过了。三)研究肿瘤抑制因子在这些形式中的作用 基因的不稳定性。抑癌基因P53的突变形式 将其导入结直肠癌细胞系以确定 它们是否会刺激多种突变的产生。和iv) 研究多重突变背后的生化机制。 检测复制保真度和错配修复作用的方法 在可选择遗传座位上形成多个突变的过程中 将结直肠癌细胞株进行传代。单股 将进行构象多态分析和多重DNA测序 用来描述自发和诱发突变的特征。体外试验 将用于检查DNA复制的完整性和保真度 和错配修复。

DESCRIPTION: Tumor development and progression are stepwise processes involving the accumulation of multiple genotypic and phenotypic alterations. These alterations include all types of genome rearrangements and point mutations that activate dominant oncogenes or eliminate tumor suppressors. While considerable progress has been made in the identification of genes altered in tumors, little is known of the mutation mechanisms affecting them. It has been argued that the rate of point mutation in tumor cells is too low to account for all mutations involved in tumor development. The goal of the work proposed here is to elucidate mechanisms of hypermutation in human tumor cells and to determine how these contribute to the genetic instability of tumor cells. In particular, mechanisms underlying the production of multiple mutations (in which a single low frequency initiating event apparently results in the production of multiple alterations in a target sequence) and microsatellite instability in human colorectal carcinoma cell lines will be investigated. Dr. Meuth proposes that these forms of hypermutation are the result of error prone DNA synthesis or altered mismatch repair and could significantly contribute to the accumulation of point mutations in tumor cells. Specific aims are i) to examine the cell and locus specificity of these mutations. if these events contribute to genome instability, then they should occur in a wide range of tumor cells and throughout the genome. The nature and rate of mutation in lines showing multiple mutations will be a particular focus. ii) to determine the structure and origin of multiple mutations. The frequency of multiple mutations relative to single point mutations and the complexity of these events (the distance over which the mutations fall and the frequency of mutations in this tract) will be determined. Such data provide a putative "patch" size for the error prone synthesis and an in vivo measurement of the fidelity of replication in this patch. The ability of DNA damaging agents to trigger these mutations will be examined. iii) to examine the role of tumor suppressors in these forms of genetic instability. Mutant forms of the tumor suppressor gene p53 will be transfected into colorectal carcinoma cell lines to determine whether they stimulate the production of multiple mutations. and iv) to investigate biochemical mechanisms underlying multiple mutations. Assays to examine the role of replicational fidelity and mismatch repair in the formation of multiple mutations at selectable genetic loci in colorectal carcinoma cell lines will be undertaken. Single strand conformation polymorphism analysis and multiplex DNA sequencing will be used to characterize spontaneous and induced mutations. In vitro assays will be used to examine the integrity and fidelity of DNA replication and mismatch repair.