MODEL SYSTEMS FOR ANALYSIS OF GENETIC SUSCEPTIBILITY TO COLON CANCER

最近的研究表明，人类的遗传突变 酿酒酵母错配修复基因msh2和msh2的同源基因 大约90%的遗传性非息肉病是由MLH1引起的 结肠癌(HNPCC)。这些结果表明，遗传缺陷在 错配修复可导致癌症，散发性肿瘤的分析 提供的证据表明相当大比例的散发性肿瘤可能 也是失配维修的瑕疵。这些结果定义了一种新的， 癌症易感性的主要途径。这项提议的目标是 开发失配修复分析模型系统及其应用 与癌症易感性的关系。为了实现这些目标， 将进行以下方面的调查。(1)小鼠 将构建MSH2突变的杂合子来发育一种动物 HNPCC的模型系统。小鼠MSH2基因已被克隆，一个外显子 编码MSH2蛋白保守区的序列已经被测序并 这一信息已被用于启动包含以下内容的小鼠的构建 破坏基因这一区域的突变。完整的序列 小鼠MSH2基因及其基因组内含子/外显子连接 轨迹将会被确定。这将有助于建造 其他小鼠突变和对MSH2突变的分析 在肿瘤中灭活MSH2基因的第二个拷贝。鼠标系统 也将被用于研究积累的其他突变的类型 在肿瘤进展过程中。(2)人类MSH2和MLH1基因高度同源 与相应的酿酒酵母基因同源，这将使 酿酒酵母基因将用于研究在人类身上发现的突变。 在人类肿瘤和HNPCC家族中发现的突变将在 通过定点突变获得相应的酿酒酵母基因。这个 由此产生的突变体将被表征以确定基因 在人类身上发现的不同突变的特性。的属性 这些突变将与HNPCC的特性相关 发现突变的家系和散发性肿瘤。(3)S. 正常酿酒酵母错配修复的酿酒酵母菌株 蛋白质在功能上已被人类蛋白质或 将开发酿酒酵母/人类嵌合蛋白。这个系统将会 用于研究在人类中发现的突变对错配的影响 修理。(4)将使用遗传和生化方法来识别基因 编码与人类MSH2和MLH1蛋白相互作用的蛋白质。 将对这些基因进行分析，以确定它们是否编码错配修复 如果HNPCC家族和肿瘤在这些基因中有突变。 这些研究的结果应该使我们能够阐明修复 降低细胞内源性突变率的途径，了解 更好地了解这些修复途径中的缺陷如何导致 癌症和定义额外的癌症易感基因。

Recent studies have demonstrated that inherited mutations in the human homologues of the Saccharomyces cerevisiae mismatch repair genes MSH2 and MLH1 are responsible for approximately 90% of hereditary nonpolyposis colon cancer (HNPCC). These results indicate that inherited defects in mismatch repair can cause cancer and analysis of sporadic tumors has provided evidence that a significant proportion of sporadic tumors may also be defective for mismatch repair. These results have defined a new, major pathway for cancer susceptibility. The goal of this proposal is to develop model systems for the analysis of mismatch repair and its relationship to cancer predisposition. To achieve these goals, the following lines of investigation will be performed. (1) Mice which are heterozygous for msh2 mutations will be constructed to develop an animal model system for HNPCC. The mouse MSH2 gene has been cloned, an exon encoding a conserved region of the MSH2 protein has been sequenced and this information has been used to initiate construction of mice containing mutations that disrupt this region of the gene. The complete sequence of the mouse MSH2 cDNA and the intron/exon junctions of the mouse genomic locus will be determined. This will facilitate the construction of additional mouse mutations and the analysis of msh2 mutations that inactivate the second copy of the MSH2 gene in tumors. The mouse system will also be used to study the types of other mutations that accumulate during tumor progression. (2) The human MSH2 and MLH1 genes are highly homologous to the corresponding S. cerevisiae genes which will allow the S. cerevisiae genes to be used to study the mutations found in humans. Mutations found in human tumors and HNPCC kindreds will be made in the corresponding S. cerevisiae genes by site specific mutagenesis. The resulting mutants will be characterized to determine the genetic properties of the different mutations found in humans. The properties of these mutations will be correlated with the properties of the HNPCC kindreds and sporadic tumors in which the mutations were found. (3) S. cerevisiae strains in which the normal S. cerevisiae mismatch repair proteins have been functionally substituted for by human proteins or chimeric S. cerevisiae/human proteins will be developed. This system will be used to study the effect of mutations found in humans on mismatch repair. (4) Genetic and biochemical methods will be used to identify genes encoding proteins that interact with the human MSH2 and MLH1 proteins. These genes will be analyzed to determine if they encode mismatch repair proteins and if HNPCC kindreds and tumors have mutations in these genes. The results of these studies should allow us to elucidate the repair pathways that reduce the endogenous mutation rates in cells, understand better how defects in these repair pathways lead to the development of cancer and define additional cancer susceptibility genes.