MODEL SYSTEMS FOR ANALYSIS OF GENETIC SUSCEPTIBILITY TO COLON CANCER

最近的研究表明，人类遗传性突变 酿酒酵母错配修复基因MSH 2和 MLH 1导致大约90%的遗传性非息肉病 结肠癌（HNPCC）。这些结果表明， 错配修复可导致癌症，散发性肿瘤的分析 提供的证据表明，很大一部分散发性肿瘤可能 也不能用于失配修复。这些结果定义了一个新的， 癌症易感性的主要途径。本提案的目的是 开发用于分析错配修复及其 与癌症易感性的关系。为了实现这些目标， 将进行以下调查。(1)老鼠， 将构建MSH 2突变杂合的动物 HNPCC的模型系统。小鼠MSH 2基因已被克隆， 编码MSH 2蛋白的保守区的基因已经测序， 该信息已被用于启动小鼠的构建， 破坏该基因区域的突变。的完整序列 小鼠MSH 2 cDNA和小鼠基因组的内含子/外显子连接 地点将被确定。这将有助于建设 另外的小鼠突变和MSH 2突变分析 肿瘤中MSH 2基因的第二个拷贝。小鼠系统 也将用于研究其他类型的突变， 在肿瘤进展过程中。(2)人类MSH 2和MLH 1基因高度表达， 与相应的S.酿酒酵母基因， S.酿酒酵母基因将用于研究在人类中发现的突变。 在人类肿瘤和HNPCC激酶中发现的突变将在 相应的S.通过位点特异性诱变获得酿酒酵母基因。的 将对所得突变体进行表征，以确定遗传 在人类中发现的不同突变的特性。的性质 这些突变将与HNPCC的特性相关 （3）S. 酿酒酵母菌株中，正常的S.酿酒酵母错配修复 蛋白质在功能上被人蛋白质取代， 嵌合长鳍篮子鱼酿酒酵母/人类蛋白质将被开发。该系统将 用于研究人类中发现的突变对错配的影响 修复. (4)遗传学和生物化学方法将被用来鉴定基因 编码与人MSH 2和MLH 1蛋白相互作用的蛋白。 这些基因将被分析以确定它们是否编码错配修复 如果HNPCC激酶和肿瘤在这些基因中有突变。 这些研究的结果应该能让我们阐明 降低细胞内内源性突变率的途径， 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.