DNA MISMATCH REPAIR GENES AND TUMORIGENESIS

DNA错配修复基因hMLH1、hPMS2和hPMS1的突变与人类遗传性结肠癌有关。微卫星不稳定性的存在决定了MMR活性的缺乏，这也是来自各种组织的相当一部分散发性肿瘤的共同特征，并与对几种化疗药物的耐药性的产生有关。我们最近培育了MLH1、PMS2和Pms1 DNA错配修复基因缺陷的小鼠并对其进行了鉴定。我们的分析表明，携带这些基因突变的小鼠的肿瘤易感性不同，肠道肿瘤的发生与肠道上皮细胞DNA复制错误的频率没有明显的相关性。这些发现表明，单个DNA错配修复基因具有独特的生物学作用，特征不佳的因素在与DNA错配修复缺陷相关的肿瘤发生中发挥了作用。这项提议的广泛目标是确定由于DNA错配修复缺陷而导致的肿瘤形成的机制(S)。我们假设在MLH1、PMS2和PMS1缺陷小鼠中不同的肿瘤易感性是由于基因在自发或化学诱导的突变中的特定作用。为了验证这一假设，我们建议通过同源重组构建携带MLH1、PMS2和PMS1突变的等基因细胞系(目标1)。这些细胞系将与以前产生的Msh2-/-细胞系一起用于许多检测，使我们能够系统地确定不同MMR基因对自发突变的相对贡献、特定位点的突变谱、简单重复序列的不稳定性以及不同DNA元件之间的基因重组(目标2)。此外，我们将确定错配修复基因是否影响化学诱导的突变，以及碱基修饰剂是否会影响错配修复缺陷小鼠的肿瘤发展(目标3)。拟议的研究计划将在细胞水平上确定不同的错配修复基因在诱变的几个方面的作用，并将结果应用于DNA错配修复缺陷小鼠在机体水平上的肿瘤形成研究。阐明DNA错配修复基因的新的和特定的作用将为携带错配修复基因突变的家系的癌症预防提供新的策略，并将为治疗DNA错配修复缺陷肿瘤提供新的治疗途径。

Mutations in the DNA mismatch repair genes hMLH1, hPMS2 and hPMS1 are involved in human hereditary colon cancer. Lack of MMR activity, as determined by the presence of microsatellite instability, is also a common feature in a significant fraction of sporadic tumors from a variety of tissues, and is associated with the development of resistance to several chemotherapeutic agents. We have recently generated and characterized mice deficient for the Mlh1, Pms2 and Pms1 DNA mismatch repair gene. Our analysis has demonstrated that mice carrying mutations in these genes differ in tumor susceptibility, and that there is no obvious correlation between intestinal tumorigenesis and the frequency of DNA replication errors in the intestinal epithelium. These findings imply that individual DNA mismatch repair genes have unique biological roles, and that poorly characterized factors play a role in tumorigenesis associated with DNA mismatch repair deficiency. The broad goal of this proposal is to define the mechanism(s) of tumor formation due to defects in DNA mismatch repair. We hypothesize that different tumor susceptibility in Mlh1, Pms2 and Pms1 deficient mice is due to gene-specific roles in either spontaneous or chemically induced mutagenesis. In order to examine this hypothesis, we propose to construct isogenic cell lines carrying mutations in Mlh1, Pms2 and Pms1 through homologous recombination (Aim 1). These cell lines will be used in conjunction with previously generated Msh2-/-cell lines in a number of assays that will allow us to systematically determine the relative contribution of different MMR genes to spontaneous mutation, mutation spectra at defined loci, instability of simple-repeat tracts, and genetic recombination between diverged DNA elements (Aim 2). Additionally, we will determine if mismatch repair genes affect chemically induced mutagenesis, and if base modifying agents can affect tumor development in mismatch repair deficient mice (Aim 3). The proposed research program will define the role of different mismatch repair genes in several aspects of mutagenesis at the cellular level, and results will be applied in the study of tumor formation at the organismal level in DNA mismatch repair deficient mice. Elucidation of novel and specific roles of DNA mismatch repair genes will provide new strategies for cancer prevention in families carrying mismatch repair gene mutations, and will suggest new therapeutic avenues for treatment of DNA mismatch repair deficient tumors.