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大量散发性肿瘤的共同特征，并且与对几种化疗药物的耐药性的发展有关。我们最近培育并鉴定了Mlh1、Pms2和Pms1 DNA错配修复基因缺陷的小鼠。我们的分析表明，携带这些基因突变的小鼠在肿瘤易感性上存在差异，肠道肿瘤发生与肠上皮DNA复制错误的频率之间没有明显的相关性。这些发现表明，个体DNA错配修复基因具有独特的生物学作用，并且特征不明确的因素在与DNA错配修复缺陷相关的肿瘤发生中发挥作用。本提案的总体目标是定义由于DNA错配修复缺陷而形成肿瘤的机制。我们假设Mlh1， Pms2和Pms1缺陷小鼠的不同肿瘤易感性是由于自发或化学诱导突变中的基因特异性作用。为了验证这一假设，我们提出通过同源重组构建携带Mlh1、Pms2和Pms1突变的等基因细胞系（目的1）。这些细胞系将与先前生成的Msh2-/-细胞系一起用于许多试验，这些试验将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，定义位点上的突变谱，简单重复序列的不稳定性以及分歧DNA元件之间的遗传重组（目的2）。此外，我们将确定错配修复基因是否会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（目的3）。拟开展的研究项目将在细胞水平上明确不同错配修复基因在诱变过程中几个方面的作用，并将研究结果应用于DNA错配修复缺陷小鼠机体水平上肿瘤形成的研究。阐明DNA错配修复基因的新和特定作用将为携带错配修复基因突变的家庭提供新的癌症预防策略，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.