) My goal is to establish a research program as an independent investigator. My long-term objective is to study basic and clinical aspects of Genetic Cancer Susceptibility, with a focus on mammalian genetics and genomics. The purpose of the proposed study is to test the hypothesis that targeted mutation of a novel mammalian DNA mismatch repair gene cloned in our laboratory, MLH3, causes genetic susceptibility to cancer, with particular reference to gastrointestinal and lymphoid cancer. Six mammalian homologues of yeast DNA mismatch repair genes associated with microsatellite instability have previously been described: MLH1, MSH2, MSH3, MSH6, PMS1, and PMS2. Each one of these genes is associated with genetic susceptibility to cancer in humans (specifically, Hereditary Non-Polyposis Colon Cancer) and/or mice. As yeast mlh3 is similarly associated with MSI, this proposal examines the role of MLH3 in mammalian DNA mismatch repair and tests whether Mlh3 knockout mice are genetically susceptible to cancer.
Specific aims are: (1) To generate antibodies against Mlh3 for immunohistochemical analyses. (2) To generate mice that are deficient in Mlh3 and mice that are compound deficient in Mlh3 and the Pms2 DNA mismatch repair genes. Experiments in yeast suggest that mlh3 and pms2 are both involved in the repair of insertion/deletion loops of mismatched genomic DNA. Mlh3/Pms2 compound mutant mice would therefore be predicted to have a more severe phenotype than mice mutant for either protein separately. These mouse lines will be analyzed for susceptibility to cancer, with special emphasis on gastrointestinal and lymphoid systems. (3) To test the hypothesis that mutations in mammalian Mlh3 cause defects in DNA mismatch repair. We will utilize both Mlh3 and Mlh3/Pms2 null mice as a source of colonic epithelial cells to analyze microsatellite instability. These data will help to prioritize families with a hereditary predisposition to colon cancer for future MLH3 candidate gene mutation analyses. (4) To examine the effect of Mlh3 deficiency on the Apc tumor suppressor gene. Colon cancer cell lines with mutations in the MLH1 gene accumulate mutations in the tumor suppressor gene APC. We will generate mouse lines that are mutant for mlh3 and heterozygous for a mutant Apc A1638N allele that predisposes to spontaneous colorectal cancers. The analysis of the tumor spectrum and the onset and progression of tumor formation will indicate the role of Mlh3 in gastrointestinal cancer susceptibility and provide a detailed analysis of the spectrum of Apc mutations resulting from mlh3 deficiency.
