MUTATOR PHENOTYPE IN MSH2-DEFICIENT MURINE EMBRYONIC FIBROBLASTS

Embryonic fibroblast cell lines were established from mice deficient, heterozygous, or proficient for Msh2, one of the three known DNA misma tch repair genes involved in hereditary nonpolyposis colon cancer (HNP CC). Cell lines were established by transfection of primary mouse embr yo fibroblasts with E7 and Ras oncogenes or mutant p53. Spontaneously immortalized cells derived from the primary cultures were also studied . To determine whether these cells developed a mutator phenotype simil ar to that found in colon cancer cells deficient in mismatch repair, m e measured mutation rates, microsatellite instability, and sensitiviti es to a range of DNA-damaging agents. The mutator phenotype detected i n the E7 and Rns or mutant p53-immortalized Msh2-/- mouse cells was si milar to that found in human mismatch repair-deficient colorectal carc inoma cell lines. Mutation rates to ouabain resistance were increased 8-12-fold relative to lines from Msh2+/+ mice, and microsatellite inst ability was detectable in 12-18% of subclones derived from the Msh2-/- line but was undetectable in subclones developed from the Msh2+/+ lin e. Furthermore, E7 and Ras or spontaneously immortalized Msh2-/- cells were significantly more resistant to the cytotoxic effects of 6-thiog uanine relative to Msh2+/+ cells. In contrast, these lines showed vari ous responses to UV light and cis-platinum, suggesting that mismatch r epair deficiency was not the sole determinant for sensitivity to these DNA-damaging agents. Particular attention was paid to the properties of cells heterozygous for the Msh2 mutant gene, which mould mimic the situation of an HNPCC carrier. However, our studies failed to reveal a ny properties of these cells that might provide a growth advantage or predispose them for the acquisition of further mutations. This observa tion is consistent with the model that inactivation of the wild-type M sh2 allele is a critical step for tumorigenesis in HNPCC patients.