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Mutator Phenotype in Msh2-deficient Murine Embryonic Fibroblasts¹

Ontario Cancer Institute/Amgen Institute, Departments of Medical Biophysics and Immunology, University of Toronto, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada [A. H. R., T. W. M.]; Departments of Oncological Sciences and Radiation Oncology, Eccles Institute of Human Genetics, University of Utah, Salt Lake City, Utah 84112 [R. R., A. G., M. M.]; Ontario Cancer Insitute, Departments of Medical Biophysics and Radiation Oncology, University of Toronto, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada [R. G. B., A. J., R. P. H.]; DNA Repair and Molecular Carcinogenesis Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [T. W., R. F.]; and Ontario Cancer Institute, Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada [J. P., S. B.]

Embryonic fibroblast cell lines were established from mice deficient, heterozygous, or proficient for Msh2, one of the three known DNA mismatch repair genes involved in hereditary nonpolyposis colon cancer (HNPCC). Cell lines were established by transfection of primary mouse embryo 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 similar to that found in colon cancer cells deficient in mismatch repair, we measured mutation rates, microsatellite instability, and sensitivities to a range of DNA-damaging agents. The mutator phenotype detected in the E7 and Ras or mutant p53-immortalized Msh2-/- mouse cells was similar to that found in human mismatch repair-deficient colorectal carcinoma cell lines. Mutation rates to ouabain resistance were increased 8–12-fold relative to lines from Msh2+/+ mice, and microsatellite instability was detectable in 12–18% of subclones derived from the Msh2-/- line but was undetectable in subclones developed from the Msh2+/+ line. Furthermore, E7 and Ras or spontaneously immortalized Msh2-/- cells were significantly more resistant to the cytotoxic effects of 6-thioguanine relative to Msh2+/+ cells. In contrast, these lines showed various responses to UV light and cis-platinum, suggesting that mismatch repair 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 would mimic the situation of an HNPCC carrier. However, our studies failed to reveal any properties of these cells that might provide a growth advantage or predispose them for the acquisition of further mutations. This observation is consistent with the model that inactivation of the wild-type Msh2 allele is a critical step for tumorigenesis in HNPCC patients.

¹ A. H. R. was supported by a stipend of the German Academic Exchange Service (DAAD-Doktorandenstipendium aus Mitteln des zweiten Hochschulsonderprogramms). This work was funded in part by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada (to R. B., S. B., R. P. H., and T. W. M.). National Cancer Institute Grants R01 CA62244 and R01 CA22188 supported the work of M. M.; Grants R01 CA56542 and R01 CA 67007 supported the work of R. F.

² These authors contributed equally to this work.

³ To whom requests for reprints should be addressed. Phone: (801) 585-5258; Fax: (801) 585-3501; E-mail: mark.meuth@genetics.utah.edu.

Received 12/18/96. Accepted 7/ 2/97.

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