The DLD-1 human colon cancer cell line displays an elevated spontaneous mutation rate. Since DLD-1 carries frameshift mutations in both alleles of the MSH6 gene and missense mutations in the POLD1 gene, either or both of these mutations were suggested to be involved in this mutator phenotype. Therefore, we examined the effect of exogenous wild-type MSH6 and POLD1 expression on the spontaneous mutation rate at the HPRT locus in DLD-1 cells. POLD1 genotypes were first determined, since four POLD1 missense mutations were previously reported in DLD-1 cells. Sequencing analyses on the genomic DNA and cDNA of the POLD1 gene revealed that DLD-1 cells are a mixture of two distinct sublines with regard to POLD1 genotypes. Moreover, the wild-type POLD1 allele was not present in either of the two DLD-1 sublines. We next established MSH6- and POLD1-transfected DLD-1 clones from both sublines, respectively. The two DLD-1 sublines exhibited HPRT mutation rates of 4.8x10−6 and 5.4x10−6 mutations/cell/generation. The mutation rates were more than 4-fold decreased in both of the MSH6-transfected DLD-1 clones examined, while they were not significantly decreased in three of four POLD1-transfected DLD-1 clones. Thus, it was indicated that mutations in the MSH6 gene, and not in the POLD1 gene, are primarily responsible for the elevated mutation rates in DLD-1 cells.

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