MICROSATELLITE MUTATION-RATES IN CANCER CELL-LINES DEFICIENT OR PROFICIENT IN MISMATCH REPAIR

A selectable system has been used to determine mutation rates within a microsatellite sequence in human cancer cell lines with or without de fects in mismatch repair. A sequence consisting of 17 repeats of poly (dC-dA).poly(dT-dG) [abbreviated as (Ca)(17)] was inserted near the 5' end of the bacterial neomycin-resistance gene in a plasmid vector, su ch that the reading frame of the neo gene is disrupted. This plasmid w as introduced into cancer cell lines, where it became integrated into the cellular genome. Clones with insertions or deletions of CA-repeats that restored the normal reading frame of the neo gene were selected in G418, and mutation rates were determined by fluctuation analysis. T he rates of reversion in LoVo cells, which are deficient for hMSH2, we re about one in a thousand per generation, which is approximately two orders of magnitude higher than in the repair-proficient HT-1080 human fibrosarcoma cell line. The mutation rates in H6 cells, which are der ived from the hMLH1-deficient HCT116 line, were more heterogeneous tha n in LoVo, but all were considerably higher than in the repair-profici ent line. Nearly all of the revertants of the repair-deficient lines h ad deletions of a single CA-repeat from the microsatellite sequence, w hereas repair-proficient cells had a broader spectrum of mutations.