Hypoxia-induced enrichment and mutagenesis of cells that have lost DNA mismatch repair

Loss of DNA mismatch repair (MMR) increases the risk of spontaneous mutatio ns. We sought to determine whether there was an interaction between hypoxia and MMR deficiency that might contribute to the phenomenon of tumor progre ssion. Human colon carcinoma HCT116+ch2 (MMR-deficient) and HCT116+ch3 (MMR -proficient) sublines were exposed for varying periods of time to an enviro nment of <0.1% O-2 and pH as low as 6.1. When a population containing 5% MM R-deficient cells and 95% MMR-proficient cells was subjected to hypoxia for 72 h, the MMR-deficient cells were enriched by a factor of 2-fold in the s urviving population, whereas no enrichment was detected in cells maintained under aerobic conditions. The potential of hypoxia to destabilize the geno me was determined by measuring the frequency of clones in the surviving pop ulation resistant to very high concentrations of 6-thioguanine or cisplatin . A 72-h exposure to hypoxia did not increase the frequency of resistant cl ones in the MMR-proficient cells but produced a 7.8-fold increase in 6-thio guanine-resistant clones and a 2.5-fold increase in cisplatin-resistant clo nes in the MMR-deficient cells. Loss of MMR increased the frequency of muta tions in a reporter vector sensitive to frameshift mutations in a microsate llite sequence. Exposure to hypoxia for a time period as short as 48 h furt her increased the number of mutations in both cell types, but the absolute number of mutants was higher in the MMR-deficient cells. These results indi cate that hypoxia and its accompanying low pH enrich for MMR-deficient cell s and that loss of MMR renders human colon carcinoma cells hypersensitive t o the ability of hypoxia to induce microsatellite instability and generate highly drug-resistant clones in the surviving population.