Drug-induced loss of unstably amplified genes

Methotrexate-resistant R500 cells slowly lose amplified dihydrofolate reductase (dhrf) genes with biphasic kinetics when grown in the absence of methotrexate. Both phases of gene loss were markedly accelerated by subcytotoxic drug treatments. R500 cells were passed in low concentrations of cytotoxic drugs (inhibitors of ribonucleotide reductase, type I and type II topoisomerases, and polyamine synthesis). At each passage, relative dhfr gene copy number was determined by slot blot analysis. All of these drugs were able to induce rapid loss of dhfr gene dosage in the R500 cell population. The ability of these treatments to cause the rapid emergence of a cell population with substantially reduced dhfr gene dosage indicates that either the amplified genes or those cells with the highest levels of gene amplification are selectively targeted by low-level cytotoxic stress. The complex kinetics of amplified gene loss are suggestive of differential targeting of resistant cell subpopulations.