Isolation of fluoropyrimidine-resistant murine leukemic cell lines by one-step mutation and selection

The effectiveness of the clinically useful fluoropyrimidines in the treatment of human cancer is often limited by the development of resistance to the drugs by the tumor. In order to systematically study the mechanisms of resistance to 5-fluorouracil and its nucleoside derivatives, several cell lines resistant to these drugs have been derived from murine leukemia cells by a one-step mutation and selection procedure. Logarithmically growing suspension cultures of L1210 and P388 cells were treated with ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, or ICR-191 at concentrations which result in 20 to 30% cell survival. After a 10-day expression time, mutagenized cells were plated into soft-agarose medium that contained 10(-5) M 5-fluorouracil, 10(-5) M 5-fluoro-2'-deoxyuridine, or 10(-6) M 5-fluorouridine. Twenty stable clones were isolated and found to be 5- to 28-fold resistant to growth inhibition by 5-fluorouracil, 4,000- to 25,000-fold resistant to 5-fluoro-2'-deoxyuridine, or 8- to 220-fold resistant to 5-fluorouridine. The clones retain their drug-resistant phenotype after repeated passaging in the absence of selection. Since the biochemical changes responsible for resistance to one drug can render the cells collaterally sensitive to other drugs, the growth-inhibitory effects of antimetabolites that inhibit other steps in pyrimidine metabolism were examined in the wild-type cells and in the fluoropyrimidine-resistant sublines. Although cross-resistance to 5-azacytidine was found in L1210 cells selected for resistance to 5-fluorouridine, none of the cell lines tested demonstrated collateral sensitivity to methotrexate, 1-beta-D-arabinofuranosylcytosine, 5-azacytidine, or N-(phosphonacetyl)-L-aspartate.