Resistance of resting 3T6 mouse fibroblasts to methotrexate cytotoxicity

We have compared the relative susceptibility of resting (GO) versus exponentially growing mouse fibroblasts (line 3T6) to methotrexate (MTX) cytotoxicity. Cultures of cells were exposed to different levels of MTX for various intervals. Cloning efficiencies of the cells were then determined in medium containing hypoxanthine and thymidine. We found that growing 3T6 cells exhibit time- as well as dose-dependent cytotoxicity. Greater than 95% of the cells were killed by a 24-hr exposure to 10(-6) M MTX. In contrast resting cells showed no loss of cloning efficiency even at drug concentrations of 10(-3) M and exposure times of 7 days. The resistance of resting cells is not the result of a MTX transport deficiency since extracellular MTX is taken up by both resting and growing cells. Furthermore, intracellular dihydrofolate reductase activity is abolished in both resting and growing cells at similar extracellular MTX dosages.