MUTATIONS LEADING TO ANTIFOLATE RESISTANCE IN CHINESE-HAMSTER OVARY CELLS AFTER EXPOSURE TO THE ALKYLATING AGENT ETHYLMETHANESULFONATE

Chinese hamster ovary cells with a single allele for dihydrofolate red uctase were used as a model system to study the effect of exposure to an alkylating agent, ethylmethanesulfonate, on rates and types of muta tions at the dihydrofolate reductase locus leading to antifolate resis tance. After overnight exposure to 400 mug/ml ethylmethanesulfonate, c ells were allowed to recover for 3 days, and resistant colonies were s elected in 8 x 10(-8) M trimetrexate. Trimetrexate, rather than methot rexate, was used as the selecting agent to increase the probability of obtaining mutations in dihydrofolate reductase, rather than in the re duced folate transport carrier protein. Seven of several hundred survi ving colonies were selected at random, and cell lines were established . Cell lines 1-3 were maintained in culture in the presence of 8 x 10( -8) M trimetrexate and were 66-170-fold resistant to the drug. Cell li nes 4-7 were initially expanded in 8 x 10(-8) M trimetrexate but were then maintained in the absence of the drug. These cell lines were 4.4- 26-fold resistant to the drug, compared with the parental cell line. C ell line 1 was found to have an increase in dihydrofolate reductase ac tivity, a corresponding increase in mRNA for dihydrofolate reductase, and amplification of this gene. Cell lines 2 and 6 had a mutated dihyd rofolate reductase with altered trimetrexate- and methotrexate-binding properties. Cell line 3 had a 3-fold increase in dihydrofolate reduct ase activity. In cell lines 4, 5, and 7 the mechanisms of resistance t o trimetrexate remain unknown.