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 reductase were used as a model system to study the effect of exposure to an alkylating agent, ethylmethanesulfonate, on rates and types of mutations at the dihydrofolate reductase locus leading to antifolate resistance. After overnight exposure to 400 micrograms/ml ethylmethanesulfonate, cells were allowed to recover for 3 days, and resistant colonies were selected in 8 x 10(-8) M trimetrexate. Trimetrexate, rather than methotrexate, was used as the selecting agent to increase the probability of obtaining mutations in dihydrofolate reductase, rather than in the reduced folate transport carrier protein. Seven of several hundred surviving 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 lines 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. Cell line 1 was found to have an increase in dihydrofolate reductase activity, a corresponding increase in mRNA for dihydrofolate reductase, and amplification of this gene. Cell lines 2 and 6 had a mutated dihydrofolate reductase with altered trimetrexate- and methotrexate-binding properties. Cell line 3 had a 3-fold increase in dihydrofolate reductase activity. In cell lines 4, 5, and 7 the mechanisms of resistance to trimetrexate remain unknown.
Fanin
R
R
Program of Molecular Pharmacology and Therapeutics, Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Banerjee
D
D
Volkenandt
M
M
Waltham
M
M
Li
W W
WW
Dicker
A P
AP
Schweitzer
B I
BI
Bertino
J R
JR
D000477:Alkylating Agents / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D016466:CHO Cells; D006224:Cricetinae; D004347:Drug Interactions; D004351:Drug Resistance / Q000235:genetics; D005020:Ethyl Methanesulfonate / Q000494:pharmacology*; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D008727:Methotrexate / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D016597:Trimetrexate / Q000494:pharmacology
D000477:Alkylating Agents; D005493:Folic Acid Antagonists; D005020:Ethyl Methanesulfonate; D013762:Tetrahydrofolate Dehydrogenase; D016597:Trimetrexate; D008727:Methotrexate