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A Phenotype Conferring Selective Resistance to Lipophilic Antifolates in Chinese Hamster Ovary Cells¹

Department of Biological Sciences, Stanford University, Stanford, California 94305

Trimetrexate, a lipid-soluble analogue of methotrexate, appears to enter mammalian cells by passive diffusion, thus circumventing the methotrexate transport system which is frequently a subject for alterations leading to methotrexate resistance. Using a single-step selection protocol with trimetrexate, we have isolated 45 clonal variants and found the majority of them to be selectively resistant to lipohilic antifolates while retaining their sensitivity to methotrexate and drugs involved in multidrug resistance. The majority of spontaneously induced trimetrexate-resistant clones showed a change in neither the mRNA levels of dihydrofolate reductase (24 of 30) and P-glycoprotein (26 of 30) nor their gene copy numbers, whereas a small fraction of clones (4 of 30) showed multidrug resistance gene amplification and P-glycoprotein mRNA overexpression. -Irradiation prior to selection markedly enhanced the frequency of trimetrexate resistance (100-fold after 1000 rads). None of the -ray-induced trimetrexate-resistant clones (0 of 15) had evidence of dihydrofolate reductase and multidrug resistance gene amplification and/or overexpression. Flow cytometry data on trimetrexate-resistant clones showed no defect in the transport of trimetrexate. Verapamil, a modulator of the multidrug resistance phenotype, had no cytotoxic effect on parental and trimetrexate-resistant clones. However, when present with trimetrexate, verapamil (0.3–0.6 µM) reversed the lipohilic antifolate-resistant phenotype in clones that had invariant levels of P-glycoprotein and dihydrofolate reductase. This selective resistance to lipid-soluble antifolates was initially unstable but became stable after continued drugselective growth. Two-dimensional gel electrophoresis showed some differences in protein(s) that may potentially be associated with this phenotype of selective resistance to lipophilic antifolates. We conclude that a -radiation-enhanceable, verapamil-reversible, stable phenotype of selective resistance to lipid-soluble antifolates frequently emerges which requires neither the amplification nor the overexpression of dihydrofolate reductase or multidrug resistance genes.

¹ This work was supported by Research Grant CA 16318 from the National Cancer Institute.

² Y. G. A. is a Postdoctoral Fellow of the John D. and Catherine T. MacArthur Foundation.

Received 10/26/90. Accepted 3/22/91.

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