Drosophila dihydrofolate reductase mutations confer antifolate resistance to mammalian cells

Antifolates, such as methotrexate, are used to inhibit dihydrofolate reductase (DHFR), an enzyme essential for the biosynthesis of thymidylate, purines, and several amino acids. DHFR sequences corresponding to mutations found in a methotrexate resistant Drosophila S3 cell line (L30Q), a methotrexate resistant fly population (K31P, Q134K), as well as predicted in silico (L22R) were expressed in Chinese Hamster Ovary (CHO) cells. The L30Q and L22R DHFRs both conferred resistance to methotrexate. L22R DHFR provided approximately 200-fold resistance to methotrexate when compared to wild-type Drosophila DHFR allowing CHO(L22R) cells to divide in 10 microM methotrexate, a level of resistance not previously observed in any mammalian system. Constructs using this substitution in combination with other Drosophila DHFR specific residues would make excellent candidates for gene therapy and genetic markers in the treatment of certain human disorders.