Abstract
Previous studies in this laboratory have identified an altered dihydrofolate reductase (DHFR) enzyme in the methotrexate (MTX) resistant Chinese hamster ovary (CHO) cell line Pro-3MtxRIII [1,2]. The alteration involved the replacement of the active site residue Leu (L, position 22) by Phe as the result of a base transition at nucleotide 67 in the DHFR gene [3]. Enzyme kinetic studies of the altered enzyme revealed that the Km for both substrates (H2folate & NADPH) were similar when compared with wild-type vertebrate enzyme, but the Ki for MTX was increased some 100-fold. Interestingly, previous studies by other researchers have also identified a mutation at residue 22 in MTX-resistant cell lines: a Leu to Arg mutation in the mouse line 3T6-R400 [4], and a Leu to Phe mutation in yet another CHO cell line [5]. Combined, these results suggest that codon 22 may be a “hot spot” which readily mutates to impart a MTX-resistant phenotype to its host cell. While definitive proof still awaits, it seems plausible that these or similar mutations in DHFR may be responsible for some instances of resistance in tumors of patients who receive antifolates therapeutically.
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