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Dihydrofolate reductase (DHFR) protein levels increase rapidly in human cells upon exposure to methotrexate (MTX), a potent inhibitor of this enzyme. A model to explain this increase proposes that DHFR protein inhibits its own translation by binding to its cognate mRNA and that methotrexate disrupts the DHFR protein-mRNA complex, allowing its translation to resume. Previously we identified three loci, E30, L22 and S118 that appear to be involved in the upregulation of DHFR protein levels by MTX and other DHFR inhibitors. Cells transfected with E30A, L22R and S118A mutants that did not respond to MTX up-regulation had also higher basal levels of DHFR. To confirm that the higher basal levels of DHFR protein were not due to increased mRNA expression of these mutants, the DNA and RNA levels were measured using quantitative RT-PCR. While the E30A mutant had same mRNA level compared to wt, the S118A and the L22R mutants had elevated levels of DHFR. To sort out whether the increased basal protein levels were be due to both higher levels of mRNA and increased translation, equal concentrations of wt and each of the mutants mRNA were translated in vitro using a rabbit reticulocyte translation assay. Indeed, all three mutants yielded more DHFR protein compared to wt DHFR, demonstrating the lack of feedback regulation of these mutant enzymes. We also kinetically characterized the mutants by preparing GST fusion protein and purifying them using Glutathione Sepharose 4B. Although the S118A mutant enzyme had catalytic activity and sensitivity to MTX similar to that of wt DHFR, cells containing these high basal levels of the S118A mutant DHFR had the same sensitivity to the cytotoxicity of MTX, as did the cells with lower basal levels of wt DHFR. This provides the first evidence that the adaptive upregulation of DHFR by MTX contributes to the decreased sensitivity to this drug.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]