Identification of Amino Acids Required for the Functional Up-regulation of Human Dihydrofolate Reductase Protein in Response to Antifolate Treatment

Nancy Skacel,Lata G Menon,Prasunkumar J Mishra,Rowayda Peters,Debabrata Banerjee,Joseph R Bertino,Emine Ercikan Abali

doi:10.1074/jbc.m500277200

Abstract

Human dihydrofolate reductase (DHFR) protein levels rapidly increase upon exposure to methotrexate, a potent inhibitor of this enzyme. A model to explain this increase proposes that DHFR inhibits its own translation by binding to its cognate mRNA and that methotrexate disrupts the DHFR protein-mRNA complex allowing its translation to resume. In the present study, Chinese hamster ovary cells lacking DHFR were transfected with wild type and mutants of human DHFR to identify amino acids that are essential for increases in DHFR in response to methotrexate. Glu-30, Leu-22, and Ser-118 were involved in the up-regulation of DHFR protein levels by methotrexate and certain other antifolates. Cells transfected with E30A, L22R, and S118A mutants that did not respond to methotrexate up-regulation had higher basal levels of DHFR, consistent with the model, i.e. lack of feedback regulation of these enzymes. Although cells containing the S118A mutant enzyme had higher levels of DHFR and had catalytic activity similar to that of wild type DHFR, they had the same sensitivity to the cytotoxicity of methotrexate, as were cells with wild type DHFR. This finding provides evidence that the adaptive up-regulation of DHFR by methotrexate contributes to the decreased sensitivity to this drug. Based on these observations, a new model is proposed whereby DHFR exists in two conformations, one bound to DHFR mRNA and the other bound to NADPH. The mutants that are not up-regulated by methotrexate are unable to bind their cognate mRNA.

Highlights

NADPH as a cofactor, catalyzes the reduction of dihydrofolate to tetrahydrofolate
Establishment of a Mammalian Cell Culture System to Study Changes in dihydrofolate reductase (DHFR) Protein Levels upon MTX Exposure—Previous studies have shown that, when DG44 cells are transfected with the coding sequence of wild type human DHFR alone, significant increases in DHFR protein levels are detected upon exposure to 1 ␮M MTX for 24 h and 48 h [22]
The levels of DHFR-enhanced green fluorescent protein (EGFP) fusion protein increased in response to MTX comparable with cells transfected with wt human DHFR alone (Fig. 1D)

Summary

EXPERIMENTAL PROCEDURES

Materials—CMRL 1066 medium, fetal bovine serum (FBS), dialyzed FBS (dFBS), G418 sulfate (Geneticin), penicillin, streptomycin, and trypsin were purchased from Invitrogen. When the cells reached 10 –15% confluency, they were transfected with 10 ␮g of plasmid DNA plus 70 ␮l of DOTAP reagent according to the manufacturer’s protocol. When probing with the rabbit anti-human DHFR polyclonal antibody, both primary and secondary phosphate-buffered saline-T contained 5% FBS, 0.1% Tween 20, and 5% nonfat dry milk powder. The product was gel-isolated, digested with HindIII and BamHI, and ligated into the pEGPN3 mammalian expression vector (Clontech, Palo Alto, CA) at the HindIII and BamHI sites using a Rapid DNA ligation kit (Roche Applied Science) according to the manufacturer’s instructions. C, wt DHFR-EGFP transfectants were exposed to 1 ␮M MTX or medium without MTX for 24 h, and cell fluorescence was viewed with fluorescent microscopy using identical camera settings. The cell-free translation products were run on a 12% SDSpolyacrylamide gel and autoradiographed for visualization

RESULTS

DISCUSSION

Steady-state kinetic properties of wt and mutant variants of DHFR wt DHFR