Comparison of antifolate induced upregulation of proteins as fusion partners of dihydrofolate reductase (DHFR) or expressed from bicistronic constructs separated by an internal ribosome entry site (IRES) element

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We have shown previously that treatment of cells with antifolates such as methotrexate (MTX) results in “induction” of DHFR. This induction is insensitive to actinomycin D treatment but is inhibited by cycloheximide suggesting that it occurs at the translational/post translational level. Mechanistic studies have revealed that DHFR binds its cognate RNA and regulates its own translation. Addition of MTX relieves this “translational control” and allows resumption of DHFR synthesis. We have also shown that fusion proteins of DHFR are similarly regulated and can be induced upon antifolate treatment of cells transfected to express DHFR-fusion proteins. Concerns regarding antigenicity of fusion proteins of DHFR, particularly as applied to gene transfer studies in vivo, prompted us to examine whether proteins separated from DHFR by an IRES element could also be regulated in a similar manner as the fusion proteins. We hypothesized that DHFR, by binding the bicistronic message can regulate translation of both itself and the partner protein. Antifolate treatment of cells, expressing these two proteins from a bicistronic construct, would thus lead to induction of both DHFR and the partner protein. In this report we demonstrate induction of a second protein, enhanced green fluorescence protein (EGFP), both as a fusion partner and separated from DHFR by an IRES element upon MTX treatment of cells transfected to express either the fusion protein or the two cDNAs contained in a bicistronic vector. Using Western blotting as well as fluorescence imaging, in vitro and in vivo, we demonstrate antifolate mediated induction of EGFP. While the fusion protein is induced within 24h of antifolate treatment, induction appears to be delayed for EGFP when present as part of the bicistronic construct DHFR-IRES-EGFP but is sustained over a period of 72h. This suggests that levels of desired proteins can be modulated by antifolate treatment even when the protein is expressed from a bicistronic construct containing DHFR rather than a fusion protein of DHFR. This is particularly important for gene transfer strategies utilizing DHFR and a second protein for purposes of myeloprotection or suicide gene therapy.