FIG. 3.

Induced expression of ODC after Raf activation. (A) Induction of ODC mRNA by Raf. NIH 3T3 cells expressing ΔRaf-1:ER were cultured in DMEM containing 0.5% serum for 16 h, and RNA samples were prepared at different times after the addition of 1 μM ICI. Expression of the mRNAs encoding ODC (upper panel), heparin-binding epidermal growth factor (HB-EGF, middle panel), and GAPDH (lower panel) was quantitated by using RNase protection assays. (B) ODC promoter activation. NIH 3T3 cells expressing ΔB-Raf:ER* were transiently transfected with reporter constructs consisting of the promoter region of human ODC linked to luciferase (ODCΔLuc) or a form of this promoter containing a point mutation in each of the two E-boxes located in the first intron of the gene (ODCΔLucS-5A). pSRαβ-Gal was cotransfected with the ODC reporter plasmids as a control for transfection efficiency. Transfected cells were treated with either ethanol (solvent control, open bars) or 500 nM 4-HT (shaded bars) for 40 h, at which time the luciferase and β-galactosidase activities were measured. Results are presented as the ratios of the luciferase to the β-galactosidase activities.