FIG. 1.
Induction of c-Myc by Raf in NIH 3T3 cells. (A) c-Myc mRNA regulation. NIH 3T3 cells expressing ΔB-Raf:ER* were cultured in 0.5% serum for 16 h, and RNA samples were prepared at different times after the addition of 1 μM ICI to activate ΔB-Raf:ER*. The expression of c-Myc (upper panel) and GAPDH (lower panel) mRNAs was detected by simultaneous RNase protection assay. The fold induction of c-Myc mRNA was quantitated by obtaining the ratio of c-Myc to GAPDH mRNAs by PhosphorImager analysis. (B) Comparison of c-Myc mRNA induction by serum versus ΔB-Raf:ER* activation. NIH 3T3 cells expressing ΔB-Raf:ER* were cultured in 0.5% serum for 16 h, and RNA samples were prepared at different times after stimulation with 10% FCS (closed diamonds) or the activation of ΔB-Raf:ER* with 1 μM ICI (open squares). c-Myc and GAPDH mRNAs were detected by RNase protection, and data were quantified by PhosphorImager analysis. Results are presented as the fold induction over the baseline level of expression. (C) Activation of the c-Myc promoter. NIH 3T3 cells expressing ΔB-Raf:ER* were transiently transfected with reporter constructs consisting of the promoter region of human c-Myc linked to luciferase (pGL2-myc) and pSRαβ-Gal as a control for transfection efficiency. Transfected cells were treated with either ethanol (solvent control, open bar) or 500 nM 4-HT (shaded bar) in 0.5% serum for 36 h, at which time the luciferase and β-galactosidase activities were measured. Results are presented as the ratios of the luciferase to β-galactosidase activities. (D) Induction of c-Myc protein expression. NIH 3T3 cells expressing ΔB-Raf:ER* were cultured in 0.5% FCS for 16 h, and cell extracts were prepared at different times after the addition of 500 nM 4-HT. The expression of p62c-Myc was detected by Western blotting with a specific antiserum (upper panel). The same Western blot was reprobed for the expression of p42 MAP kinase as a control for equal loading in each lane (lower panel). (E) Effect of increasing ΔB-Raf:ER* activity on c-Myc expression. NIH 3T3 cells expressing ΔB-Raf:ER* were cultured in modified DSFM for 24 h and stimulated with different concentrations of 4-HT, and cell extracts were prepared 4.5 h later. MAP kinase activity was measured by performing p42MAPK immune complex kinase assays with MBP as a substrate (upper panel). c-Myc expression was analyzed by Western blotting.