Figure 1.

Effects of 8Br-cAMP on the activities of MAPK-1,2 and DNA synthesis in MCF-7 cells. (A) Immunoblot of phospho-MAPK-1,2 in extracts from cells treated with or without (C) EGF (10 ng/ml) after incubation with 8Br-cAMP (10 mM). (B) EGF stimulated DNA synthesis in cells incubated with and without 8Br-cAMP. Serum-starved cells were incubated with or without 8Br-cAMP (10 mM) for 12 h and then with 1 mCi [³H]-thymidine, without or with EGF (10 ng/ml), for another 16 h. Cells then were washed and extracted with trichloroacetic acid, and the trichloroacetic acid precipitate was counted (9). Values are mean ± SD of triplicate determinations. (C) Effect of 8Br-cAMP on the basal MAPK activity of serum-starved MCF-7 cells. Cell extracts were resolved on Mono-Q columns by using fast protein liquid chromatography. Aliquots of column fractions were assayed. Total counts under the peak are plotted as a bar graph. For controls, extracts that had been heat inactivated were resolved on Mono-Q columns by fast protein liquid chromatography and assayed. Control activity was 50–75 per column aliquot. (D, left) Immunoblot of total ERK1 and ERK2 from serum-starved cells treated without any additives (C) or with EGF (10 ng/ml) or 8Br-cAMP (10 mM). (D, right) Immunoblot of phospho-MAPK-1,2 in extracts from serum-starved cells treated without any additives (C) or with EGF (10 ng/ml) or 8Br-cAMP (10 mM). Equal aliquots were used in both assays.