FIG. 7.
Cdc25A activation in vivo is inhibited by p27Kip1 and by dominant-negative Ras. (A, top panel) Effects of transduction with Ad.p16, Ad.p27, and Ad.RasN17 on generation of Cdc25A activity in MCF-7 cells. MCF-7 cells were infected with Ad.Con, Ad.p16, Ad.RasN17, or Ad.p27 as indicated, and Cdc25A activity was assayed following growth arrest and a 20-h estrogen treatment. Results are presented in graph form with input cyclin B1-Cdc2 activity taken as a value of 1. (A, lower panel) Western blotting (WB) analysis of Cdc25A expression in MCF-7 cells. Cdc25A was immunoprecipitated from 600 μg of the same lysates assayed above. Relative Cdk2 activities assayed in the same lysates are given below the panel based on densitometric analysis. (B) Expression of p27Kip1 was assayed by Western blotting of lysates from MCF-7 cells infected with Ad.Con and Ad.rasN17 vectors following growth arrest and 20-h treatment with estrogen as indicated. (C) Pim-1-associated histone kinase activity (upper panel) was determined as given in Materials and Methods with lysates of control and Ad.p16-infected MCF-7 cells 20 h after E2 treatment. Western blot analysis of Pim-1 protein expression in the same lysates is given in the lower panel.