FIG. 1.

Cytoskeletal integrity regulates cyclin A gene transcription. (A) Diagram of the cyclin A promoter-luciferase constructs used in this study. (B and C) NIH 3T3 cells were transiently transfected with the cyclin A promoter-luciferase constructs and pRL-SV40 Renilla luciferase. Transfected cells were G₀ synchronized and stimulated with mitogens (see Materials and Methods) in the absence or presence of cytochalasin D (CCD) for 18 h (B) or the various times indicated (C). Luciferase activity was normalized to a constant activity of Renilla luciferase. In panel B, normalized luciferase activity is plotted relative to the normalized activity obtained with the 1.3-kb cyclin A promoter from cells cultured in the absence of cytochalasin D (defined as 100%). In panel C, luciferase activity is plotted as the fold induction relative to G₀. In panel D, NIH 3T3 cells were G₀ synchronized by serum starvation and then stimulated with mitogens in the absence or presence of cytochalasin D for the times shown. Lysates were prepared and analyzed by Western blotting for the phosphorylation of pRb and p107 and the expression of cyclin A. The upper and lower arrows, respectively, in panel D show the positions of phosphorylated and unphosphorylated pRb and p107.