FIG. 5.

Characterization of p53 in E6 mutant-immortalized MECs. (A) G₁ arrest in response to DNA damage. Exponentially growing cells (16E6, F2V II, and Y54H IV) were treated with 0.5 nM actinomycin D (Act.D) for 24 h or left untreated. DNA content was analyzed by propidium iodide staining followed by flow cytometric analysis. The G₁/S ratios determined with ModFit software (Becton Dickinson) from a representative experiment are shown. (B) Activation of a p53-responsive reporter in response to DNA damage. Immortal MECs (16E6, F2V II, and Y54H IV), normal 76N cells, and p53-null 76R-30 cells were transfected with a p53-responsive luciferase reporter construct, pPG, along with β-galactosidase and green fluorescence protein reporter constructs. At 40 h posttransfection, the cells were changed to medium containing or lacking 0.5 nM actinomycin D. Twenty-four hours later, cell extracts were made and luciferase and β-galactosidase activities were measured. Luciferase activities were corrected by normalizing β-galactosidase activities. Values shown are the means and standard deviations from three independent experiments, each performed in duplicate.