FIG. 5.

p53’s role in the G₂ checkpoint appears to be mediated by the transcriptional down regulation of cyclin B and CDC2. (A) E6-HFFs and LXSN-HFFs were synchronized and pulsed with 2 μM ADR for 1 h at 5 to 6 h after aphidicolin release. Cells were harvested for RNA at the indicated hour. Northern blotting was performed, and blots were probed with radiolabeled cyclin B, CDC2, and the loading control, 36B4. Lanes 1 to 8 indicate the hour of release from synchronization into complete medium; lanes 12 to 14 indicate the hours after aphidicolin release in the cells pulsed with ADR. (B) Cotransfection experiments show p53 to be a transcriptional repressor of cyclin B and CDC2 promoters. p53^−/− MEFs were cotransfected with a CMV-driven expression vector (p53) or without p53 (−) and the various promoter-reporter constructs shown and described in Materials and Methods. Luciferase assays were performed on the protein lysates, and RLU values are normalized for transfection efficiency represented per microgram of protein. Error bars represent standard errors of the means of triplicate experiments. The untransfected control represents p53^−/− MEFs that have been mock transfected. (C) Identical cotransfections were performed in p21^−/− MEFs.