Forskolin and rolipram stimulation leads to sequential induction of p53 and p21 proteins.

CEM cells were stimulated with 20 μM forskolin and 20 μM rolipram, and cell extracts obtained at various time points were analyzed by parallel immunoblots using anti-p53, anti-p21, and antiactin antibodies. (A) An early time course shows that induction of p53 protein is detected by 15 minutes of treatment with forskolin and rolipram, with p21 protein subsequently detected by 1 hour. The actin control demonstrates equivalent loading in all lanes. (B) Prolonged induction of p21 protein is seen during a longer time course in cells treated with forskolin and rolipram (FR), with a peak at 12 hours, but sustained elevation is still seen at 72 hours. Extracts collected from negative control cells at parallel time points (Neg) show only very low level increases in p21 levels compared to the robust induction in treated cells. The magnitude of p21 protein induction is comparable to that seen in positive control extracts from NIH 3T3 fibroblasts treated with etoposide for 8 hours (first lane, +).