Figure 6.

PGE₂-promoted rise in p53 mRNA is not explained by PGE₂-induced mRNA stabilization. In order to assess whether p53 mRNA stability was affected by a pulse of exogenous PGE₂, sets of replicate cultures were pulsed with PGE₂ (50 nM) or vehicle alone. After 8 h of additional culture (for peak PGE₂-induced elevation in p53 mRNA; Fig. 3), a series of replicates within each of the above subsets received actinomycin C (5 μM) or DMSO vehicle. RNA was isolated at varying time points after the actinomycin D or DMSO (0.5, 1, 3, 8, and 20 h). q-PCR of cDNA measured p53 transcript levels in each culture, expressed as a fraction of that present in the respective PGE₂-untreated or PGE₂-treated cultures with DMSO vehicle (means±se of 4 experiments). Dotted line represents the normalized level of transcript present in cultures with or without PGE₂ but without actinomycin D. Data were statistically analyzed (see Materials and Methods) to compare mean fold change of p53 mRNA between PGE₂-pulsed and nonpulsed cultures to their respective controls across time after actinomycin D. There was no significant interaction between time and presence of PGE₂ (P<0.72), indicating that the slope of p53 mRNA expression after actinomycin D (mRNA decay) was not significantly different with or without PGE₂. Nevertheless, when the interaction term was removed from the model, the main effects of cell type and time were significant. Cells without PGE₂ supplement had a significantly greater p53 mRNA, relative to their controls, as compared to those pulsed with PGE₂ (P<0.0001). Overall, p53 mRNA expression decreased over time for all cells relative to controls (P<0.0002). Taken together, these experiments show that the elevated p53 mRNA levels in PGE₂-prepulsed cultures do not reflect diminished degradation of the p53 transcript.