Upregulation of cyclooxygenase-2 (Cox-2) expression by p53. (a) p53-dependent induction of a COX-2 reporter construct. Cells were co-transfected with COX-2-LUC reporter, increasing amounts of an expression vector for p53 protein (pC53-SN3), and RSV-βGAL reporter as internal transfection standard. LUC activity was normalized to βGAL and expressed as fold induction relative to the activity observed with COX-2-LUC reporter alone. (b) Increased Cox-2 expression after transfection of p53. Left, HCT116 cells were transfected either with empty vector (pcDNA3, CTRL) or with p53 expression vector (pC53-SN3, p53). Right, a tetracyclin-inducible p53 HCT116 cell line (tet-on) was used (p53-ind). Cells were treated with tetracyclin (1 mg/ml) for 8, 16 and 24 h and levels of p53, Cox-2 and p21waf−1 were detected by Western blot using β-actin as a loading control. (c) Enhanced COX-2 expression and enzymatic activity in cells expressing a temperature-sensitive p53 mutant. TE-1, a cell line expressing the temperature-sensitive p53 mutant V272M, was used. Left, reverse transcription reverse transcripition–polymerase chain reaction (RT–PCR) detection of COX-2 mRNA in cells cultured at 37°C (p53 mostly in ‘mutant’, inactive form) and 32°C (p53 mostly in ‘wild-type’, active form). Middle, real-time (TaqMan) RT–PCR analysis of COX-2 expression levels. Right, PGE2 production at both temperatures.