Figure 2 | Oncogene

Figure 2

From: Transcriptional activation of cyclooxygenase-2 by tumor suppressor p53 requires nuclear factor-kappaB

Figure 2

Requirement of (NF-kappaB) for transactivation of COX-2 by p53. (a) Effect of NF-kappaB protein components on COX-2 promoter activation by p53. Cell lines HCT116 (upper panel) and TE-13 (lower panel) were transfected with COX-2-LUC reporter and increasing amounts of p53, p65 and p50 expression vectors alone or in combination, and RSV-βGAL as transfection standard. LUC activity was expressed as fold induction relative to the activity observed with reporter alone, normalized to βGAL. (b) A dominant-negative NF-kappaB inhibitor reduces COX-2 promoter activation by p53. HCT116 and HCT116 MT9 (expressing a dominant-negative IkappaBalpha mutant) were transfected with COX-2 LUC reporter, increasing amounts of p53 expression vector (pc53-SN3) and RSV-βGAL as transfection standard. (c) Association of p53 and NF-kappaB with the COX-2 promoter. HCT116 cells were transfected with pC53-SN3 (0.1, 0.3, 1 μg). Left, cross-linked chromatin was immunoprecipitated with anti-p53, anti-NF-kappaB antibodies or control antibody as negative control, and analysed by polymerase chain reaction (PCR) with primers specific for COX-2 promoter. MW: molecular weight markers. Input: non-precipitated cross-linked chromatin. Right, Western blot analysis showing protein levels of Cox-2, p53 and p21 in nontransfected and transfected HCT116, using Ku80 as loading control. (d) Inhibition of NF-kappaB p65 by siRNA prevents p53-dependent induction of COX-2 expression. Left panel: HCT116-p53ind cells were transfected with control or with p65 siRNA, followed by tetracycline treatment. WAF-1 and COX-2 mRNA expression was determined by real-time reverse transcription-PCR (RT–PCR) as in Figure 1c. Right panel: Western blot analysis of p65 expression in HCT116 cells transfected with control, p53 and p65 siRNA, or not transfected (none). Ku80: loading control. (e) The NF-kappaB inhibitor BAY117082 decreases p53-dependent COX-2 expression. Upper panel: TE-1 cells cultured at 37°C were switched to 32°C, treated with BAY117082 (10 μ M) for 12 h, and COX-2 mRNA expression was determined by real-time RT–PCR as in Figure 1c. Lower panel: cross-linked chromatin was immunoprecipitated with anti-p53, anti-NF-kappaB antibodies or control antibody as negative control, and analysed by PCR as in (c).