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Manganese superoxide dismutase (MnSOD) is a primary antioxidant enzyme necessary for the survival of aerobic life. Previously, we demonstrated that the specificity protein 1 (Sp1) is essential for the basal transcription of the MnSOD gene. We also identified Nucleophosmin (NPM), an RNA binding protein as an important co-activator of NF-κB in the induction of MnSOD by cytokine and tumor promoter. Here, we demonstrate Sp1 and NPM interact in vivo to enhance NF-κB-mediated MnSOD induction using Chromatin Immuno-Precipitation (ChIP) analysis. Interaction between NPM and Sp1 or NF-κB at the promoter and enhancer of the MnSOD gene in vivo, were verified by the presence of the PCR products from the promoter and enhancer elements in the ChIP assay. Unexpectedly, we also found p53 in the transcription complex detected by ChIP assay. The presence of p53 in this transcription complex was verified by immunoprecipitation of p53 proteins with antibody to Sp1 in nuclear extracts. Using a vector expressing full-length p53 cDNA, we demonstrate that p53 overexpression suppresses MnSOD mRNA and protein levels. Consistent with the negative role of p53 in the expression of MnSOD gene, expression of small interfering RNA for p53 leads to an increase of MnSOD mRNA levels. Using ChIP assays and immunoprecipitation we further demonstrate that p53 interact with Sp1 and NPM but not NF-κB upon treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Inhibition of MnSOD gene by p53 was abolished when Sp1 sites on the MnSOD promoter were mutated or the Sp1 protein was reduced by siRNA approaches. Overexpression of wild type p53 suppresses Sp1 mediated MnSOD gene transcription. These results suggest that p53-mediated MnSOD gene suppression is Sp1 dependent. Since the expression of MnSOD is essential for cell survival, our findings that p53, an apoptotic inducing protein suppresses MnSOD expression reveals a previously unrecognized mechanism of p53-mediated apoptosis and demonstrates an intricate relationship between the prosurvival and proapoptotic mechanism.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]