INDUCTION OF P53 DNA-BINDING ACTIVITY BY TUMOR-NECROSIS-FACTOR-ALPHA

Tumor necrosis factor-alpha (TNF-alpha) has been reported to activate the murine p53 promoter, which was mediated by activation and binding of the NF-kappa B transcription factor to the intact p53 consensus bin ding site. We show in this report that TNF-alpha at concentrations of 0.1 nM induced the DNA-binding activity of wild-type p53 fivefold in t he H460a human lung carcinoma cell line. The increases in p53 DNA-bind ing activity by TNF-alpha in H460a cells resulted from a post-translat ional activation mechanism, since levels of the p53 protein remained u nchanged in TNF-alpha-treated cells. Furthermore, Northern and nuclear run-off analyses ruled out an increase in transcriptional activity of the promoter of the human p53 gene in TNF-alpha-treated cells. These results are in contrast to those reported for the murine p53 gene, who se promoter is induced by TNF-alpha through activation of the NF-kappa B transcription factor. To determine the molecular basis for the lack of activation of the human p53 gene promoter by TNF-alpha, the bindin g of NF-kappa B to the kappa B-like sites in the murine and human p53 promoters was compared in nuclei of TNF-alpha-treated NSCLC cells by e lectrophoretic mobility shift assay. The human kappa B-like site diffe rs from the murine by the substitution of two base pairs. With the exc eption of the H460a cell line, all NSCLC cell lines treated with 0.1 n M concentrations of TNF-alpha exhibited increased NF-kappa B DNA-bindi ng activity. The kappa B-like site located in the murine p53 promoter bound strongly to NF-kappa B complexes in p53-negative H1299 cells. Ho wever, the kappa B-related site in the human p53 promoter showed a muc h lower affinity for NF-kappa B complexes. In addition, p50 homodimers specifically interacted with the human kappa B site, whereas p65/p50 heterodimers bound selectively to the mouse kappa B site. We conclude that TNF-alpha can activate human wild-type p53 in NSCLC cells by a po st-translational mechanism. Moreover, the inability of TNF-alpha to ac tivate the human p53 gene promoter, in contrast to that reported for t he murine p53 gene, may be the result of differences in the interactio ns of the murine and human kappa B sites with NF-kappa B complexes.