Additive eect between NF-kB subunits and p53 protein for transcriptional activation of human p53 promoter Valerie Benoit 1,2 , Anne-CeÂcile Hellin 1,2 , Sandra Huygen 1 , Jacques Gielen 1 , Vincent Bours 1 and Marie-Paule Merville* ,1 1 Laboratory of Medical Chemistry and Medical Oncology, Pathology B23, University of LieÁge, Sart-Tilman, 4000 LieÁge, Belgium The tumor suppressor p53 plays a pivotal role in the cellular response to DNA damage as it controls DNA repair, cell cycle arrest and apoptosis. We studied the autoregulation of human p53 gene transcription in colon cancer cell lines. Wild-type p53 has been shown to autoregulate its own transcription either positively or negatively and probably in a cell-type-speci®c manner. Indeed, a p53 binding site has been described in the human and murine p53 promoters, but a direct binding of wild-type p53 protein to this site has never been reported. In this study, we demonstrated a transactivation of human p53 promoter by wild-type p53 in human colon cancer cells. We identi®ed in the human p53 promoter a novel potential p53-responsive element that binds wild- type p53. Moreover, wild-type p53 protein transactivated a reporter plasmid containing a luciferase gene driven by a minimal promoter harboring this p53 binding site. Finally, as the p53 promoter contains an NF-kB binding site, we demonstrated an additive eect when NF-kB subunits and p53 protein combined to transactivate the human p53 promoter. Oncogene (2000) 19, 4787 ± 4794. Keywords: p53; autoregulation; NF-kB; transcription factors Introduction Over the past several years, the role of the tumor suppressor gene p53 and its product in human cancer development has been clearly demonstrated (for review, see May and May, 1999). Indeed, the p53 gene is one of the most frequently mutated genes in human malignancies (Hollstein et al., 1991; Levine et al., 1991; Vogelstein and Kinzler, 1992). Its product, the wild-type (WT) p53 protein, plays an important role in the cellular response to DNA damage by controlling cell cycle and allowing repair of the altered genome (Hartwell, 1992; Lane, 1992). The responses of the p53- dependent signaling pathways to dierent stresses such as anticancer drugs or irradiations have been exten- sively studied. Following DNA damage, p53 is modi®ed by a number of post-translational mechan- isms such as phosphorylations, acetylations, glycosyla- tions, protein interactions, redox mechanisms, subcellular localization, etc. These structural changes dramatically increase the half-life of p53, which accumulates rapidly in the nucleus. Moreover, the level of p53 can also be modulated at the transcriptional level after mitogenic stimulation, dierentiation induc- tion and genotoxic stress (Reich and Levine, 1984; Sun et al., 1995; Balint and Reisman, 1996; Kirch et al., 1999). The WT-activated p53 acts as a transcriptional factor and regulates the expression of target genes including p21(WAF1/CIP1), gadd45, bax or bcl2, which in turn control cell cycle checkpoints, DNA repair and apoptosis (Kern et al., 1991; Farmer et al., 1992; Miyashita et al., 1994). Surprisingly, little is known about the transcriptional autoregulation of p53 expression. Mosner et al. (1995) showed a negative feedback regulation of p53 on its own synthesis. The p53 mRNA can form a stable stem- loop structure that involves the 5'-untranslated region (5'-UTR) as well as some 280 nucleotides of the coding sequence. The p53 protein binds to the 5'-UTR region and inhibits the translation of its mRNA. Another negative regulation of p53, in normal and cancer cells as well as after exposure to genotoxic agents, involved the product of the protooncogene Mdm2. Indeed, the Mdm2 gene is transcriptionally activated by p53. Mdm2 binds the N-terminal transactivation domain of p53, interferes with the recruitment of basal transcription machinery components and inhibits the transcriptional activity of p53. More importantly, Mdm2 binding can also lead to complete proteolytic degradation of p53 through the ubiquitin-proteasome pathway (Honda et al., 1997; Honda and Yasuda, 1999). Several oncoproteins, functioning as transcriptional factors, have been shown to autoregulate, negatively (C-Myc and C-Fos) or positively (C-Myb), the transcription of their encoding gene (Schonthal et al., 1989; Nicolaides et al., 1991; Facchini et al., 1997). p53 gene transcriptional autoregulation has been studied by three dierent groups, who reported con¯icting results. Ginsberg et al. (1991) demonstrated that p53 protein down-modulated its own promoter. Dee et al. (1993), on the other hand, showed that p53 transactivated its own promoter and identi®ed in the murine p53 promoter a domain responsive to the wild-type but not mutant p53. Finally, Hudson et al. (1995) suggested that p53 autoregulated its own transcription indirectly and in a cell-type-speci®c manner. None of these authors, however, could demonstrate a direct binding of p53 to its promoter, raising the possibility that WT p53 autoregulates its own transcription indirectly by interacting with other transcription factors. The existence of such a mechanism would also explain the fact that p53 regulated its own promoter in a cell-type-speci®c manner. Oncogene (2000) 19, 4787 ± 4794 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc *Correspondence: MP Merville 2 The ®rst two authors contributed equally to this work Received 29 March 2000; revised 20 July 2000; accepted 1 August 2000