Wild-type p53 transactivates the KILLER/DR5 gene through an intronic sequence-specific DNA-binding site

KILLER/DR5, a tumor necrosis factor-related apoptosis-inducing Ligand (TRAI L) death receptor gene, has been shown to be induced by DNA damaging agents and radiation in a p53-dependent manner. Although TRAIL is a potential the rapeutic agent for cancer, the induction mechanism of its receptors is poor ly understood, Here we show the identification of three p53 DNA-binding sit es in the KILLER/DR5 genomic locus located upstream (BS1; -0.82 Kb) of the ATG site, within Intron 1 (BS2; + 0.25 Kb downstream of the ATG) and within Intron 2 (BS3; + 1.25 Kb downstream of the ATG), A modified p53-binding an d immunoselection protocol using a wildtype p53-expressing adenovirus vecto r (Ad-p53) was used to identify the binding sites and to show that each bin ding site can bind specifically to wild-type p53 protein (wt-p53), A report er assay revealed that only BS2 could enhance luciferase expression driven by a basal promoter. We constructed a reporter plasmid carrying the genomic regulatory region of KILLER/DR5 including the three p53 DNA-binding sites but no additional basal promoter. The genomic fragment showed basal transcr iptional activity which was induced by wt-p53 but not by mutant p53, and hu man papilloma virus E6 inhibited the p53-dependent activation. Mutation of BS2 abrogated not only the binding activity of wt-p53 but also the inductio n of the KILLER/DR5 genomic promoter-reporter gene, indicating that BS2 is responsible for the p53-dependent transactivation of KILLER/DR5, In p53-wil d-type but not -mutant or -null cell lines, doxorubicin treatment stabilize d p53 protein, and increased specific binding to BS2 as revealed by EMSA, a nd upregulated the KILLER/DR5 promoter-luciferase reporter gene. These resu lts suggest that the transactivation of KILLER/DR5 is directly regulated by exogenous or endogenous wt-p53 and establishes KILLER/DR5 as a p53 target gene that can signal apoptotic death.