Abstract
KILLER/DR5, a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) 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 therapeutic agent for cancer, the induction mechanism of its receptors is poorly understood. Here we show the identification of three p53 DNA-binding sites 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 and immunoselection protocol using a wild-type p53-expressing adenovirus vector (Ad-p53) was used to identify the binding sites and to show that each binding site can bind specifically to wild-type p53 protein (wt-p53). A reporter 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 transcriptional activity which was induced by wt-p53 but not by mutant p53, and human papilloma virus E6 inhibited the p53-dependent activation. Mutation of BS2 abrogated not only the binding activity of wt-p53 but also the induction of the KILLER/DR5 genomic promoter-reporter gene, indicating that BS2 is responsible for the p53-dependent transactivation of KILLER/DR5. In p53-wild-type but not -mutant or -null cell lines, doxorubicin treatment stabilized p53 protein, and increased specific binding to BS2 as revealed by EMSA, and upregulated the KILLER/DR5 promoter-luciferase reporter gene. These results 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.
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Acknowledgements
The authors thank Gen Sheng Wu for providing DNA sequence data for the 1.7 kb region upstream of the KILLER/DR5 initiating methionine, in the early stages of this project. The authors also thank Nesrin Özören for providing the 3.6 Kb PstI KILLER/DR5 clone used initially to map and sequence the location of the novel p53 DNA binding sites. This work was supported in part by a grant provided by the Japan Health Sciences Foundation (R Takimoto) and by NIH grant # P01 CA75138-01 (WS El-Deiry). WS El-Deiry is an Assistant Investigator of the Howard Hughes Medical Institute.
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Takimoto, R., El-Deiry, W. Wild-type p53 transactivates the KILLER/DR5 gene through an intronic sequence-specific DNA-binding site. Oncogene 19, 1735–1743 (2000). https://doi.org/10.1038/sj.onc.1203489
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DOI: https://doi.org/10.1038/sj.onc.1203489
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