Unc5D regulates p53-dependent apoptosis in neuroblastoma cells

Abstract

The mechanism of apoptosis via the p53-dependent pathway remains to be fully understood. In the present study, a novel p53 target gene, Unc5D, was identified and its possible function in human neuroblastoma cells was investigated. The apoptotic effects of Unc5D in SK-N-BE (p53-/-) and SH-SY5Y (p53+/+) cells were measured by an 3-(4,5-dimethylthiazol-2- yl)2,5-diphenyltetrazolium bromide solution assay. Reverse transcription- polymerase chain reaction (RT-PCR) was also performed to detect the endogenous expression of Unc5D. In H1299 (p53-/-) cells, following overexpression of p53, RT-PCR and western blot analysis were used to detect the Unc5D mRNA and protein levels. In order to detect the promoter activity in the Unc5D gene, a luciferase assay was performed. Finally, to confirm the activate site of p53 subsequent to DNA damage, western blot analysis was used to analyze the phosphorylation site of Unc5D stable and mock clones in H1299 cells by co-expression of p53. Unc5D-induced apoptosis may be largely dependent on the p53 status. Notably, Unc5D was found to be a direct transcriptional target of p53. During adriamycin-mediated apoptosis, Unc5D was significantly induced in p53-proficient SH-SY5Y cells but not in p53-deficient SK-N-BE cells. Overexpression of p53 resulted in an increase in the expression levels of endogenous Unc5D. Additionally, two elements were identified in the sequence of Unc5D. Notably, Unc5D expression also induced phosphorylation of p53 at serine-15. Unc5D is thus a newly identified transcriptional target of pro-apoptotic p53 and may also act upstream of p53 to induce p53-dependent apoptosis by phosphorylation at ser-15.