Delta Np63 alpha transcriptionally represses p53 target genes involved in the radiation-induced DNA damage response

Background The DNA damage response (DDR) is a mechanism that protects cells against radiation-induced oxidative DNA damage by causing cell cycle arrest and apoptosis. TP63 is a member of the tumour suppressor TP53 gene family, and Delta Np63 alpha, a TP63 splicing variant, is constitutively expressed in the stem cell-containing basal layer of stratified epithelial tissues, including the mammary gland, where it plays a critical role in stemness and tissue development. Delta Np63 alpha has been reported to transcriptionally inhibit the tumour suppression protein p53. This p53-repressive activity may cause genomic instability in epithelial stem cells exposed to radiation. In this study, we analysed the inhibitory effect of Delta Np63 alpha on radiation-induced DDR. Methods To elucidate the role of the p53-repressive effect of Delta Np63 alpha in radiation response, we performed a p63-siRNA knockdown experiment using human mammary epithelial cells (HMECs) expressing Delta Np63 alpha and then performed ectopic and entopic expression experiments using human induced pluripotent stem cells (hiPSCs). After irradiation, the expression of DDR-related genes and proteins in Delta Np63 alpha-expressing and control cells was analysed by RT-qPCR, Western blotting, and flow cytometry. Results The mRNA/protein expression levels of BAX and p21 were significantly increased in p63-siRNA-treated HMECs (sip63) after X-ray irradiation (4 Gy, 0.7 Gy/min) but not in scramble-siRNA treated HMECs (scr). Transcriptomic analysis showed decreased RNA expression of cell cycle-related genes and increased expression of programmed cell death-related genes in sip63 cells compared to scr cells. Furthermore, flow cytometric analysis revealed an increase in apoptotic cells and a decrease in 5-ethynyl-2'-deoxyuridine uptake in sip63 cells compared to scr cells. On the other hand, both the ectopic and entopic expression of Delta Np63 alpha in apoptosis-sensitive hiPSCs reduced the expression levels of BAX after irradiation and significantly decreased the number of apoptotic cells induced by radiation. Conclusion Taken together, these results indicate that Delta Np63 alpha represses p53-related radiation-induced DDR, thereby potentially causing genomic instability in epithelial stem cells.