학술논문
Np63[alpha] transcriptionally represses p53 target genes involved in the radiation-induced DNA damage response
Document Type
Report
Author
Source
Radiation Oncology. November 15, 2022, Vol. 17 Issue 1
Subject
Language
English
ISSN
1748-717X
Abstract
Author(s): Ken-ichi Kudo[sup.1] , Naohiro Tsuyama[sup.1] , Kento Nagata[sup.2] , Tatsuhiko Imaoka[sup.2] , Daisuke Iizuka[sup.2] , Misaki Sugai-Takahashi[sup.1] , Moe Muramatsu[sup.3] and Akira Sakai[sup.1] Background Ionizing radiation is well known [...]
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 [DELA]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. [DELA]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 [DELA]Np63[alpha] on radiation-induced DDR. Methods To elucidate the role of the p53-repressive effect of [DELA]Np63[alpha] in radiation response, we performed a p63-siRNA knockdown experiment using human mammary epithelial cells (HMECs) expressing [DELA]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 [DELA]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 [DELA]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 [DELA]Np63[alpha] represses p53-related radiation-induced DDR, thereby potentially causing genomic instability in epithelial stem cells. Keywords: [DELA]Np63[alpha], TP63, p53, DNA damage response, Mammary epithelial cells, Stem cells, Genomic instability, Radioresistance
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 [DELA]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. [DELA]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 [DELA]Np63[alpha] on radiation-induced DDR. Methods To elucidate the role of the p53-repressive effect of [DELA]Np63[alpha] in radiation response, we performed a p63-siRNA knockdown experiment using human mammary epithelial cells (HMECs) expressing [DELA]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 [DELA]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 [DELA]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 [DELA]Np63[alpha] represses p53-related radiation-induced DDR, thereby potentially causing genomic instability in epithelial stem cells. Keywords: [DELA]Np63[alpha], TP63, p53, DNA damage response, Mammary epithelial cells, Stem cells, Genomic instability, Radioresistance