Background
Ionizing radiation is well known to induce oxidative DNA damage, such as DNA double-strand breaks (DSBs), and consequently trigger the DNA damage response (DDR), including cell cycle arrest and apoptosis. The DNA guardian protein p53 plays the most important role in DDR: it promotes DNA repair and the elimination of cells that are unable to repair the damage caused by oxidative stresses, including radiation. The p53 protein is normally degraded by the ubiquitin–proteasome system through the E3 ubiquitin ligase MDM2 and is maintained at low levels in a steady state [
1]. However, when DSBs are induced in the nucleus by radiation, p53 is acetylated and phosphorylated by proteins such as p300/CBP and ataxia telangiectasia mutated (ATM), preventing its ubiquitination and consequent degradation and enabling its activation: it then binds to the promoter region of DDR-related genes, including
CDKN1A, and upregulates their gene expression to promote DNA strand repair [
2‐
4]. When DNA repair fails, the cell undergoes various forms of death including apoptosis. Notably, p53 induces apoptosis through the upregulation of pro-apoptotic genes, including
BAX, and the subsequent activation of pro-apoptotic proteins, including Caspase-3 [
5]. Thus, p53 is the key protein that directs the DDR.
TP63 was discovered as a
TP53 family gene in 1998 and has high homology with
TP53 in its transactivation (TA), DNA-binding, and tetramerization domains [
6,
7]. It has two main isoforms: TAp63 has an N-terminal TA homologous to that of p53, while ΔNp63 has a truncated but specific TA domain, the expression of which depends on selective promoters. In addition, these two isoforms each have three further isoforms, α, β, and γ, with different C-termini generated through alternative RNA splicing. In particular, the α-type of p63 (p63α) has an additional region in its C-terminus, which includes the sterile alpha motif (SAM) domain and plays an important role in protein–protein interactions [
8,
9]. The TAp63 and ΔNp63 isoforms have opposing functionalities: TAp63 acts like p53, enhancing apoptosis and cell cycle arrest in the DDR, while ΔNp63 acts as a dominant negative regulator of TAp63; the α-type of ΔNp63 (ΔNp63α) is the most potent p53 repressor among all isoforms [
6,
8,
10]. ΔNp63α is expressed only inside the nucleus, appearing as foci at replication factories upon immunostaining [
11]. It has been elucidated that ΔNp63α cannot form hetero-oligomers with p53, unlike mutant p53 [
12], and thus, its p53 repressor activity is attributed to competitive inhibition, which results from the high homology between its DNA-binding domain and that of p53, and gene regulations [
10].
Yang et al. [
6] and Westfall et al. [
8] confirmed the transcriptional inhibitory effect of ΔNp63α on p53 through competitive binding to the p53 response element (RE) in the p21 promoter region by luciferase reporter assay and chromatin immunoprecipitation (ChIP). Interestingly, the magnitude of its inhibitory effect depends on the C-terminus; among all isoforms ΔNp63α has the greatest inhibitory effect on p53, while ΔNp63β and ΔNp63γ have only a weak effect. In addition, some studies have shown an inverse relationship between ΔNp63α expression and apoptosis [
13,
14]. On the other hand, Woodstock et al. [
10] recently noted that transcriptome analyses show very little overlap in the target sequences of ΔNp63α and p53 [
15]; this observation presents a problem, as it is inconsistent with the competitive inhibition theory. Min et al. [
14] reported that ΔNp63α promotes the expression of the follistatin (Fst) gene, which inhibits apoptosis by blocking the binding of Activin to its receptor located on the cell membrane. Thus, the inhibitory effect of ΔNp63α may involve two pathways: competitive inhibition and antagonistic gene expression.
ΔNp63α is highly expressed in the basal cell layer of the epithelium and plays an important role in stemness maintenance and cell repopulation [
6,
16]. The basal cells in epithelial tissues contain stem cells and have relatively high reproductive ability and stemness [
17‐
19]. These cells differentiate into other cell types as ΔNp63α expression decreases [
20]. On the other hand, stem cells are considered favoured candidates for transformation into cancerous cells because of their inherent capacity for self-renewal and their longevity, which may allow the accumulation of genetic mutations induced by oxidative stresses over long periods [
21,
22]. Radiation-induced carcinogenesis has also been found to occur with higher frequency in the epithelium, including the mammary gland and epidermis [
23]. Recently, its immunostaining has also been regarded as a marker for the diagnosis of squamous cell carcinoma [
24,
25]. Thus, ΔNp63α is thought to confer stem cell properties, while its p53 repressor activity is expected to be an important factor for elucidating the mechanism of radiation-induced carcinogenesis.
Radiation is one of the best stimulants with which to study DDR, as it leads to the oxidation and breakage of DNA strands through the generation of reactive oxygen species (ROS) in the vicinity of the DNA. Thus, radiation biology is one of the most appropriate fields for elucidating the DDR-related function of ΔNp63α. However, few studies have focused on the transcriptional repressor activity of ΔNp63α in this field. Two studies have previously reported that mammary basal cells are less responsive to radiation than mammary luminal cells, differentiating cells from basal stem/progenitor cells [
26,
27]. If basal stem cells that have genetic mutations and chromosomal aberrations continue to divide and differentiate for a long time, cancerous cells may be expected to develop. In this study, we aim to clarify transcriptional inhibition mediated by ΔNp63α during the radiation response.
Methods
Materials
To analyse the function of ΔNp63α, human mammary epithelial cells (HMECs, Thermo Fisher Scientific, Waltham, MA, USA) were purchased and cultured in HuMEC Ready Medium (1X) (HuMEC, Thermo Fisher Scientific) supplemented with gentamicin/amphotericin solution (Thermo Fisher Scientific). DeltaNp63alpha-FLAG (#26,979; RRID:Addgene_26979) was obtained from Addgene (
www.addgene.org), and pRetro-X-Tight-Pur and pRetro-X-Tet-Off Advanced vectors were purchased from TaKaRa Bio Inc. (Otsu, Japan). Polymerase chain reaction (PCR) primers were obtained from Merck (St. Louis, MO, USA) (Table S1). Knockdown experiments were carried out by lipofection with siRNA targeting the DNA-binding domain of
TP63 mRNA with siLentFect (Bio-Rad, Hercules, CA, USA). Scramble siRNA (scr) was used as a negative control. All siRNA double strands were synthesized by NIPPON GENE (Tokyo, Japan) (Table S1). G418 sulfate (Nacalai Tesque, Kyoto, Japan) and puromycin (TaKaRa Bio Inc.) were used for cell selection. Doxycycline (Dox, TaKaRa Bio Inc.) was used to regulate gene expression using an inducible Tet-OFF system.
Irradiation
Cells in microtubes, plates, and dishes were irradiated with X-rays using an MBR-1605R X-ray generator (HITACHI, Hitachi, Japan) at 150 kVp and 5 mA with a 0.5 mm Al + 0.2 mm Cu filter. The dose rate was 0.7 Gy/min.
iPSC cell culture, transfection, and virus packaging
Human induced pluripotent stem cells (hiPSCs, HiPS-RIKEN-2 A; RRID:CVCL_B512), which were derived from human fibroblasts and have high radiosensitivity, were obtained from the Riken Cell Bank (Tsukuba, Japan). hiPSCs were seeded on a plate coated with iMatrix-511 (Nippi, Tokyo, Japan) and cultured with StemFit AK02N (REPROCELL, Yokohama, Japan) containing 10 µM Y27632. hiPSC-ΔNp63α (iPS-DN) with Dox-dependent ΔNp63α expression was generated by retrovirus infection as described below. DeltaNp63alpha-FLAG plasmids were digested with BamHI and NotI, and the fragment containing the ΔNp63α coding sequence was separated by agarose gel electrophoresis and purified with a FastGene Gel/PCR Extraction Kit (NIPPON Genetics, Tokyo, Japan). The ΔNp63α fragment was ligated downstream of the pRetro-X-Tight-Pur vector (puromycin-resistant), which has a tight TRE promoter, with the Ligation high reagent (TOYOBO, Osaka, Japan). For retrovirus packaging, plasmids were transfected into gp293 cells (RRID:CVCL_E072) using PEImax 40,000 (Polyscience Inc., Warrington, PA, USA) following the manufacturer’s protocol. Virus particles were recovered by centrifugation of culture supernatant supplemented with PEG6000 and NaCl [
28]. Virus particles were also produced from the pRetro-X-Tet-Off Advanced vector (Neomycin-resistant, TaKaRa Bio Inc.). Then, hiPSCs were coinfected with these virus suspensions in the presence of polybrene (4 µg/mL) and cultured with medium containing 1 µg/mL puromycin, 200 µg/mL G418 sulfate, and 10 ng/mL Dox. After incubation for 14 days, Dox was removed from the medium, and ΔNp63α expression was confirmed by both reverse transcription-quantitative PCR (RT–qPCR) and Western blotting. Subsequently, 5 passages were performed to obtain cells for use in experiments.
Organoid culture and immunohistochemistry (IHC)
Human mammary organoids were generated as described previously [
27]. HMECs were mixed on ice with HuMECs containing 2 mg/mL rat tail collagen I (Corning Inc., Corning, NY, USA). Four hundred microlitres of this mixture was plated in the wells of an ultralow-attachment 24-well plate (Corning Inc.) and then incubated at 37 °C and 5% CO
2 to allow collagen gelation. After 1 h, the gels were immersed in HuMECs supplemented with 2.5% FBS, 10 µM forskolin (Enzo Biochem Inc., Farmingdale, NY, USA), and 0.5 µg/mL hydrocortisone (STEMCELL Technologies, Vancouver, Canada). The medium was changed every 4 days for 11–14 days. Thereafter, mammary organoids embedded in gels were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature (RT) and processed for regular paraffin embedding. Immunofluorescence and haematoxylin–eosin (HE) staining for mammary organoids were performed on paraffin-embedded sections. Antigen retrieval was accomplished by autoclaving at 120 °C in 10 mM sodium citrate buffer (pH 6.0) for 15 min. After blocking with 5% normal goat serum (Vector Laboratories, Burlingame, CA, USA), sections were treated with primary antibodies overnight, followed by secondary antibodies at RT for 1 h (Table S2). Finally, sections were mounted with Vectashield mounting medium containing 4´,6-diamidino-2-phenylindole (DAPI, Vector Laboratories). Images were captured using an Axio Imager. Z2 (RRID:SCR_018856; Carl Zeiss, Oberkochen, Germany).
Immunocytochemistry (ICC)
Cells were inoculated on a sterile coverslip in dishes and incubated at 37 °C and 5% CO2. After X-irradiation, the cells were fixed with 4% PFA for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 30 min, and incubated with 5% normal goat serum to block nonspecific binding to the target sites in the cells. Cells were then incubated with primary antibodies at RT overnight, followed by incubation with secondary antibodies for 1 h at RT. Cells were mounted with Vectashield containing DAPI. Images were captured using an Axio Imager Z2. The primary and secondary antibodies used in this study are listed in Table S2.
Neutral comet assay
To quantify the DSBs induced by radiation, neutral comet assays were performed. The cells were mixed with 0.5% low-melting-point agarose (Nacalai Tesque) and then seeded on microscope slides coated with 0.8% normal-melting-point agarose gel (Nacalai Tesque). After the agarose gel was solidified on ice for 15 min, the slides were immersed in alkaline lysis buffer (1% Triton X-100, 10% DMSO, 100 mM EDTA, 2.5 M NaCl, 10 mM Tris-HCl, 1% sodium lauryl sarcosine, pH = 10) for 1 h at 4 °C to release DNA from the cell and remove proteins. Then, slides were electrophoresed (13–15 V, 70–100 mA) in TAE buffer at 1 h and fluorescently stained with SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific) for 30 min. To estimate the quantity of DSBs generated in a single cell, the %tail DNA was calculated using Comet Assay Software Project ver. 1.2.3b2 (RRID:SCR_007249; CaspLab).
Flow cytometry (FCM)
For the measurement of DNA synthesis ability and cell cycle analysis, 5-ethynyl-2´-deoxyuridine (EdU) incorporated in cellular DNA was stained by using a Click-iT Plus EdU Flow Cytometry Assay kit (Thermo Fisher Scientific). The cells were treated with medium containing 10 µM EdU for 30 min before cell recovery, and EdU was incorporated into S-phase cells during DNA synthesis. After centrifugation at 300 x g for 5 min at 4 °C, the cells were washed with HBSS, fixed in 4% PFA and permeabilized with saponin-based solution according to the manufacturer’s protocol. Then, the RNA in the cells was degraded by 100 µg/mL RNase A. Finally, the cells were stained with 3 µM propidium iodide (PI, Thermo Fisher Scientific) for 15 min at RT.
For apoptosis analysis, FCM measurement by anti-Cleaved Caspase3 (CC3) antibody (RRID:AB_2341188; Cell Signaling Technology, Danvers, MA, USA) was performed on the irradiated cells. After fixation using 4% PFA and permeabilization with 0.1% Triton X-100, the cells were treated with CC3 antibody for 2 h on ice, followed by secondary antibody (Table S2).
To determine the ROS induced in X-irradiated cells, 2´,7´-dichlorodihydrofluorescein diacetate (DCFH-DA, Dojindo Laboratories, Kumamoto, Japan) was used as a colorimetric cell-permeable probe. HMECs were seeded on 60 mm dishes with a cell density of 1 × 106 cells/dish, and DCFH-DA dye solution (λex = 505 nm, λem = 525 nm) was added for 30 min before X-irradiation. Then, the cells were trypsinized, detached from the dish and analysed by FCM.
All FCM analyses were performed using an S3e cell sorter (RRID:SCR_019710; Bio-Rad). Data were analysed with FlowJo software version 10.8 (RRID:SCR_008520; BD Biosciences, Franklin Lakes, NJ, USA).
RT–qPCR
Total RNA was extracted from cells by using Sepasol-RNA I Super G (Nacalai Tesque) and then reverse-transcribed into complementary DNA using SuperScript IV VILO Master Mix reverse transcriptase (Thermo Fisher Scientific) according to the manufacturer’s protocol. RT–qPCR was performed using SYBR Premix Ex Taq (TaKaRa Bio Inc.) and the LightCycler Nano (Roche Diagnostics, Basel, Switzerland). Primer sequences are listed in Table S1. The cycling profile included a hot start at 95 °C for 120 s; 40 cycles consisting of a denaturation step at 95 °C for 10 s, annealing at 60 °C for 20 s, and extension at 72 °C for 10 s; and fluorescent signal acquisition at 72 °C, with a final dissociation curve analysis. All gene expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The relative mRNA expression level of each gene was defined based on the threshold cycle (Ct) and calculated by the 2−ΔCt formula.
RNA-seq
Total RNA was extracted from HMECs at 24 h post-irradiation by using a FastGene RNA Premium Kit (NIPPON Genetics). To ensure the accuracy of the data, each sample was mixed with total RNA obtained from two independent experiments. RNA quality was checked using agarose electrophoresis (total RNA > 1.0 µg, OD
260/280 = 1.8–2.2, RIN > 6.5). RNA-seq was performed via next-generation sequencing using DNBSEQ-G400RS (RRID:SCR_017980; MGI Tech Co., Ltd., Shenzhen, China). All analyses were performed using integrated Differential Expression and Pathway analysis (iDEP) version 0.95 (
http://bioinformatics.sdstate.edu/idep95/) [
29].
Western blotting
The cells were lysed in RIPA buffer with a protease inhibitor cocktail (Nacalai Tesque). After 30 min on ice, the cell lysates were centrifuged at 12,000 x g for 20 min at 4 °C, and the supernatants were recovered. The protein content of every sample was determined by the Bradford method, and equal amounts of protein from each sample were separated by SDS–PAGE and then transferred onto a nitrocellulose membrane (Bio-Rad) or a PVDF membrane (ATTO Corporation, Tokyo, Japan). The membrane was incubated with primary antibody and horseradish peroxidase-labelled secondary antibody. Then, the signal was visualized with Chemi-Lumi One Super (Nacalai Tesque) and detected with a ChemDox XRS+ (Bio-Rad). All antibodies used in this experiment are listed in Table S2.
ChIP–qPCR
ChIP–qPCR was carried out using a SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) according to the manufacturer’s protocol. Briefly, iPS-DNs (4 × 106 cells/sample) were treated with 2% PFA for 15 min at RT. The chromatin was harvested and fragmented using enzymatic digestion. Chromatin solutions were subjected to immunoprecipitation with anti-p53 (clone 7F5) antibody (RRID:AB_10695803; Cell Signaling Technology) overnight at 4 °C. Then, the immunoprecipitated complex was treated with protease. ChIP DNA was subjected to qPCR assay with amplification of the BAX and CDKN1A promoters using the primers listed in Table S1. Rabbit immunoglobulin G (IgG) was used as a negative control for nonspecific immunoprecipitation of DNA. The data were analysed by the following formula: %Recovery = 100 × 2(input Cq − Target sequence Cq).
Differentiation of hiPSCs into keratinocytes
Human iPSC-derived keratinocytes (iPS-KCs) were generated as described previously [
30]. Briefly, hiPSCs were seeded and cultured under feeder-free conditions as described above, and then the culture medium was replaced with defined keratinocyte serum-free medium (DKSFM, Thermo Fisher Scientific) supplemented with 1 µM retinoic acid (Sigma–Aldrich) and 10 ng/mL human bone morphogenetic protein 4 (Peprotech, Cranbury, NJ, USA). Three days later, the culture medium was replaced with DKSFM supplemented with 20 ng/mL epidermal growth factor (FUJIFILM, Tokyo, Japan) and 10 µM Y27632. After an incubation for 7–14 days, the cells were reseeded on dishes coated with iMatrix-511.
Discussion
In this study, we experimentally dissected p53 repression by ΔNp63α during the radiation response. TP63 is a member of the TP53 gene family and is homologous to TP53 in its TA, DNA-binding domain, and tetramerization regions. ΔNp63 is one of the proteins generated through selective alternative splicing after TP63 transcription. Therefore, since its discovery, ΔNp63, especially ΔNp63α, has been thought to work competitively with p53 against target genes, thereby inhibiting the typical function of p53. On the other hand, radiation is highly cell permeant and induces DNA oxidative damage, including DSBs, which causes gene mutations and chromosomal aberrations through the generation of ROS, such as hydroxyl radicals and singlet oxygen, in the vicinity of DNA in the nucleus. This in turn leads to the activation of kinases such as ATM to induce the p53-derived DDR, including cell cycle arrest and apoptosis. Therefore, radiation biology provides the environment necessary to determine how ΔNp63α affects DDR in these signalling transductions and whether it is a competitive inhibitor of p53.
We first performed siRNA knockdown experiments using HMECs, which exhibit mammary basal cell characteristics, and observed changes in the expression of DDR-related genes downstream of p53 by RT–qPCR. After the X-irradiation to HMECs, genes related to cell cycle arrest and apoptosis showed little response in the scr-treated group but exhibited a marked increase in expression after ΔNp63α knockdown. This was further confirmed by Western blotting and RNA-seq. Although these DDR-related genes are upregulated through p53 binding to the promoter regions after irradiation, the differential expression of these genes even in the absence of irradiation suggests that ΔNp63α suppresses the expression of these genes at all times.
To investigate whether the transcriptional repression of DDR-related genes is independent of the amount of DNA damage, we directly quantified the DSBs generated in HMECs after irradiation. We found that the amounts of DSBs and ROS generated after irradiation were increased in the sip63-treated group compared to the scr-treated group. However, similar increases were observed in the nonirradiated group, suggesting that the difference between groups was due to the upregulation of antioxidant genes, such as
GPX2 and
CYBG, by ΔNp63α [
36,
37], resulting in a decrease in long-lived ROS, such as H
2O
2. Hence, ΔNp63α-expressing cells may be resistant to less reactive long-lived ROS. However, the ΔNp63α-mediated cellular antioxidant system is not sufficiently potent to protect DNA from more reactive short-lived ROS, such as hydroxyl radicals, generated in the vicinity of DNA by radiation, which thus did not explain the impact of ΔNp63α in radiation-induced DDR.
The RNA-seq analysis of HMECs revealed that ΔNp63α upregulates genes related to the cell cycle and cell division while downregulating genes related to apoptosis and cell death. These findings are consistent with those of previous studies [
13,
14] and suggest that ΔNp63α acts as a transcription factor that maintains the stemness/inhibits the differentiation of mammary stem cells, keeps them alive by preventing cell death, and turns on genes that promote proliferation. The properties of ΔNp63α are opposite those of cancer suppressor genes such as
TP53 and
PTEN, and it is thus thought that ΔNp63α initially inhibits the typical radiation responses triggered upon radiation-induced DNA damage. In addition, RNA-seq analysis suggests that ΔNp63α downregulates
TP53 and
PTEN. These findings suggest that ΔNp63α suppresses the expression of
TP53 and other tumour suppressor genes and counteracts their effects. On the other hand, the protein analysis of HMECs showed that the p53 protein was upregulated by ΔNp63α expression, which was not observed in iPS-DNs. This suggest that ΔNp63α may increase the lifetime of the p53 protein in HMECs. Indeed, since the SAM domain in the C-terminus of ΔNp63α interacts with p300/CBP [
9], it is possible that p53 undergoes acetylation, resulting in low expression but a longer lifetime. Taken together, the results indicate that the relationship between ΔNp63α and tumour suppressor genes requires further investigation.
In the three cell types used in this study, HMECs, iPS-DNs, and iPS-KCs, RT–qPCR analysis results showed that ΔNp63α significantly inhibited the expression of
BAX. Consistent with this, the detection of apoptotic cells by FCM showed that ΔNp63α suppressed radiation-induced apoptosis, since the proportion of apoptotic cells decreased in ΔNp63α-expressing cells. Furthermore, ChIP–qPCR assays of iPS-DNs confirmed that the binding of p53 to its target gene promoter region was reduced by ΔNp63α expression. These results all suggest that ΔNp63α inhibits radiation-induced apoptosis by suppressing the expression of apoptosis-related genes such as
BAX by p53. On the other hand, although ΔNp63α transcriptionally repressed
CDKN1A expression and reduced p21 at the protein level, cell cycle analysis by FCM showed that cell cycle arrest occurred after irradiation regardless of ΔNp63α expression. A potential explanation for this result is that G1 arrest is regulated by genes or pathways independent of p53, such as ATM-CHK2-CDC25A [
45]. Consistent with this model, the analysis of mammary organoids also showed that p21 is expressed at the same level in ΔNp63α-positive cells as in ΔNp63α-negative cells after irradiation. With regard to cell cycle arrest, this study supports the results of Westfall et al. [
8]: ΔNp63α transcriptionally represses
CDKN1A, but p21 is still expressed in certain amounts after transcriptional repression; moreover, the results of the cell cycle analysis by FCM suggest that the transcriptional repressive effect is limited during cell cycle arrest. At the same time,
GADD45A in X-irradiated iPS-DNs was not transcriptionally inhibited, although it is a p53 downstream gene that has been shown to play a minor role in G2 arrest compared to ATM/CHK2 and ATR/CHK1 [
46]. Thus, these results indicate that the transcriptional repression of ΔNp63α preferably affects proapoptotic p53 target genes. Understanding whether this transcriptionally repressive effect is due to competitive inhibition against p53 RE, as conventionally suggested, or to gene expression regulated by ΔNp63α will require further discussion addressing a wider range of p53-related genes.
It is problematic that stem cells contained in basal cells may all have this DDR vulnerability. If epithelial stem cells expressing ΔNp63α are vulnerable to DDR, especially apoptosis, it can be inferred that not only the mammary gland but also the prostate, lung, skin, and other epithelial tissues are at risk. Since caspase-3, which is activated by the p53 pathway, is also inhibited in ΔNp63α-expressing cells, it is conceivable that stem cells, which are long-lived and capable of differentiating into other cells [
21,
22], may survive the failure to repair radiation-induced DNA damage, leaving DNA damage and mutations behind, and then differentiate into other cells, including cancerous cells. BRCA1/2 genes are involved in DNA repair, especially homologous recombination repair, which precisely repairs DSBs. Women with BRCA1 or 2 gene deficiency are more likely to develop breast cancer. Thus, this may suggest an additive or synergistic effect of BRCA gene deficiency and p53 repression by ΔNp63α. Indeed, BRCA1-deficient breast cancers are positive for CK14, which seems to be associated with the development of triple-negative breast cancer [
47].
Epithelial stem cells such as mammary stem cells still lack definitive markers, and therefore, single-cell analysis, including single-cell RNA-seq and whole-genome sequencing, will be needed to elucidate DDR inhibition by ΔNp63α in epithelial stem cells and then characterize the mutational signatures and chromosomal aberrations induced by irradiation. The process of long-term mutation accumulation in cancer-initiating cells should be investigated in detail by further analysing the characteristics of DDR inhibition by ΔNp63α in epithelial stem cells.
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