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P53-participated cellular and molecular responses to irradiation are cell differentiation-determined in murine intestinal epithelium
Archives of Biochemistry and Biophysics 542 (2014) 21–27
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P53-participated cellular and molecular responses to irradiation are cell differentiation-determined in murine intestinal epithelium q Fengchao Wang a,1, Jin Cheng b,1, Dengquan Liu a, Huiqin Sun a, Jiqing Zhao b, Junping Wang a, Junjie Chen c, Yongping Su a,⇑, Zhongmin Zou b,⇑ a Institute of Combined Injury, State Key Laboratory of Trauma, Burns and Combined Injury, Department of Radiation Medicine, School of Preventive Medicine, The Third Military Medical University, 30 Gaotanyan Street, Shapingba District, Chongqing 400038, China b Department of Chemical Defense, School of Preventive Medicine, The Third Military Medical University, 30 Gaotanyan Street, Shapingba District, Chongqing 400038, China c Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Room Number Y3.6006, 1515 Holcombe Blvd, Houston, TX 77030, USA
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Article history: Received 15 October 2013 and in revised form 18 November 2013 Available online 4 December 2013 Keywords: DNA damage response Small intestine epithelium Apoptosis DNA repair Signaling pathway
a b s t r a c t Aim: Cells respond differently to DNA damaging agents, which may related to cell context and differentiation status. The aim of present study was to observe the cellular and molecular responses of cells in different differentiation status to ionizing irradiation (IR). Methods: Crypt-villus unit of murine small intestine was adopted as a cell differentiation model. DNA damage responses (DDRs) of crypt and villus were observed 1–24 h after 12 Gy IR using gene expression microarray analysis, immunohistochemical staining, Western blotting and Electrophoretic Mobility Shift Assay. Results: Microarray analysis revealed that most differentially expressed genes were related to p53 signaling pathway in crypt 4 h after IR and in both crypt and villus 24 h after IR. In crypt stem cells/progenitor cells, H2AX was phosphorylated and dephosphorylated quickly, Ki67 attenuated, cell apoptosis enhanced, phosphorylated P53 increased and translocated into nuclear with the ability to bind p53specific sequence. In upper crypt (transit amplifying cells) and crypt-villus junction, cells kept survive and proliferate as indicated by retained Ki67 expression, suppressed p53 activation, and rare apoptosis. Conclusions: DDRs varied with cell differentiation status and cell function in small intestinal epithelium. P53 signaling pathway could be an important regulatory mechanism of DDRs. Ó 2013 Elsevier Inc. All rights reserved.
Cell differentiation-determined cellular and molecular responses to irradiation in murine crypt and villus in vivo.
Introduction Histologically, small intestinal epithelial cells form a cell differentiation hierarchy along the crypt-villus axis. Small intestinal stem cells/progenitor cells locate in the crypt base, immediately above or in between of the Paneth Cells [1]. Transit amplifying cells, the immediate descendant cells of crypt stem cells, locate in the middle and upper part of crypt and differentiate into the epithelial cells while migrating upwards. Differentiated and functional epithelial cells cover the whole surface of villus. These cells continuously migrate along the surface of villus and q
finally shed off at the villus tip, finishing the course of epithelial cell turnover. This self-refresh process of epithelial cells takes as short as 3–5 days with a clear migration route [2]. Thus, small intestinal epithelium is a good model for studying cell proliferation, differentiation, apoptosis as well as DNA2 damage and repair [3,4]. Damaged DNA in a stem cell may cause gene mutation or canceration if neither the DNA damage is repaired nor the cell goes into apoptosis [5]. So, DNA repair in stem cells is of importance in both carcinogenesis and stemness maintenance. DNA repair ability is evolutionally conserved among species, but different DNA repair processes are involved in embryonic stem cells and differentiated cells [6,7]. P53 protein has been recognized as a key regulator of DNA damage and repair responses. It is unclear that if the activation of p53 signaling pathway relies on cell
Grant support: Natural Science Foundation of China (30828007).
⇑ Corresponding author. Fax: +86 23 68752285 (Z. Zou), fax: +86 23 68752009 (Y. Su). E-mail addresses: [email protected] (Y. Su), [email protected] (Z. Zou). 1 These authors contributed equally to this work. 0003-9861/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2013.11.012
2 Abbreviations used: DDR, DNA damage response; DSB, double strand break; H&E, hematoxylin and eosin; IHC, immunohistochemistry; IR, ionizing irradiation; p-,phosphorylated; PI3K, phosphatidylinositol-3-kinase; TBI, total body irradiation; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; EMSA, Electrophoretic Mobility Shift Assay.
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differentiation status, and why cells respond with different levels of p53 activation. We presume that cellular and molecular responses to DNA damage may vary with cell differentiation status, and p53 is critical in regulating the responses. Adult stem cells, such as hematopoietic stem cells and small intestinal crypt cells [8–10], are cell sources that replace aged and damaged cells, and very sensitive to ionizing radiation (IR). With the development of stem cell transplantation and hematopoietic growth factor production, hematopoietic type of acute radiation syndrome (ARS) becomes curable. But, there is no successful rescue in human with intestinal type of ARS. Local IR on abdomen is common for radiotherapy of abdominal tumor, and high dose exposure sometimes occurs in accidental irradiation. As the hematopoiesis can be successfully maintained under such situation, the protection and treatment of intestinal injury become the key questions. Understanding the early response of intestinal cells to irradiation may help clinical intervention on IR-induced intestinal injury. In this study, mice were challenged with 12 Gy total body irradiation (TBI), a dose that can cause intestinal type of ARS with half crypt survived [11]. We investigate the characteristics of DNA damage response (DDR), especially p53 signaling pathway-related events, of adult stem cells and their differentiated descendants in cyprt-villus unit in vivo in order to find the commons and the differences among different cell populations.
Gene chip hybridization and analysis The isolated crypts and villi at 0 h, 4 h, 8 h and 24 h after IR were homogenated in Trizol for total RNA extraction, and the extracts of the same group were pooled for RNA quality control test and microarray. The quality and quantity of the extracted RNAs were checked to ensure a convincible microarray comparison of gene expression profiling. The reverse transcription, fluorescence labeling, and hybridization on Mouse Genome 430 2.0 chip (Affymetrix) were conducted by Beijing Biocapital Company. The call values of Perfect Match (PM) and MisMatch (MM) were detected by AffymetrixÒ GeneChipÒ Scanner 3000. The difference degree between these two values and Computed P value was discriminated. Significant level was exactly represented by a margin region (0.04–0.06) to reduce false positive result, P < 0.04 demonstrates present gene and P > 0.06 demostrates absent gene. P value in the margin region means an uncertified result. Comparative analyses of expressed genes were carried out using AffymetrixÒ GeneChipÒ Operating SoftwareVersion1.4. Gene expression was considered increased or decreased only if the levels changed by greater than twofold with P < 0.0025 for increased expression and P > 0.9975 for decreased expression to reduce false discovery rate. Upregulated or downregulated genes were also clustered by Cluster 3.0 to get the category distribution. H&E, TUNEL, IHC staining and Western blot
Materials and methods Materials Mouse monoclonal anti p-P53 antibody were products of Cell Signal Technology (German). Other antibodies included mouse monoclonal anti-phospho-H2AX (cH2AX) antibody (Abcam, USA), Anti-Lamin B (Abcam, USA), rabbit monoclonal anti-Ki67 antibody (LabVision, Fremont, CA, USA) and corresponding secondary antibodies. Mouse Genome 430 2.0 chip (Affymetrix, Santa Clara, CA, USA), RIPA buffer (Beyotime Inc. Nantong, China), LightShiftÒ Chemiluminescent EMSA Kit (Pierce, Rockford, IL, USA), Tissue DNA Kit (Omega, Norcross, USA) were purchased from vendors in China.
Polyformaldehyde fixed 4 lm-thick deparaffinized sections was prepared for hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and immunohistochemistry (IHC) staining. TUNEL staining was processed according to the procedure of ROCH Company. After retrieving antigen activity and inhibiting endogenous peroxidase activity, standard IHC protocol was followed with primary antibodies (mouse monoclonal anti-cH2AX, rabbit monoclonal anti-Ki67, overnight at 4 °C. All slides were read in a blinded manner by specialists. Crypt and villus were lyzed in RIPA buffer containing protease inhibitors, and the extracted protein was subjected to standard Western blotting. For the quantification of protein bands, Image J (1.38e, NIH, USA) was used to analyze the densitometry of each band. The adjusted gene expression level was presented as the ratio between the interest gene and the corresponding internal control, such as b-actin and Lamin B.
Animals and irradiation DNA ladder Adult male C57/BL6 mice (LD50/30 = 8 Gy) [12] weighted 20– 22 g were irradiated with cobalt source, at a dose rate of about 0.77 Gy/min. The absorption dose of TBI was 12 Gy, which causes gastrointestinal type of acute radiation sickness. Mice were sacrificed by cervical dislocation at 0, 1, 4, 8 and 24 h after TBI (n = 6) for small intestine sampling.
The extraction of genomic DNA from isolated crypts and villi was performed according to the procedure of Tissue DNA Kit (Omega). The extracted genomic DNA was resolved with agarose gel electrophoresis and analyzed by gel reader (Bio-Rad, USA). Electrophoretic Mobility Shift Assay (EMSA)
Separation of crypt and villus Method was referred to Neil’s report [13] and a modification [14]. Briefly, the jejunum of normal and irradiated mice was dissected, washed with cold PBS, inverted to expose the mucous membrane, washed again, and cut into 2–3 cm segments. The segments were digested in cold chelator buffer [14] at 4 °C with shaking. Villi and crypts in the supernatant of digested jejunum were separated by gravity precipitation. The isolated villus and crypt compartments were respectively pooled and store in liquid nitrogen for further study. The intestine segments as well as the isolated villus and crypt collections were also fixed in 4% polyformaldehyde for paraffin embedding.
The double strand probes for P53 [15] was synthesized, 50 endlabeled with biotin and purified by TaKaRa Biotechnology Co., Ltd. (Dalian, China). The cold probe contained the same sequence of oligoDNAs except the omission of biotin conjugation. EMSA assay was conducted according to the instruction of LightShiftÒ Chemiluminescent EMSA Kit. In brief, the crypt cell protein was incubated with biotin-conjugated probe (20 fmol/ll) in the presence or absence of corresponding cold probe (2000 fmol/ll) for 20 min. The product was resolved in non-denatured polyacrylamide gel. Standard reaction of biotin and horseradish peroxidase-conjugated streptavidin combination and chemiluminescence development were performed. The image was analyzed with Bio-Rad gel scanning imaging system (Bio-Rad Laboratories, Hercules, CA, USA).
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Statistical analysis Gene chip data were analyzed as described above. Other experiment data are evaluated by one-way ANOVA and presented as mean ± standard derivation of measurement.
Results H2AX was extensively phosphorylated in epithelium early after the IR damage, but eliminated faster in crypts DNA damage usually causes quick H2AX phosphorylation (cH2AX), which is critical for DNA damage-induced foci formation. Therefore, cH2AX foci analysis is a sensitive indicator of double– strand breaks (DSBs), and the kinetics of cH2AX foci loss strongly correlate with the time course of localized chromatin decondensation [16] and DSB repair [17]. In the small intestine of normal control mice, only a few cH2AX foci located in the upper part of crypts and crypt-villus junction area, where the cells are in active DNA replication and recombination due to cell proliferation and differentiation, and at the tips of villi, where the cells are aged to shed off (Fig. 1A and D). At 1 h after 12 Gy TBI, both crypts and villi, displayed very strong cH2AX staining (P > 0.05, Fig. 1B and D), indicating extensive DNA damage and H2AX phosphorylation in the damaged epithelial cells. Interestingly, the cH2AX foci in crypts were more effectively eliminated than that in villi 4 h after IR (P < 0.05, Fig. 1C and D), which suggested higher DNA repair ability and/or faster elimination of damaged cells in crypts (see below). To quantitatively confirm the above result, Western blot was applied to detect cH2AX level. Consistently, normal crypt and villus contained a little cH2AX. In contrast, cH2AX level was significantly increased in both crypt and villus at 1 h after IR. However, the cH2AX
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level in crypt decreased significantly faster than that in villus at 4 h (P < 0.05, Fig. 1E and F). Apoptosis occurred early after IR was mainly in crypts It is commonly accepted that cells with failed DNA repair are usually eliminated through apoptosis or other cell death pathways to avoid neoplasma development. In normal intestinal epithelium, TUNEL staining revealed that apoptosis occurred only at the tips of villi (Fig. 2A). Very early after IR, there was no obvious increase in cell apoptosis (1.8 ± 0.8 cells/crypt, Fig. 2B). However, the apoptotic cells abruptly appeared at 4 h and were mainly presented at the crypt bases (11.8 ± 2.9 cells/crypt), where the stem cells and progenitor cells reside. Some apoptotic cells were also presented in lamina propria, which could be endothelial cells and blood cells, such as IR-sensitive lymphocytes (Fig. 2C). The amount of the apoptotic cells decreased at 8 h in the epithelium (6.2 ± 2.2 cells/ crypt), but still kept at high level. The much less apoptotic cells left in lamina propria might result from the physiological clearance of apoptotic cells in blood flow and in mesenchyme as well (Fig. 2D). H&E staining showed similar result to TUNEL staining. Although 1–8 h after IR, the whole mucous membrane maintained the normal histological structure, there were many apoptotic cells in crypt 4 h after IR. The apoptotic crypt cells exhibited karyopyknosis, karyorrhexis and cell body shrinkage, which gave a heavy staining in nuclei and cytoplasm (7.2 ± 1.6 cells/crypt, Fig. 2F) compared with normal control (1.3 ± 0.6 cells/crypt, Fig. 2E). Genomic DNA electrophoresis showed a typical DNA ladder pattern of apoptosis in crypt cells but was not detectable in villus cells at 4 h after IR (Fig. 2G). Colonal cells were used as positive control to show DNA ladder although they are not as sensitive to IR as the crypt cells. IR significantly suppressed the proliferation of crypt cells Physiologically, the crypt cells are in active proliferation to continuously provide new cells for the quick turnover of intestinal epithelium. In normal intestinal epithelium, most of the crypt cells showed positive staining for Ki67, a marker for proliferating cells (Fig. 3A and B). Interestingly, cells in between the Paneth Cells, according to the histological structure, were also Ki67 positive, which is consistent with the recent finding of the location of intestinal stem cells [1]. Four hours after IR, the average numbers of Ki67 positive cells per crypt were significantly decreased when compared with normal control (19.2 ± 2.3 vs 26.8 ± 3.0, P < 0.05), and the staining intensity was also decreased. The Ki67 positive cells were mainly confined to transit amplifying cells, the upper part of crypt (Fig. 3C and D). The results suggested that IR preferentially arrested the proliferation of stem/progenitor cells, which was well consistent with the result of TUNEL staining. p53 pathway played important roles in IR-induced response in crypts
Fig. 1. H2AX phosphorylation in small intestinal epithelium of normal and irradiated mice. (A) IHC staining revealed weak cH2AX signals in the middle and upper part of crypts, as well as the villus tips of normal small intestine. (B) At 1 h after 12 Gy total body irradiation, there was an extensive cH2AX staining in the whole intestinal epithelium, i.e., from crypt to villus. (C) At 4 h after IR, cH2AX level in crypt was notably reduced while high level remained in villus. (D) Quantification of the IHC staining on phosphorylate H2AX. (E) The cH2AX level in crypts and villi after IR assessed by Western blot using whole cell lystes. (F) Quantification of Western blot by densitometric analysis, which was consistent with IHC results. Bar = 100 lm in A–C.
To precisely define the different molecular and biochemical responses of crypt and villus to IR-induced DNA damage, we isolated these two cell compartments with well-preserved morphology and satisfying purity [14]. The isolated villi showed a relatively intact fingerstall-like structure (Fig. 4A), and the crypts a caterpillar-like structure (Fig. 4B), both resembled their in situ histological morphology in vivo. As the whole procedure was taken at 4 °C, the molecular events happened in vivo in the epithelium could be further explored by in vitro analysis on the isolated crypt and vilus compartments [13–15]. To get an overview of cell response after IR stress, the gene expression profiles of crypt and villus were assessed by microarray. A total of about 1200 expressed sequence tags (ESTs) expressed
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Fig. 2. Apoptosis of small intestine epithelial cells after IR detected by TUNEL staining. There were some apoptotic cells at the tip of villus and few positive cells scattered in epithelium of (A) normal mice and of (B) mice 1 h post-irradiation. (C) Many cells in crypt bases and some cells in lamina propria exhibited positive staining at 4 h after IR, indicating cell apoptosis occurred. (D) Apoptotic cell number decreased at 8 h after IR, but still significantly higher than the control. (E and F) In 5 H&E stained tissue sections from normal (E) or irradiated (F) mice, many cells showed karyopyknosis and karyorrhexis in crypts 4 h after IR (arrows) comparing with normal crypt. (G) To quantify the apoptotic cells, 50 crypts were randomly chosen to count apoptotic cells by two independent investigators. a, P < 0.05 vs control. (H) Genomic DNA electrophoresis of crypt cells showed a characteristic DNA ladder pattern of apoptosis 4 h after IR, but not in villus cells. Whole colon cells were used as a parallel control in the whole process of sample preparation. A–D: Bar = 50 lm; E and F: Bar = 20 lm.
Fig. 3. Expression of cell proliferation marker Ki67 in small intestinal epithelium. (A) Nuclei of normal crypt cells were positive for Ki67 in the whole crypt bases except Paneth Cells, and deeply stained in the middle part of crypt. (C) Four hours after IR, the Ki67 positive cells decreased and confined to the upper part of crypt, where the transit amplifying cells reside. The cells in the middle part of crypt turn to Ki67 negative, indicating their exit from cell cycle. (B and D) were the enlarged areas of boxes in (A and C) respectively. Arrows indicated Ki67 positive cells. A and C: Bar = 100 lm; B and D: Bar = 30 lm.
differentially, either upregulated or downregulated for more than 2-fold, at least at one of the checked time points in crypt compared with that of the normal control (Fig. 4C and D). In the timematched villus, about 820 different ESTs expressed differentially (Fig. 4C and D). In both crypt and villus, cell signal pathway cluster analysis (Table 1 and Supplemental Table 1) showed that the most important signaling pathway involved in IR-induced responses was p53 signaling pathway (including p53, ataxia telangiectasia and Rad3 related [ATR], transformation related protein 53 inducible nuclear protein 1 [Trp53inp1], transformation related protein 53 regulating kinase, transformation related protein 53 binding protein 1 [trp53bp], p21), followed by cell cycle (including cyclin G1,
D2, D1, B1, and E2, cyclin-dependent kinase 6), MAPK and cell adhesion pathways. The activation of p53 signaling pathway in crypt was the most predominant at 4 h and 24 h after IR. Although p53 pathway was also activated in villus, it was not the major one until 8 h after IR (Table 1). This may indicate that, when DNA damage occurred, crypt cells responded quickly on cell cycle control because the crypt cells, especially in the crypt base, are normally in active proliferation. In contrast, the villus cells primarily responded in the aspects of nuleotide synthesis and biochemical metabolism instead of cell cycle regulation since the villus cells usually are not in cell cycle any more.
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Fig. 4. The gene expression profiles of intestinal crypts and villi from normal and irradiated mice. Three independent replications were done for isolation. Total RNA was extracted from the well preserved and isolated crypts (A) and villi (B), labeled with Cyt3 and Cyt5, and hybrided with probe sets on microarray chips. (C) An example of cluster analysis showed the gene expression patterns of crypts at different time points after IR. (D) Gene expression changes in crypt and villus at different times after IR were plotted. Among them, about 1200 ESTs in crypts and 820 in villi were differentially expressed, either upregulated or downregulated for more than 2-fold.
Table 1 Signalling pathways involved in IR-induced response of crypt and villus after IR. Time after IR
Order
Crypt
Villus
4h
1 2 3 4 5 6 7 8 9 10
p53 signaling pathway Focal adhesion Metabolism of xenobiotics by cytochrome P450 Cell adhesion molecules (CAMs) Drug metabolism–cytochrome P450 Systemic lupus erythematosus Glioma MAPK signaling pathway Cell cycle Chronic myeloid leukemia
Type I diabetes mellitus Allograft rejection Graft-versus-host disease Antigen processing and presentation PPAR signaling pathway Autoimmune thyroid disease Cell cycle p53 signaling pathway Cell adhesion molecules (CAMs) Biosynthesis of unsaturated fatty acids
8h
1 2 3 4 5 6 7 8 9 10
Antigen processing and presentation Cell adhesion molecules (CAMs) Allograft rejection Graft-versus-host disease Type I diabetes mellitus Autoimmune thyroid disease MAPK signaling pathway Systemic lupus erythematosus Prostate cancer GnRH signaling pathway
Cell cycle DNA polymerase p53 signaling pathway Pyrimidine metabolism PPAR signaling pathway Systemic lupus erythematosus Small cell lung cancer Purine metabolism Mismatch repair Antigen processing and presentation
24 h
1 2 3 4 5 6 7 8 9 10
p53 signaling pathway Pyrimidine metabolism MAPK signaling pathway Antigen processing and presentation Cell adhesion molecules (CAMs) Cell cycle Colorectal cancer Allograft rejection Pancreatic cancer Glycan structures–biosynthesis 1
p53 signaling pathway Metabolism of xenobiotics by cytochrome P450 Antigen processing and presentation Type I diabetes mellitus Drug metabolism - cytochrome P450 Cell adhesion molecules (CAMs) Allograft rejection Graft-versus-host disease Autoimmune thyroid disease Glutathione metabolism
Note: p53 activation is induced by a number of stress signals, including DNA damage, oxidative stress and activated oncogenes. The p53 protein is employed as a transcriptional activator of p53-regulated genes. This results in three major outputs; cell cycle arrest, cellular senescence or apoptosis. Other p53-regulated gene functions communicate with adjacent cells, repair the damaged DNA or set up positive and negative feedback loops that enhance or attenuate the functions of the p53 protein and integrate these stress responses with other signal transduction pathways.
P53 was activated and translocated to nuclear in crypt at the early stage of IR-induced response To confirm the early involvement of p53 signaling pathway in IR-induced response indicated by the microarray analysis, the protein extracted from crypt and villus was examined by Western
blot. In normal crypt and villus, p53 activation was not obvious, if any. IR-induced DNA damage quickly activated p53 in crypt from 1 h to 4 h, which receded at 8 h. In contrast, no obvious p-P53 protein was detected in villus during the same period (Fig. 5A). As already known, P53 protein takes its effect by translocating to nuclear and functioning as a transcription factor. When nuclear
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protein extracted from crypt cells was probed for p-P53 protein, the P53 content was extremely high at 1 h after IR challenge, and gradually attenuated from 4 h to 8 h (Fig. 5B). The EMSA result showed an increased P53 protein in the nuclear of crypt cells and the ability to bind oligoDNA with P53-specific sequence. The mobility shift of DNA: P53 complex was detectable for normal level of P53 in the crypt (Fig. 5C, lane 1). Irradiation resulted in a heavy shift band at 1 h (lane 2), a less intensified band at 4 h (lane 3) and almost invisible band after 8 h (lane 4). The competitive binding test of the cold probe confirmed the specific binding of nuclear P53 protein with the DNA probe (lane 5). IHC staining demonstrated that the activated P53 protein was not detected in normal crypt (Fig. 5E), and was dramatically increased in crypt, especially in the lower part, but not in the villus (Fig. 5D). Discussion Adult stem cells are vulnerable to DNA damaging agents. DNA repair defects are associated with premature aging, stem cell exhaustion and carcinogenesis, indicating the indispensable role of DNA repair in stem cell self-renew and genomic stabilization [18]. To testify our assumption that the cells in different differentiation status may respond differently to DNA damage in vivo, the well-defined crypt-villus axis of small intestinal epithelium was adopted in this study. The compartments of crypt and villus were purified to study the IR-induced molecular changes by microarray analysis and protein blot. The isolated crypts was heterogeneous and contained stem cells, progenitor cells and transit
Fig. 5. P53 activation in crypt after IR. (A) Phosporylated P53 protein was not detectable in normal crypt and villus (0 h). However, high expression of p-p53 appeared 1–4 h after IR in crypt but not in villus. NS indicated the non-specific bands of the blotting. (B) The level of phosporylated P53 protein in the nuclear of irradiated crypt cells was significantly increased 1–4 h after IR. (C) EMSA assay confirmed that nuclear p-P53 in crypt cells 1–4 h after IR was able to bind to a sequence-specific oligoDNA and resulted in the shift bands. The competitive binding of cold probe abrogated the shift. (D) IHC staining allocated the activated P53 to the crypt base of irradiated mice, while the normal crypt did not obvious P53 activation (E). Bar = 25 lm.
amplifying cells, while the histopathological study can provide more detail information related with cell differentiation status and can determine the exact location where the molecular events happened in crypt-villus unit after immunostaining. The applications of these methods are mutually complementary. Based on our data, the IR-induced response of the crypt stem cells and progenitor cells can be summarized as (1) quick H2AX phosphorylation, and faster elimination compared with that in villus; (2) the transition of these proliferating cells from Ki67 positive to negative, indicating their exit from cell cycle; (3) the occurrence of extensive apoptosis, which effectively avoid gene mutation and may partially contribute to the quicker elimination of cH2AX foci in crypts; (4) high p-P53 expression in crypt base, an area of negative Ki67, which may control cell cycle arrest and apoptosis. Taken together, these results suggest that crypt stem cells and progenitor cells respond faster and stronger to IR in terms of DNA repair, p53 activation, exit of cell cycle, and apoptosis. There are two types of stem cells in murine small intestine. One locats below the +4 position in the ‘‘stem cell zone’’ and the other locates at the +4 position from the crypt bottom [19]. The rapidly cycling daughter cells, i.e. the transit amplifying cells, undergo a limited number of cell divisions before terminally differentiating into functional epithelial cells. The different response of crypt cells and villus cells to irradiation we observed at very early time may be oriented by their physiological roles. For example, in order to secure genome integrity and stability, the cells in stem cell zone quickly exhibited cell cycle arrest either repair DNA damage or undergo apoptosis if the damage is too severe to be repaired. In contrast, the transit amplifying cells, which are usually in active proliferation, did not response as fast as stem cells. The lag of p53 induction and cell cycle arrest in transit amplifying cells could be explained by (1) the limited cell division ability made these cells less possibility of canceration even if they carried DNA damage or error repair; (2) the maintenance of Ki67 expression early after TBI may reflect these cells still functioned as a cell source for villus epithelium; (3) these cells could undergo late cell death in a p53independent way as Merritt et al. suggested [20]. Potten’s group and others have done much work on titering the frequencies of crypt cell apoptosis and proliferation with different doses of irradiation. Their observing time points included 4.5 h [21], 4.5–40 h [20], 1–3.5 d [22] after TBI, while we focused on 4–8 h for apoptosis and proliferation. We observed the crypt cell response at very early phase, and the characterized cell responses to IR were not only consistent with their conclusions but also added more chronological information of the responses. Merritt et al. [20] proposed two phases of crypt cell death after 8 Gy irradiation, the p53-dependent early phase (mainly 4.5–12 h) and p53independent late phase (>24 h). The apoptotic cells we observed at 4 and 8 h is more frequently located at the lower part of crypt, where p53 is induced and Ki67 expression diminished. Our observation on intestinal epithelium of mice with 12 Gy TBI provided new supportive evidence to the p53-dependent early cell death, which is consistent with 5 Gy irradiated mice [23]. Gene expression profiling showed that genes related to apoptosis (p53) and cell cycle were extensively activated in crypt. These results agree with the pathological features of IR-treated epithelial cells, especially the stem cells/progenitors in crypt. P53 is considered as a universal sensor of genotoxic stress, and plays roles in different manners of DNA repair [24,25] and cell cycle checkpoint regulation after IR [26] through various signal pathways, such as ERK [27] and NF-kappaB [28]. It has been reported that P53 primes DNA repair, apoptotic cell death and H2AX activation [29]. In our study, at 1–4 h of DNA damage, p53 protein is activated quickly in crypt, and its location is confined to the lower part of crypt. We further confirmed that activated P53 protein enters into nucleus and possesses the DNA binding ability as a transcription
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factor. Miyoshi-Imamura et al. found that the apoptosis wave of irradiated small intestinal crypts peaked at 3 h accompanied by increased p53 expression [30]. The augmented p53 nuclear retention may sensitize crypt stem cells to p53-dependent apoptosis [31]. The differentially expressed genes in microarray analysis are highly correlated with p53 pathway, indicating that p53 signaling is the most important one in IR response (i.e., DNA repair and cell cycle control) of crypt, especially at the early stage. In contrast, villus cells are less sensitive to IR since they show a slower response compared with crypt cells. The early response of villus cells to IR primarily limits to nucleotide metabolic events and biochemical adjustment. Potten has suggested that stem cells in the crypts are intolerant of radiation and they commit an altruistic TP53dependent cell suicide (apoptosis) [4]. In summary, present study demonstrates the cell differentiation-oriented response to DNA damage in small intestinal epithelium early after irradiation. The crypt stem cells/progenitor cells displayed p53 signal pathway activation along with cell cycle arrest, DNA repair, and apoptosis to prevent gene mutation. The transit amplifying cells, which was the cell supply for epithelial turnover, kept proliferating. The villus cells conducted metabolic response with lagged and weak p53 response. All these data suggested that DNA damage responses are coupled with cell differentiation status. Differentiation of the primitive enterocytes is usually completed before the cells reach the crypt-villus junction, a gate once the cells migrate through they will exposed to the lumen environment of intestine. It is noteworthy that the cells in the region of crypt-villus junction may be a special population since they do not proliferate anymore and do not exhibit early p53 response under IR challenge. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.abb.2013.11.012. References [1] L.G. van der Flier, H. Clevers, Annu. Rev. Physiol. 71 (2009) 241–260.
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P53-participated cellular and molecular responses to irradiation are cell differentiation-determined in murine intestinal epithelium q Fengchao Wang a,1, Jin Cheng b,1, Dengquan Liu a, Huiqin Sun a, Jiqing Zhao b, Junping Wang a, Junjie Chen c, Yongping Su a,⇑, Zhongmin Zou b,⇑ a Institute of Combined Injury, State Key Laboratory of Trauma, Burns and Combined Injury, Department of Radiation Medicine, School of Preventive Medicine, The Third Military Medical University, 30 Gaotanyan Street, Shapingba District, Chongqing 400038, China b Department of Chemical Defense, School of Preventive Medicine, The Third Military Medical University, 30 Gaotanyan Street, Shapingba District, Chongqing 400038, China c Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Room Number Y3.6006, 1515 Holcombe Blvd, Houston, TX 77030, USA
a r t i c l e
i n f o
Article history: Received 15 October 2013 and in revised form 18 November 2013 Available online 4 December 2013 Keywords: DNA damage response Small intestine epithelium Apoptosis DNA repair Signaling pathway
a b s t r a c t Aim: Cells respond differently to DNA damaging agents, which may related to cell context and differentiation status. The aim of present study was to observe the cellular and molecular responses of cells in different differentiation status to ionizing irradiation (IR). Methods: Crypt-villus unit of murine small intestine was adopted as a cell differentiation model. DNA damage responses (DDRs) of crypt and villus were observed 1–24 h after 12 Gy IR using gene expression microarray analysis, immunohistochemical staining, Western blotting and Electrophoretic Mobility Shift Assay. Results: Microarray analysis revealed that most differentially expressed genes were related to p53 signaling pathway in crypt 4 h after IR and in both crypt and villus 24 h after IR. In crypt stem cells/progenitor cells, H2AX was phosphorylated and dephosphorylated quickly, Ki67 attenuated, cell apoptosis enhanced, phosphorylated P53 increased and translocated into nuclear with the ability to bind p53specific sequence. In upper crypt (transit amplifying cells) and crypt-villus junction, cells kept survive and proliferate as indicated by retained Ki67 expression, suppressed p53 activation, and rare apoptosis. Conclusions: DDRs varied with cell differentiation status and cell function in small intestinal epithelium. P53 signaling pathway could be an important regulatory mechanism of DDRs. Ó 2013 Elsevier Inc. All rights reserved.
Cell differentiation-determined cellular and molecular responses to irradiation in murine crypt and villus in vivo.
Introduction Histologically, small intestinal epithelial cells form a cell differentiation hierarchy along the crypt-villus axis. Small intestinal stem cells/progenitor cells locate in the crypt base, immediately above or in between of the Paneth Cells [1]. Transit amplifying cells, the immediate descendant cells of crypt stem cells, locate in the middle and upper part of crypt and differentiate into the epithelial cells while migrating upwards. Differentiated and functional epithelial cells cover the whole surface of villus. These cells continuously migrate along the surface of villus and q
finally shed off at the villus tip, finishing the course of epithelial cell turnover. This self-refresh process of epithelial cells takes as short as 3–5 days with a clear migration route [2]. Thus, small intestinal epithelium is a good model for studying cell proliferation, differentiation, apoptosis as well as DNA2 damage and repair [3,4]. Damaged DNA in a stem cell may cause gene mutation or canceration if neither the DNA damage is repaired nor the cell goes into apoptosis [5]. So, DNA repair in stem cells is of importance in both carcinogenesis and stemness maintenance. DNA repair ability is evolutionally conserved among species, but different DNA repair processes are involved in embryonic stem cells and differentiated cells [6,7]. P53 protein has been recognized as a key regulator of DNA damage and repair responses. It is unclear that if the activation of p53 signaling pathway relies on cell
Grant support: Natural Science Foundation of China (30828007).
⇑ Corresponding author. Fax: +86 23 68752285 (Z. Zou), fax: +86 23 68752009 (Y. Su). E-mail addresses: [email protected] (Y. Su), [email protected] (Z. Zou). 1 These authors contributed equally to this work. 0003-9861/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2013.11.012
2 Abbreviations used: DDR, DNA damage response; DSB, double strand break; H&E, hematoxylin and eosin; IHC, immunohistochemistry; IR, ionizing irradiation; p-,phosphorylated; PI3K, phosphatidylinositol-3-kinase; TBI, total body irradiation; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; EMSA, Electrophoretic Mobility Shift Assay.
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differentiation status, and why cells respond with different levels of p53 activation. We presume that cellular and molecular responses to DNA damage may vary with cell differentiation status, and p53 is critical in regulating the responses. Adult stem cells, such as hematopoietic stem cells and small intestinal crypt cells [8–10], are cell sources that replace aged and damaged cells, and very sensitive to ionizing radiation (IR). With the development of stem cell transplantation and hematopoietic growth factor production, hematopoietic type of acute radiation syndrome (ARS) becomes curable. But, there is no successful rescue in human with intestinal type of ARS. Local IR on abdomen is common for radiotherapy of abdominal tumor, and high dose exposure sometimes occurs in accidental irradiation. As the hematopoiesis can be successfully maintained under such situation, the protection and treatment of intestinal injury become the key questions. Understanding the early response of intestinal cells to irradiation may help clinical intervention on IR-induced intestinal injury. In this study, mice were challenged with 12 Gy total body irradiation (TBI), a dose that can cause intestinal type of ARS with half crypt survived [11]. We investigate the characteristics of DNA damage response (DDR), especially p53 signaling pathway-related events, of adult stem cells and their differentiated descendants in cyprt-villus unit in vivo in order to find the commons and the differences among different cell populations.
Gene chip hybridization and analysis The isolated crypts and villi at 0 h, 4 h, 8 h and 24 h after IR were homogenated in Trizol for total RNA extraction, and the extracts of the same group were pooled for RNA quality control test and microarray. The quality and quantity of the extracted RNAs were checked to ensure a convincible microarray comparison of gene expression profiling. The reverse transcription, fluorescence labeling, and hybridization on Mouse Genome 430 2.0 chip (Affymetrix) were conducted by Beijing Biocapital Company. The call values of Perfect Match (PM) and MisMatch (MM) were detected by AffymetrixÒ GeneChipÒ Scanner 3000. The difference degree between these two values and Computed P value was discriminated. Significant level was exactly represented by a margin region (0.04–0.06) to reduce false positive result, P < 0.04 demonstrates present gene and P > 0.06 demostrates absent gene. P value in the margin region means an uncertified result. Comparative analyses of expressed genes were carried out using AffymetrixÒ GeneChipÒ Operating SoftwareVersion1.4. Gene expression was considered increased or decreased only if the levels changed by greater than twofold with P < 0.0025 for increased expression and P > 0.9975 for decreased expression to reduce false discovery rate. Upregulated or downregulated genes were also clustered by Cluster 3.0 to get the category distribution. H&E, TUNEL, IHC staining and Western blot
Materials and methods Materials Mouse monoclonal anti p-P53 antibody were products of Cell Signal Technology (German). Other antibodies included mouse monoclonal anti-phospho-H2AX (cH2AX) antibody (Abcam, USA), Anti-Lamin B (Abcam, USA), rabbit monoclonal anti-Ki67 antibody (LabVision, Fremont, CA, USA) and corresponding secondary antibodies. Mouse Genome 430 2.0 chip (Affymetrix, Santa Clara, CA, USA), RIPA buffer (Beyotime Inc. Nantong, China), LightShiftÒ Chemiluminescent EMSA Kit (Pierce, Rockford, IL, USA), Tissue DNA Kit (Omega, Norcross, USA) were purchased from vendors in China.
Polyformaldehyde fixed 4 lm-thick deparaffinized sections was prepared for hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and immunohistochemistry (IHC) staining. TUNEL staining was processed according to the procedure of ROCH Company. After retrieving antigen activity and inhibiting endogenous peroxidase activity, standard IHC protocol was followed with primary antibodies (mouse monoclonal anti-cH2AX, rabbit monoclonal anti-Ki67, overnight at 4 °C. All slides were read in a blinded manner by specialists. Crypt and villus were lyzed in RIPA buffer containing protease inhibitors, and the extracted protein was subjected to standard Western blotting. For the quantification of protein bands, Image J (1.38e, NIH, USA) was used to analyze the densitometry of each band. The adjusted gene expression level was presented as the ratio between the interest gene and the corresponding internal control, such as b-actin and Lamin B.
Animals and irradiation DNA ladder Adult male C57/BL6 mice (LD50/30 = 8 Gy) [12] weighted 20– 22 g were irradiated with cobalt source, at a dose rate of about 0.77 Gy/min. The absorption dose of TBI was 12 Gy, which causes gastrointestinal type of acute radiation sickness. Mice were sacrificed by cervical dislocation at 0, 1, 4, 8 and 24 h after TBI (n = 6) for small intestine sampling.
The extraction of genomic DNA from isolated crypts and villi was performed according to the procedure of Tissue DNA Kit (Omega). The extracted genomic DNA was resolved with agarose gel electrophoresis and analyzed by gel reader (Bio-Rad, USA). Electrophoretic Mobility Shift Assay (EMSA)
Separation of crypt and villus Method was referred to Neil’s report [13] and a modification [14]. Briefly, the jejunum of normal and irradiated mice was dissected, washed with cold PBS, inverted to expose the mucous membrane, washed again, and cut into 2–3 cm segments. The segments were digested in cold chelator buffer [14] at 4 °C with shaking. Villi and crypts in the supernatant of digested jejunum were separated by gravity precipitation. The isolated villus and crypt compartments were respectively pooled and store in liquid nitrogen for further study. The intestine segments as well as the isolated villus and crypt collections were also fixed in 4% polyformaldehyde for paraffin embedding.
The double strand probes for P53 [15] was synthesized, 50 endlabeled with biotin and purified by TaKaRa Biotechnology Co., Ltd. (Dalian, China). The cold probe contained the same sequence of oligoDNAs except the omission of biotin conjugation. EMSA assay was conducted according to the instruction of LightShiftÒ Chemiluminescent EMSA Kit. In brief, the crypt cell protein was incubated with biotin-conjugated probe (20 fmol/ll) in the presence or absence of corresponding cold probe (2000 fmol/ll) for 20 min. The product was resolved in non-denatured polyacrylamide gel. Standard reaction of biotin and horseradish peroxidase-conjugated streptavidin combination and chemiluminescence development were performed. The image was analyzed with Bio-Rad gel scanning imaging system (Bio-Rad Laboratories, Hercules, CA, USA).
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Statistical analysis Gene chip data were analyzed as described above. Other experiment data are evaluated by one-way ANOVA and presented as mean ± standard derivation of measurement.
Results H2AX was extensively phosphorylated in epithelium early after the IR damage, but eliminated faster in crypts DNA damage usually causes quick H2AX phosphorylation (cH2AX), which is critical for DNA damage-induced foci formation. Therefore, cH2AX foci analysis is a sensitive indicator of double– strand breaks (DSBs), and the kinetics of cH2AX foci loss strongly correlate with the time course of localized chromatin decondensation [16] and DSB repair [17]. In the small intestine of normal control mice, only a few cH2AX foci located in the upper part of crypts and crypt-villus junction area, where the cells are in active DNA replication and recombination due to cell proliferation and differentiation, and at the tips of villi, where the cells are aged to shed off (Fig. 1A and D). At 1 h after 12 Gy TBI, both crypts and villi, displayed very strong cH2AX staining (P > 0.05, Fig. 1B and D), indicating extensive DNA damage and H2AX phosphorylation in the damaged epithelial cells. Interestingly, the cH2AX foci in crypts were more effectively eliminated than that in villi 4 h after IR (P < 0.05, Fig. 1C and D), which suggested higher DNA repair ability and/or faster elimination of damaged cells in crypts (see below). To quantitatively confirm the above result, Western blot was applied to detect cH2AX level. Consistently, normal crypt and villus contained a little cH2AX. In contrast, cH2AX level was significantly increased in both crypt and villus at 1 h after IR. However, the cH2AX
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level in crypt decreased significantly faster than that in villus at 4 h (P < 0.05, Fig. 1E and F). Apoptosis occurred early after IR was mainly in crypts It is commonly accepted that cells with failed DNA repair are usually eliminated through apoptosis or other cell death pathways to avoid neoplasma development. In normal intestinal epithelium, TUNEL staining revealed that apoptosis occurred only at the tips of villi (Fig. 2A). Very early after IR, there was no obvious increase in cell apoptosis (1.8 ± 0.8 cells/crypt, Fig. 2B). However, the apoptotic cells abruptly appeared at 4 h and were mainly presented at the crypt bases (11.8 ± 2.9 cells/crypt), where the stem cells and progenitor cells reside. Some apoptotic cells were also presented in lamina propria, which could be endothelial cells and blood cells, such as IR-sensitive lymphocytes (Fig. 2C). The amount of the apoptotic cells decreased at 8 h in the epithelium (6.2 ± 2.2 cells/ crypt), but still kept at high level. The much less apoptotic cells left in lamina propria might result from the physiological clearance of apoptotic cells in blood flow and in mesenchyme as well (Fig. 2D). H&E staining showed similar result to TUNEL staining. Although 1–8 h after IR, the whole mucous membrane maintained the normal histological structure, there were many apoptotic cells in crypt 4 h after IR. The apoptotic crypt cells exhibited karyopyknosis, karyorrhexis and cell body shrinkage, which gave a heavy staining in nuclei and cytoplasm (7.2 ± 1.6 cells/crypt, Fig. 2F) compared with normal control (1.3 ± 0.6 cells/crypt, Fig. 2E). Genomic DNA electrophoresis showed a typical DNA ladder pattern of apoptosis in crypt cells but was not detectable in villus cells at 4 h after IR (Fig. 2G). Colonal cells were used as positive control to show DNA ladder although they are not as sensitive to IR as the crypt cells. IR significantly suppressed the proliferation of crypt cells Physiologically, the crypt cells are in active proliferation to continuously provide new cells for the quick turnover of intestinal epithelium. In normal intestinal epithelium, most of the crypt cells showed positive staining for Ki67, a marker for proliferating cells (Fig. 3A and B). Interestingly, cells in between the Paneth Cells, according to the histological structure, were also Ki67 positive, which is consistent with the recent finding of the location of intestinal stem cells [1]. Four hours after IR, the average numbers of Ki67 positive cells per crypt were significantly decreased when compared with normal control (19.2 ± 2.3 vs 26.8 ± 3.0, P < 0.05), and the staining intensity was also decreased. The Ki67 positive cells were mainly confined to transit amplifying cells, the upper part of crypt (Fig. 3C and D). The results suggested that IR preferentially arrested the proliferation of stem/progenitor cells, which was well consistent with the result of TUNEL staining. p53 pathway played important roles in IR-induced response in crypts
Fig. 1. H2AX phosphorylation in small intestinal epithelium of normal and irradiated mice. (A) IHC staining revealed weak cH2AX signals in the middle and upper part of crypts, as well as the villus tips of normal small intestine. (B) At 1 h after 12 Gy total body irradiation, there was an extensive cH2AX staining in the whole intestinal epithelium, i.e., from crypt to villus. (C) At 4 h after IR, cH2AX level in crypt was notably reduced while high level remained in villus. (D) Quantification of the IHC staining on phosphorylate H2AX. (E) The cH2AX level in crypts and villi after IR assessed by Western blot using whole cell lystes. (F) Quantification of Western blot by densitometric analysis, which was consistent with IHC results. Bar = 100 lm in A–C.
To precisely define the different molecular and biochemical responses of crypt and villus to IR-induced DNA damage, we isolated these two cell compartments with well-preserved morphology and satisfying purity [14]. The isolated villi showed a relatively intact fingerstall-like structure (Fig. 4A), and the crypts a caterpillar-like structure (Fig. 4B), both resembled their in situ histological morphology in vivo. As the whole procedure was taken at 4 °C, the molecular events happened in vivo in the epithelium could be further explored by in vitro analysis on the isolated crypt and vilus compartments [13–15]. To get an overview of cell response after IR stress, the gene expression profiles of crypt and villus were assessed by microarray. A total of about 1200 expressed sequence tags (ESTs) expressed
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Fig. 2. Apoptosis of small intestine epithelial cells after IR detected by TUNEL staining. There were some apoptotic cells at the tip of villus and few positive cells scattered in epithelium of (A) normal mice and of (B) mice 1 h post-irradiation. (C) Many cells in crypt bases and some cells in lamina propria exhibited positive staining at 4 h after IR, indicating cell apoptosis occurred. (D) Apoptotic cell number decreased at 8 h after IR, but still significantly higher than the control. (E and F) In 5 H&E stained tissue sections from normal (E) or irradiated (F) mice, many cells showed karyopyknosis and karyorrhexis in crypts 4 h after IR (arrows) comparing with normal crypt. (G) To quantify the apoptotic cells, 50 crypts were randomly chosen to count apoptotic cells by two independent investigators. a, P < 0.05 vs control. (H) Genomic DNA electrophoresis of crypt cells showed a characteristic DNA ladder pattern of apoptosis 4 h after IR, but not in villus cells. Whole colon cells were used as a parallel control in the whole process of sample preparation. A–D: Bar = 50 lm; E and F: Bar = 20 lm.
Fig. 3. Expression of cell proliferation marker Ki67 in small intestinal epithelium. (A) Nuclei of normal crypt cells were positive for Ki67 in the whole crypt bases except Paneth Cells, and deeply stained in the middle part of crypt. (C) Four hours after IR, the Ki67 positive cells decreased and confined to the upper part of crypt, where the transit amplifying cells reside. The cells in the middle part of crypt turn to Ki67 negative, indicating their exit from cell cycle. (B and D) were the enlarged areas of boxes in (A and C) respectively. Arrows indicated Ki67 positive cells. A and C: Bar = 100 lm; B and D: Bar = 30 lm.
differentially, either upregulated or downregulated for more than 2-fold, at least at one of the checked time points in crypt compared with that of the normal control (Fig. 4C and D). In the timematched villus, about 820 different ESTs expressed differentially (Fig. 4C and D). In both crypt and villus, cell signal pathway cluster analysis (Table 1 and Supplemental Table 1) showed that the most important signaling pathway involved in IR-induced responses was p53 signaling pathway (including p53, ataxia telangiectasia and Rad3 related [ATR], transformation related protein 53 inducible nuclear protein 1 [Trp53inp1], transformation related protein 53 regulating kinase, transformation related protein 53 binding protein 1 [trp53bp], p21), followed by cell cycle (including cyclin G1,
D2, D1, B1, and E2, cyclin-dependent kinase 6), MAPK and cell adhesion pathways. The activation of p53 signaling pathway in crypt was the most predominant at 4 h and 24 h after IR. Although p53 pathway was also activated in villus, it was not the major one until 8 h after IR (Table 1). This may indicate that, when DNA damage occurred, crypt cells responded quickly on cell cycle control because the crypt cells, especially in the crypt base, are normally in active proliferation. In contrast, the villus cells primarily responded in the aspects of nuleotide synthesis and biochemical metabolism instead of cell cycle regulation since the villus cells usually are not in cell cycle any more.
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Fig. 4. The gene expression profiles of intestinal crypts and villi from normal and irradiated mice. Three independent replications were done for isolation. Total RNA was extracted from the well preserved and isolated crypts (A) and villi (B), labeled with Cyt3 and Cyt5, and hybrided with probe sets on microarray chips. (C) An example of cluster analysis showed the gene expression patterns of crypts at different time points after IR. (D) Gene expression changes in crypt and villus at different times after IR were plotted. Among them, about 1200 ESTs in crypts and 820 in villi were differentially expressed, either upregulated or downregulated for more than 2-fold.
Table 1 Signalling pathways involved in IR-induced response of crypt and villus after IR. Time after IR
Order
Crypt
Villus
4h
1 2 3 4 5 6 7 8 9 10
p53 signaling pathway Focal adhesion Metabolism of xenobiotics by cytochrome P450 Cell adhesion molecules (CAMs) Drug metabolism–cytochrome P450 Systemic lupus erythematosus Glioma MAPK signaling pathway Cell cycle Chronic myeloid leukemia
Type I diabetes mellitus Allograft rejection Graft-versus-host disease Antigen processing and presentation PPAR signaling pathway Autoimmune thyroid disease Cell cycle p53 signaling pathway Cell adhesion molecules (CAMs) Biosynthesis of unsaturated fatty acids
8h
1 2 3 4 5 6 7 8 9 10
Antigen processing and presentation Cell adhesion molecules (CAMs) Allograft rejection Graft-versus-host disease Type I diabetes mellitus Autoimmune thyroid disease MAPK signaling pathway Systemic lupus erythematosus Prostate cancer GnRH signaling pathway
Cell cycle DNA polymerase p53 signaling pathway Pyrimidine metabolism PPAR signaling pathway Systemic lupus erythematosus Small cell lung cancer Purine metabolism Mismatch repair Antigen processing and presentation
24 h
1 2 3 4 5 6 7 8 9 10
p53 signaling pathway Pyrimidine metabolism MAPK signaling pathway Antigen processing and presentation Cell adhesion molecules (CAMs) Cell cycle Colorectal cancer Allograft rejection Pancreatic cancer Glycan structures–biosynthesis 1
p53 signaling pathway Metabolism of xenobiotics by cytochrome P450 Antigen processing and presentation Type I diabetes mellitus Drug metabolism - cytochrome P450 Cell adhesion molecules (CAMs) Allograft rejection Graft-versus-host disease Autoimmune thyroid disease Glutathione metabolism
Note: p53 activation is induced by a number of stress signals, including DNA damage, oxidative stress and activated oncogenes. The p53 protein is employed as a transcriptional activator of p53-regulated genes. This results in three major outputs; cell cycle arrest, cellular senescence or apoptosis. Other p53-regulated gene functions communicate with adjacent cells, repair the damaged DNA or set up positive and negative feedback loops that enhance or attenuate the functions of the p53 protein and integrate these stress responses with other signal transduction pathways.
P53 was activated and translocated to nuclear in crypt at the early stage of IR-induced response To confirm the early involvement of p53 signaling pathway in IR-induced response indicated by the microarray analysis, the protein extracted from crypt and villus was examined by Western
blot. In normal crypt and villus, p53 activation was not obvious, if any. IR-induced DNA damage quickly activated p53 in crypt from 1 h to 4 h, which receded at 8 h. In contrast, no obvious p-P53 protein was detected in villus during the same period (Fig. 5A). As already known, P53 protein takes its effect by translocating to nuclear and functioning as a transcription factor. When nuclear
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protein extracted from crypt cells was probed for p-P53 protein, the P53 content was extremely high at 1 h after IR challenge, and gradually attenuated from 4 h to 8 h (Fig. 5B). The EMSA result showed an increased P53 protein in the nuclear of crypt cells and the ability to bind oligoDNA with P53-specific sequence. The mobility shift of DNA: P53 complex was detectable for normal level of P53 in the crypt (Fig. 5C, lane 1). Irradiation resulted in a heavy shift band at 1 h (lane 2), a less intensified band at 4 h (lane 3) and almost invisible band after 8 h (lane 4). The competitive binding test of the cold probe confirmed the specific binding of nuclear P53 protein with the DNA probe (lane 5). IHC staining demonstrated that the activated P53 protein was not detected in normal crypt (Fig. 5E), and was dramatically increased in crypt, especially in the lower part, but not in the villus (Fig. 5D). Discussion Adult stem cells are vulnerable to DNA damaging agents. DNA repair defects are associated with premature aging, stem cell exhaustion and carcinogenesis, indicating the indispensable role of DNA repair in stem cell self-renew and genomic stabilization [18]. To testify our assumption that the cells in different differentiation status may respond differently to DNA damage in vivo, the well-defined crypt-villus axis of small intestinal epithelium was adopted in this study. The compartments of crypt and villus were purified to study the IR-induced molecular changes by microarray analysis and protein blot. The isolated crypts was heterogeneous and contained stem cells, progenitor cells and transit
Fig. 5. P53 activation in crypt after IR. (A) Phosporylated P53 protein was not detectable in normal crypt and villus (0 h). However, high expression of p-p53 appeared 1–4 h after IR in crypt but not in villus. NS indicated the non-specific bands of the blotting. (B) The level of phosporylated P53 protein in the nuclear of irradiated crypt cells was significantly increased 1–4 h after IR. (C) EMSA assay confirmed that nuclear p-P53 in crypt cells 1–4 h after IR was able to bind to a sequence-specific oligoDNA and resulted in the shift bands. The competitive binding of cold probe abrogated the shift. (D) IHC staining allocated the activated P53 to the crypt base of irradiated mice, while the normal crypt did not obvious P53 activation (E). Bar = 25 lm.
amplifying cells, while the histopathological study can provide more detail information related with cell differentiation status and can determine the exact location where the molecular events happened in crypt-villus unit after immunostaining. The applications of these methods are mutually complementary. Based on our data, the IR-induced response of the crypt stem cells and progenitor cells can be summarized as (1) quick H2AX phosphorylation, and faster elimination compared with that in villus; (2) the transition of these proliferating cells from Ki67 positive to negative, indicating their exit from cell cycle; (3) the occurrence of extensive apoptosis, which effectively avoid gene mutation and may partially contribute to the quicker elimination of cH2AX foci in crypts; (4) high p-P53 expression in crypt base, an area of negative Ki67, which may control cell cycle arrest and apoptosis. Taken together, these results suggest that crypt stem cells and progenitor cells respond faster and stronger to IR in terms of DNA repair, p53 activation, exit of cell cycle, and apoptosis. There are two types of stem cells in murine small intestine. One locats below the +4 position in the ‘‘stem cell zone’’ and the other locates at the +4 position from the crypt bottom [19]. The rapidly cycling daughter cells, i.e. the transit amplifying cells, undergo a limited number of cell divisions before terminally differentiating into functional epithelial cells. The different response of crypt cells and villus cells to irradiation we observed at very early time may be oriented by their physiological roles. For example, in order to secure genome integrity and stability, the cells in stem cell zone quickly exhibited cell cycle arrest either repair DNA damage or undergo apoptosis if the damage is too severe to be repaired. In contrast, the transit amplifying cells, which are usually in active proliferation, did not response as fast as stem cells. The lag of p53 induction and cell cycle arrest in transit amplifying cells could be explained by (1) the limited cell division ability made these cells less possibility of canceration even if they carried DNA damage or error repair; (2) the maintenance of Ki67 expression early after TBI may reflect these cells still functioned as a cell source for villus epithelium; (3) these cells could undergo late cell death in a p53independent way as Merritt et al. suggested [20]. Potten’s group and others have done much work on titering the frequencies of crypt cell apoptosis and proliferation with different doses of irradiation. Their observing time points included 4.5 h [21], 4.5–40 h [20], 1–3.5 d [22] after TBI, while we focused on 4–8 h for apoptosis and proliferation. We observed the crypt cell response at very early phase, and the characterized cell responses to IR were not only consistent with their conclusions but also added more chronological information of the responses. Merritt et al. [20] proposed two phases of crypt cell death after 8 Gy irradiation, the p53-dependent early phase (mainly 4.5–12 h) and p53independent late phase (>24 h). The apoptotic cells we observed at 4 and 8 h is more frequently located at the lower part of crypt, where p53 is induced and Ki67 expression diminished. Our observation on intestinal epithelium of mice with 12 Gy TBI provided new supportive evidence to the p53-dependent early cell death, which is consistent with 5 Gy irradiated mice [23]. Gene expression profiling showed that genes related to apoptosis (p53) and cell cycle were extensively activated in crypt. These results agree with the pathological features of IR-treated epithelial cells, especially the stem cells/progenitors in crypt. P53 is considered as a universal sensor of genotoxic stress, and plays roles in different manners of DNA repair [24,25] and cell cycle checkpoint regulation after IR [26] through various signal pathways, such as ERK [27] and NF-kappaB [28]. It has been reported that P53 primes DNA repair, apoptotic cell death and H2AX activation [29]. In our study, at 1–4 h of DNA damage, p53 protein is activated quickly in crypt, and its location is confined to the lower part of crypt. We further confirmed that activated P53 protein enters into nucleus and possesses the DNA binding ability as a transcription
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factor. Miyoshi-Imamura et al. found that the apoptosis wave of irradiated small intestinal crypts peaked at 3 h accompanied by increased p53 expression [30]. The augmented p53 nuclear retention may sensitize crypt stem cells to p53-dependent apoptosis [31]. The differentially expressed genes in microarray analysis are highly correlated with p53 pathway, indicating that p53 signaling is the most important one in IR response (i.e., DNA repair and cell cycle control) of crypt, especially at the early stage. In contrast, villus cells are less sensitive to IR since they show a slower response compared with crypt cells. The early response of villus cells to IR primarily limits to nucleotide metabolic events and biochemical adjustment. Potten has suggested that stem cells in the crypts are intolerant of radiation and they commit an altruistic TP53dependent cell suicide (apoptosis) [4]. In summary, present study demonstrates the cell differentiation-oriented response to DNA damage in small intestinal epithelium early after irradiation. The crypt stem cells/progenitor cells displayed p53 signal pathway activation along with cell cycle arrest, DNA repair, and apoptosis to prevent gene mutation. The transit amplifying cells, which was the cell supply for epithelial turnover, kept proliferating. The villus cells conducted metabolic response with lagged and weak p53 response. All these data suggested that DNA damage responses are coupled with cell differentiation status. Differentiation of the primitive enterocytes is usually completed before the cells reach the crypt-villus junction, a gate once the cells migrate through they will exposed to the lumen environment of intestine. It is noteworthy that the cells in the region of crypt-villus junction may be a special population since they do not proliferate anymore and do not exhibit early p53 response under IR challenge. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.abb.2013.11.012. References [1] L.G. van der Flier, H. Clevers, Annu. Rev. Physiol. 71 (2009) 241–260.
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