|Year : 2015 | Volume
| Issue : 2 | Page : 403-408
The effect of mesenchymal stem cells on the p53 methylation in irradiation-induced thymoma in C57BL/6 mice
Hai B Zheng1, Yan T Fu2, Zhen X Pan1, Tong W Yang1, Li H Sun1, Yu B Chen3, Ti Tong1
1 Department of Thoracic Surgery, Second Hospital of Jilin University, Changchun, China
2 Department of Thyroid Surgery, China-Japan Union Hospital of Jilin University, Changchun, China
3 Department of Rediotherapy, Second Hospital of Jilin University, Changchun, China
|Date of Web Publication||7-Jul-2015|
Second Hospital of Jilin University, 218 Ziqiang Street, Changchun-130041
Source of Support: None, Conflict of Interest: None
Context: Our previous studies showed that mesenchymal stem cells (MSCs) preferentially migrated to irradiation-damaged thymus tissue to maintain the thymus integrity and simultaneously decrease the incidence of thymoma from 57 to 37.5%.
Objective: This study was designed to investigate the mechanisms by which MSCs decrease the irradiation-induced thymoma formation.
Materials and Methods: Thymus genome DNA was extracted, treated with sulfite, and amplified by polymerase chain reaction (PCR) using bisulfite sequencing PCR (BSP) as primers. The PCR productions were sequenced after recovery from 1.5% agarose gel electrophoresis. These sequences were analyzed using ClustalW2-Multiple Sequence Alignment. p53 expression in thymus or thymoma was measured using immunohistochemistry.
Results: Study showed the p53 methylation in irradiation alone group took place at loci +143 and -1190, which are beyond known binding motif of transcription factors. However, Matlnspector Professional Database revealed that locus -1190 is located in binding region of E2A transcription factor. In the non tumor thymus tissues from MSCs-treated irradiated mice, p53 promoter methylation existed at four loci. Three loci of them located at either negative regulation regions or their vicinity. The methylation affects the interaction between transcription factors and p53 promoter to increase the expression of p53. Indeed, an increased p53 expression was detected by immunohistochemistry in thymus tissues from MSCs-treated irradiated mice as compared to irradiated alone mice.
Conclusion: MSCs decrease the incidence of irradiation-induced thymoma, which may be mediated by improving thymus microenvironment and changing the methylation of p53 promoter, and subsequently maintaining genome's stability.
Keywords: Irradiation, MSCs, methylation, p53, thymoma
|How to cite this article:|
Zheng HB, Fu YT, Pan ZX, Yang TW, Sun LH, Chen YB, Tong T. The effect of mesenchymal stem cells on the p53 methylation in irradiation-induced thymoma in C57BL/6 mice. J Can Res Ther 2015;11:403-8
|How to cite this URL:|
Zheng HB, Fu YT, Pan ZX, Yang TW, Sun LH, Chen YB, Tong T. The effect of mesenchymal stem cells on the p53 methylation in irradiation-induced thymoma in C57BL/6 mice. J Can Res Ther [serial online] 2015 [cited 2019 Nov 12];11:403-8. Available from: http://www.cancerjournal.net/text.asp?2015/11/2/403/160105
| > Introduction|| |
Mesenchymal stem cells (MSCs) transfused back to veins preferentially migrate into damaged tissues and organs to participate in wound repair (such as spinal cord, cartilage, skin, and intestines). , Our previous studies have also evidenced the ability of MSCs in wound repair. , [4,]5[,]6 Numerous investigations showed the carcinogenic effects of irradiation. This yields a question: Whether MSCs reduces the irradiation-induced tumorigenesis through tissue wound repair. We applied the traditional Kaplan irradiation to induce thymoma in mice. These mice received the irradiation once per week for 4 weeks. Then these mice were injected MSCs through caudal vein once per week for 2 weeks. We found that the incidence of thymoma was decreased to 37.5 from 57%.  This demonstrates the protective role of MSCs in irradiation-induced thymoma formation.
Previous studies revealed the correlation between the inhibitive effect of MSCs on tumors and molecules expression. Lu et al.,  reported that MSCs upregulated the expression of p21, a negative regulation factor in cell cycle, and mRNA expression of apoptosis-related protease caspase 3. The upregulation of p21 and caspase 3 caused cell cycle arrest in G0/G1 stages and then suppressed the growth of tumor cells. Khakoo et al.,  found that MSCs inhibited tumor growth via the activity suppression of Akt protein kinasein Kaposi's sarcoma model. Wang et al.,  reported that MSCs reduced the incidence of thymoma through down regulating β-catenin expression, a key regulation factor in Wnt signaling pathway, and the expression of c-myc and cyclin D1, two downstream target genes of β-catenin. However, the underlying regulation mechanisms by which MSCs affect the expression of related molecules are still not elucidated.
Interestingly, Brathwaite et al.,  irradiated C57BL/6J mice using g-ray to induce thymoma. In these thymoma tissues, the mutation rate of p53 was 13%. But not even one case of p53 mutation was found in the early stage of tumorigenesis. This indicates p53 mutation does not play a key role in irradiation-induced thymoma formation. As we know, abnormal methylation of CpG island in gene promoter region, which is non methylated in normal condition, causes gene inactivation and then tumorigenesis. Methylation of p53 was seen in some malignant tumors. ,, The methylation rates were 16% in breast tumor,  32% in acute lymphoblastic leukemia,  and 51.5% in ovarian tumor. 
Here, we randomly selected three thymus tissues per group from our previous research samples.  These samples were identified by pathology, including normal thymus tissues in normal control group, thymoma tissues inirradiation alone group, and non tumor thymus tissues in MSCs-treated irradiation group. Our results showed methylation existed in one sample in irradiation alone groups and MSCs-treated irradiation group. Another two samples in the two groups were not methylated. The detailed results are reported as following.
| > Materials and methods|| |
Preparation of irradiation-induced thymoma model
C57BL/6 female mice (13 ± 2 g) were supplied by the Experimental Animal Center of Jilin University. The X-ray therapy machine linear accelerator (VARIAN 23EX-354, USA) was used to irradiate the whole mice body at dosage rate of 300 cGy/s. The dosage was set up 1.75 Gy/time according to the description by Kaplan.  The irradiation was given once per week for 4 weeks. The total dosage was 7 Gy.
MSCs administration in irradiated mice
MSCs were isolated from C57BL/6 suckling mice. These mice were sacrificed, and put in 75% ethanol for 5 min. The femurs and humerus were taken out and metaphyses were cut off in sterile condition. The medullary cavity was repeatedly washed with Dulbecco's modified Eagle's medium (DMEM; Life Technologies Inc, USA) culture medium. The collected DMEM medium was filtered with 200 mesh screen. The filtered medium was centrifuged at 1,500 rpm/min for 5 min. The collected cells were resuspended in DMEM to make single cells. The single nucleus cells were isolated with Percoll isolation solution. These cells were counted and (4 ± 2)×10 6 cells were transferred to 25 cm 2 culture flask containing 10 ml DMEM including 15% fetal bovine serum (FBS; Hyclone Inc, USA) and cultured at 37°C in an atmosphere of 5% CO 2 . The culture medium was changed twice per week. When up to 80-90% full of the flask bottom, the cells were digested with 0.25% trypsin (Sigma Inc, USA) and pass aged at 1:2. The third passage of cells was used for further experiments.
On the day following the last irradiation, MSCs at the third passage were collected and adjusted to 2.0 × 10 6 cells/ml. 0.5 ml MSCs were injected into irradiated mice through caudal vein once per week for 4 weeks. The mice in irradiation alone (M) and normal control (C) groups were given 0.5 ml saline as control.
Pathological examination of thymus tissues
The thymus were taken out and fixed in 10% formaldehyde. Then the tissues were embedded in paraffin and sectioned. The sections were deparaffinized, dehydrated, stained with hematoxylin and eosin (H and E), and examined under microscope. From our previous research samples,  we randomly selected three pathologically identified thymus tissues from each group: Normal thymus tissues from control mice, thymoma tissues from irradiated alone mice, and non tumor thymus tissues from MSCs-treated irradiated mice.
Evaluation of p53 promoter methylation with bisulfite sequencing PCR
BSP primers were designed with MethPrimer (http://www.urogene.org/methprimer/index1.html) software based on the sequence of mouse p53 promoter from GenBank (reference No: AF287146.1) [Table 1]. The thymus genomic DNA was extracted using DNA Extraction Kit (Biotechnology (Dalian) Co., Ltd., China), and bisulfite treatment was performed using EZ DNA methylation-Gold Kit (Zymo, USA) and polymerase chain reaction (PCR) using BSP primers. The PCR system included 10 × PCR buffer 2.5 μl, dNTPs 1.0 μl, DNA 1.0 μl, sense primer 0.5 μl, antisense primer 0.5 μl, and Taq DNA polymerase (Biotechnology (Dalian) Co., Ltd., China) 0.2 μl, ddH 2 O, 9.3 μl. It was 25 μl of total volume. After an initial denaturation at 94°C for 2 min, the PCR cycling was performed as follows: 94°C for 30 s, 55-60°C for 30s, and 72°C for 30s. Amplification was performed for 30 cycles, followed by extension at 72°C for 5 min. The PCR products were electrophoresed in 1.5% agarose gel. Then the gel was recycled for sequencing. The sequence was analyzed using ClustalW2-Multiple Sequence Alignment (www.ebi.ac.uk/Tools/msa/clustalw2/). Primer synthesis and DNA sequencing were completed in Sangon Biotech (Shanghai).
Measurement of p53 expression with immunohistochemistry
The thymus tissues were embedded in paraffin and sectioned. After deparaffinization and dehydration, the sections were incubated in antigen repair solution for 15 min, endogenous peroxidase blocker for 10 min, and 2% bovine serum albumin (BSA) for 20 min at room temperate. The sections were then incubated in rabbit anti-mouse p53 antibody (Proteintech Inc, USA) at 4°C overnight, followed by horseradish peroxidase (HRP)-labeled goat anti-rabbit antibody (Santa Cruz Biotechnology Inc, USA) for 20 min at room temperate. Sections were thoroughly washed with PBS after each incubation. The sections were developed with 3,3'-diaminobenzidine (DAB), and contrast stained with hematoxylin, cleaned with xylene, and covered for examination under microscope.
| > Results|| |
Based on the pathological results of our previous research samples, we randomly selected three normal thymus tissues from control mice, three thymoma tissues from radiated alone mice, and three nontumor thymus tissues from MSCs-treated irradiated mice. The MethPrimer software was used to predict p53 promoter methylation. BSP primers targeting to four different regions were designed to analyze p53 promoter methylation.
Methylation of p53 promoter in irradiation-induced thymoma tissues
Methylation was evaluated in the sequence acquired from BSP based on that DNA methylation more frequently happens at CpG or CpNpG. p53 promoter methylation was not observed in all three thymus tissues in normal control group [Figure 1]b-Co. Among the three thymoma tissues in irradiation alone group, two thymoma tissues did not show p53 promoter methylation [Supplementary information, S1 [Additional file 1]], only another thymoma tissues was found p53 promoter methylation. This methylation was located at +143 of the first exon and −1190 of the promoter distal end [Figure 1]b-M. These two methylation loci were not included in the known transcription factors binding motif. However, Matlnspector Professional Database (www.genomatix.de/) analysis showed −1190 located in binding motif of transcription factor E2A [Figure 1]d and e. The methylation sequencing is shown in S2 [Additional file 2].
|Figure 1: Mesenchymal stem cells (MSCs) promote p53 promoter methylation in thymus tissue from irradiated mice. (a) p53 promoter methylation loci predicted by MethPrimer software and CpG island. = Methylation loci, = CpG island, /// = omitted bases, = exon 1, = bisulfite sequencing PCR (BSP) primers. (b) Distribution of p53 promoter methylation loci. (c) PCR was amplified using BSP primers. m= DNA marker, Co = Control group, M = irradiation alone group, T =MSCs-treated irradiation group. (d) Transcript factors binding motif of p53 promoter according to previous reports.[16,17] (e) Location relationship between p53 promoter methylation loci and transcription regulation regions, and the effect of methylation on transcription. Blue = Methylation locus in irradiation alone group, green = methylation loci in MSCs-treated irradiation group, purple = transcript factor binding motif, TSS = transcription initiation site, PCR = polymerase chain reaction|
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MSCs promotes p53 promoter methylation in thymus tissue from irradiated mice
BSP showed that in three nontumor thymus tissues from MSCs-treated irradiated mice, two did not show p53 promoter methylation (S1), only one thymus tissues exhibited methylation. The methylation located at +198 of the first exon and −1043, −1090, and −1158 of the promoter distal end [Figure 1]b-T. +198 located in the binding motif of transcription factor Pax (+195/+201). −1043 and −1090 located at the either side of the negative regulation region − 1065/−1084 [Figure 1]d and e. The sequence numbering of p53 promoter was based on Reisman et al.'s report in 2001.  Methylation sequencing is listed in S2.
MSCs upregulate p53 expression in thymus tissues
expression in thymus tissues in the three groups was measured with immunohistochemistry. The results showed that the p53 expression in thymoma tissues in irradiation alone group was higher than in normal control group, and it was higher in nontumor thymus tissues in MSCs-treated irradiation group than in irradiation alone group [Figure 2]b.
|Figure 2: MSCs upregulate p53 expression in thymus tissues.(a) Pathological analysis of thymus structure. (b) p53 expression in thymus tissues by immunohistochemistry. (c) β-actin expression in thymus tissues. (d) Quantification of p53 expression levels|
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| > Discussion|| |
DNA methylation is an important epigenetic modification that regulates genome function without changing the original DNA sequence. In mammal genome, the main locus of DNA methylation is CpG dinucleotide. CpG exists as sporadic and highly aggregation form. In the latter, CpG island is rich in promoter region and/or the first exon. According to the statistics, CpG content is high in 60% human gene promoters. 
Commonly, CpG of promoter region is presented as non-methylation state. CpG non-methylation can bind to transcription factor and transcription cofactor to regulate gene transcription.  Thus, promoter methylation may block the binding with transcription factors and interfere in gene transcription activity. Generally, DNA methylation inhibits gene transcription and causes gene expression decrease or stop, including cell cycle regulation gene CDKN2A,  apoptosis-related gene DAPK1,  DNA wound repair gene MGMT,  and subsequently promotes tumorigenesis. DNA methylation can also activate gene expression, such as melanoma-related tumor/testis antigen MAGE gene. Kim et al., tested 32 colorectal cancer cell lines.  Of them, 66% cell lines expressed MAGE-A3, and cell lines with low methylation of MAGE-A3 gene promoter were up to 81%. After treatment with methylation inhibitor 5-aza-2' -deoxycytidine, nine cell lines without MAGE-A3 expression re-express MAGE-A3. Thus Kim et al., thought that low methylation of MAGE-A3 gene promoter upregulated or reactivated MAGE-A3 expression. Definitely, epigenetics is closely related to the genesis and progression of tumors. Epigenetics is also affected by environmental factors.
MSCs maintain normal thymus structure in irradiated mice through participating in the reconstruction of thymus microenvironment.  However, no p53 mutation has been found in the early stage of irradiation-induced thymoma formation.  Therefore, p53 promoter methylation possibly accounts for the decreased irradiation-induced thymoma formation by MSCs. In the present study, BSP showed no p53 promoter methylation took place in all thymus tissues from control group, and only one thymus sample exhibited p53 promoter methylation in both irradiation alone and MSCs-treated irradiation groups.
It has been reported that there are at least nine conservative transcription factor binding motifs in mouse p53 promoter [Figure 1]d.  Further analysis of the samples with methylation showed that the loci of p53 methylation were + 143 and − 1190 in irradiation alone group. However, these two loci do not locates in the known transcription regulation region. Furthermore, the analysis via Matlnspector Professional Database showed that −1190 is located in the binding motif of transcription factor E2A (−1181/−1197). E2A is one of the members of transcription factor bHLH family, and is necessary for B-cell formation. The formation of B-cells is inhibited in E2A gene knockout mice.  E2A promotes p21 expression but suppresses PUMA expression, and controls the fate of cells which dependonp53 activation.  However, more experimental evidences are needed to confirm what role E2A plays in p53 expression: positive regulation or negative regulation.
There were four p53 promoter methylation loci in nontumor thymus tissue from MSCs-treated irradiated mice. One of them located at + 198 of the first exon, and in the binding region of transcription factor Pax (+195/+201) [Figure 1]d and e.  Pax inhibits the activation of p53 promoter. The methylation in this region blocks the inhibitive effect of Pax and then increases p53 expression. The other three loci are located at −1043, −1090, and −1158 at the distal end of the promoter. Boggs and Reisman  disclosed that there were three regulation regions at the upstream of p53 transcription initial site: −403/−432, −601/−620, and −1065/−1084 [Figure 1]d and e. Among them, −601/−620 was positive regulation region with which C/EBPβ and RBP-Jk bound to increase p53 expression. The other two regions (−403/−432 and −1065/−1084) were negative regulation region. No transcription factor is found to bind to the regions so far. Two methylation loci (−1043 and −1090) of p53 promoter were close to and located at either side of negative regulation region −1065/−1084 [Figure 1]d and e.
The influence of the promoter methylation in gene transcription is caused by the change of promotersteric configuration induced by CpG island methylation. CpG island methylation blocks the binding of transcription factor to recognition site and then directly alters gene expression. Moreover, CpG island methylation causes methyl-CpG-binding proteins to bind to methylated CpG sites, interact with other transcription compound inhibition factors, and enrollhistone modification enzymes to change chromatin structure. This blocks the binding of transcription factors to regulation sequence, and indirectly altersgene expression. 
Additionally, DNA methylation can cause the agglutination of chromatin structure,  reduce the activity of RNA polymerases, and then suppress gene expression.  Comprehensive analysis of p53 promoter methylation in our samples suggests that the methylation in Pax regulation region and near −1065/−1084 negative regulation region p53 promoter interfere in the interaction between regulation factors and p53 promoter, and subsequently lead to increased p53 expression. This was proved by immunohistochemistry that p53 expression was higher in MSCs-treated irradiation group than in irradiated group. Furthermore, gene sequencing showed there was no any base mutation in p53 promoter (S3 [Additional file 3]), excluding the influence of gene mutation.
Immunohistochemistry also showed that irradiation alone group had higher p53 expression than control group. It is still hard to speculate the change of p53 expression just through p53 promoter methylation in irradiation alone group. When cells undergo irradiation stress, p53 is responsively activated to increase the expression. High p53 expression exerts its effect as transcription regulation factor through blocking cell cycle progress, repairing the damaged DNA or inducing cell apoptosis. Thus, the DNA methylation and altered gene expression are not the independent events, but the comprehensive consequence of numerous factors.
In summary, our results suggest that MSCs participate in the repair of radiation damage to reduce the incidence of thymoma possibly via improving thymus microenvironment and interfering in p53 promoter methylation, and then maintaining genome stability. However, it is still not clear that the improvement of microenvironment and change of p53 promoter methylation are achieved via the direct cells touch or exosome secretion. Further explorations are needed.
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[Figure 1], [Figure 2]