Background
Posttranslational modifications of histone, such as methylation, acetylation, phosphorylation and ubiquitination, are known to play an important role in modulating chromatin structure and regulating gene expression [
1]. Phosphorylation of histone H3 at Ser10 is crucial for chromosome condensation and traditionally regarded as a marker of mitosis [
2]. Conversely, phosphorylation of histone H3 at Ser10 was observed in interphase after cell stimulation with growth factor, stresses and chemical compounds, and associated with the transcriptional activation of immediate-early (IE) genes, including proto-oncogenes
c-
fos and
c-
jun[
3,
4]. The IE gene response has been implicated in proliferation, differentiation and diseases, such as inflammation and cancer [
5]. Constitutive activation of Ras-mitogen-activated protein kinase (MAPK) pathway in oncogene-transformed (e.g. H-
ras) mouse fibroblasts elevated the level of phosphorylated histone H3 at Ser10, accompanying with the aberrant expression of c-
fos, c-
myc and uPA gene [
6,
7]. However, much less is known about the role of histone H3 phosphorylation at Ser10 in neoplastic cell transformation and carcinogenesis.
Accumulating evidences have demonstrated that phosphorylation of histone H3 at Ser10 is involved in different signaling pathways depending on specific stimulation and stress. Fibroblasts with mutations in ribosomal subunit protein S6 kinase 2 (RSK2) gene failed to exhibit epidermal growth factor (EGF)-stimulated phosphorylation of histone H3 at Ser10, suggested that RSK2 is required for EGF-induced phosphorylation of histone H3 [
8]. Mitogen- and stress-activated kinase (MSK1) has been shown to mediate EGF, 12-O-tetradecanoyl phorbol-13-acetate (TPA), ultraviolet and oncogene-induced phosphorylation of histone H3 at Ser10 [
9‐
11]. Our previous studies indicated that RSK2, but not MSK1, was involved in arsenite-induced phosphorylation of H3 at Ser10 [
12]. All these studies showed that various stimuli probably trigger different kinases to phosphorylate histone H3, thus, it’s very important to identify the responsible kinases and the circumstances mediated histone H3 phosphorylation.
Nasopharyngeal carcinoma (NPC) is a most common malignant tumor in southern China and some regions in Southeast Asia. Its occurrence involves the interaction of host genetic alterations with environmental factors, especially infection by Epstein-Barr virus (EBV) [
13]. EBV-encode latent membrane protein 1 (LMP1) is the only latent gene product with transformation properties. It has been shown that LMP1 is crucial for EBV-induced transformation and immortalization of B lymphocytes [
14]. Similar oncogenic properties were displayed in rodent fibroblasts and transgenic mice [
15]. When expressed in tumorigenic epithelial cells, LMP1 potentiated anchorage-independent growth and greatly promoted migration and invasion [
16‐
18]. Many of the oncogenic effects of LMP1 are attributed to constitutively triggering a plethora of signaling pathways including NF-κB, AP-1 and STAT pathways, which regulates the expression of downstream target genes, thereby mediating tumorigenesis of NPC [
17‐
19]. It has been shown that increased phosphorylation of histone H3 at Ser10 may contribute to the aberrant gene expression and promote oncogene-mediated transformation [
6,
7]. However, there is no evidence whether phosphorylation of histone H3 at Ser10 is involved in LMP1-induced cell transformation in NPC.
In this study, the expression of histone H3 phosphorylation at ser10 and its correlation with EBV-LMP1 expression in NPC are investigated. Then, we further explore the role of histone H3 phosphorylation at Ser10 in LMP1-induced CNE1 cell transformation and its regulatory kinase.
Methods
Patients and tissue specimens
Nasopharyngeal carcinoma tissue microarray (catalog No. NPC961) was from US Biomax (Rockville, MD), including 33 cases of poorly differentiated NPC tissues, 26 cases of adjacent normal tissues, and 10 cases of normal nasopharyngeal tissues. In addition, 15 cases of poorly differentiated NPC tissues and 15 cases of chronic nasopharyngitis tissues were obtained from the First Affiliated Hospital of Guangdong Medical College, Zhanjiang, China. The patients were not pretreated with radiotherapy or chemotherapy prior to surgery. All cases were confirmed by pathological examination and staging was performed according to the 1997 NPC staging system of the WHO. In the 48 NPC cases, there were 37 male and 11 female with age ranging from 26 to 62 years (median, 43.6 years). For the use of these clinical materials for research purposes, prior consent of the patients and approval from the Institutional Ethics Committee of Guangdong Medical College were obtained.
Cell culture and plasmids
CNE1 cells, an EBV-negative cell line derived from a well-differentiated Chinese NPC patient, were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (GIBCO-Invitrogen, Carlsbad, CA) and antibiotics (100 U/ml penicillin,100 g/ml streptomycin). CNE1G (CNE1 stably transformed with PAT-GFP) and CNE1GL(CNE1 stably transfected with PAT-GFP-LMP1) cells were provided by Dr. Xiaoyi Chen, Guangdong Medical College [
20], and were maintained in completed RPMI 1640 medium described above, containing 0.5 μg/ml puromycin (Sigma-Aldrich, St. Louis, MO). The pcDNA3.0 and pcDNA3.0-LMP1 vectors were kindly provide by Dr Ellen Cahir-McFarland, Brigham and Women’s Hospital, Boston, Massachusetts, USA. The mU6pro vector was provided by Dr. Zigang Dong, Hormel Institute, University of Minnesota, Austin, Minnesota, USA. The AP-1 reporter vector pRTU14 was kindly provided by Dr ArndKieser, Helmholtz ZentrumMünchen, Munich, Germany [
21].
A cDNA fragment encoding human histone H3 was inserted in-frame into the XbaI/EcoRI sites of the pcDNA6.0/myc-His B vector (Invitrogen, Carlsbad, CA) to produce the myc and His epitope-tagged construct, pcDNA6.0-H3. The vector of histone H3 S10A mutant was generated by replacing Ser10 of histone H3 with alanine using the KOD-Plus-Mutagenesis kit (Toyobo Co., Ltd, Osaka, Japan), and named as pcDNA6.0-H3S10A. To construct the siRNA-H3 (si-H3) or siRNA-MSK1 (si-MSK1), the mU6pro vector was digested with XbaI and BbsI. The annealed synthetic primers (H3 siRNA sense: 5′-TTTGCAGACAGCTCGGAAATCCATTCAAGAGATGGATTTCCGAGCTGTCTGTTTTTT-3′ and antisense: 5′-CTAGAAAAAACAGACAGCTCGGAAATCCATCTCTTGAATGGATTTCCGAGCTGTCTG-3′; MSK1 siRNA sense: 5′-TTTGAGACCTAATTCAGCGTCTTTTCAAGAGAAAGACGCTGAATTAGGTCTTTTTT-3′ and antisense: 5′-CTAGAAAAAAGACCTAATTCAGCGTCTTTCTCTTGAAAAGACGCTGAATTAGGTCT-3′) were then introduced into the mU6pro vector. The recombinant plasmids were confirmed by agarose gel electrophoresis and DNA sequencing.
Antibodies and reagents
Antibodies against phosphorylated or total histone H3, phosphorylated or total ERK1/2 and MSK1 were purchased from Cell Signaling Technology (Beverly, MA). Anti-EBV LMP1 (CS1-4) antibody was purchased from DAKO (Glostrup, Denmark). Infrared-dye-conjugated secondary antibodies were purchased from Rockland Immunochemicals (Gilbertsville, PA). PD98059 and H89 were purchased from Cell Signaling Technology (Beverly, MA). Pure histone H3 was from NEB (Beverly, Mass). JetPEI transfection reagent was from Polyplus (llkirch, France).
Immunohistochemistry analysis
Formalin-fixed and paraffin-embedded (FFPE) specimens were cut into 4-μm sections, mounted onto the polylysine-coated slides, deparaffinized in xylene, and rehydrated in a graded ethanol series. Heat-mediated antigen retrieval was performed with sodium citrate buffer (0.01M, pH 6.0). Endogenous peroxidase activity and non-specific antigen were blocked with 3% hydrogen peroxide and normal goat serum. The sections were incubated with the primary antibodies against LMP1 or phosphorylated histone H3 (Ser10) overnight at 4°C. HRP-conjugated secondary antibodies (ChemMate Envision Detection Kit, Dako) were applied onto the sections and incubated for 30 min at room temperature. 10% normal goat serum was used to replace primary antibodies as a negative control. Staining of LMP1 appeared on the cell membrane or/and in the cytoplasm. The percentage of stained cells was determined in 3 representative fields contained at least 200 tumor cells. The expressions of LMP1 were then scored as positive (≥10%) and negative (<10%) based on the percentage of stained cells [
22]. The immunoreactivity to histone H3 phosphorylation was localized in the cell nucleus. The number of nuclear stained cells was determined by the examination of at least 1000 cells in 3 representative fields, named as positive labeling index (PLI) for histone H3 phosphorylation [
23].
In order to detect the expression of LMP1 and histone H3 phosphorylation at Ser10 in CNE1G and CNE1GL cells, they were immunohistochemically stained using the same staining method as for the clinical specimens.
Protein extraction and western blot analysis
Extraction of histone protein was performed as described previously [
24]. In brief, approximately 1×10
6 cells were resuspended in 1 mL lysis buffer [10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 1.5 mmol/L MgCl
2, 0.65% NP40, 0.5mmol/L DTT, 1.5mmol/L PMSF]. The lysates were centrifuged at 10000×g for 10 minutes to pellet the intact nuclei. The Nuclei were extracted with 0.4 N H
2SO
4 and were incubated on a rotator for at least 30 min. Extraction solutions were centrifuged at 10000×g for 10 min, and acid-insoluble pellets were discard. Supernatant fractions were precipitated with 5 volumes of ice-cold acetone for overnight. The acid-soluble protein was dissolved in 100 μl double-distilled H
2O. As described elsewhere, total protein was extracted with RIPA lysis buffer (Beyotime Ins. Bio, China).
Protein concentration was determined by the bicinchoninic acid (BCA) assay (Pierce, USA). Samples containing equal amount of protein were resolved by SDS-PAGE and transferred to PVDF membranes (millipore, Billerica, MA). The membranes were blocked with 5% not-fat dried milk for 2 hours, and then probed with the primary antibodies overnight at 4°C. After washing with 0.1% Tween-20 in TBS, membranes were incubated with infrared-dye-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized by Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE, USA).
Cell counting kit-8 (CCK-8) assay
The cell proliferative ability was evaluated by CCK-8 (Dojindo Laboratories, Kumamoto, Japan) assay. CNE1G or CNE1GL cells were transfected with si-mock or si-H3 plasmids and then seeded in 96-well plates (3×103 per well). After culturing for various periods of time, CCK-8 solution (10 μl per 100 μl medium) was added to each well, and cells were then incubated for 1 hour at 37°C. Absorbance was measured at 450 nm using Synergy2 Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA). The assay was conducted in five replicate wells for each sample and three parallel experiments were performed.
The transformation potential of the introduced genes in cells was evaluated by Focus-forming assay. CNE1 cells were transiently transfected with various combinations of expression vectors and seeded in six-well plates (500 per well). After culturing for 2 weeks, foci were fixed with methanol and stained with 0.5% crystal violet. Foci containing more than 50 cells were considered, and the mean values from three replicate wells were calculated. Data are representative of at least three independent experiments.
Reporter gene assay
Activator protein-1 (AP-1) activation was determined by the luciferase reporter gene assay. Cells were transiently cotransfected with AP-1 reporter gene and pRL-TK vector (Promega, China). The pRL-TK vector expressing Renilla luciferase was cotransfected to calibrate the firefly luciferase activity. Cells were lysed with passive lysis buffer (Promega) for 20 min with gently shaking. Luciferase activities were measured with cell lysates using the Dual-Luciferase assay system (Promega) in FB12 Luminometer (Berthold detection system). The firefly luciferase activity was normalized against Renilla luciferase activity. Data were derived from the mean of triplicate samples and recorded as relative luciferase activity (fold or %). All experiments were done at least in triplicate.
Histone H3 Kinase Assay in vitro
Cell extracts (20 μg) of CNE1G and CNE1GL cells were incubated in 1×kinase buffer supplemented with 1 μg of pure histone H3, 200 μM ATP, and presence or absence of 10 μM H89 for 30 min at 30°C. Reactions were terminated with 6×SDS sample buffer. The samples were denatured at 95–100°C for 5 min before they were separated by 15% SDS-PAGE. The phosphorylation of histone H3 at Ser10 and total histone H3 protein were detected by western blot with specific antibodies.
MSK1 kinase assay in vitro
Cell extracts (200 μg) of CNE1G and CNE1GL cells were incubated with immobilized Phospho-MSK1 (Thr581) monoclonal antibody overnight at 4°C. Then protein A/G agarose beads (20 μl) were added and incubated for 2 hours at 4°C. These samples were washed three times with 500 μl of 1×cell lysis buffer, and then washed twice with 500 μl of 1×kinase buffer. The pellets were suspended in 40 μl of 1×kinase buffer supplemented with 1 μg of histone H3 protein and 200 μM ATP, and incubated for 30 min at 30°C. Reactions were terminated with 6×SDS sample buffer, and then samples were separated by 15% SDS-PAGE. MSK1 kinase activity for histone H3 was analyzed by western blot using anti-phosphorylated histone H3 antibody.
Statistical analysis
Quantitative values were expressed as means ± SD. The SPSS version 16.0 software package and GraphPad Prism were used for the statistical analysis and data plotting. Student t-test was used to compare the mean value of each group. The relationship between LMP1 and histone H3 phosphorylation expression was analyzed using Chi-square test. p<0.05 was considered statistically significant.
Discussion
Phosphorylation of histone H3 at Ser10 is correlated closely with chromosome condensation, mitosis and gene expression. Many tumor promotion agents, such as EGF, TPA, or ultraviolet, and transformation by oncogene H-
ras or v-Src can elevate the level of phosphorylated histone H3 at Ser10 [
6,
10,
11]. Increased phosphorylation of histone H3 as a result of AIM-1/Aurora B overexpression contributed to chromosome instability and was observed in many tumor cell lines, including colorectal and hepatocellular carcinomas [
23,
27]. These observations implied that the deregulation of histone H3 phosphorylation may play a role in carcinogenesis. In this study, using immunostaining analysis, we found that the p-H3Ser10 positive index in poorly differentiated NPC was significantly higher than that in chronic nasopharyngitis and normal nasopharynx tissues. It is indicated that the increasing phosphorylation of histone H3 might be an important event in NPC pathogenesis and promoted the malignant transformation of nasopharyngeal epithelium. Compared with normal nasopharynx tissues, chronic nasopharyngitis exhibited a higher level of phosphorylated histone H3 at Ser10. It might be associated with chronic stimulation of the nasopharynx from various factors, such as chemical agents, cigarette smoking and viral or bacterial infection, which were shown to induce the phosphorylation of histone H3 at Ser10 [
28‐
30]. However, the specific mechanism remains to be further studied.
LMP1 is the only EBV-encoded latent gene with classical transforming properties, which is closely associated with the carcinogenesis of NPC [
17,
18]. LMP1 functions as a viral mimic of tumor necrosis factor receptor (TNFR) family member, CD40, and thus triggers a number of cellular signaling pathways, which participates in regulation of cell proliferation, apoptosis, malignant transformation, invasion and metastasis [
31,
32]. In this study, we found that the elevated expression level of histone H3 phosphorylation in NPC tissues was closely related to LMP1 expression. Moreover, the phosphorylation of histone H3 at Ser10 was more frequently observed in LMP1-transfected CNE1 cells compared with mock control cells in the serum-starved condition. It was found that the most CNE1GL cells with p-H3Ser10 expression did not belong to the G2/M phase of cell cycle. Similar result was also observed in v-Src-transformation mouse fibroblasts [
11]. The findings suggested that EBV-LMP1 can constitutively activate phosphorylation of histone H3 at Ser10 in interphase and may contribute to the aberrant expression of IE genes.
Recent studies showed that histone H3, especially the Ser10 motif, has oncogenic effects and directly regulated EGF- or TPA-induced neoplastic cell transformation and cell proliferation [
10,
33]. Here, we used the knockdown and mutant of histone H3 to explore the role of histone H3 phosphorylation at Ser10 in regulating LMP1-promoted cell transformation of CNE1 cells. The results showed that the knockdown of histone H3 by siRNA suppressed the LMP1-induced cell proliferation and foci formation. Moreover, we found that overexpression of mutation histone H3 (H3S10A) also inhibited foci formation promoted by LMP1 in CNE1 cells compared with overexpressing H3 WT cells. These observations indicated that the phosphorylation of histone H3 at Ser10 might be a crucial regulatory mechanism for LMP1-induced cell transformation in NPC.
In vitro histone H3 kinase assay showed that H3 kinase activity in the LMP1-transfected CNE1 cells was greater than that in the mock control cells. But the presence of H89, an inhibitor of MSK1, significantly reduced the H3 kinase activity. We surmised that increasing MSK1 kinase activity may account for the increasing phosphorylation level of histone H3 at Ser10. MSK1 is a nuclear kinase which is activated by the ERK and p38 MAPKs in response to extracellular stimuli. MSK1 has been shown to activate various transcription factors, including cyclic AMP (cAMP)-response element-binding protein (CREB), ATF1, STAT3 and NF-κB, and alters their target DNA-binding capacity or promotes the recruitment of their coactivators [
34‐
36]. Persistent activation of Ras-MAPK pathway and elevated MSK1 activity were observed in many human cancers and tumor cell lines [
37,
38]. MSK1 has also been reported to phosphorylate the chromatin protein histone H3 and high mobility group 14 (HMG-14) when induced by mitogen- and stress- stimuli [
39]. The Ras-MAPK pathway and MSK1 appear to play a critical role in the phosphorylation of histone H3 and oncogenic growth of v-Src transformed cells [
11]. In this study, we found that LMP1 increased the phosphorylation level of MSK1 at Thr581 and enhanced the MSK1 kinase activity. ERK1/2 inhibitor PD98059 and MSK1 inhibitor H89 obviously suppressed LMP1-induced phosphorylation of histone H3 at Ser10. Similar results were obtained with MSK1-specific siRNA. These results strongly suggested that LMP1 induced phosphorylation of histone H3 at Ser10 via activation of Ras-MAPK pathway and MSK1 kinase.
Previous studies suggested the AP-1 signaling pathway played an important role in LMP1-mediated tumorigenesis of NPC [
17,
18]. LMP1 activated c-Jun N-terminal kinases (JNK) and promoted the formation of c-Jun/JunB heterodimers leading to expression of AP-1 regulated gene [
40,
41]. In present study, we showed the relationship of MSK1-mediated histone H3 phosphorylation and AP-1 transactivation promoted by LMP1 in CNE1 cells. MSK1 inhibitor H89 or knockdown of MSK1 by siRNA significantly suppressed LMP1-promoted AP-1 activation. Furthermore, histone H3, especially the Ser10 motif, also regulated AP-1 activation promoted by LMP1. It was revealed that c-
jun or c-
fos gene was a common target of histone H3 leading to induction of AP-1 activity [
33]. The activation of the
c-
fos serum responsive element (SRE) by histone H3 phosphorylation might promote c-Fos expression and stabilize the c-Fos/c-Jun heterodimer [
42]. The increasing AP-1 transactivation activity coupled with histone H3 phosphorylation may contribute to elucidate the mechanism of neoplastic cell transformation mediated by post-translational modification of histone H3. Take together, these results indicated that histone H3 phosphorylation at Ser10 mediated by MSK1 was required for AP-1 activation promoted by LMP1, which was very much associated with LMP1-induced cell transformation. In addition, MSK1-mediated phosphorylation of transcription factors CREB and ATF1 has been shown to induce
c-
fos and
junB transcription [
35], and thereby might regulate AP-1 transactivation.
Acknowledgments
This work was supported by National Natural Science Foundation of China (No: 30973374 to Z H), Medical Science Research Foundation of Guangdong Province (No: A2011424 to B L) and National Natural Science Foundation of China (No: 81102048 to G-L H). The authors are grateful for the support from Dr. Xiaoyi Chen, Cancer Research Institute, Guangdong Medical College, Guangdong, China; Dr. Ellen Cahir-McFarland, Brigham and Women’s Hospital, Boston, Massachusetts, U. S.A., Dr Arnd Kieser, Helmholtze Center, Munich, Germany; Dr. Zigang Dong, Hormel Institute, University of Minnesota, Austin, Minnesota, USA.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BB L designed and performed the experiments, analyzed data and drafted the manuscript. G H and X Z contributed reagents, performed experiments and analyzed data. R L and J W performed experiments and analyzed data. Z D and Z H designed the experiments, analyzed data and drafted the manuscript. All authors read and approved the final manuscript.