Aims and background The mechanisms of Epstein-Barr virus (EBV)-associated tumor development are incompletely understood. The aim of this study was to investigate the gene expression of EBV-associated lymphomas in hu-PBL/SCID mice. Methods Human peripheral blood lymphocytes (hu-PBL) from EBV-seropositive donors were transplanted into severe combined immunodeficiency (SCID) mice. In situ hybridization was used to detect EBV-encoded small RNA-1 (EBER1) in tumor tissues. Mutation of TP53 exons 5–8 in EBV-induced lymphomas was analyzed by PCR-SSCP. Immunohistochemical staining was used to examine EBV gene products and cellular oncoproteins. Results Twenty-one of 29 mice developed tumors. EBER1 was positive in the nuclei of almost all tumor cells. Immunohistochemistry showed positive staining of LMP1, EBNA2 and ZEBRA in a small number of tumor cells. Immunohistochemically detectable p53 protein expression was common (85.7%), but TP53 gene mutations were identified in only four cases (19.1%) of EBV-associated lymphomas. Positivity rates of C-myc, Bcl-2 and Bax expression were 100%, 95.2%, and 90.5%, respectively, in the 21 cases of EBV-associated lymphomas. Conclusions Our preliminary findings suggest that EBV-associated lymphomas in hu-PBL/SCID chimeras show EBV infection, expression of oncogenic viral genes, and overexpression of cellular oncogenes. TP53 gene mutations are rare but p53 protein is commonly expressed in EBV-associated lymphomas.
Although the Epstein-Barr virus (EBV) is a well-known human oncogenic virus, its molecular mechanisms involved in the transformation of healthy human cells remain poorly understood. In this study, human lymphocytes were isolated from the peripheral blood of healthy adults, and lymphocytes were transformed in vitro by EBV. Agilent human whole genome microarrays were used to detect the differential gene expression profiles of EBV-transformed lymphoblasts and healthy peripheral blood lymphocytes (PBLs). By constructing the gene functional network of EBV-induced lymphocyte transformation, we screened out candidate key genes in this process and verified their expression levels by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. In the EBV-transformed lymphoblasts, 2335 differentially expressed genes, including 1328 up-regulated and 1007 down-regulated, were screened out. Five candidate key genes, namely, PLK1, E2F1, PTPN11, BIRC5 and FYN were mainly screened out according to the results of LIMMA, String, Cytoscape software analysis. RT-qPCR and Western blot showed that PLK1, E2F1, PTPN11, BIRC5 genes had increased expression levels, and FYN gene was down-regulated in EBV-transformed lymphoblasts. Silencing of PLK1 gene in Raji cells could inhibit cell proliferation and invasion, and induce cell cycle arrest and apoptosis. In conclusion, PLK1, E2F1, PTPN11, BIRC5 and FYN are the candidate key molecules of EBV-transformed lymphocytes.
// Lielian Zuo 1, 2 , Haibo Yu 2, 4 , Lingzhi Liu 2 , Yunlian Tang 2, 5 , Hongzhuan Wu 3 , Jing Yang 1, 2 , Meijuan Zhu 2 , Shujuan Du 2 , Lian Zhao 2, 6 , Li Cao 2 , Guiyuan Li 1, 2 , Jianhong Lu 1, 2 1 Central Laboratory, Hunan Provincial Tumor Hospital, the Affiliated Tumor Hospital of Xiangya Medical School, Central South University, Changsha, Hunan 410013, China 2 Cancer Research Institute, the Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, School of Basic Medical Science, Central South University, Changsha, Hunan 410078, China 3 Department of Biological Sciences, Alabama State University, Montgomery, AL 36101, USA 4 Department of Metabolism and Endocrinology, the Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China 5 Current address: Cancer Research Institute, University of South China, Hengyang, Hunan 421001, China 6 Current address: Department of Gastroenterology, the Third Xiangya Hospital, Central South University, Changsha, Hunan 410013, China Correspondence to: Jianhong Lu, e-mail: jianhlu@csu.edu.cn Keywords: Epstein–Barr virus, latent genome, copy number, latent membrane protein 1, oncogenicity Received: May 28, 2015 Accepted: September 15, 2015 Published: October 16, 2015 ABSTRACT A tumor model that Epstein-Barr virus (EBV) latent infection facilitated the tumorigenicity was previously established using the Maxi-EBV system. In the present approach, EBV-lost cell clones demonstrated significantly decreased tumorigenesis. On the other hand, the LMP1 gene in Maxi-EBV genome was replaced by that of nasopharyngeal carcinoma origin. The resultant cell line, 293–1/NL showed much lower malignancy than the original 293-EBV. The result was opposite to our expectation. The change of 293 sublineage cells for EBV harboring also got similar result. To seek the underlying reason, the copy number of EBV genome in all the cell lines was detected. The result indicated that 293-EBV contained about 4.5-fold higher EBV copies than 293–1/NL did. Parallel EBV genomes led to relatively stable copies in different 293 sublineages, suggesting the viral genome structure is a factor for the sustainability of EBV's copy number. Moreover, the LMP1 transcription in high copy-containing cells showed abnormally high level. Furthermore, the main LMP1-driven pathway, transcription factor NF-κB, was highly activated in high-copy cells. Here we first manifest by experimental model that the copy number of EBV latent genome correlates with the viral pathogenesis, which depends on the activation level of LMP1 and NF-κB. Overall, both the presence and amount of EBV genome are crucial for the viral oncogenicity.
Background: Cathepsin D is a lysosomal aspartyl protease. Our previous studies have suggested that cathepsin D plays an important role in invasion and metastasis of nasopharyngeal carcinoma (NPC).Methods: To identify proteins that interact with cathepsin D and gain an insight into the role of cathepsin D in invasion and metastasis of NPC, co-immunoprecipitation (co-IP) combined with mass spectrometry (MS)-based proteomics approach was used. Cathepsin D associated proteins were identified by MS. Protein-protein interaction network were analyzed by bioinformatics including gene ontology (GO), function clustering. And co-IP and western blotting confirmation were for protein-protein interaction. The expression of cathepsin D, epidermal growth factor receptor (EGFR) and heat-shock protein 90A (HSP90A) in NPC were detected by immunohistochemistry (IHC). And the invasion and metastasis capability were detected by Transwell invasion assay.Results: One hundred and forty-one cathepsin D associated proteins were identified, including EGFR and HSP90A, proteins clearly associated with tumor invasion and metastasis. The interaction of these two proteins with cathepsin D was further validated by co-IP followed by western blotting. The 141 proteins were classified into 12 function-related groups. Protein-protein interaction network analysis indicated that cathepsin D might have a significance on invasion and metastasis of NPC by interacting with EGFR and HSP90A. Indeed, cathepsin D/EGFR/HSP90A could form complexes in NPC cells. In addition, overexpression of cathepsin D in NPC cells not only up-regulated EGFR and HSP90A, but also increased the invasive ability of NPC cells.Conclusions: Cathepsin D could enhance the invasion and metastasis capability of NPC cells may through binding to EGFR and HSP90A and triggering the activation of the signaling pathways.
To analyze the association of several types of malignant lymphomas in different anatomical sites with the Epstein-Barr virus (EBV) infection status, 127 cases of formalin-fixed paraffin-embedded samples of malignant lymphomas were investigated with in situ hybridization detecting EBV-encoded small RNA (EBER) in tumor cells. Forty-six out of 108 non-Hodgkin lymphoma (NHL) cases were positive for EBER (42.6%). The EBER-positivity rate of NHL in the nasal cavity and nasopharynx (35/60 cases, 58.3%) was higher than that of NHL in stomach (9/30 cases, 30%) and in the superficial lymph nodes (2/18 cases, 11.1%) (P<0.05). The EBER-positivity rate of Hodgkin lymphoma in the superficial lymph nodes was 26.3% (5/19 cases). These findings suggest that the EBV-positivity rate in lymphomas is related to their histological types and locations.
The NAG7, an estrogen receptor repressor, is a negative regulator of nasopharyngeal carcinoma cell growth. Here, we report that NAG7 promotes human nasopharyngeal carcinoma invasion, and we identify the mechanisms underlying this function. As a consequence of elevated NAG7 expression, the adhesion, migration, and invasive capabilities of HNE1 cells in vitro and in vivo were enhanced. NAG7 was a significant negative regulator of protein expression of estrogen receptor alpha (ERalpha), and activated both the JNK2/AP-1/MMP1 and the upstream H-Ras/p-c-Raf pathways. None of these effects induced by NAG7 over-expression could be counteracted by estrogen. These observations indicate that NAG7 plays a potential role in promoting nasopharyngeal carcinoma invasion by regulation of ERalpha and the H-ras/p-c-Raf and JNK2/AP-1/MMP1 signaling pathways.
Infection with Epstein-Barr virus (EBV) induces activation and proliferation of B lymphocytes. Detection of latent membrane protein (LMP)-1 is used to identify the proliferative ability of B cells. However, changes in the expression levels of the three LMPs during EBV-induced B lymphocyte transformation, have not yet been reported. In the present study, the expression levels of LMP-1, LMP-2A and LMP-2B were compared between EBV-transformed B lymphocytes and paired normal lymphocytes. Seven lymphoblast cell lines were established by EBV infection of normal human lymphocytes in vitro. The expression levels of LMP genes and LMP-1 protein were determined using quantitative (q)PCR and western blotting in lymphoblasts and normal lymphocytes, respectively. The expression of LMP1, LMP-2A and LMP-2B genes was significantly upregulated in EBV-induced lymphoblasts compared with the normal lymphocytes. The LMP-1 protein level was also significantly increased in EBV-transformed B lymphocytes. Expression of LMP1, LMP-2A and LMP-2B genes was significantly upregulated in EBV-induced lymphoblasts, suggesting LMP genes are important in the transformation of human lymphocytes.
To explore the effect of all-trans-retinoic acid (ATRA) on the growth inhibition and cellular differentiation of C6 glioma cells.Human glioma C6 cells were treated with 5 mg/L ATRA,and the inhibition of cell growth was assessed by methyl thiazolyl tetrazolium assay. The differentiation of C6 cells was determined by flow cytometry, microscopy,transmission electron microscope, and immunohistochemical technique.Treatment of ATRA could result in the growth inhibition of C6 cells, and the cell density significantly decreased(P<0.01). The cell cycle distribution was changed, G0/G1 phase was prolonged, and cells at S phase decreased(P<0.01). The C6 glioma cells displayed normal fibroblast-like morphology under the microscope before the induction, and the ATRA-treated C6 cells became slightly long, turned into round in the middle, and had protrusions at both ends. The ATRA-treated C6 cells did not display obvious apoptosis by flow cytometry(P>0.05).Whereas, early apoptosis was observed under the transmission electron microscope, the vacuoles increased, the mitochondria and endoplasmic reticulum were abundant in the cytoplasm, and the cellular structures tended to be normal.The expression of glial fibrillaryacidic protein in C6 cells increased in the treatment group.ATRA can inhibit the proliferation, and induce the differentiation of C6 glioma cells.
BRD7 is a novel bromodomain gene. It plays critical role in cell growth, cell cycle progression, and signal-dependent gene expression. Overexpression of the BRD7 gene in nasopharyngeal carcinoma cells is effective to inhibit cell growth and cell cycle progression from G1 to S phase. However, little is known about its bio-functions because of the unavailability of a specific BRD7 antibody. In this study, for the first time, we generated a highly specific BRD7 antibody. It is able to specifically recognize recombinant GST-BRD7N protein with a molecular mass of 65 kDa and recognize BRD7-Myc and endogenously expressed BRD7 protein with an approximate molecular mass of 75 kDa, which corresponds well with the calculated molecular mass of the BRD7 protein. More importantly, with these antisera, we analyzed BRD7 distribution in the human fetus by Western blot and immunohistochemistry assays. Obvious nuclear expression of BRD7 protein presents in human cerebellum, pancreas, intestines, liver, and kidney. Cardiomyocyte shows high cytoplasm expression of the BRD7 protein. Weak nuclear expression of the BRD7 protein is found in human cerebrum, lung, and stomach. These data may help to further study the cellular role of the BRD7 gene. In particular, the prepared BRD7 antibody will be helpful for studying the bio-functions of endogenously expressed BRD7 protein.
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