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Journal of Virology logoLink to Journal of Virology
. 2013 Oct;87(19):10515–10523. doi: 10.1128/JVI.00955-13

Epstein-Barr Virus-Encoded BARF1 Promotes Proliferation of Gastric Carcinoma Cells through Regulation of NF-κB

Mee Soo Chang a,b,✉, Dong Ha Kim c, Jin Kyung Roh c, Jaap M Middeldorp d, Yang Soo Kim c,e, Sunghan Kim c,e, Seungbong Han f, Chul Woo Kim b,h, Byung Lan Lee g,h, Woo Ho Kim b,h, Jun Hee Woo c,e,✉
PMCID: PMC3807382  PMID: 23824821

Abstract

In Epstein-Barr virus (EBV)-infected gastric carcinoma, EBV-encoded BARF1 has been hypothesized to function as an oncogene. To evaluate cellular changes induced by BARF1, we isolated the full-length BARF1 gene from gastric carcinoma cells that were naturally infected with EBV and transfected BARF1 into EBV-negative gastric carcinoma cells. BARF1 protein was primarily secreted into culture supernatant and only marginally detectable within cells. Compared with gastric carcinoma cells containing empty vector, BARF1-expressing gastric carcinoma cells exhibited increased cell proliferation (P < 0.05). There were no significant differences in apoptosis, invasion, or migration between BARF1-expressing gastric carcinoma cells and empty vector-transfected cells. BARF1-expressing gastric carcinoma cells demonstrated increased nuclear expression of nuclear factor kappa B (NF-κB) RelA protein and increased NF-κB-dependent cyclin D1. The expression of p21WAF1 was diminished by BARF1 transfection and increased by NF-κB inhibition. Proliferation of naturally EBV-infected gastric carcinoma cells was suppressed by BARF1 small interfering RNA (siRNA) (P < 0.05). Immunohistochemical analysis of 120 human gastric carcinoma tissues demonstrated increased expression of cyclin D1 and reduced expression of p21WAF1 in EBV-positive samples versus EBV-negative gastric carcinomas (P < 0.05). In conclusion, the secreted BARF1 may stimulate proliferation of EBV-infected gastric carcinoma cells via upregulation of NF-κB/cyclin D1 and reduction of the cell cycle inhibitor p21WAF1, thereby facilitating EBV-induced cancer progression.

INTRODUCTION

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that has been implicated in the etiology of many human lymphoid (1, 2) and epithelial (2, 3) malignancies. EBV-positive gastric carcinoma was first reported in 1990 (4), and EBV-positive carcinomas comprise 2 to 16% of all gastric carcinomas worldwide (5–9). Gastric carcinoma is not only the most common EBV-associated malignancy in South Korea but the most common cancer overall in South Korea (8, 9, 41). EBV-positive gastric carcinomas show distinct clinicopathological characteristics, including lymphoid stroma (7, 10), a higher prevalence in male patients and poorly differentiated WHO-type and diffuse Lauren-type tumors (7, 11), less frequent metastasis to lymph nodes (10), predominant localization to the proximal stomach (7, 10–12), unique expression of many cancer-related genes (7, 9, 12), and global CpG island methylation of cancer-related gene promoters (6).

The oncogene responsible for EBV-driven gastric carcinoma has not been identified. Latent membrane protein 1 (LMP1) is an EBV-encoded oncoprotein that is thought to be responsible for the development of EBV-associated lymphomas and nasopharyngeal carcinomas (2, 13–15). However, LMP1 is not expressed in EBV-positive gastric carcinomas (2). The EBV-carried BARF1 gene has been proposed to function as an oncogene (16–26). However, little is known about BARF1-induced changes in human gastric carcinoma cells (22). We previously reported that the BARF1 transcript is expressed in the human gastric carcinoma cell line SNU719, which is naturally infected with EBV (9).

Endogenous expression of BARF1 leads to secretion of BARF1 from cells (17–19, 27–35). The secreted form of BARF1 is partly responsible for the growth-promoting and antiapoptotic functions, which, however, remain to be confirmed (9, 31). Secreted BARF1 binds to human colony-stimulating factor 1 (hCSF-1) in a manner similar to that in which hCSF-1 binds to hCSF-1 receptor (c-fms or FMS). This interaction may be related to the oncogenic role of BARF1 (29). The hCSF-1 cytokine has pleiotropic effects, including promoting differentiation and growth of macrophages (29). Recently, the interaction between macrophage CSF and secreted BARF1 was studied (33, 35). This interaction may mediate CSF-stimulated effects on the immune system (33) and BARF1-induced effects on cellular growth (33, 35).

Previously, we reported increased immunopositive staining for nuclear factor kappa B (NF-κB) RelA in EBV-positive human gastric carcinoma tissues compared with EBV-negative gastric carcinoma tissues (9). In unstimulated cells, NF-κB interacts with inhibitory proteins, such as IκBα, and is sequestered in the cytoplasm in an inactive form. Upon stimulation by LMP1 or other factors, IκBα is phosphorylated, ubiquitinated, and degraded. Degradation of IκBα permits translocation of NF-κB to the nucleus. Nuclear NF-κB activates transcription of numerous genes that inhibit apoptosis, metastasis, or proliferation, including bcl-2, c-Myc, and cyclin D1 genes (36, 37). Cyclin D1 is an NF-κB target in the interleukin-1 receptor-associated kinase 1 (IRAK1)/IκBα/NF-κB/cyclin D1 pathway (36, 37) and a key regulator of the G1/S cell cycle checkpoint (37). The cyclin D1/cyclin-dependent kinase 4 (Cdk4) complex promotes cell proliferation. Conversely, inhibition of Cdk4 by p21WAF1 promotes cell cycle arrest (38).

To assess the role of BARF1 in gastric cancer progression, we generated BARF1-expressing gastric carcinoma cells and investigated changes in the molecular and biological properties of these cells.

MATERIALS AND METHODS

Cell culture and reagents.

SNU719, which is a naturally EBV-infected gastric carcinoma cell line, and SNU601, an EBV-negative gastric carcinoma cell line, were purchased from the Korean Cell Line Bank (Seoul, South Korea). Cells were maintained in RPMI 1640 medium (Gibco BRL, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in an atmosphere of 5% CO2. The IκB kinase β (Ikkβ) inhibitor peptide (Ikk-2 inhibitor IV) and brefeldin A were purchased from Millipore (Bedford, MA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Ikkβ inhibitor is an upstream inhibitor of NF-κB, and brefeldin A is a transport blocker that disrupts export from distal Golgi compartments.

Construction of BARF1-stable transfectants in SNU601 cells.

The full-length BARF1 gene was amplified from SNU719 genomic DNA. The resulting PCR fragment (666 bp) was cloned into the BamHI and XhoI sites of the pCMV-Tag 2B/flag vector (Stratagene, La Jolla, CA, USA). To generate stable transfectants, SNU601 cells were transfected with pCMV-Tag 2B/flag or pCMV-Tag 2B/flag/BARF1 with the Lipofectamine reagent according to the manufacturer's protocol (Invitrogen). Selection was initiated 2 days later with medium containing 0.5 mg/ml G418 (Invitrogen).

Concentration of secreted BARF1.

SNU601 cells transfected with BARF1 or SNU719 cells were cultured in a 100-mm plate to 70% confluence. Cells were washed four times with serum-free medium and incubated in 10 ml serum-free medium for 24 h. Culture medium was stored at −80°C after eliminating cellular debris by 10 min of centrifugation at 3,000 × g. After being cleared, 300 ml culture medium was concentrated to 50 μl or 100 μl with an Ultra-15 centrifugal filter (Amicon Ultra 10k device; Millipore, Amsterdam, Netherlands) followed by centrifugation in Ultra-2 and Ultra-0.5 centrifugal filters (Millipore). The final volume was concentrated by 6,000- or 3,000-fold. BARF1 expression was examined in this concentrated fraction.

Measurement of cell proliferation.

Cell proliferation was measured by WST-1 and trypan blue exclusion assays. Briefly, cells in logarithmic growth phase were harvested and seeded in 96- or 12-well plates overnight. Cells were incubated for the indicated times. For the WST-1 assay, cells were incubated with 10 μl WST-1 for another 2 h and measured at A450 on a spectrophotometer (Spectramax 190; Molecular Devices Corp., Sunnyvale, CA, USA). The number of viable cells was counted on a hemocytometer by trypan blue dye exclusion.

Apoptosis assay.

Apoptosis was quantified with the annexin V-fluorescein isothiocyanate (FITC) apoptosis kit (BD Biosciences, San Diego, CA, USA) in accordance with the manufacturer's instructions. Briefly, cells were cultured in serum-free medium, trypsinized (Invitrogen), spun down by centrifugation, and resuspended in annexin V-binding buffer (150 mmol/liter NaCl, 18 mmol/liter CaCl2, 10 nmol/liter HEPES, 5 mmol/liter KCl, and 1 mmol/liter MgCl2). FITC-conjugated annexin V (1 μg/ml) and propidium iodide (50 μg/ml) were added to cells and incubated for 30 min at room temperature in the dark. Analyses were performed on a FACScan flow cytometer (Becton, Dickinson, Mountain View, CA, USA). Data were analyzed with CellQuest software (Becton, Dickinson).

Cell invasion and migration assays.

Invasion assays were performed on 24-well BioCoat Matrigel invasion chambers (BD Biosciences) according to the manufacturer's protocol with minor modifications. Briefly, cells (1 × 105) in 200 μl medium were placed in the upper chamber, and the lower chamber was filled with 1 ml serum-free medium supplemented with 10% bovine serum albumin. After incubation of cells for 48 h at 37°C, cells that invaded the lower chamber were stained with 8 μg/ml calcein AM (BD Biosciences) in Hanks' buffered saline at 37°C for 1 h. Invasive cells were counted on a fluorescence microscope (Olympus IX71; Tokyo, Japan). To detect migration, a scratch or wound was made in a plate of confluent cells with the tip of a micropipette. Images were captured 24 h later on an inverted photomicroscope (Olympus IX71). Movements of individual cells were analyzed with NIH ImageJ software (http://rsb.info.nih.gov/ij/index.html).

RT-PCR.

Total cellular RNA samples were prepared with an RNeasy minikit (Qiagen, Hilden, Germany). Extracted RNA was treated for 1.5 h at 37°C with 10 units of DNase I (Roche, Basel, Switzerland) in the presence of RNase inhibitor (Roche) to remove remaining genomic DNA. After inactivation at 75°C for 10 min, RNA samples were purified with an RNeasy minikit (Qiagen) according to the manufacturer's recommendations. cDNA was synthesized from 1 μg total RNA with a high-fidelity reverse transcriptase PCR (RT-PCR) system. The forward and reverse primers for cDNA amplification were as follows: BARF1, forward, 5′-CGGGATCCATGGCCAGGTTCATC-3′; reverse, 5′-CCGCTCGAGTCATTGCGACAAGTAT-3′; β-actin, forward, 5′-GACAGGATGCAGAAGGAGATTACT-3′; reverse, 5′-TGATCCACATCTGCTGGAAGGT-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward, 5′-GAGTCAACGGATTTGGTCGT-3′; reverse, 5′-TTGATTTTGGAGGGATCTCG-3′. The PCR conditions were 30 to 35 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 1 min. PCR products were analyzed on 2% agarose gels.

Western blotting and densitometric analyses.

Proteins were measured with a bicinchoninic acid assay kit (Merck & Co., Gibbstown, NJ, USA). BARF1 protein was extracted from cell culture supernatants. Other proteins were derived from whole-cell extracts. Proteins were separated on 12% SDS-PAGE gels with a 5% stacking gel and transferred onto reinforced polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking of nonspecific sites, blots were incubated with primary antibodies against NF-κB RelA (A, sc-109, 1:500; Santa Cruz Biotechnology, CA, USA), IRAK1 (F-4, sc-5288, 1:1,000; Santa Cruz Biotechnology), IκBα (6A920, 1:500; Abcam, Cambridge, United Kingdom), phospho-IκBα (39A1431, sc-52943; 1:500; Santa Cruz Biotechnology), cyclin D1 (H-295, sc-753, 1:1,000; Santa Cruz Biotechnology), FLAG (D-8, sc-807, 1:500; Santa Cruz Biotechnology), transcription factor IIB (TF-IIB, sc-23875, 1:2,000; Santa Cruz Biotechnology), β-actin (AC-15, 1:10,000; Abcam), and p21WAF1 (F-5, sc-6246, 1:500; Santa Cruz Biotechnology) overnight at 4°C. BARF1 antibody (monoclonal antibody [MAb] 4A6, 1:100) was supplied by Middeldorp (28, 30, 32–34). Blots were washed and incubated for 120 min at room temperature with horseradish peroxidase-conjugated anti-mouse secondary antibody (Abcam). Antigen-antibody complexes were visualized by ECL staining (Amersham, Arlington Heights, IL, USA) and exposure to X-ray film. Western blots were digitized with a GS-700 imaging densitometer (Bio-Rad Laboratories, Richmond, CA, USA) and processed with Corel Photo Paint 7.0 to adjust image brightness and contrast. Densitometric evaluations were performed with Molecular Analyst software (Bio-Rad) and normalized relative to controls.

Immunofluorescence assay.

Cells were grown on glass coverslips and treated with brefeldin A (3 μg/ml) for 24 h to block BARF1 secretion. Cells were fixed for 10 min in 4% paraformaldehyde in 10 mmol/liter piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), pH 6.8, 10 mmol/liter NaCl, 300 mmol/liter sucrose, 3 mmol/liter MgCl2, and 2 mmol/liter EDTA. Cells were permeated for 10 min in Tris-buffered saline (TBS) with 0.75% Triton X-100 and blocked for 10 min in 5% bovine serum albumin and 0.1% Triton X-100 in TBS. BARF1 was detected with BARF1 antibody (MAb 6F4, 1:100) and Alexa Fluor 488 goat anti-mouse IgG(H+L) secondary antibody (Invitrogen). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/ml).

Preparation of nuclear extracts.

We used the method performed in our previous study (8). Cells were resuspended in 100 μl lysis buffer A (10 mmol/liter Tris [pH 8.0], 60 mmol/liter NaCl, 1 mmol/liter EDTA, 1 mmol/liter dithiothreitol [DTT], 0.1% Nonidet P-40, 1 mmol/liter phenylmethylsulfonyl fluoride [PMSF]) and incubated on ice for 5 min. Cell membranes were spun down by centrifugation at 1,000 × g for 10 min at 4°C, and supernatants containing cytoplasmic extracts were transferred to fresh tubes. Glycerol was added to a final concentration of 20%, and cytoplasmic extracts were stored at −80°C. Nuclear pellets were immediately washed in 1 ml lysis buffer A without Nonidet P-40, centrifuged, and resuspended in 50 μl buffer B (200 mmol/liter HEPES [pH 7.9], 0.75 mmol/liter spermidine, 0.15 mmol/liter spermine, 0.2 mmol/liter EDTA, 2 mmol/liter EGTA, 2 mmol/liter DTT, 20% glycerol, 1 mmol/liter PMSF, and 0.4 M NaCl). Nuclear lysates were incubated on ice for 10 min with occasional vortexing. Extracts were centrifuged at 14,000 × g for 20 min at room temperature, and supernatants containing nuclear extracts were collected. Samples were stored at −80°C.

siRNA transfection.

A small interfering RNA (siRNA) specific for NF-κB RelA (L-003533-00-0005, human Rel A; four siRNAs combined into a single pool) and three siRNAs for BARF1 (type 1, 5′-GGGUUUAUGUUUCUGGAUAUU-3′; type 2, 5′-CUGGAUACUUGUCGCAAUAUU-3′; type 3, 5′-GAGCCUCGGUCCAGAGAUUUU-3′) were synthesized by Dharmacon RNA technologies (Dharmacon, Lafayette, CO, USA). A scrambled siRNA (Dharmacon) containing a random sequence of nucleotides with no known specificity was used as a negative control. Cells were transfected with 50 pmol siRNA using Lipofectamine 2000 (Invitrogen) in 500 μl serum-free medium for 4 h at 37°C in a CO2 incubator according to the manufacturer's recommendations. Without removal of the transfection mixture, 500 μl RPMI with 20% FBS was added. Cells were incubated for an additional 24 h. Transfected cells were examined by cell proliferation assays and Western blotting.

Immunohistochemistry.

Immunohistochemistry for cyclin D1 and p21WAF1 was performed with a BenchMark XT automated stainer (Ventana Medical Systems, Tucson, AZ, USA). Tissue was incubated with anti-cyclin D1 antibody (SP4-R; Ventana) or anti-p21WAF1 antibody (sc-817; Santa Cruz Biotechnology) at 37°C for 28 min. Antigen was detected with an ultraView Universal diaminobenzidine (DAB) detection kit. Positive staining for cyclin D1 was defined as positive staining that exceeded 10% of the total cancer cell nuclei. Immunostaining for p21WAF1 is found in normal glandular epithelial cell nuclei of the gastric mucosa. Loss of p21WAF1 was considered significant if staining for p21WAF1 was lost in more than 90% of cancer cell nuclei.

Statistical analysis.

The slopes of graphs and levels of apoptosis and proliferation were compared with linear mixed models incorporating R statistical software package nlme (http://cran.r-project.org/bin/windows/base/). The χ2 test, Pearson's test, and Kendall's Tau-b correlation analyses were conducted with IBM SPSS Statistics version 20.0 (IBM Corp., Somers, NY, USA) to evaluate correlations between EBV status and immunoexpression in gastric carcinoma tissues. A P value of less than 0.05 was considered statistically significant.

RESULTS

BARF1 protein is primarily secreted and weakly observed within SNU601 BARF1-transfected cells.

EBV-negative SNU601 gastric carcinoma cells were transfected with pCMV-Tag 2B/flag/BARF1 plasmid (SNU601 BARF1). BARF1-transfected cell clones were isolated by G418 selection. The recombinant plasmid pCMV-Tag 2B/flag/BARF1 was confirmed to be correct by restriction endonuclease digestion with BamHI/XhoI (Fig. 1A) and DNA sequencing (Fig. 2). Sequencing revealed SNU719 BARF1 to be virtually identical to the prototype B95-8 sequence, except for an A-to-G mutation at position 165710 (Fig. 2). Specific full-length transcripts of BARF1 were restricted to SNU719 cells (a naturally EBV-infected gastric carcinoma cell line) and SNU601 BARF1 cells, whereas BARF1 transcripts were not detected in SNU601 mock-transfected cells (here called SNU601 mock) (Fig. 1B).

Fig 1.

Fig 1

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Generation of BARF1 stable transfectants in SNU601 cells. (A) The recombinant plasmid pCMV-Tag 2B/flag/BARF1 was digested with BamHI and XhoI. (B) BARF1 mRNA was detected in SNU719 (naturally EBV-infected gastric carcinoma cells) and SNU601 BARF1 (stably transfected with BARF1) but not in SNU601 mock (stably transfected with an empty vector). (C and D) BARF1 protein was observed mainly in the supernatant but also weakly in cell lysates by Western blotting. Treatment with the transport blocker brefeldin A (3 μg/ml) for 24 h blocked BARF1 secretion, BARF1 protein accumulated in cell lysates of SNU601 BARF1 and was not detected in culture supernatants. All cell culture supernatants were concentrated as described in Materials and Methods. (E) Immunofluorescence microscopic examination showed that treatment with brefeldin A intensified cytosolic and nuclear localization of BARF1 protein. Brefeldin A-treated BARF1-expressing cells were labeled with BARF1 antibody (MAb 6F4) (green), and nuclei were counterstained with DAPI (blue).

Fig 2.

Fig 2

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Cloning of the EBV-carried BARF1 gene. Full-length BARF1 was amplified with primers F and R, which introduced BamHI and XhoI recognition sites, respectively. The 666-bp BARF1 gene was cloned from a cDNA library of SNU719, which is a naturally EBV-infected gastric carcinoma cell line. The BARF1 from SNU719 showed 1 silent mutation at position 165710 (A to G). Sequence data were searched against mRNA sequences extracted from the GenBank database (http://www.ncbi.nlm.nih.gov/gene/3783772).

BARF1 protein expressed in SNU601 BARF1 cells was detected as a 23- to 26-kDa band on Western blots with an anti-Flag antibody and the 4A6 anti-BARF1 antibody (Fig. 1C). BARF1 protein was secreted and was observed mainly in culture supernatants and only marginally detectable in cell lysates of SNU601 BARF1. To confirm that BARF1 is secreted via the classical pathway, we first examined BARF1 protein expression in cell culture supernatants treated with brefeldin A, which hinders export from distal Golgi compartments to the cell surface (Fig. 1D and E). BARF1 was detected in cell lysates upon treatment with brefeldin A, and BARF1 secretion was almost completely inhibited (Fig. 1D). These results indicate that BARF1 is a classically secreted protein. Immunofluorescence analysis showed that brefeldin A blocked secretion and caused retention of the BARF1 protein in the cytoplasm and nucleus (Fig. 1E).

BARF1-expressing SNU601 cells show increased proliferation.

SNU601 BARF1 cells showed higher proliferation than did SNU601 mock cells (P < 0.05) (Fig. 3B). There were no statistical differences in apoptosis, invasion, or migration between SNU601 BARF1 and SNU601 mock cells (Fig. 3A, C, and D). The effect of BARF1 knockdown on cell proliferation was investigated in SNU719 cells, which express high endogenous levels of BARF1. SNU719 cells, which are naturally infected with EBV, were transfected with different siRNA oligonucleotides against BARF1 type 1, 2, or 3. Knockdown was verified by RT-PCR. BARF1 mRNA expression was markedly inhibited by the siRNA against BARF1 type 3 (Fig. 4A). Interestingly, we observed a significant reduction in the growth rate of SNU719 cells transfected with siRNA against BARF1 type 3 compared to cells transfected with scrambled siRNA (P < 0.05) (Fig. 4B).

Fig 3.

Fig 3

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Biological properties of BARF1-expressing gastric carcinoma cells. (A) Apoptosis assay. Plots of flow cytometric data indicated no difference in apoptosis between SNU601 BARF1 and SNU601 mock. The percentage of apoptotic cells was determined by calculating annexin V-positive/propidium iodide-negative cells (bottom right quadrant). (B) Cell proliferation was measured with the WST-1 assay (upper graph) and trypan blue exclusion assay (lower graph). SNU601 BARF1 cells showed higher proliferation than did SNU601 mock cells (P < 0.05). (C) Matrix invasion assay showed that SNU601 BARF1 and SNU601 mock cells did not have statistically significant differences in invasive activities. Invaded cells were photographed with a fluorescence microscope (left panel). The means ± standard deviations of three independent experiments are shown (right panel). (D) Cell migration assessed using a scratch assay. SNU601 BARF1 and SNU601 mock cells did not have statistically significant differences in migration distance. (Left) Representative images of SNU601 BARF1 and SNU601 mock cells at the indicated time points after wound scratching (original magnification, ×400). (Right) Graphical presentation of the percentage of wound healing, which was calculated as 0-, 24-, and 72-h distances of wound/0-h distance. The distance was measured in at least three randomly selected fields. All experiments were done in triplicate.

Fig 4.

Fig 4

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Analysis of SNU719 (naturally EBV-infected gastric carcinoma cells) transfected with siRNA against BARF1. (A) BARF1 expression was almost completely inhibited upon transfection of 20 μM siRNA against BARF1 type 3. (B) Cell proliferation was lower in SNU719 cells treated with siRNA against BARF1 type 3 than in cells treated with scrambled siRNA (P < 0.05). All experiments were performed in triplicate.

BARF1-induced NF-κB RelA increment is associated with cyclin D1 and p21WAF1 expression.

In this study, we focused on the IRAK1/IκBα/NF-κB/cyclin D1 pathway (31, 33). We observed increased expression of the NF-κB RelA protein in nuclear extracts of SNU601 BARF1 cells compared with SNU601 mock cells (Fig. 5). Increased nuclear expression of NF-κB RelA correlated with increased expression of cyclin D1 and reduced expression of p21WAF1. Interestingly, expression of NF-κB RelA did not correlate with levels of the NF-κB-modulator IRAK1.

Fig 5.

Fig 5

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Alteration of NF-κB RelA, cyclin D1, and p21WAF1 protein levels in BARF1-expressing gastric carcinoma cells. Western blotting showed that SNU601 BARF1 cells had increased NF-κB RelA in nuclear extracts and cyclin D1 in total cell lysates, and p21WAF1 was reduced in total cell lysates (*, P < 0.05). Regarding expression of molecules in the NF-κB signaling pathway, pIκBα increased in SNU601 BARF1 cells. TF-IIB and β-actin were loading controls. All experiments were conducted in triplicate.

NF-κB inhibition neutralizes the increased proliferation observed in SNU601 BARF1 cells.

Transfection of siRNA against NF-κB RelA potently suppressed expression of NF-κB RelA protein in SNU601 BARF1 cells within 72 h following transfection (Fig. 6A). Further, RelA knockdown overcame the increased cellular proliferation that had been conferred by BARF1 stable overexpression (Fig. 6B). In fact, the growth rate of SNU601 BARF1 cells transfected with NF-κB RelA siRNA was similar to that of SNU601 mock-transfected cells. Additionally, we treated cells with IKKβ inhibitor to determine if proliferation was dependent upon NF-κB signaling. As a result, IKKβ inhibitor-treated SNU601 BARF1 cells did not show increased cell proliferation, in contrast to dimethyl sulfoxide (DMSO)-treated SNU601 BARF1 cells (Fig. 6C). These results suggest that activation of NF-κB signaling is important for the increased proliferation of BARF1-expressing cells.

Fig 6.

Fig 6

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Effect of NF-κB RelA inhibition. (A) NF-κB RelA in total cell lysate was almost totally inhibited by siRNA against NF-κB RelA (20 μM) (**, P < 0.01). (B) Proliferation was reduced in BARF1-expressing cells treated with siRNA against NF-κB RelA (solid squares) relative to BARF1-expressing cells treated with scrambled siRNA (open squares) or BARF1-expressing cells (open triangles; P < 0.05, respectively). (C) BARF1-expressing cells treated with IKKβ inhibitor (5 μM) (solid squares) had reduced proliferation relative to that of BARF1-expressing cells treated with DMSO (open squares; P < 0.05). (D) Increased expression of cyclin D1 was dependent upon NF-κB RelA in BARF1-expressing cells (*, P < 0.05). (E) p21WAF1 was decreased by BARF1, and NF-κB RelA inhibition led to p21WAF1 increase (*, P < 0.05). All experiments were executed in triplicate.

We examined cyclin D1 and p21WAF1 in NF-κB-inhibited cells (SNU601 BARF1 and SNU601 mock). NF-κB RelA siRNA suppressed cyclin D1 expression in SNU601 BARF1 cells (P < 0.05) but not in SNU601 mock cells (Fig. 6D). Additionally, p21WAF1 expression was reduced by BARF1 transfection and increased by NF-κB inhibition (P < 0.05) (Fig. 4E). These results suggest a probable link between cell proliferation, NF-κB/cyclin D1, and p21WAF1 in SNU601 BARF1 cells.

EBV-positive gastric carcinoma tissues demonstrate more frequent NF-κB and cyclin D1 immunopositivity and p21WAF1 loss.

We previously described immunohistochemistry (IHC) results for NF-κB RelA in 120 paraffin-embedded human gastric carcinoma tissues obtained following surgical resection. There were 23 cases of EBV-positive gastric carcinomas according to in situ hybridization for EBV-carried small RNAs (9). In the present study, IHC analysis of 120 cases of gastric carcinomas displayed a greater frequency of positive staining for cyclin D1 and negative staining for p21WAF1 in EBV-positive gastric carcinomas than in EBV-negative carcinomas (P < 0.05) (Fig. 7 and Table 1). Positivity for NF-κB was also more common in EBV-positive tissues than in EBV-negative gastric carcinoma tissues (39% versus 29%, respectively). However, IHC results for NF-κB did not reach statistical significance (Table 1).

Fig 7.

Fig 7

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Representative microscopic images of gastric carcinoma tissues. (A) EBV infection in gastric carcinoma tissue was confirmed by in situ hybridization for EBV-carried small RNAs. Black signals were seen in almost all cancer cell nuclei. (B to E) Examples of immunohistochemical staining: NF-κB RelA immunopositivity (B), cyclin D1 immunopositivity (C), p21WAF1 preservation (D), and p21WAF1 loss (E). Magnification, ×400. Panels A, B, C, and E were obtained from EBV-positive gastric carcinomas, whereas panel D was from an EBV-negative gastric carcinoma.

Table 1.

Differential immunoexpression of cyclin D1, p21WAF1, and NF-κB RelA between EBV-positive gastric carcinomas and EBV-negative gastric carcinomas

Protein and status No. (%) of samples
 P value
EBV positive (n = 23) EBV negative (n = 97)
Cyclin D1 0.006
    Positive 15 (65) 33 (34)
    Negative 8 (35) 64 (66)
p21WAF1 0.005
    Lost 18 (78) 44 (45)
    Preserved 5 (22) 53 (55)
NF-κB RelA NSa
    Positive 9 (39) 28 (29)
    Negative 14 (61) 69 (71)
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a

NS, not significant.

We categorized these 120 tumors into diffuse type (poor glandular formation) and intestinal type (well-developed glandular formation) in the Lauren classification. Of 23 EBV-positive gastric carcinomas, 21 were diffuse type (91%), whereas 57 of 97 (59%) of EBV-negative gastric carcinomas were diffuse type.

DISCUSSION

The present study highlights the important role of the EBV-encoded BARF1 gene in proliferation of EBV-infected gastric carcinoma. This is consistent with a recent report that cell growth was activated in BARF1-transfected HaCaT immortalized human keratinocytes (39). We did not find any effects of BARF1 expression on apoptosis in the present study. However, others suggested that the intracellular N-terminal fragment of BARF1 contributes to its antiapoptotic function as an oncogene in rodent fibroblasts (21). This conflict might be due to context, as different cell types may show different biological effects from BARF1 expression. Alternatively, additional factors may be required to enhance the antiapoptotic effect of BARF1 in gastric cancer cells. For example, in studies that examined the anticancer drug paclitaxel (originally named taxol), the antiapoptotic role of BARF1 was associated with increased expression of bcl-2 (22). However, we have previously shown that bcl-2 is rarely expressed in surgically resected EBV-positive gastric carcinoma tissues (9).

Secreted BARF1 may contribute to the increased proliferation observed in BARF1-expressing cells. This is consistent with previous suggestions that secreted BARF1 contributes to viral oncogenesis (17–19, 27–35). Additionally, in order to verify the autocrine/paracrine effect of BARF1, we put supernatant from SNU601 BARF1 (50-fold concentration) or supernatant from SNU601 (50-fold concentration) into original SNU601 cells, respectively. Then, in comparing cell proliferation rates, cells with BARF1 supernatant appeared to show greater proliferation than did cells with supernatant (minus BARF1), but the finding was not significant statistically (P = 0.06) (data not shown). There might be some obstacles to the proliferation effect of BARF1 supernatant, such as influence of pH and adsorption or deformation of macromolecules like growth-related factors during membrane penetration for concentration, or no persistency of BARF1 secretion, etc.

The present study also showed that BARF1 protein was mainly secreted into culture supernatants and only marginally detectable within the cells (Fig. 1D). This is consistent with recent observations in transfected HaCaT cells (39). Meanwhile, most papers in the past 16 years have described BARF1 as being almost completely secreted. The functions of intracellular BARF1 protein were not addressed in the present study. We believe that intracellular BARF1 may have little effect on promoting cellular proliferation, because the proliferation rates were similar in brefeldin A-treated SNU601 BARF1 cells and SNU601 mock-transfected cells but quite different from that of SNU601 BARF1 cells (data not shown). Further study is needed to confirm it. Also, we did not detect secreted BARF1 protein in naturally EBV-infected SNU719 gastric carcinoma cells, although BARF1 protein was observed in the lysates of SNU719 cells treated with the transport blocker brefeldin A. It is possible that the secreted amount is too small to detect in our assays.

The present study suggests that EBV-encoded BARF1 promotes cell proliferation through upregulation of NF-κB signaling in EBV-infected gastric carcinoma cells. NF-κB RelA inhibition neutralized BARF1-induced proliferation. This finding may shed light on the puzzling interaction between EBV and human stomach cells. Induction of NF-κB signaling by EBV-encoded LMP1 has already been reported in EBV-infected lymphoma, implicating LMP1 in cancer progression (2, 13–15). Similarly, increased NF-κB expression may contribute to progression of EBV-infected gastric carcinoma.

The NF-κB/cyclin D1 system appears to be important for EBV-infected gastric carcinoma, but its role in EBV-negative gastric carcinoma is less clear. The present study showed an increase in NF-κB and cyclin D1 expression in BARF1-expressing cells but not in mock-transfected cells. This is consistent with the results of Wiech et al. (40). They reported BARF1-induced cyclin D1 upregulation in gastric carcinoma, citing our previous data that cyclin D1 immunostaining was more frequently positive in EBV-positive gastric carcinomas than in EBV-negative gastric carcinomas (9). It was unknown if BARF1 affects MUC1 or beta-catenin, although interactions between MUC1 and beta-catenin have been reported to stimulate cyclin D1 expression and cell proliferation in helicobacter-induced gastric carcinoma (42). We suggest that BARF1 does not affect MUC1 or beta-catenin, because BARF1 transfection did not alter beta-catenin levels (data not shown). Furthermore, in our previous reports, MUC1 immunostaining was more frequently positive in EBV-negative gastric carcinoma tissues than in EBV-positive gastric carcinoma tissues (12), and the frequencies of beta-catenin alteration were similar in EBV-positive and EBV-negative gastric carcinoma tissues (12, 43).

BARF1-induced reduction in p21WAF1 expression was observed in the present study. To the best of our knowledge, there are no other reports regarding the relationship between BARF1 and p21WAF1. We demonstrated increased nuclear expression of NF-κB RelA along with p21WAF1 reduction in BARF1-expressing gastric carcinoma cells. This concurs with our previous paper, which showed that EBV infection correlated with p21WAF1 loss in immunohistochemical studies of gastric carcinomas (43). The present study showed NF-κB-dependent regulation of p21WAF1 regardless of the presence or absence of BARF1. NF-κB-dependent p21WAF1 expression has been reported in human cancers, such as colon cancer (44).

In conclusion, EBV-encoded BARF1 promotes proliferation of EBV-infected gastric carcinoma cells through autocrine/paracrine pathways activated by the secreted BARF1 protein, in which the signaling implies upregulation of NF-κB/cyclin D1 and reduction of the cell cycle inhibitor p21WAF1.

ACKNOWLEDGMENTS

This work was supported by a Korea Research Foundation grant funded by the Korean Government (MOEHRD) (KRF-2007-313-E00103).

We thank SuperBioChips Laboratories (Seoul, South Korea) for their technical assistance with tissue array production.

The authors have no conflict of interest to declare.

Footnotes

Published ahead of print 3 July 2013

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