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Cancer Research 68, 1030, February 15, 2008. doi: 10.1158/0008-5472.CAN-07-5252
© 2008 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Reconstitution of Nasopharyngeal Carcinoma–Type EBV Infection Induces Tumorigenicity

Eri Seto1, Tadamasa Ooka2, Jaap Middeldorp3 and Kenzo Takada1

1 Department of Tumor Virology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan; 2 Laboratoire de Virologie Moleculaire, Centre National de la Recherche Scientifique, Faculte de Medecine R.T.H. Laennec, Universite Claude Bernard Lyon-1, Lyon, France; and 3 Department of Pathology, Vrije Universiteit Medical Center, Amsterdam, the Netherlands

Requests for reprints: Kenzo Takada, Department of Tumor Virology, Institute for Genetic Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo 060-0815, Japan. Phone: 81-11-706-5071; Fax: 81-11-706-7540; E-mail: kentaka{at}igm.hokudai.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Several reports have shown that the EBV-encoded BARF1 gene has oncogenic activity. We have recently reported that BARF1 is expressed as a latent gene in most nasopharyngeal carcinomas (NPC), suggesting that BARF1 may have an important role in NPC oncogenesis. However, we found that when the NPC-derived EBV-negative cell lines, HONE-1 and CNE-1, were infected with EBV in vitro, BARF1 was not expressed, although the expression of other latent genes was identical to that of NPC tumors. Therefore, we generated a recombinant EBV (rEBV) carrying the BARF1 gene (BARF1-rEBV) under the SV40 promoter to reconstitute the NPC-type EBV infection. NPC-derived EBV-negative cell lines were stably infected with either a wild-type rEBV (wild-rEBV) or BARF1-rEBV. The resultant BARF1-rEBV–infected NPC cell clones represented NPC-type EBV expression, and BARF1 expression was similar to that observed in NPC tissues. BARF1-rEBV–infected cell clones grew to a higher cell density and were more resistant to apoptosis than wild-rEBV–infected counterparts. BARF1 protein was quickly secreted into the culture medium, and secreted BARF1 contributed to the increase of cell densities in NPC cells, but it had no effect on resistance to apoptosis. Furthermore, BARF1-rEBV–infected cell clones became tumorigenic in nude mice. These results suggest that BARF1 plays an important role in NPC development. [Cancer Res 2008;68(4):1030–6]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nasopharyngeal carcinoma (NPC) is a common cancer in areas of southern China and Southeast Asia, reaching a peak incidence of 20 to 30 cases per 100,000 individuals (1). The disease is a poorly differentiated carcinoma and the mechanism underlying its development remains largely unknown. Although the primary tumors are sensitive to radiotherapy, 60% to 85% of NPC patients already have clinically detectable metastases in the regional lymph nodes or distant organs at the time of diagnosis (2). There is no effective treatment for NPC metastases, and the prognosis remains poor with a 5-year survival of ~50% (3). EBV is present in 100% of NPC cells (4), but the role of EBV in the oncogenic transformation of NPC cells is unclear.

The EBV-encoded BARF1 gene is located in the BamHI-A fragment of the EBV genome and has oncogenic activity (5). BARF1 induces malignant transformation of rodent fibroblasts (6), and a 54-amino acid region of the NH2 terminus, which is capable of activating expression of the antiapoptotic protein bcl-2, is essential for the transforming activity (7). Transfection with the BARF1 gene enhances the tumorigenicity of EBV-negative Burkitt's lymphoma (BL)-derived cell lines (8, 9). BARF1 can also immortalize primary monkey kidney epithelial cells (10), but the immortalized cells were not tumorigenic in nude mice (11).

Two distinct types of EBV latent gene expression have been described in NPC cells (12). About half of the NPC cases are type I latency, which is characterized by expression of the EBV-determined nuclear antigen 1 (EBNA1), EBV-encoded RNA, and BamHI-A rightward transcripts. The remaining NPC cases express these genes and additionally express latent membrane protein 1 (LMP1), LMP2A, and LMP2B and are classified as type II latency. Several reports have shown that the EBV-encoded BARF1 gene is expressed in a high proportion of NPC tissues, suggesting that BARF1 plays an important role in the pathogenesis of NPC (13–16). Because expression of the BARF1 gene is induced on the induction of the lytic cycle in EBV-positive cell lines (14, 17), expression of BARF1 in NPC tissues is thought to reflect spontaneous induction of the lytic cycle in carcinoma cells. We previously performed a comparative analysis of the expression of BARF1 and three EBV-encoded lytic genes (18) in NPC tissues (19) and showed that the BARF1 gene was expressed in the absence of lytic gene expression, indicating that BARF1 was expressed as a latent gene in those tissues.

We also have previously developed an infection system of epithelial cells in vitro using a recombinant EBV (rEBV) carrying a selectable marker (20). However, BARF1 was not expressed in EBV-converted epithelial cells, in contrast to its expression in NPC tissues. Therefore, these cell lines could not be considered as a proper in vitro model of NPC, although they showed type I latency.

In this study, we generated a rEBV carrying the BARF1 gene under the control of the SV40 promoter to reconstitute a NPC-type EBV infection, which is characterized by type I or type II latency with BARF1 expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and culture. EBV-negative and EBV-positive Akata cell lines were grown in RPMI 1640 (Sigma) at 37°C in a 5% CO2 incubator. HONE-1 (21) and CNE-1 (22), which are EBV-negative NPC-derived cell lines, were cultured in DMEM (Sigma). All media were supplemented with 10% fetal bovine serum (FBS; Invitrogen), penicillin (40 units/mL), and streptomycin (50 µg/mL).

Generation of a rEBV expressing the BARF1 gene. The targeting construct TXneoV-BARF1, used for the generation of BARF1-rEBV in Akata cells, is essentially the same as the construct used to generate a rEBV with the enhanced green fluorescent protein (EGFP-rEBV; ref. 23), with the BARF1 open reading frame driven by the SV40 promoter replacing the EGFP expression cassette. Briefly, TXneoV-BARF1 contains a 2,010-bp EcoRV-SalI fragment [containing the neomycin resistance gene (NeoR gene)] from pcDNA3 (Invitrogen) and a 1,889-bp NotI fragment (containing the BARF1 gene driven by the SV40 promoter) from pBS-KS (+)-BARF1 inserted into the BamHI-X fragment. This insertion site corresponds to nucleotide 131,290 of the wild-type EBV sequence (GenBank accession number AJ507799), where the SmaI site is located. The HindIII fragment (10.2 kb) of TXneoV-BARF1, which was 4.2 kb longer than the wild-type fragment due to the insertion of the NeoR gene and the BARF1 gene, was excised and introduced into EBV-positive Akata cells by electroporation, as described previously (24). Transfected cells were cultured for 2 days and plated in 96-well plates at 104 per well in medium containing 700 µg/mL neomycin for selection. Half of the culture medium was replaced with fresh medium containing neomycin every 5 days. To obtain cell clones harboring BARF1-rEBV, a total of 185 neomycin-resistant cell clones were screened for the existence of homologously recombined EBV by Southern blotting.

Virus infection and drug selection. Targeted Akata cells harboring wild-rEBV and BARF1-rEBV (2 x 106) were resuspended in 1 mL of fresh medium containing 0.5% rabbit anti-human IgG (DakoCytomation) and incubated for 6 h to induce lytic replication. The culture medium was replaced with fresh medium, and 2 days later, the culture supernatant was harvested, filtered through a 0.45-µm pore size membrane, and used as the virus stock solution. For infection, EBV-negative Akata cells (106) were suspended in 1 mL of virus stock solution and incubated at 37°C for 90 min with continuous gentle mixing. After removal of the virus solution, the infected cells were cultured for 2 days and then plated in 96-well plates at 104 per well in medium containing 700 µg/mL G418 for selection. Half of the culture medium was replaced with medium containing G418 every 5 days. Drug-resistant clones were screened by Southern blotting to obtain cell clones harboring BARF1-rEBV.

Southern blot analysis. DNA was extracted by using the AquaPure genomic DNA isolation kit (Bio-Rad Laboratories) according to the manufacturer's instructions. Extracted DNA was digested with BamHI or XbaI, separated by electrophoresis in a 0.8% agarose gel, and transferred to Hybond N+ nylon membrane (Amersham Biosciences). The BamHI-X fragment of Akata EBV DNA (nucleotides 142,740–144,861 of the EBV B95-8 strain) was used as a probe. Probe labeling was carried out using the AlkPhos direct labeling kit (Amersham Biosciences) and the signals were detected with the CDP-Star detection reagent (Amersham Biosciences).

Establishment of rEBV-infected NPC cell clones. HONE-1 or CNE-1 cells were cocultured with anti–Ig-treated Akata cells infected with either wild-rEBV (a rEBV carrying only the NeoR gene) or BARF1-rEBV. rEBV-infected NPC cell clones were isolated by neomycin selection as described previously (20). Cell clones were maintained in culture medium containing 500 µg/mL neomycin.

Immunofluorescence. Expression of EBNA was examined in cells fixed with acetone/methanol (1:1) by anticomplement immunofluorescence with a reference human serum (titer, x640).

Reverse transcription-PCR analysis. Total RNA was extracted from cells using the Trizol reagent (Life Technologies) and then treated with DNase I (Life Technologies). One microgram of total RNA was reverse transcribed with ReverTra Ace (TOYOBO) using 100 pmol of random hexamer (TaKaRa) for cDNA synthesis. cDNA aliquots were then subjected to PCR analyses using primer pairs specific for BARF1 (5'-GGCTGTCACCGCTTTCTTGG-3' and 5'-AGGTGTTGGCACTTCTGTGG-3') or glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 5'-GCCTCCTGCACCACCAACTG-3' and 5'-CGACGCCTGCTTCACCACCTTCT-3'). Each PCR cycle consisted of denaturation at 94°C for 30 s, annealing at 64°C for 30 s (BARF1) or 55°C for 30 s (GAPDH), and extension at 72°C for 1 min for 26 cycles (BARF1) or 21 cycles (GAPDH).

Immunoblot analysis. Wild-rEBV–infected or BARF1-rEBV–infected NPC cell clones were cultured in serum-free medium for 2 days and whole-cell lysates and culture supernatants were then harvested. Supernatants were concentrated by ultrafiltration using Ultrafree-MC 10,000 NMWL filter units (Millipore Co.). BARF1 protein was precipitated from the concentrated supernatants in a 4-fold volume of acetone at –20°C. Equal volumes of cell lysates (7.5 x 105 cells) and the protein precipitate from supernatants were dissolved in SDS-PAGE loading buffer, separated using 15% polyacrylamide gels, and transferred to a nitrocellulose membrane (Schleicher & Schuell). Membranes were blocked with 2% nonfat dry milk in TBS (pH 7.6) and probed with a rabbit polyclonal anti-BARF1 antibody, which was raised against a purified BARF1-GST fusion protein. After incubation with the primary antibody for 2 h at room temperature, membranes were washed in TBS-Tween 20 (0.1%) followed by incubation with anti-rabbit peroxidase-conjugated IgG. Membranes were visualized with an enhanced chemiluminescence Western blotting kit (Amersham Biosciences).

Cell growth assays. NPC cell clones in log phase were plated in six-well tissue culture plates at a density of 1 x 105 cells per well (HONE-1) or 2 x 104 cells per well (CNE-1) in 3 mL of medium containing 0.1% FBS. The number of viable cells was counted every day after plating.

Apoptosis assay. For induction of apoptosis, NPC cell clones were plated in six-well tissue culture plates at a density of 4 x 105 cells per well (HONE-1) or 3 x 104 cells per well (CNE-1) in 2.5 mL of serum-free medium. After 4 days, total cells were harvested and fixed in 1 mL PBS containing 70% ethanol for 1 h at 4°C. The fixed cells were washed with PBS, treated with 100 µg/mL RNase A for 30 min at 37°C, and stained with 50 µg/mL propidium iodide for 30 min at 37°C. The percentage of apoptotic cells with hypodiploid DNA was measured using a FACSCalibur instrument (Becton Dickinson).

Tumorigenicity assay. NPC cell clones were grown to log phase and then harvested by trypsinization and washed thrice in serum-free DMEM. Cells (3 x 106) were resuspended in 100 µL of serum-free DMEM and injected s.c. into 4-week-old Swiss nude mice. Tumor development was monitored for 30 days.

Preparation of conditioned medium. Cells (106) were cultured for 48 h after plating and then cultured in serum-free medium for an additional 48 h. The culture medium (i.e., conditioned medium) was collected and filtered through a 0.2-µm pore size membrane.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BARF1 expression in EBV-converted NPC cell lines. The NPC-derived EBV-negative cell lines HONE-1 and CNE-1 were infected with wild-rEBV (carrying only the NeoR gene) and EBV-converted cell clones were isolated by G418 selection. Figure 1 shows the expression of the BARF1 gene in EBV-converted NPC cell clones, as detected by reverse transcription-PCR (RT-PCR). Anti–Ig-treated Akata cells (25), which express levels of BARF1 transcripts equivalent to that of NPC tissues (19), were used as a positive control. BARF1 is a lytic gene and its expression was therefore induced in EBV-positive Akata cells after treatment with anti-Ig for 24 h (Fig. 1, lane 3). BARF1 was not expressed in EBV-converted NPC cell clones (Fig. 1, lanes 5–7).


Figure 1
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  		Figure 1. RT-PCR analysis of BARF1 expression in EBV-converted HONE-1 and CNE-1 cells.

 
Generation of a rEBV expressing BARF1. To establish EBV-infected NPC-derived cells expressing BARF1, we generated a rEBV carrying the BARF1 gene driven by the SV40 promoter. EBV-positive Akata cells were transfected with a targeting construct containing the NeoR gene and the BARF1 gene, which had been inserted into the BXLF1 locus of the EBV genome (Fig. 2A ). This region encodes viral thymidine kinase, which is dispensable for viral replication. After transfection, the cells were cultured in selection medium and drug-resistant cell clones were screened by Southern blot analysis for the presence of cell clones with homologously recombined EBV, designated BARF1-rEBV. Of the 185 cell clones assayed, 1 clone contained BARF1-rEBV as well as wild-rEBV (Fig. 2B, lane 2). Subsequently, the obtained cell clone was treated with anti-Ig for virus production, and the mixture of wild-rEBV and BARF1-rEBV was used to infect EBV-negative Akata cells. The infected cells were subjected to G418 selection, and only cell clones carrying BARF1-rEBV alone were thus obtained (Fig. 2B, lanes 3–5).


Figure 2
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  		Figure 2. Generation of a rEBV carrying the BARF1 gene driven by the SV40 promoter in Akata cells. A, map of the EBV genome surrounding the targeted region before and after insertion of NeoR gene and BARF1 gene driven by the SV40 promoter. The positions of the BamHI (B) and XbaI (X) sites are indicated. Arrowheads, positions of HindIII sites located at the end of the linear targeting construct. The BamHI-X fragment was used as a probe for Southern blot analysis. B, Southern blot analysis using BamHI- or XbaI-digested genomic DNA. The bands derived from the BARF1-rEBV (white arrowheads) and the bands derived from wild-type EBV (black arrowheads) are indicated. C, RT-PCR analysis of BARF1 expression. The results from an EBV-negative Akata cell clone (lane 1), an EBV-positive Akata cell clone (lane 2), an anti–Ig-treated Akata cell clone (lane 3), and several BARF1-rEBV–infected Akata cell clones (lanes 4–7) are shown.

 
Next, we examined BARF1 expression in BARF1-rEBV–infected Akata cell clones by RT-PCR. BARF1 expression was detected in all cell clones examined and the levels of expression were similar to that of anti–Ig-treated Akata cells (Fig. 2C).

Establishment of wild-rEBV–infected and BARF1-rEBV–infected NPC cell clones. HONE-1 and CNE-1 cells were cocultured with anti–Ig-treated Akata cells infected with either wild-rEBV or BARF1-rEBV, and rEBV-infected NPC cell clones were isolated by G418 selection. Immunostaining revealed that all rEBV-infected NPC cell clones expressed EBNA (Fig. 3A ). Immunoblot analyses indicated that only EBNA1 was expressed in the rEBV-infected cell clones (Fig. 3B, top). Two wild-rEBV–infected CNE-1 cell clones weakly expressed LMP1, but other cell clones were negative for LMP1 expression (Fig. 3B, middle). We further examined BARF1 gene expression in wild-rEBV–infected and BARF1-rEBV–infected cell clones. RT-PCR indicated that all BARF1-rEBV–infected cell clones showed BARF1 expression at levels similar to that of anti–Ig-treated Akata cells, whereas wild-rEBV–infected cell clones did not express BARF1 (Fig. 3C).


Figure 3
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  		Figure 3. Establishment of wild-rEBV–infected or BARF1-rEBV–infected HONE-1 and CNE-1 cell clones. A, immunofluorescent staining of EBNA in BARF1-rEBV–infected NPC cells. EBV-uninfected parental cells are shown as negative controls. B, immunoblot analysis of EBNA and LMP1 in rEBV-infected NPC cells. Lysates of LCL, parental EBV-negative cells, three independent wild-rEBV–infected clones, and three independent BARF1-rEBV–infected clones were used for analysis. β-Actin served as a loading control. C, RT-PCR analysis of BARF1 expression in rEBV-infected NPC cells. D, immunoblot analysis of BARF1 protein in rEBV-infected HONE-1 or CNE-1 cells. Cell lysates and the concentrated culture medium from a wild-rEBV–infected cell clone and two independent BARF1-rEBV–infected cell clones were analyzed.

 
We have previously reported that BARF1 protein is quickly secreted into the culture supernatant of BARF1-trasfected CNE-1 cells and is only weakly detected in cell lysates (19). Therefore, we examined whether BARF1 protein was secreted into the culture supernatant by BARF1-rEBV–infected cell clones. Two bands corresponding to the BARF1 protein were detected in the culture supernatant, but not the cell lysates of BARF1-rEBV–infected cell clones (Fig. 3D). These results are consistent with a previous report showing that the BARF1 gene encodes two different isoforms (29 and 23 kDa) of the secreted protein (26).

Taken together, these data show that we have successfully established BARF1-rEBV–infected NPC cell clones. The resultant cell clones represent NPC-type EBV expression, and the level of expression of BARF1 was comparable with that of NPC tissues, indicating that BARF1-rEBV reconstitutes a NPC-type EBV infection in vitro.

Effect of BARF1 on growth characteristics and resistance to apoptosis in NPC cell clones. The establishment of BARF1-rEBV–infected NPC cell clones enabled us to examine the effects of BARF1 on the characteristics of NPC-derived cells in the context of a NPC-type EBV infection. As shown in Fig. 4A , under low serum culture conditions, three of four BARF1-rEBV–infected HONE-1 cell clones reached higher cell densities than wild-rEBV–infected HONE-1 cell clones. In CNE-1 cells, there was more variation, but BARF1-rEBV–infected cell clones tended to reach a higher density than their wild-rEBV–infected counterparts.


Figure 4
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  		Figure 4. Effect of BARF1 on growth characteristics and resistance to apoptosis in NPC cells. A, comparison of growth between wild-rEBV–infected and BARF1-rEBV–infected HONE-1 or CNE-1 cell clones. Cell growth was measured by counting viable cells using trypan blue. B, the effect of BARF1 on resistance to apoptosis. Wild-rEBV–infected and BARF1-rEBV–infected HONE-1 or CNE-1 cells were induced to undergo apoptosis by serum deprivation. The frequency of apoptotic cells was determined using a sub-G1 apoptosis assay.

 
Next, we examined the effect of BARF1 on resistance to apoptosis. Wild-rEBV–infected and BARF1-rEBV–infected cell clones were cultured in serum-free medium for 4 days and then subjected to a sub-G1 apoptosis assay. BARF1-rEBV–infected HONE-1 cell clones were more resistant to apoptosis than their wild-rEBV–infected counterparts (Fig. 4B). In contrast, differences in resistance to apoptosis were not observed between wild-rEBV–infected and BARF1-rEBV–infected CNE-1 cell clones.

Tumorigenicity of BARF1-rEBV–infected NPC cell clones in nude mice. Several reports have shown that BARF1 transfection induced malignant transformation in mouse fibroblasts, as well as BL-derived cell lines, suggesting that BARF1 plays a role in oncogenesis. However, the effect of BARF1 expression in EBV-infected NPC-derived cell lines on malignant transformation has never been determined. Therefore, we examined the effect of BARF1 expression on the tumorigenicity of NPC-derived cells. Parental HONE-1 and CNE-1 cells, their NeoR gene-transfected cell clones (two clones from each cell line), wild-rEBV–infected cell clones (two clones from each cell line), and BARF1-rEBV–infected cell clones (three clones from each cell line) were inoculated into nude mice. As shown in Fig. 5 , all BARF1-rEBV–infected cell clones developed tumors by as early as the second week after inoculation, in all mice assayed, and the tumors reached 600 to 1,900 mm3 in size by 30 days. In contrast, other cell clones, including wild-rEBV–infected cell clones, were not tumorigenic. These results clearly show that BARF1 contributes to the tumorigenicity of NPC.


Figure 5
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  		Figure 5. Tumor formation in nude mice. The tumorigenicity of the indicated cells was evaluated in vivo by injecting cells s.c. into nude mice. The size of tumors at the inoculation site was measured using a standard procedure on the indicated days after inoculation. Points, mean average of four independent mice for HONE-1 cell clones or three independent mice for CNE-1 cell clones; bars, SD.

 
Role of secreted BARF1 in growth and resistance to apoptosis in HONE-1 cells. The mechanism by which BARF1 induces malignant transformation of NPC cells remains to be determined and we thus examined the role of secreted BARF1 in this process. As described above, BARF1 protein was quickly secreted into the culture medium of BARF1-rEBV–infected NPC cells. To determine the role of the secreted form of BARF1, uninfected HONE-1 cells were cultured in control medium or conditioned medium prepared from EBV-uninfected, wild-rEBV–infected, or BARF1-rEBV–infected HONE-1 cells and then examined for growth and resistance to apoptosis. HONE-1 cells cultured in conditioned medium of BARF1-rEBV–infected cells attained higher cell densities than those cultured in other medium (Fig. 6A ). On the other hand, neither conditioned medium of BARF1-rEBV–infected cells nor that of wild-rEBV–infected cells affected resistance to apoptosis in HONE-1 cells (Fig. 6B).


Figure 6
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  		Figure 6. Role of the secreted BARF1 protein. A, growth of HONE-1 cells in the presence or absence of secreted BARF1. HONE-1 cells were cultured in control medium or in conditioned medium from EBV-uninfected, wild-rEBV–infected, or BARF1-rEBV–infected HONE-1 cells. Growth curves were generated as described in Fig. 4A. Points, mean of three independent experiments; bars, SE. B, resistance to apoptosis of HONE-1 cells in the presence or absence of secreted BARF1. HONE-1 cells were cultured in control medium or conditioned medium from the indicated cells for 4 d. The frequency of apoptotic cells was determined using a sub-G1 apoptosis assay. Columns, mean of three independent experiments; bars, SE. C, effect of anti-CSF-1 antibody on growth of HONE-1 cells. Purified rabbit anti-human CSF-1 antibody (PeproTech EC), at various concentrations, was added to the culture medium containing 0.1% FBS. Points, mean of three independent experiments; bars, SE.

 
Previous studies have shown that BARF1 is a homologue of the human colony-stimulating factor-1 receptor (CSF-1R), the product of the proto-oncogene c-fms (27). The secreted form of BARF1 protein binds human CSF-1 (26) and neutralizes the proliferative effects of CSF-1 on mouse macrophage progenitor cells. We also observed that the preincubation of recombinant CSF-1 with conditioned medium prepared from BARF1-rEBV–infected HONE-1 cells abolished tyrosine phosphorylation of CSF-1R in mouse NIH3T3 cells stably expressing human CSF-1R (Supplementary Fig. S1). These findings indicate that secreted BARF1 functions as an antagonist of CSF-1. Because CSF-1 and its receptor were expressed in HONE-1 cells (data not shown), we examined whether inhibition of CSF-1 by anti-human CSF-1 neutralizing antibody increased the growth of HONE-1 cells. As shown in Fig. 6C, the addition of anti-human CSF-1 antibody to the culture medium did not increase growth of HONE-1 cells, suggesting that CSF-1 is not involved in growth promotion of HONE-1 cells by secreted BARF1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we generated BARF1-rEBV carrying the BARF1 gene under the SV40 promoter using an Akata cell system and then established BARF1-rEBV–infected NPC cell clones. All established BARF1-rEBV–infected NPC cell clones showed type I latency and BARF1 expression at levels similar to that of NPC tissues. Thus, BARF1-rEBV provides a model system for studying the role of BARF1 in NPC oncogenesis by reconstituting NPC-type EBV infection in vitro.

Importantly, BARF1-rEBV–infected HONE-1 and CNE-1 cell clones produced tumors in nude mice, whereas tumor formation was rarely observed in control mice inoculated with wild-rEBV–infected cell clones, as well as EBV-negative cell clones, suggesting that BARF1 was important for NPC tumorigenicity. Although several studies have indicated that BARF1 expression endows EBV-uninfected BALB/c3T3, Louckes, and Akata cells with tumorigenicity, our data first showed that BARF1 contributes to the tumorigenicity of NPC-derived cells in the context of a NPC-type EBV infection.

We also observed that BARF1-rEBV–infected NPC cell clones reached higher cell densities and were more resistant to apoptosis than wild-rEBV–infected clones under low serum culture conditions. As reported previously, we confirmed that the BARF1 protein was quickly secreted into the culture medium and was not detectable in cell lysates in BARF1-rEBV–infected NPC cells. This result is consistent with an earlier report showing that BARF1 protein was only weakly detected in most NPC tissues, although the tissues were strongly positive for BARF1 expression at the transcriptional level (19). The addition of conditioned medium from BARF1-rEBV–infected HONE-1 cells to EBV-uninfected HONE-1 cell cultures revealed that secreted BARF1 contributed to growth of HONE-1 cells. This is consistent with a previous report that the secreted form of BARF1 protein induces cell cycle activation of rodent fibroblasts, human BL-derived cell lines, and primary monkey kidney epithelial cells (28).

In contrast with the effect of the increase of cell densities, secreted BARF1 did not increase resistance to apoptosis in HONE-1 cells. BARF1 has been reported to confer resistance to apoptosis in rodent fibroblasts through up-regulation of bcl-2 (7). However, we did not detect any differences in the expression of bcl-2 between wild-rEBV–infected and BARF1-rEBV–infected HONE-1 cell clones (data not shown). The mechanism of the antiapoptotic effect of BARF1 remains to be clarified.

BARF1 inhibits phosphorylation of CSF-1R by CSF-1. However, this activity is not related to the growth-promoting activity of secreted BARF1 because the inhibition of CSF-1 by anti-CSF-1 antibody did not increase the growth of HONE-1 cells.

EBV-converted NPC-derived cell clones established in vitro do not express BARF1 in the absence of lytic infection. Although most NPC tissues express BARF1, the level of BARF1 expression is variable. Among three transplantable NPC tumors (29), C17 and C18, which were derived from metastatic NPC tissues, expressed BARF1, but C15, which was derived from a primary NPC tumor, did not express BARF1, as reported in our previous study (19). These results suggest that BARF1 expression in NPC tumors is a consequence of selection of cells having the highest rate of proliferation, although the precise mechanisms regulating BARF1 expression remain to be clarified.

LMP1 is important for NPC oncogenesis (30), but expression of LMP1 is variable and 50% of NPC tumors as well as all the BARF1-rEBV–infected NPC cell clones established in this study did not express LMP1. Therefore, BARF1 seems to contribute to tumorigenicity in NPC more universally than LMP1.

In conclusion, the present findings indicate that the reconstitution of NPC-type EBV infection induces NPC cell tumorigenicity in nude mice. Our study is the first description of the role of BARF1 in tumorigenicity of NPC cells in the context of NPC-type EBV infection. Secreted BARF1 seems to play an important role in NPC through growth promotion. A recent study has shown that BARF1 protein exists in the serum and saliva of patients with NPC (31). The detection of secreted BARF1 in the serum would be a useful diagnostic marker of NPC. Moreover, BARF1 protein should be a target of therapeutics against NPC.


   Acknowledgments
 
Grant support: Grants-in-aid from the Ministry of Education, Science, Sports, Culture, and Technology, Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 9/10/07. Revised 11/ 1/07. Accepted 11/20/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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