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J Virol. Mar 2003; 77(6): 3859–3865.
PMCID: PMC149494

Malignant Transformation of Epstein-Barr Virus-Negative Akata Cells by Introduction of the BARF1 Gene Carried by Epstein-Barr Virus

Abstract

Spontaneous loss of the Epstein-Barr virus (EBV) genome in the BL cell line Akata led to loss of tumorigenicity in SCID mice, suggesting an important oncogenic activity of EBV in B cells. We previously showed that introduction of the BARF1 gene into the human B-cell line Louckes induced a malignant transformation in newborn rats (M. X. Wei, J. C. Moulin, G. Decaussin, F. Berger, and T. Ooka, Cancer Res. 54:1843-1848, 1994). Since 1 to 2% of Akata cells expressed lytic antigens and expressed the BARF1 gene, we investigated whether introduction of the BARF1 gene into EBV-negative Akata cells can induce malignant transformation. Here we show that BARF1-transfected, EBV-negative Akata cells activated Bcl2 expression and induced tumor formation when they were injected into SCID mice. In addition, when EBV-positive Akata cells expressing a low level of BARF1 protein were injected into SCID mice, the expression of BARF1, as well as several lytic proteins, such as EA-D, ZEBRA, and a 135-kDa DNA binding protein, increased in tumor cells while no latent LMP1 and late gp220-320 expression was observed in tumor cells. These observations suggest that the BARF1 gene may be involved in the conferral of tumorigenicity by EBV.

Epstein-Barr virus (EBV), a ubiquitous human herpesvirus that causes infectious mononucleosis, is likely to be involved in the pathogenesis of endemic Burkitt's lymphoma (BL) and lymphoma induced in AIDS patients. Almost 100% of NPCs (nasopharyngeal carcinomas) (1, 24) and about 10% of gastric carcinomas are associated with EBV (2, 17). The EBV genome is present in more than 90% of endemic BL cases in equatorial Africa and in about 10% of sporadic BL cases (11). In vitro, EBV is able to infect resting B lymphocytes and create the immortalized lymphoblastoid cell line (LCL).

EBV-positive Akata BL cells spontaneously lost the EBV genome during long-term culture, becoming nontumorigenic, while parental EBV-positive Akata cells induced tumors in SCID mice, suggesting an important oncogenic role of EBV in these cells (8). In fresh BL biopsy tissues and early-passage BL-derived cell lines, only EBNA1, EBERs, and BARF0 are expressed and these are classified as type I latency genes. In the EBV-positive Akata BL cell line, three of the genes mentioned above plus LMP2A were expressed (7, 12). Transfection of the EBNA1 gene into EBV-negative Akata cells did not restore tumorigenicity to Akata cells. Recent data showed that transfection of EBERs, the second viral gene expressed in EBV-positive Akata cells and in EBV-negative Akata cells, induced tumor formation in SCID mice (7, 11). In these EBERs-expressing cells, activation of the antiapoptotic Bcl2 gene was observed (7) but this activation was not confirmed by another laboratory (12). In our previous study, we reported that about 1 to 2% of EBV-positive Akata cells expressed a lytic antigen (22). In these cells, we observed expression of the BARF1 gene localized in the BamHI A fragment of the viral genome (23).

The BARF1 gene, encoding a 31-kDa early protein, has been shown to be able to induce malignant transformation in BALB/c3T3 cells and in the human B-cell line Louckes (15, 19, 20). Injection of BARF1-expressing BALB/c3T3 cells into newborn rats resulted in the induction of aggressive tumors, while injection of BARF1-expressing Louckes human EBV-negative B cells into the same mouse induced the formation of a small tumor that regressed after 3 weeks. In addition, this viral gene was capable of immortalizing primary epithelial cells (20) and was expressed in NPC biopsy tissues (4). Thus, the BARF1 gene has oncogenic activity (9). Expression of the BARF1 gene in the EBV-negative Louckes cell line induces expression of the proto-oncogene c-myc and B-cell activation antigens CD23 and CD21 (10). However, the known activities of BARF1 are not limited to its oncogenic ability; BARF1 has also been shown to serve as a target for antibody-dependent cytotoxicity (18). Recent reports have shown that this viral protein could play a role as a soluble receptor for human colony-stimulating factor 1 (16) and could regulate the immune response by inhibiting alpha interferon secretion from mononuclear cells (3).

We wondered if BARF1 is capable of inducing a tumoral transformation when it is expressed in EBV-negative Akata cells.

Activation of BARF1 gene expression in tumors.

Since 1 to 2% of EBV-positive Akata cells expressed early proteins (22), we asked whether BARF1 is expressed in this cell line. To detect BARF1 expression, we carried out reverse transcription RT-PCR and an immunoblot assay. RT-PCR analysis was carried out with RNA extracted from EBV-positive Akata, M81, B95-8, and P3HR-1 cells. The RT-negative control came from a PCR without reverse transcriptase (Fig. (Fig.11 right part). The presence of BARF1 transcription was clear not only in EBV-positive Akata cells but also in other EBV-positive B-cells (Fig. (Fig.1,1, left part). We were then interested in determining whether the BARF1 gene is expressed in tumors induced by injection of EBV-positive Akata cells in SCID mice. After 3 months of Akata cell injection, we harvested tumor biopsy tissues and examined the expression of early proteins like BARF1, ZEBRA, EA-D, and BALF2 by both the Western blot and immunofluorescence assay methods. Figure Figure22 shows representative immunoblots of protein extracted from tumors induced by EBV-positive Akata cells. Translational expression of BARF1 was very weak in EBV-positive Akata cells (Fig. (Fig.2A,2A, lane 2) and absent in EBV-negative Akata cells (Fig. (Fig.2A,2A, lane 1), as well as in another EBV-negative B-cell line, Louckes (Fig. (Fig.2A,2A, lane 5), while its expression became more important in two EBV-positive Akata cell-derived tumors (Fig. (Fig.2A,2A, lanes 3 and 4). Expression of EA-D, ZEBRA, and BALF2 was weakly positive in cell cultures but became significant in tumors, while expression of the EBV gp220-320 late protein was never detected in tumors or in Akata cell cultures (Fig. (Fig.2B).2B). Expression of the early proteins was also analyzed by using an immunofluorescence assay (6) with anti-BARF1, anti-ZEBRA, anti-EA-D, and anti-BALF2 antibodies (Fig. (Fig.3).3). Surprisingly, a large proportion of cells from tumors effectively expressed the above-mentioned early proteins. Before injection, about 1% of EBV-positive Akata cells expressed lytic proteins (BARF1, EA-D, BALF2, and ZEBRA) while a large proportion of tumor-derived cells became positive for early antigens (more than 50% for BARF1, ZEBRA, and EA-D and 30% for BALF2). EBV-positive Akata cells do not expressed LMP1 as a type I latency protein. We also observed no reactivation of LMP1 in tumors derived from EBV-positive Akata cells. Neither RT-PCR nor immunoblot analysis showed any positive response in cell cultures or in tumors (Fig. (Fig.2B2B).

FIG. 1.
Transcriptional expression of BARF1 in EBV-positive Akata cells. BARF1 expression was examined by RT-PCR in EBV-positive Akata cells, as well as in other EBV-positive B-cell lines. Total RNA was extracted from EBV-positive Akata, M81 (B cells immortalized ...
FIG. 2.
Expression of BARF1 and latent, early, and late antigens encoded by EBV in tumors induced after injection of EBV-positive Akata cells. (A) Western blot analysis of BARF1 expression in tumors A and B produced by distinct SCID mice (lanes 3 and 4) and in ...
FIG. 3.
Immunofluorescence analysis of EBV proteins in tumors. Akata cells were extracted from tumors, washed twice with PBS, deposited onto a slide, and fixed with acetone for 15 min (right side). EBV-positive Akata cells before injection are on the left side. ...

Expression of BARF1 and Bcl2 in BARF1-transfected, EBV-negative Akata cells.

We previously showed that introduction of the BARF1 gene into the human B-cell line Louckes led to the induction of malignant transformation in newborn rats (21). We addressed the question of whether introduction of the BARF1 gene into EBV-negative Akata cells can induce malignant transformation. Four neomycin-resistant clones (pZC55A, pZC55B, pZC55C, and pZC55D) were isolated after transfection of a recombinant BARF1 plasmid into EBV-negative Akata cells by an electric field-mediated DNA transfer method. The expression of p31 BARF1 was examined by both the RT-PCR and Western blot methods. For RT-PCR analysis, we treated the extracted RNA with DNase I (Gibco) in order to eliminate any contaminant DNA present in the RNA sample. The cDNA was synthesized from RNA and amplified by Taq polymerase with primers corresponding to the entire BARF1 sequence (Fig. (Fig.1).1). The specificity of the amplified fragment was verified by hybridization with the radiolabeled BARF1 sequence used as probe. In this experiment, P3HR-1 cells expressing a lytic phase were used as a positive control. The four transfectants transcribed BARF1 RNA with an expected size of 668 bp (Fig. (Fig.4A).4A). BARF1 gene transcription was also observed in EBV-positive Akata cells, while EBV-negative Akata cells transfected with the vector plasmid did not give any positive response. BARF1 expression was also analyzed at the translational level by immunoblotting with anti-Pep-1 as previously done in immunoblot and FACS analyses (4, 18) (Fig. (Fig.44 B). We used two xenografts of NPC, C17 and C18, that express a high level of BARF1 protein as positive controls. Three transfectants (pZC55A, pZC55C, and pZC55D) translated the p31 BARF1 protein, while pZC55B showed a much lower level of expression (Fig. (Fig.3B).3B). EBV-negative Akata cells transfected with vector DNA (pZip-Akata) gave a negative response. The expression of BARF1 was almost undetectable in EBV-positive Akata cells in this experiment. Immunofluorescence analysis showed that 1 to 2% of EBV-positive Akata cells spontaneously entered a lytic phase (22). In fact, when BARF1 expression was analyzed with anti-BARF1 antibody in EBV-positive Akata cells, only 0.5 to 1% of the cells became positive (Fig. (Fig.3).3). The small proportion of EBV-positive Akata cells expressing the lytic phase makes BARF1 expression easy to detect by the RT-PCR method but difficult to detect by the immunoblot method.

FIG. 4.
Expression of BARF1 and Bcl2 in EBV-positive Akata cells and BARF1 transfectants. (A) BARF1 expression was analyzed by the RT-PCR method. P3HR-1 induced by TPA-SB was used as a positive control. RT-PCR was carried out as described in the legend to Fig. ...

Our previous results showed that BARF1 is able to activate Bcl2 expression in rodent cells and its activation is likely associated with the oncogenesis of BARF1 (15). We therefore examined whether Bcl2 expression is upregulated by BARF1 in B cells. In this analysis, Jurkat cells were used as a positive control. As illustrated in Fig. Fig.4C,4C, a slight increase in Bcl2 expression was observed in three of four BARF1-transfected clones (pZC55A, pZc55C, and pZC55D) in which BARF1 expression is higher than in pZ vector-transfected cells, as well as in Akata cells. When the expression of BARF1 was low, as in the case of the pZC55B clone, the level of Bcl2 expression was also low. The Bcl2 expression in EBV-positive Akata cells was detected at a level comparable to that observed in EBV-negative Akata cells and vector DNA-transfected, EBV-negative Akata cells. Transfection of individual EBV latent genes into EBV-negative BL cells has shown that LMP1, EBNA2, and EBNA3B can up-regulate Bcl2 expression. All three of these EBV genes are not expressed, however, in EBV-positive Akata cells.

Growth in soft agar.

We analyze first the growth properties of four BARF1 transfectants by examining their ability to form colonies in soft agar (SeaPlaque; FMC Bioproducts). Colony formation efficiency was determined by three independent experiments for each BARF1 transfectant. Cells (0.2 × 106) were cultured in complete medium containing 0.33% agarose as already described (15). High cloning efficiency was observed in three BARF1-expressing clones. The averages of these experiments were 52% for pZC55A, 71% for pZC55C, and 64% for pZC55D(Fig. pZC55D(Fig.5,5, right side). The average obtained for pZC55B was 14%. No colony formation was observed in cells transfected by vector DNA under the same condition (Fig. (Fig.5,5, left side).

FIG. 5.
Soft-agar assay. Anchorage dependence of cell growth was tested in 0.33% agarose in RPMI 1640 medium with 10% FCS as previously described (15). Seven milliliters of 0.66% agarose (SeaPlaque FMC) in RPMI 1640 medium was layered into a 10-cm-diameter dish. ...

Tumorigenicity of BARF1-expressing Akata cells in SCID mice.

Our previous observation showed that BARF1 expression in EBV-negative BL cells (Louckes) is able to induce tumors in newborn rats (21). To determine whether BARF1 expression contributes tumorigenicity to EBV-negative Akata cells, all four BARF1 transfectants (20 × 107 cells/mouse) were injected into SCID mice (eight SCID mice for each transfectant). In parallel, three control cell lines (EBV-positive Akata cells, EBV-negative Akata cells, and vector DNA-transfected, EBV-negative Akata cells) were also injected into six SCID mice. Two BARF1 transfectants (pZC55C and pZC55D) and EBV-positive Akata cells induced tumor formation 12 weeks after infection, while pZC55A produced tumors at 14 weeks. Tumors resulting from pZC55B developed 16 weeks after injection. Moreover, only one of eight of mice developed a tumor. This weak tumorigenicity probably comes from the low level of p31 BARF1 expression in the pZC55B clone (Fig. (Fig.4B).4B). In contrast, EBV-negative Akata cells and vector DNA-transfected Akata cells were unable to induce any tumors in SCID mice (Table (Table11).

TABLE 1.
Tumorigenicity of EBV-negative Akata cells transfected with BARF1 in SCID micea

BARF1 expression confers apoptosis resistance on B cells.

Bcl2 is the prototype of a family of related proteins that can be considered apoptotic death antagonists. In subsequent experiments, these various transfectants were subjected to examination for sensitivity to death by apoptosis following culture for 4 days in medium containing either 10 or 0.1% fetal calf serum (FCS). As illustrated in Fig. Fig.6,6, all cell samples maintained good viability (about 90%) in standard medium containing 10% FCS after 4 days (Fig. (Fig.6,6, histogram, left side). When the growth kinetics of all cell clones were measured for 4 days in 0.1% FCS, EBV-positive Akata calls, EBV-negative Akata cells, and vector DNA-transfected cells lost viability rapidly after 3 days of culture. In contrast, three clones with high-level BARF1 expression (pZC55A, pZC55C, and pZC55D) showed significant survival (60 to 65% viability after 4 days) while pZC55B showed a decrease in viability similar to that of EBV-positive Akata cells (Fig. (Fig.6,6, right side). These experiments suggest that BARF1 contributes apoptosis resistance by up-regulation of Bcl2 expression. An apoptosis assay based on the visualization of DNA fragmentation was carried out. Cells were cultured for 72 h in medium containing 0.1% FCS. For analysis of apoptotic cell death, 106 cells were washed twice with phosphate-buffered saline (PBS) and harvested by centrifugation. The pellet was lysed in a solution containing 1% (wt/vol) sodium dodecyl sulfate, 0.5 mg of proteinase K (Boehringer, Mannheim, Germany) per ml, and 10 mM Tris-HCl (pH 8.0); incubated for 12 h at 37°C; and then incubated with RNase for a further 1 h. Protein was subsequently precipitated by addition of 5 volumes of a saturated NaCl solution. The DNA was precipitated with ethanol at −20°C. A 1.0-μg DNA sample was loaded onto a 1.5% agarose gel containing ethidium bromide for examination of DNA fragmentation as previously described (5).

FIG. 6.
Sensitivity to apoptosis of EBV-negative Akata cells expressing the BARF1 gene. Cells were washed twice in serum-free medium and resuspended at a concentration of 5 × 103/ml in medium containing 10 or 0.1% FCS. Cells were then harvested from the ...

Figure Figure77 illustrates the electrophoretic analysis of DNA extracted from cells cultured for 3 days in 0.1% serum. At this serum concentration, all cultures showed weak degradation of DNA compared with that containing 10% serum, in which no degradation of DNA was observed (Akata EBV− 10%, last lane). The presence of fragmented low-molecular-weight DNA was observed particularly in EBV-positive and -negative Akata (lane 5 and 6) and pZC55B (lane 2) cells, showing that the decline in the viability of these cells in 0.1% FCS was likely due to apoptotic death.

FIG. 7.
Gel electrophoretic analysis of DNAs extracted from cells. EBV-negative (lane 6) and -positive (lane 5) Akata cells and BARF1 transfectants (pZC55A, pZC55B, pZ55C, and pZC55D) were cultured for 3 days in either 0.1% FCS (lanes 1 to 6) or 10% FCS (lane ...

In EBV-positive Akata cells, four EBV genes (EBNA1, EBERs, BARF0, and LMP2A) are expressed (7, 13). Here we demonstrate that additional BARF1 gene expression was observed in EBV-positive Akata cells by RT-PCR analysis. As the BARF1 gene belongs to the early gene group, the detection of BARF1 expression in B cells probably comes from the 1 to 2% of Akata cells expressing lytic genes (22). The detection of BARF1 protein in EBV-positive Akata cells was, however, difficult to visualize on an immunoblot. Interestingly, a large population of EBV-positive Akata cells isolated from tumor biopsy tissues expresses the BARF1 gene. This was confirmed by immunoblotting with an intensified band of p31 BARF1 protein and by immunofluorescence analysis. Surprisingly, other early proteins, like EA-D, ZEBRA, and the BALF2 DNA binding protein, also increased their expression in tumors while latent LMP1 protein expression was never detected in Akata cell cultures or Akata tumor cells. No expression of a late protein like gp220/350 was observed either. Expression of lytic proteins was therefore likely limited to early proteins. The expression of several early genes (those for DNase, EA-D, BZLF1, BARF1, and the BALF2 DNA binding protein) in NPC biopsy tissues in which no late protein or viral particles were detected has been reported previously (4, 9, 14). There is, however, no information about the expression of EBV genes in tumors developed after injection of EBV-positive B cells. It is therefore difficult to interpret our results in the light of other results. We recently reported that the LCL expressing 2 to 8% lytic antigens produced a tumor when injected into newborn rats, while the LCL expressing only latent antigens did not (21). These observations suggest that these early genes could intervene in tumor development. The increase in EA-expressing, EBV-positive Akata cells in tumors is highly interesting. However, it remains to be seen whether viral control is different in vitro and in vivo.

From our results obtained with BARF1-transfected, EBV-negative Akata cells, the subclones expressing a high level of p31 BARF1 protein become tumorigenic and resistant to apoptosis induced by serum starvation. A subclone expressing very low p31 levels was less tumorigenic and entered into apoptosis, as did EBV-positive Akata cells. EBV-negative Akata cells and EBV-negative Akata cells transfected with the pZip vector alone entered into apoptosis more rapidly than the others. Thus, BARF1 put EBV-negative Akata cells into an antiapoptotic state under serum starvation conditions. Bcl2 activation, apoptotic resistance, and tumorigenicity in SCID mice are therefore likely due to BARF1 expression. In particular, the activation of Bcl2 expression could come from the BARF1 gene because a similar activation of Bcl2 expression was observed in BARF1-transfected BALB/c3T3 cells (15). In contrast to BARF1-transfected subclones expressing a high level of p31, one of the BARF1 subclones (pZC55B) expressing a very low level of p31 protein activates Bcl2 less efficiently. These cells were less resistant to apoptosis in 0.1% serum, were poorly tumorigenic in SCID mice, and had more fragmentation of DNA in 0.1% serum. These results reinforce the antiapoptotic and oncogenic roles of BARF1 in Akata cells. In our experiments, it was difficult to show a significant difference in Bcl2 expression between EBV-positive and -negative Akata cells while we did observe significant activation of Bcl2 protein in BARF1-transfected cells. The level of Bcl2 activation with the BARF1 gene was about three- to fourfold (values estimated after scanning with a densitometer) in three BARF1-transfected, EBV-negative Akata cells compared with those obtained from EBV-positive control Akata cells. These results suggest that BARF1 is able to activate Bcl2 not only in B cells but also in rodent fibroblasts (15). It is worth noting that it was very difficult to obtain tumors when we injected EBV-positive and BARF1-transfected Akata cells into nude mice. Even with SCID mice, the development of tumors with these Akata cells took at least 12 to 16 months. A similar phenomenon was also observed with EBERs-transfected Akata cells (7). Since EBERs and BARF1 are both tumorigenic in SCID mice, it will be important to examine their oncogenic role by using a recombinant virus defective for one or both of these genes.

Acknowledgments

We thank E. Kieff (Harvard Medical School, Boston, Mass.), D. A. Thorley-Lawson (Tufts University, Boston, Mass.), and J. Middeldorp (Free University of Amsterdam, Amsterdam, The Netherlands) for the kind gifts of antibodies.

This work was supported by the Association pour la Recherche sur le Cancer (ARC) and the Programme de Recherche Fondamentale en Microbiologie, Maladies Infectieuses et Parasitaires (Ministère de l'Education National de la Recherche et de la Technologie, France).

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