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[Cancer Research 60, 5584-5588, October 1, 2000]
© 2000 American Association for Cancer Research

Virology

Expression of BARF1 Gene Encoded by Epstein-Barr Virus in Nasopharyngeal Carcinoma Biopsies1

Gisèle Decaussin, Fatima Sbih-Lammali, Mireille de Turenne-Tessier, Abdelmadjide Bouguermouh and Tadamasa Ooka2

Laboratoire de Virologie Moléculaire, UMR5537, Centre National de la Recherche Scientifique, Faculté de Médecine R.T.H. Laënnec, Université Claude Bernard Lyon-1, 69372 Lyon Cedex 08, France [G. D., F. S-L., M. D. T-T., T. O.], and Département de Virologie Humaine, Institut Pasteur d’Alger, Sidi-Frejd, Tipaza, Algeria [A. B.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We reported previously that the EBV BARF1 open reading frame encodes a Mr 31,000–33,000 protein (p31) with potential transforming and oncogenic properties. This gene was found capable of transforming both: (a) the rodent fibroblast lines Balbc/3T3 and NIH3T3 into cells producing aggressive tumors in newborn rats; and (b) the human EBV-negative B-cell line Louckes into cells leading to small tumors, which disappeared 3 weeks after injection. Our recent study showed that BARF1 ORF expression may confer the property of immortalization to primary kidney epithelial cells (M. X. Wei et al., Oncogene, 14: 3073–3081, 1997). Because this suggested that BARF1 could be involved in epithelial malignancy, we investigated its transcriptional and translational expressions in Algerian nasopharyngeal carcinoma (NPC) biopsies by reverse transcription-PCR and immunoblotting using rabbit polyclonal antisera prepared against two synthetic peptides corresponding to distinct, predicted epitopes of the BARF1 protein (NGGVMKEKD, amino acids 172–180, and GKNDKEE, amino acids 203–209). The BARF1 ORF was found to be transcribed and translated in >85% of our NPC biopsies, with high p31 protein level detected in several NPC patient biopsies as well as in NPC-derived xenografts. Our observation of BARF1 expression in a large proportion of NPC epithelial cells suggests that this EBV gene might play an important role in the malignant transformation of human epithelial cells in vivo.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NPC3 is a serious problem of public health in Southern China, much of South Asia, and North Africa (1, 2, 3) . NPC has been closely associated with EBV because its genome is present in close to 100% of tumor biopsies (4, 5, 6, 7) , but little is known about the oncogenic functions of EBV in nasopharyngeal epithelial cells. As a lymphotropic virus, EBV has been shown to induce the immortalization of human and simian primary B cells in vitro (8) and the development of lethal B lymphoma in the Tamarin monkey in vivo (9) . In regard to the epitheliotropism of EBV, it has been first reported that the transfection of either a 40-kb viral fragment containing BARF1 (10) or the BARF1 gene alone (11) can lead primate primary epithelial cells to be immortalized but not to be able to induce tumors after injection into rodents. More recently, it has been observed that primary epithelial cells from either human stomach (12) or monkey kidney4 can be immortalized in vitro by EBV particles.

In tumoral biopsies from NPC, although EBV particles have not been detected thus far, viral lytic or latent transcripts have been observed (13) . The expression of several known EBV genes, either latent (14, 15, 16) or early (17, 18, 19, 20) , has been identified. The transcription of both the latent BARF0 and the early BARF1 genes in NPC biopsies could be demonstrated by Northern hybridization (14 , 21) , whereas that of other genes, such as LMP1, LMP2, EBNA1, EBERs, and BZLF1, was only detected by either RT-PCR (22 , 23) or in situ hybridization (24) . As for the translational expression of EBV in NPC biopsies, viral latent proteins, such as EBNA1 and LMP1, have been observed in a majority of the tumor cells (5 , 16 , 17) , whereas early proteins, such as ribonucleotide reductase, DNase, EA-D, EA-R, and Zta, have been detected in occasional tumor cells using monoclonal antibodies (17, 18, 19, 20 , 25 , 26) . However, one report disproves a significant production of lytic proteins (as exemplified by EA-D) in NPC biopsies (16) . By reference to the three major types of EBV latency in B cells, the viral expression in NPC has been assimilated to latent type II, where only EBNA1, EBERs, BARF0, and LMP1 are expressed.

Among the viral genes expressed in tumor biopsies, two are known to have oncogenic activity: LMP1 and BARF1. LMP1 has a transforming activity in rodent cell lines (26 , 27) and is indispensable to B-cell immortalization. The biological functions of LMP1 are rather well documented in B cells, where this viral protein has been found similar to CD40 and involved in the activation of the cellular transcriptional factor nuclear factor-{kappa}B (28 , 29) . On the other hand, in vitro experiments suggest that LMP1 suppresses epithelial cell differentiation (30) , whereas LMP-1 RNA and protein were detected by RT-PCR and immunoblot in about 50–60% of NPC biopsies (16, 17, 18) . At present the role(s) of LMP1 in epithelial oncogenesis remain(s) unknown. Concerning the BARF1 gene, we previously identified its translation product as a polypeptide of Mr 31,000–33,000 named p31 (31 , 32) . Because the expression of this gene was not inhibited by the presence of phosphonoacetic acid and p31 was immunoprecipitated with EA-positive NPC sera, this protein appears as an early EBV antigen (31) . The BARF1 gene was capable of inducing oncogenic transformation in two rodent fibroblast lines as well as in a human B-cell line (33 , 34) . We further showed that the introduction of BARF1 into simian primary epithelial cells induced their immortalization (11) . Recently, a possible biological role of the BARF1-coded protein was reported; it might function as a receptor for the human CSF-1, because recombinant BARF1 protein was found able to bind and neutralize soluble human CSF-1 in vitro, whereas some subtle amino acid sequence homology was predicted between BARF1 and the proto-oncogene c-fms encoding the cellular receptor for CSF-1 (35) . Moreover, an inhibitory activity of BARF1 on IFN-{alpha} secretion has just been demonstrated in B cells (36) . These observations suggest that BARF1 biological activity might concern both immunomodulation and oncogenicity.

Because the BARF1 protein was associated with malignant transformation in various cell lines and with immortalization in primate epithelial cells, we thought of much interest to investigate BARF1 expression in NPC. We thus collected North-African NPC biopsies and observed that BARF1 was both transcribed and translated into p31 product in >85% of the samples analyzed. This result has to be discussed with regard to BARF1 role(s) in epithelial malignancy.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Clinical Samples.
Thirty-nine tumor biopsies were collected in Algeria from clinically diagnosed NPC patients and analyzed in Mustapha Hospital (Alger). Thirty undifferentiated and nine poorly differentiated tumor biopsies were collected as follows. Tumors from primary tissue (0.1–0.5 g) were snap-frozen in liquid nitrogen immediately after surgical removal and then stored at -80°C. Each sample was cut into two pieces; one half was used for RNA extraction, whereas the second half was divided again for DNA and protein extractions. We also examined biopsies kindly given by Dr. Busson from his C15, C17, and C18 NPC xenografts propagated in nude mice (37) . The detection of the BARF1 ORF by PCR amplification attested to the presence of EBV genome in all of the biopsies analyzed.

Cell Culture.
The EBV-carrying P3HR-1 and Rael lines (the last one being a kind gift from Dr. Fu, Karolinska Institute, Stockholm, Sweden) were maintained in RPMI 1640 containing 10% fetal bovine serum and antibiotics as described previously (38) . To induce the lytic cycle, P3HR-1 cells were treated with 50 ng/ml of TPA and 2 mM SB (39) . The EBV-negative human epithelial cell line HaCaT (a gift from Dr. Fusenig, Institut für Biochemie, Heidelberg, Germany; Ref. 40 ) was maintained in DMEM supplemented with 10% fetal bovine serum.

RT-PCR.
RT-PCR analysis was carried out as described previously (11) . Briefly, 5 µg of total RNA were used for first-strand cDNA synthesis using oligo(dT)15 as primer. Reverse transcription was done with Superscript reverse transcriptase according to the instructions of the manufacturer (Life Technologies, Inc.). Amplifications of cDNA were performed in a DNA thermal cycler (Hybaid) using the primers described previously (11) . A 697-bp amplified fragment corresponding to an entire EBV BARF1 sequence was detected by hybridization using a 32P-labeled BARF1 probe prepared with a random-primer DNA-labeling kit (Stratagene).

BARF1-specific Antibodies.
Several rabbit antisera were prepared against two synthetic peptides corresponding to distinct presumed epitopes of the COOH-terminal end of the BARF1 protein. Anti-Pep1 antibodies were obtained from one rabbit injected with the peptide GKNDKEE (named Pep1), corresponding to amino acids 203–209. Anti-Pep2A and anti-Pep-2B antibodies were produced by two rabbits (A and B) injected with the peptide NGGVMKEKD (named Pep2), corresponding to amino acids 172–180 (41) . The antibodies used in the present study were purified with an affinity column of Ultrogel coupled to either Pep1 or Pep2.

Protein Extraction and Immunoblot Analysis.
As described previously by Sbih-Lammali et al. (18) , biopsies were directly dissociated in RIPA buffer, and protein concentration of cleared extracts was measured by a Bio-Rad protein assay (Bio-Rad Laboratories, Inc.). After dilution with one volume of gel sample buffer [125 mM-Tris-HCl (pH 6.8), 4% SDS, 200 mM DTT, 20% glycerol, and 0.05% bromphenol blue] and 5 min of denaturation at 100°C, samples of 50 µg of protein per lane were loaded on top of 12% polyacrylamide gels for SDS-PAGE separation. After electrophoresis, proteins were transferred onto reinforced nitrocellulose by semidry blotting as described previously (11) . Nonspecific sites on the blots were blocked by 1-h incubation in PBS containing 0.1% Tween 20 and 5% of either dried skimmed milk or Blot-QuickBlocker (Chemicon). The detection of the BARF1 protein was performed by overnight incubation at 4°C with the above-described rabbit polyclonal antibodies raised against BARF1 epitope sequences. After extensive washing, the blots were incubated for 1 h at room temperature with peroxidase-labeled antirabbit antibodies and then the antigen-antibody complexes were visualized by enhanced chemiluminescence as instructed by the manufacturer (SuperSignal; Pierce).

Immunohistochemistry.
Immunohistochemistry was performed on paraffin-embedded NPC biopsies as described previously (18) . In use as primary antibodies are: our anti-Pep2A rabbit polyclonal antibodies or nonimmune rabbit antibody (both diluted 1:20); L26, a mouse monoclonal antibody directed against the human B-cell CD20 antigen (Dako; diluted 1:100); or a polyclonal rabbit antiserum directed against human T-cell CD3 antigen (Dako; diluted 1:100). Specific protein detection was revealed with the avidin-biotin-peroxidase complex method using a StreptABComplex/HRP Duet, Mouse/Rabbit kit (Dako).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transcriptional Expression of BARF1 in North African NPC Biopsies and in NPC Xenografts.
BARF1 transcription was analyzed by RT-PCR in 36 NPC biopsies, including 27 undifferentiated and 9 poorly differentiated tumors, and also in 3 NPC xenografts developed in mice (C15, C17, and C18; Ref. 37 ). Extraction of total RNA was followed by two treatments with DNase I to eliminate any contamination by residual DNA before cDNA synthesis by reverse transcriptase, and possible residual contamination of RNA with DNA was controlled by performing PCR on RNA samples not submitted to cDNA synthesis. The primers used for PCR were chosen in view of amplifying the whole sequence of BARF1 ORF (11) .

The autoradiogram presented in Fig. 1Citation shows that the expected fragment of 697 bp, clearly detected from TPA-SB-induced P3HR1 cells (used as a positive control, Lane 13), but not from the EBV-negative HaCaT epithelial cells (Lane 12), was revealed in 19 representative biopsies (of the 36 samples examined), with great differences in response intensity, suggesting much variation of transcription level between biopsies. Although some samples revealed an amplified doublet, the major band corresponded to the expected 697-bp fragment. We have no explanation about the second band (with a lower molecular weight) identified in some samples. Possibly, this band comes from other mRNAs transcribed in this region, because this region transcribed at least six different mRNAs (31) . Interestingly, contrary to both C15 and C18 xenografts (Lanes 1 and 3, respectively), the C17 xenograft (Lane 2) gave a rather low response at 697 bp, suggesting a BARF1 transcription level contrasting with the apparent abundance of p31 protein in this tumor (see below). On the other hand, BARF1 transcription was found in one biopsy (Lane 15) where no p31 was detected (see below). As summarized in Table 1Citation showing our results on a total of 36 biopsies tested, BARF1 transcription was detected in 87% of our North African NPC biopsies: 23 of 27 undifferentiated tumors and 8 of 9 poorly differentiated tumors, as well as in all 3 NPC xenografts tested.



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  		Fig. 1. RT-PCR analysis of BARF1 transcription in NPC biopsies. Total RNA treated twice with DNase I was subjected to cDNA synthesis, and PCR was carried out using primers permitting the amplification of a 697-bp DNA fragment. No trace of residual DNA contaminated any of the RNA samples tested because no 697-bp amplified fragment was detected when they were directly incubated with Taq polymerase (RT-minus negative controls, data not shown). We used EBV negative human HaCaT epithelial cells as a negative control (Lane 12) and TPA-SB-treated cells of the human P3HR1 lymphoblastoid line as a positive control (Lane 13). Lanes 1, 2, and 3, respectively, correspond to C15, C17, and C18 xenografts, whereas Lanes 4–11 and Lanes 14–21 refer to NPC biopsies.

 

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  		Table 1 Summary of BARF1 expression detected by RT-PCR and immunoblot in NPC biopsies

 
Translational Expression of BARF1 in NPC Biopsies Compared with Lymphoblastoid Cell Lines.
Crude protein extracts from biopsies or culture cells were analyzed on Western blots in using purified polyclonal rabbit antibodies raised against two predicted antigenic sequences from the COOH-terminal region of the BARF1 protein, named Pep1 (amino acids 203–209) and Pep2 (amino acids 172–180) as detailed in "Materials and Methods."

BARF1 translational expression was first examined in the Burkitt’s lymphoma cell line P3HR1 before and after induction of the viral lytic cycle. As shown in Fig. 2Citation , anti-Pep2 antibodies detected a Mr 31,000 protein in the EBV-productive lymphoblastoid cell line B95-8 used as a positive control but not in EBV-negative HaCaT epithelial cells. Interestingly, the level of this protein in P3HR1 cells significantly increased after induction of the EBV lytic cycle in correlation with the proportion of EA-expressing cells; the p31 level was significant in noninduced P3HR1 TK-negative cells containing about 0.02% only of EA-expressing cells, and high expression was registered at 72 h of induction (before excessive cell mortality because of viral replication) when the percentage of EA-positive cells reached 30%. On the other hand, no p31 protein was detected in Rael cells (Fig. 3ACitation , Lane 1) in which EBV expression is characteristic of type I latency. All of these observations confirm our previous identification of BARF1 as an early gene in EBV-productive B cells (31) .



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  		Fig. 2. Western blot analysis of BARF1 protein expression in P3HR1 TK- cells using an antipeptide rabbit serum. P3HR-1 TK- cells (2 x 107 cells) were collected every 24 h from 0–72 h of treatment with TPA and SB. Fifty µg of total protein extracted from each cell pellet were loaded per lane on a 12% polyacrylamide gel, and Western blot analysis was performed with our anti-Pep2-A rabbit serum (described in "Materials and Methods") as the primary antibody. HaCaT epithelial cells were used as an EBV-negative control, and TPA-SB-induced cells of the EBV-productive B-cell line B95-8 were used as a positive control. Arrow, p31 polypeptide.

 


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  		Fig. 3. Western blot analysis of BARF1 protein expression in NPC xenografts using three anti-peptide rabbit sera. A, p31 expression was first investigated with anti-Pep2-A primary antibodies (diluted 1:1000) in the NPC xenografts C15, C17, and C18, compared with the EBV latency type I B-cell line Rael and EBV-producing P3HR1 cells. Fifty µg of protein from Rael (Lane 1), P3HR-1 (Lane 2), C18 (Lane 3), and C15 (Lane 4), but 5 µg only from C17 (Lane 5), were loaded per lane. B, although never detected in human 293 cell extract used as a negative control (Lanes 1, 3, and 5), p31 was recognized from C17 extract (Lanes 2, 4, and 6) by three rabbit polyclonal antibodies raised to two distinct epitopes of the BARF1 protein: anti-Pep2-A (Lanes 3 and 4; dilution 1:10,000) and anti-Pep2-B (Lanes 1 and 2; dilution 1:70 000) from two rabbits (A and B) immunized with the same antigen (Pep2), and anti-Pep-1 (Lanes 5 and 6; dilution 1:10,000) from another rabbit injected with Pep1.

 
Biopsies from C15, C17, and C18 xenografts were then compared with TPA-SB-treated P3HR1 and latent Rael cells for BARF1 protein expression (Fig. 3)Citation . Western blotting was first performed in using anti-Pep2A as primary antibodies. As shown in Fig. 3ACitation , BARF1 was found translated in all three xenografts; C18 (Lane 3) seemed to express more p31 protein than C15 (Lane 4), and interestingly, C17 (Lane 5) revealed a much higher expression of the protein than both C18 and C15, because p31 detection from C17 in our conditions required one-tenth (5 µg) only of the total cell protein load necessary in the case of both of the other tumors and of our control B cells (50 µg). The high p31 level thus detected in C17 led us to the identification of the protein as a BARF1 product by performing Western blot analysis of C17 extract with other primary antibodies than anti-Pep2-A. As shown in Fig. 3BCitation , the same protein band was detected in C17 but not in the human embryonic kidney cell line 293 used as negative control, in using either anti-Pep2-A (Lanes 3 and 4) or anti-Pep2-B (from a second rabbit injected with Pep2, Lanes 1 and 2), or anti-Pep1 (from one rabbit injected with Pep1, Lanes 5 and 6). An identical result (not shown) was obtained with antibodies from another rabbit immunized with both Pep1 and Pep2. The clear response obtained from C17 using high dilutions of affinity-purified fractions of antibodies raised to either Pep1 or Pep2 strongly support the BARF1 origin of the p31 protein detected in the three NPC xenografts tested.

Our observations on the xenografts led us to examine BARF1 protein expression in our North African NPC biopsies, compared with human epithelial cells HaCaT as a negative control and both of the C17 xenograft and P3HR1 cells (treated for 48 h with TPA-SB) as positive controls. As shown in Fig. 4Citation on 17 samples, immunoblot analysis of NPC biopsies with anti-Pep2-A revealed different levels of p31 expression.



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  		Fig. 4. Western blot analysis of BARF1 protein expression in NPC biopsies using an antipeptide rabbit serum. Fifty µg of total protein from crude extracts of sliced biopsies were loaded per lane. The blot was treated with anti-Pep2-A (diluted 1:1000) as primary antibodies. Human epithelial HaCaT cells were used as a negative control (Lane 1), and P3HR1 cells were treated for 48 h with TPA-SB as a positive control (Lane 2). Lanes 3–21 were loaded with extracts from NPC biopsies, except for Lane 15, which was loaded with C17 xenograft extract.

 
The Western blot results obtained from all of our NPC biopsies are summarized in Table 1Citation . In total, p31 protein was detected in 85% of the biopsies, whereas BARF1 mRNA was found in 87% of them, but no strict correlation was observed between immunoblotting and RT-PCR responses. p31 was detected in 23 of 27 undifferentiated biopsies tested, 3 of the 4 negative biopsies gave no positive signal on RT-PCR analysis, 7 of 9 poorly differentiated biopsies expressed p31, and 1 of the 2 negative biopsies responded positively in RT-PCR. All three xenografts analyzed here expressed p31. Although no relationship was observed between BARF1 protein expression and either patient ages or tumor types, it is worth noting that p31 was detected in all 15 NPC biopsies (38% of our samples), which were from young patients between 13 and 21 years of age.

Immunohistochemistry.
Our previous immunohistochemical analysis showed that EBV-encoded early protein DNase was highly expressed in a NPC biopsy with the histological aspect of a typical undifferentiated NPC, characterized by the presence of a large number of tumoral cordons in the stroma lesions, where T and B lymphocytes were present, sometimes under infiltrating form. No EBERs were detected in lymphocytes around epithelial tumor cordons, and the presence of EBV was likely limited to the epithelial tumor cells (see data in Ref. 19 ). The same paraffin-fixed slide used previously for DNase detection was further analyzed for BARF1 protein expression using our rabbit polyclonal antibodies anti-Pep2-A. As illustrated in Fig. 5Citation , although no staining was observed with nonimmune rabbit serum (Fig. 5A)Citation , a positive response was obtained in almost all tumoral epithelial cells (but not in normal epithelium adjacent to tumor region) with a pattern suggesting a cytoplasmic/membrane (nonnuclear, although sometimes perinuclear) localization (Fig. 5B)Citation .



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  		Fig. 5. Immunohistochemical analysis of BARF1 protein expression in a NPC biopsy. Immunoperoxidase staining of NPC tumor cells was performed on paraffin section with rabbit preimmune serum (A) or with anti-Pep2-A polyclonal rabbit antibodies (B). The presence of infiltrating T and B lymphocytes was revealed with anti-CD3 and anti-CD20, respectively [data already published by Sbih-Lammali et al. (18) ].

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although the presence of the EBV genome and its expression in NPC biopsies are somewhat documented, data remain insufficient, and the role of EBV in the oncogenic transformation of nasopharyngeal epithelial cells is not yet known. Although the tumoral epithelial cells are known to express not only latent (EBNA1, EBERS, LMP1, LMP2, and BARF0) but also early (DNase, EA-D, BZLF1, and BARF1) EBV genes, the implication of these viral functions in epithelial oncogenesis is not yet demonstrated. Several in vitro observations support an oncogenic activity of EBV in epithelial cells: (a) the transfection with a 40-kb fragment from the EBV genome was able to immortalize primate primary epithelial cells in vitro (10) ; (b) we have demonstrated that the EBV early gene BARF1 was sufficient to immortalize monkey kidney primary epithelial cells in vitro (11) ; and (c) primary epithelial cell immortalization with EBV particles has been obtained in vitro recently (12) .4 It is not yet known whether epithelial cells immortalized with EBV particles are capable of inducing tumors after injection in nude mice, but because no tumors were observed after injection of epithelial cells immortalized either with the 40-kb subfragment of the EBV genome (10) or with the BARF1 gene (11) , the role of EBV in epithelial oncogenesis might rather be limited to the immortalization process, with other factors being required for malignant transformation.

In North African NPC biopsies, BARF1 expression was not correlated to any histological status because it was detected in a large majority of either undifferentiated or poorly differentiated tumors. In agreement with our present results, a recent study using the NASBA method showed that BARF1 was transcribed in NPC epithelial cells but not in other EBV-related tumors, such as Burkitt’s or Hodgkin’s lymphomas (42) . Concerning the known EBV oncogenic latent protein LMP1, its presence was found in ~50% of NPC tumors (16, 17, 18) , and its total absence was reported in EBV-positive gastric adenocarcinoma cells (43) . It is relevant to mention that LMP1 is not detectable in the xenograft C17,5,6whereas we observed a particularly high BARF1 protein level in this sole xenograft; the meaning of this observation remains to be determined. All together, actual data suggest that the BARF1 protein rather than LMP1 could play an important role in epithelial oncogenesis.

The significance of EA expression in NPCs is yet difficult to understand. Indeed, although the BARF1 protein was detected in ~85% of North African NPC biopsies, the EBV-coded DNase was found in almost 100% of them (18) . Some expression of EA-D (19) , of the EBV major DBP,7 and of Zta (20) was also observed in NPC. Interestingly, immunohistochemical methods revealed the presence of EBV DNase and BARF1 protein in a large proportion of tumor epithelial cells (Ref. 19 ; Fig. 5Citation ) but detected DBP and Zta expression in a small part of the tumors only8 (20) . Such different expression patterns might reveal that just a minority of NPC tumor cells (those expressing Zta and DBP) are in a starting phase of viral cycle reactivation and then enter into apoptosis. Because neither viral particles nor late viral proteins have been detected so far in NPC biopsies, the EBV replication cycle may be limited in most tumor cells to early stage, thus preventing cell death attributable to virion production. In fact, the cells expressing the EA DBP after gene transfection could remain alive for a long period without any cell damage (44 , 45) . The significance of early protein expression (Zta, EA-D, DBP, and DNase) in NPC remains to be determined. Although EBV expression in NPC epithelial cells was first assimilated to type II latency in B cells (46 , 47) , such a classification merits a reexamination to account for our observation of an extensive expression of both early DNase and BARF1 protein in Algerian NPC tumors.

We observed previously a correlated transcriptional expression of several proto-oncogenes with EBV genes in NPC biopsies (5) , and more recently Strockbine et al. (35) reported a possible functional similitude between the BARF1 protein and the CSF-1 receptor (coded by c-fms); these observations support the hypothesis of a cooperation between viral and cellular (onco)genes in EBV-associated malignant transformation of epithelial cells, as already shown in B cells (34) . Because our data show that the BARF1 protein is both able to immortalize primate primary epithelial cells in vitro and is widely expressed in vivo in NPC tumoral cells, we suggest that BARF1 could play a key role in an early step of epithelial oncogenesis. The biological functions of BARF1 in epithelial cell immortalization will be further examined to account for the above-mentioned observations.


   FOOTNOTES
 
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.

1 This work was supported by Grant Contract No. 9528 from the Association pour la Recherche contre le Cancer and a grant from the Fédération Nationale des Groupements des Entreprises Françaises dans la Lutte contre le Cancer, Grant Contract No. 95 MDU 319 from the Coopération Interuniversitaire Franco-Algérienne, and grants from the Fondation de France, the Programme de Recherche Fondamentale en Microbiologie, Maladies Infectieuses et Parasitaires (Ministère de l’Education Nationale de la Recherche et de la Technologie, France). Back

2 To whom requests for reprints should be addressed, at Laboratoire de Virologie Moléculaire, UMR5537, Centre National de la Recherche Scientifique, Faculté de Médecine R. T. H. Laënnec, Rue G. Paradin, 69372 Lyon Cedex 08, France. Phone: 04-78-01-18-36; Fax: 04-78-74-96-68; E-mail: ooka{at}laennec.univ-lyon1.fr Back

3 The abbreviations used are: NPC, nasopharyngeal carcinoma; RT-PCR, reverse transcription-PCR; EA, early antigen; CSF, colony-stimulating factor; ORF, open reading frame; DBP, DNA-binding protein; TPA, 12-O-tetradecanoylphorbol-13-acetate; SB, sodium butyrate. Back

4 C. Danve, G. Decaussin, P. Busson, and T. Ooka. Establishment of a monkey kidney epithelial cell line with NPC-derived Epstein-Barr virus, submitted for publication. Back

5 Unpublished observations. Back

6 P. Busson, personal communication.<./> Back

7 Unpublished data. Back

8 Unpublished observations. Back

Received 1/26/00. Accepted 8/ 3/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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