Synergism of BARF1 with Ras Induces Malignant Transformation in Primary Primate Epithelial Cells and Human Nasopharyngeal Epithelial Cells

Abstract

Although it is well known that nasopharyngeal carcinoma (NPC) is closely related with Epstein-Barr virus (EBV), few data are available about which and how EBV-expressed gene is involved in the carcinogenesis of human nasopharyngeal epithelial cells. EBV-encoded BARF1 (BamH I-A right frame 1) gene has been shown to be oncogenic and capable of inducing malignant transformation in BALB/c3T3 and NIH3T3 cells as well as in human B-cell lines Louckes and Akata. It remains unclear, however, whether BARF1 can transform primate or human epithelial cells. Here, we have shown that overexpression of H-Ras gene transformed BARF1-immortalized PATAS cells into malignant cell line. Furthermore, we found that cooperation of BARF1 with H-Ras and SV40 T antigens was sufficient to transform nonmalignant human nasopharyngeal epithelial NP69 cells when serially introduced BARF1 and H-Ras into the SV40 T antigens-immortalized NP69 cells. Taken together, these results demonstrated that the cooperation of BARF1 with Ras suffices to transform primary primate epithelial cell PATAS. Similarly, BARF1 together with H-Ras and SV40 T can transform human epithelial cell NP69, thereby indicating that BARF1 could be involved in the NPC pathogenesis in combination with additional genetic changes.

Introduction

Epstein-Barr virus (EBV) is a ubiquitous human γ herpes virus that has infected more than 90% of the world's adult population and has closely been linked to the development of nasopharyngeal carcinoma (NPC) [1–3]. However, despite the intensive studies of EBV for the past years, the precise role of EBV infection in NPC carcinogenesis remains poorly understood. EBV adopts a specific form of latent infection, termed latency II, in NPC cells. EBV gene expression in this state is usually limited to EBV-encoded RNA [4], nuclear antigen 1 (EBNA1) [5], latent membrane proteins 1 and 2 (LMP1 and LMP2) [6,7], BamHI-A right frame 1 (BARF1), and several BamHI A transcripts [8,9]. Among the EBV-encoded genes, LMP1 and BARF1 have important effects on cellular gene expression [10] and may be involved in EBV-mediated tumorigenesis.

LMP1 is considered an EBV oncogene because it can efficiently induce anchorage-independent growth in the rodent fibroblast cell line Rat-1 in vitro and tumor formation in nude mice in vivo [11,12]. However LMP1 expression was detected in only approximately 50% of EBV-positive NPC [13] and rarely in EBV-positive gastric cancers [14]. In contrast, the expression of BARF1 was detected in up to 90% of cases of invasive NPC [15] and EBV-positive gastric cancers [16]. Another interesting observation is that, in primary primate kidney epithelial cells immortalized by EBV infection, all cells expressed EBNA1 and BARF1 in the absence of detectable LMP1 or lytic proteins [17].

BARF1 has been shown to be able to induce malignant transformation in BALB/c3T3 and NIH3T3 cells as well as in human B-cell lines Louckes and Akata [18–21]. In our previous study, we showed that introduction of BARF1 gene into primary primate kidney epithelial PATAS cells led to morphologic changes, continuous cell growth (>100 passages) and the capacity to grow in highly diluted culture condition. However, contact inhibition was conserved in these cells, and no tumor formation was observed after injection into nude mice [22]. Thus, BARF1 by itself could only immortalize but could not transform the primate primary epithelial cells. Meanwhile, the cellular function of BARF1 in human nasopharyngeal epithelial cells, which are the natural host cells of EBV infection, remains largely unknown because of the lack of a suitable cell model. Therefore, whether BARF1 can transform primate or human epithelial cells remains unclear.

In this study, to further explore the role of BARF1 in the pathogenesis of NPC, we examined the transformation function of BARF1 in both BARF1-immortalized primary primate kidney epithelial PATAS cells and SV40 T antigens-immortalized nasopharyngeal epithelial NP69 cells. Here, we showed that in cooperation with H-Ras gene, BARF1 is able to induce malignant transformation of nontumorigenic PATAS cells. Furthermore, we demonstrated here that the cooperation of BARF1, SV40 T antigen (LT and ST), and H-Ras sufficed to transform human nasopharyngeal epithelial NP69 cell and induce tumorigenicity in nude mice.

Materials and Methods

Cell Culture

The PATAS, BARF1-transfected PATAS subclones [22], 293, 293T, and HeLa cells were cultured in Dulbecco's modified Eagle medium (DMEM; Sigma, Lyon, France) supplemented with 10% fetal calf serum, penicillin (120 µg/ml), and streptomycin (120 µg/ml) in a 5% CO₂ incubator at 37°C. Tumor cell lines (LT1, LT2, and LT3) were established from tumor induced by injection of P-BA-R cells and cultured in normal DMEM.

The NP69, NP69 pLNSX, and NP69 pLNSX-LMP1 cells were kindly provided by Dr. Sai-Wah Tsao of University of Hong Kong, PR China. The establishment and characterization of the immortalized nasopharyngeal epithelial cells NP69 have been previously described [23,24]. Cells were cultured in keratinocyte-SFM (Invitrogen, Lyon, France) supplemented with growth factor and bovine pituitary extract.

Transfection and Selection of Stable Clones

The PATAS cells were seeded at a density of 2 x 10⁵ cells per 35-mm plate 1 day before transfection. Transfections were performed with Lipofectin (Invitrogen) according to the manufacturer's instructions using 2 µg of pBABE-puro-Ras-v12 or pBABE-hygro-hTERT (gift from Dr. Robert A.Weinberg,Whitehead Institute for Biomedical Research, Cambridge,MA) per plate. Vectors carrying only drug resistance genes were used as controls. After selection with 2 µg/ml puromycin or 100 µg/ml of hygromycin, respectively, antibiotic-resistant colonies were recovered with stainless steel cloning cylinders (Bellco Glass, Inc, Vineland, NJ) and continued to be cultured.

pZIP-Neo-SV(X), pBABE-puro, BARF1-expressing vector pZIP55 [19], or pBABE-puro-Ras-v12 was transfected into NP69 or NP69 pLNSX-LMP1 cells by the Lipofectin reagent as previously described. pZIP55 and pBABE-puro-Ras-v12 were cotransfected into NP69 cells. After selection with puromycin (0.8 µg/ml) for pBABE, 400 µg/ml of G418 (Gibco, Lyon, France) for pZIP and pLNSX, the resistant colonies were recovered with stainless steel cloning cylinders and continued to be cultured.

Detection of SV40 Large T, Small T, and BARF1 with Polymerase Chain Reaction and Reverse Transcription-Polymerase Chain Reaction in NP69 Cells

DNA and RNA were extracted from control and BARF1-expressing NP69 cells with Trizol reagent (Invitrogen) according to the manufacturer's instruction. Total RNA was treated with amplification-grade Deoxyribonuclease I (Invitrogen). Reverse transcription-polymerase chain reaction (RT-PCR) was carried out as previously described [25]. SV40 large T was amplified with primers 5′-CATCCTGATAAAGGAGGAGATG-3′ and 5′-CATGCTCCTTTAACCCACCT-3′. SV40 small T was amplified with primers 5′-CATCCTGATAAAGGAGGAGATG-3′ and 5′-CGAAGCAGTAGCAATCAACC-3′. The presence of the BARF1 transcripts in transfected cells was then examined by PCR with specific primers for BARF1 (5′-AGGCTGTCACCGCTTTCTT-3′ and 5′-GGCTTCCTCCTTGTCATTTT-3′).

Telomerase Activity Assays

Telomerase activity in BARF1-transfected PATAS cells was measured using a protocol adapted from Intergen (TRAPeze Telomerase Detection Kit). Three hundred nanograms of total cellular extract was used in each telomerase activity assay. 293 cell extract was used as a positive control. PCR cycling conditions were 94°C for 30 seconds and 59°C for 30 seconds for 30 cycles. As a negative control, every sample extract has been heat-inactivated at 85°C for 10 minutes before assay.

Growth Rate Determination

For studies of growth capacity of PATAS cells, 1 x 10⁵ cells of PATAS, PATAS + BARF1 (P-BA), PATAS + BARF1 + Ras (P-BA-R), PATAS + TERT (P-TE), and PATAS + TERT + Ras (P-TE-R) were cultured in a 35-mm dish for 24 hours in DMEM supplemented with 10% serum. Cells were then trypsinized and centrifuged for 10 minutes at 1000 rpm. Cell pellet was washed twice with serum-free DMEM. A total of 0.1 x 10⁵ cells were seeded in a 35-mm dish with 10%, 1%, or 0.2% serum in DMEM. Each cell line tested was plated in triplicate. The culture medium was changed every 3 days with DMEM containing 10%, 1%, or 0.2% of serum. Cells were trypsinized at the seventh day, and the cell number was determined. For P-BA, P-BA-R, P-TE, and P-TE-R, three subclones for each transfectant were also tested in the same condition.

For studies of growth capacity of NP69 cells, control and BARF1-expressing cells were seeded onto a 24-well plate (5 x 10⁴ cells per well). The cells were trypsinized, and viable cells were counted every 24 hours until 144 hours with Trypan blue exclusion assay. For studies of growth factor dependence, 5 x 10⁴ cells were cultured in 24-well plates for 24 hours in keratinocyte-SFM supplemented with growth factor and bovine pituitary extract. Cells were then washed twice with PBS and the medium was changed to keratinocyte-SFM medium without growth factor and bovine pituitary extract. The cells were observed daily and fed every 4 days until 13 days. Viability was determined every 2 days by Trypan blue dye exclusion. Each time point was counted in triplicate. Growth curves were plotted with means of each experiment, and error bars represented SEM.

Dense Focus Formation and Anchorage-Independent Growth Assay

Focus formation assays were performed as described [26]. In brief, we seeded 5 x 10⁵ cells per well of a six-well plate in DMEM supplemented with 10% of serum for PATAS cells and in keratinocyte-SFM supplemented with growth factor and bovine pituitary extract for NP69 cells and incubated them for 2 weeks with medium changes twice per week.

The anchorage-independent growth ability of the BARF1-expressing PATAS and NP69 cells was examined by soft agar colony assay as described [18]. Cells were suspended in 0.35% agarose (Seaplaque, FMC Bioproducts, Rockland, ME) and then added on top of a base-solidified base layer of 0.6% agarose at a density of 3 x 10³ cells per well of a six-well plate. Each cell line tested was plated in triplicate. Cultures were fed once a week with two to three drops of medium. The cell colonies became visible microscopically after 1 week, and after 2 weeks, each cell population was photographed with a Nikon phase-contrast microscope (Nikon, Lyon, France) equipped with a camera.

Tumorigenicity Assay

As previously described [18], PATAS and NP69 cells were grown in log-phase for 24 hours. Then, cells were harvested, counted, and washed twice with serum-free medium. A total of 10 x 10⁶ cells for each cell line were injected subcutaneously into nude mice with Matrigel. Equal volumes of serum-free DMEM with Matrigel were injected as a negative control. The mice were then examined weekly and observed for approximately 3 months for any sign of tumorigenic growth.

Immunofluorescence Analysis of Keratin in Tumors

Primary PATAS cells, PT8 cell line, tumor cells cultured after 4 days (TL1), and 30th passage were cultured in microchamber for 72 hours and fixed with acetone for 15 minutes, then anti-AE1/AE3 with a dilution of 50 was treated on each cells.

Western Blot Analysis

Immunoblot analysis was performed as previously described [18]; after separation by 8% to 12% polyacrylamide gel electrophoresis, the following antibodies were used to confirm protein expression: primary antibody against Bcl-2 (sc-7382; Santa Cruz, Germany), c-myc (sc-764; Santa Cruz), BARF1 (anti-Pep 2A serum, a polyclonal rabbit antiserum prepared against a synthetic peptide corresponding to a presumed epitope, amino acids (aa) 172 to 180 [NGGVMKEKD], of the BARF1 protein), Ras (sc-29; Santa Cruz), SV40 Tantigen (sc-148; Santa Cruz; N-terminal epitope mapping within residues 1 to 82 of SV40 large Tantigen for detection of 94-kDa SV40 LT antigen and 21-kDa SV40 ST antigen), LMP1 (BD Biosciences Pharmingen, San Diego, CA), h-TERT (SC-7212; Santa-Cruz), and tubulin (sc-5286; Santa Cruz).

Results

BARF1 Induced the Activation of Telomerase in Immortalized PATAS

To become immortalized, human cells must bypass two barriers: replicative senescence and crisis. Replicative senescence can be avoided by the expression of SV40 large Tantigen (LT) in presenescent human cells [27,28]. Crisis can be averted by additional expression of the telomerase catalytic subunit (hTERT), thereby yielding immortalized cells [29,30]. Our previous work showed that introduction of the BARF1 gene could immortalize PATAS cells, which are primary kidney epithelial cells derived from the monkey Erythrocebus patas and which senesced after about three passages in DMEM containing 10% fetal calf serum [22]. We therefore asked whether the BARF1-induced immortalization of PATAS cells was due to the activation of telomerase. As illustrated in Figure 1A, in BARF1-transfected PATAS cell populations, strong telomerase activity was detected by telomeric repeats amplification protocol (TRAP) as early as the sixth passage (Ba 6). However, no telomerase activity was observed in primary cells transfected with the control vector at the same passage (Ve 6). Consistent with these results, a weak expression of TERT protein was detected at sixth passage and became more evident by 10th passage (Figure 1B, lanes 2 and 3). By the 30th passage, BARF1-transfected cell populations exhibited a similar intensity of TERT band (Figure 1B, lane 4) to cells transfected with hTERT gene (Figure 1B, lanes 5–7). Interestingly, the increased TERT activity in BARF1-transfected cells was accompanied by a parallel up-regulation of BARF1 and c-myc expression (Figure 1C). Moreover, by cotransfection of different hTERT promoter-luciferase reporter vectors together with BARF1-expressing vector into PATAS and HeLa cells, we confirmed that BARF1 could directly induce telomerase activation by targeting initiator (Inr) elements at positions +13 and +43 in hTERT promoter region (data not shown). To determine whether elevated telomerase expression alone suffices to immortalize primary PATAS epithelial cells, we transfected hTERT-expressing vector into primary PATAS cells and followed the growth kinetics of the resulting cells every 3 or 4 days for 5 months. As shown in Figure 1D, hTERT-transfected cells exhibited constant exponential growth during this period, whereas empty vector-transfected control cells underwent senescence within 36 days (approximately six passages). Notably, hTERT-transfected clones showed similar growth rate to BARF1-transfected cell clones in vitro (Figure 1E). This observation suggested BARF1 induced immortalization of PATAS cells might be mediated by telomerase activation.

Figure 1.

(A) Telomerase activity in BARF1-transfected PATAS cells. 293 cell extract was used as a positive control, and PATAS cell transfected with empty vector (Ve) was used as a negative control. Cell extracts from 6th, 10th, and 30th passages after BARF1 transfection (Ba) were tested for telomerase activity by using the PCR-based TRAP assay. Heat-inactivated (HI) samples were included as controls. IC refers to an internal PCR standard to demonstrate the absence of PCR inhibitors in the cellular extracts. (B) Detection of TERT expression in PATAS cell immortalization by BARF1. Protein was extracted from the indicated passage after BARF1 transfection, and TERT expression was analyzed byWestern blot. (C) Expression of BARF1 and c-myc by Western blot analysis in PATAS cells transfected with BARF1 (Ba) or control vector (Ve). Actin protein was used to confirm even loading. (D) Expression of hTERT immortalizes primary PATAS cells. Cells were transfected with a control vector expressing a drug resistance marker alone or with hTERT gene. Cell proliferation (Population doublings) was measured during 150 days. Cells lacking hTERT (◆) entered crisis and could no longer be passaged. Squares (□) indicate cells expressing hTERT. (E) Comparative study on cell growth between BARF1- and hTERT-transfected PATAS cells. Cells were seeded at a 35-mm plate in DMEM supplemented with 10% serum. Cell number was monitored daily to assess growth rate. Each experiment was performed in triplicate, and results represented the mean ± SD of three experiments.

BARF1 Could Induce Malignant Transformation by Cooperating with a Limited Number of Oncogenes

Previous studies revealed that in combination with EBV latent infection, the collaborative disruption of RB, p53, c-myc, Bcl-2, and Ras pathways and telomerase activity may be involved in the pathogenesis of NPC [9,31,32]. Our previous data indicated that pro-oncogenes of the Ras family (K-Ras and H-Ras) were activated in NPC tumor epithelial cells [33]. Therefore, we introduced H-Ras oncogene (mutated in valine to glycine at codon 12) into BARF1- or hTERT-immortalized PATAS cells as well as BARF1- or LMP1-transfected NP69 cells to investigate whether the cooperation of these different oncogenes could induce malignant transformation.

We first examined the cooperative effect of these oncogenes on focus formation ability. No H-Ras expression was detected in either primary PATAS cells or hTERT-immortalized cells (P-TE), but BARF1-immortalized (P-BA) cells had slightly increased expression of H-Ras. H-Ras expression was much higher in H-Ras-transfected P-BA cells (P-BA-R) and H-Ras-transfected P-TE (P-TE-R; Figure 2A). Consistent with our previous results [18], elevation of c-myc and Bcl-2 expression was also detected in BARF1-expressing cells. These cell lines were cultured for 2 weeks after confluence without passage to assess focus formation ability. Surprisingly, P-BA-R but not P-TE-R cells lost their contact inhibition and displayed a large number of foci (Figure 2B, b and d). As expected, no such foci were observed in primary cells, P-BA cells (Figure 2B, a and c), or P-TE cells (data not shown). In addition, primary cells expressing H-Ras could not be passaged because they entered a senescent state within few population doublings after transfection.

Figure 2.

(A) Detection of H-Ras, c-myc, and Bcl-2 by Western blot analysis in primary PATAS cells, BARF1-transfected PATAS (P-BA), BARF1 and Ras-transfected PATAS (P-BA-R), hTERT-transfected PATAS (P-TE), and hTERT and Ras-transfected PATAS cells (P-TE-R). (B) Foci formation was detected by light microscopy. Whereas BARF1-transfected PATAS cells showed an organized growth (photographed at x50 [a] and x100 [c]), PATAS expressing both BARF1 and Ras genes formed a higher number of large foci (photographed at x50 [b] and x100 [d]). Cells were cultured in DMEM supplemented with 10% serum. (C) Schematic diagram of the SV40 ER and the alternatively spliced SV40 viral transcripts coding for large T, small T, and 17K T proteins. Numbering refers to the SV40 genomic nucleotide numbers. Origin of replication was indicated as Ori. (D) PCR for the detection of the alternatively spliced messenger RNA encoding the SV40 ST. Positions of PCR primers are indicated in panel A. m, marker. a, PCR with NP69 cell DNA to amplify SV40 ER including LT exons and intron. b, RT-PCR with NP69 cell RNA to amplify SV40 LT transcript. c, RT-PCR with NP69 cell RNA to amplify SV40 ST transcript. d, RT-PCR with 293T cell RNA to amplify SV40 LT transcript. e, PCR with 293 cell DNA as negative control.

Next, we examined the effect of BARF1 in NP69 cells by focus formation assay. NP69 cell line is an immortalized human nasopharyngeal epithelial cell line that cannot proliferate in soft agar or induce tumor formation in immunocompromised mice [23,34]. In addition to NP69 vector (pLNSX) and NP69-LMP1 (pLNSX-LMP1) cells (from Dr. Sai-Wah Tsao), we established different monoclonal cells including NP69 vector (pZIP-Neo-SV(X)), NP69 vector (pBABE-puro), NP69-H-Ras (pBABE-puro-Ras-v12), NP69-BARF1 (pZIP55 [19]), NP69-BARF1 + H-Ras, and NP69-LMP1 + H-Ras. Because we observed no differences in cell growth rate, focus formation or soft agar colony formation among NP69-pZIP-Neo-SV(X), NP69-pBABE-puro, or NP69-pLNSX cells, we chose NP69-pZIP-Neo-SV(X) cells to represent empty vector control cells. Previous studies reported that the NP69 cell line was immortalized only by the SV40 large Tantigen [23].However, we noted that the vector that was used to establish NP69 cell line might have been constructed from the SV40 genome DNA fragment, so we doubted that SV40 early region (ER) was introduced. The SV40 ER encodes three distinct proteins by alternative splicing, including the 708-aa large Tantigen, the 174-aa small Tantigen (ST), and the 135-aa 17K Tantigen (Figure 2C) [35,36]. Both LT and ST have been intensively studied not only because they play important roles in viral transcription and replication but also because they participate in cell immortalization and transformation [37]. Therefore, we designed two pairs of primers to test the possible existence of ST. One spanned the 346-bp intron and was located in LT exons 1 and 2 to confirm the existence of the intron (Figure 2C), and the other one was located in LT exon 1 and intron (both of them in the CDS region of ST) to detect the transcript of ST. As shown in Figure 2D, we found that the transcripts of both LT and ST existed in NP69 cells. Furthermore, by immunoblot analysis, we also detected that both LT and ST proteins were expressed in NP69 cells (Figure 4, C and D). These observations indicated that the NP69 cell line was immortalized by at least SV40 LT and ST.

Figure 4.

(A) Expression of BARF1 protein and H-Ras in cell lines, tumor biopsies, and tumor cell lines. Cell extracts were prepared from PATAS (primary cells), P-BA (PATAS transfected by BARF1), P-BA-R (P-BA plus Ras), P-BA-R-T (tumor biopsy induced by injection of P-BA-R), and P-BA-R-TL (established cell line from tumor biopsy induced by injection of P-BA-R). For BARF1 detection, A20, BARF1 protein produced by BARF1 recombinant adenovirus system was used as a positive control. For H-Ras detection, we used protein extracted from NPC biopsy expressing a high Ras protein (NPC26) as a positive control. (B) Detection of cytokeratin AE1/AE3 in cell lines. Cells were cultured in microchamber for 72 hours and fixed with acetone for 15 minutes, then anti-AE1/AE3 with a dilution of 50 was treated on each cell. a, Primary PATAS cells. b, P-BA-R cell line. c, P-BA-R tumor cell culture after 4 days (TL1). d, The 30th passage of P-BA-R tumor cell line (TL1). (C) Western blot analysis of associated signaling molecules in BARF1-expressing, LMP1-expressing, and control cell populations. Different cell populations were indicated at the top of the panel. The detected proteins are indicated on the right side. (D) RT-PCR detection of BARF1 and Western blot analysis of Ras and SV40 LT and ST expressions in cloned cell line and tumors. Different tumors formed by NP69-BARF1 + Ras cloned cell line were indicated at the top of the panel. The detected molecules are indicated on the right side.

Within transfected NP69 cells, we also observed that NP69-BARF1 and NP69-BARF1 + Ras cells continued to proliferate to form foci on the surface of monolayer cells, but no obvious foci were detected in the other cells. However, compared with NP69-BARF1 cells, there were denser, larger, and more foci in NP69-BARF1 + Ras cells. Because most of the NP69-LMP1 cells peeled off the dish surface during the longtime culture without passage, we could not observe any foci formation at the same time point.

Next, we analyzed the cooperative effects of different oncogenes on cell growth capacity. PATAS cells were evaluated by culturing differently transformed PATAS cells in DMEM supplemented with 10%, 1%, or 0.2% of serum. As shown in Figure 3A, primary PATAS cells grew slowly in culture medium with 10% serum and almost could not grow with 1% or 0.2% serum, whereas P-BA, P-TE, and P-TE-R cells could proliferate consistently with 10% or 1% serum. However, when these clonal cells were cultured with 0.2% serum, they could only grow no more than 3 days. Only P-BA-R cells could proliferate in all three serum concentrations and grew in a much higher rate than other transfectants with 10% and 1% serum. These results showed that the introduction of H-Ras gene rendered serum-independent growth advantage to BARF1-immortalized PATAS cells but not to hTERT-immortalized PATAS cells in low serum conditions.

Figure 3.

(A) BARF1 and Ras rendered PATAS cells resistant to serum deprivation. Cells were seeded at 0.1 x 10⁵ cells per 35-mm plate in DMEM supplemented with 10% serum. After 24 hours, cultured were washed twice with serum-free medium then incubated with DMEM supplemented with 10%, 1%, and 0.2% serum for 7 days. Viable cells were counted by Trypan blue staining on day 7. Each cell line tested was plated in triplicate. a, Primary PATAS cells (PATAS). b, hTERT-immortalized PATAS cells (P-TE). c, BARF1-immortalized PATAS cells (P-BA). d, Ras-transfected P-BA cells (P-BA-R). e, Ras-transfected P-TE cells (P-TE-R). (B) Growth curves of BARF1-expressing NP69 cells and control cells that were cultured in medium with growth factors and bovine pituitary extract. Each experiment was performed in triplicate, and results represented the mean ± SD of three experiments. (C) Growth curves of BARF1-expressing NP69 cells and control cells in medium without growth factors and bovine pituitary extract. Each experiment was performed in triplicate, and results represented the mean ± SD of three experiments: NP69 vector (filled squares), NP69-BARF1 (open triangles), NP69-BARF1 + Ras (filled triangles), NP69-LMP1 (open diamonds), NP69-LMP1 + Ras (filled diamonds), and NP69-Ras (open squares). (D) Soft agar colony formation assays. a, hTERT-immortalized PATAS cells (P-TE). b, BARF1-immortalized PATAS cells (P-BA). c, Ras-transfected P-BA cells (P-BA-R). d, Ras-transfected P-TE cells (P-TE-R). The colonies were photographed after 2 weeks. (E) Photomicrographs of anchorage independent growth in soft agar. a, NP69 vector cells. b, NP69-Ras cells. c, NP69-LMP1 cells. d, NP69-LMP1 + Ras cells. e, NP69-BARF1 cells. f, NP69-BARF1 + Ras cells. Original magnifications: a1, b1, c1, d1, and e1, x50; for a2, b2, c2, d2, and e2, x100. All the colonies were photographed after 2 weeks.

The cooperative effects of different oncogenes on NP69 cell growth were assessed by comparison of growth curve and growth factor dependence among different cells populations. As seen in Figure 3B, the additional coexpression of BARF1 and H-Ras (in BARF1 + Ras NP69 cells) had a significant effect on the rate of proliferation, which resulted in a doubling time 20% shorter than that of empty vector control cells. Other cell lines grew in a similar way to the empty vector control cells. After 6 days, the maximum cell number of BARF1 + Ras, BARF1, and empty vector control cells was 4.8 ± 0.27 x 10⁵, 4.0 ± 0.20 x 10⁵, and 3.2 ± 0.05 x 10⁵, respectively. Morphologic differences were found among the BARF1 cells and other cells; BARF1 cells were round or in oviform shape and in larger size in comparison with the empty vector control NP69 cells. LMP1-expressed NP69 cells exhibited an elongated fibroblastlike shape as previously reported [34], whereas H-Ras-only introduced NP69 cells exhibited no morphologic difference from the empty vector control NP69 cells.

To examine the growth factor dependence of different NP69 cells (Figure 3C), growth factor and bovine pituitary extract were withdrawn from culture medium. As expected, BARF1-expressing cells (BARF1 + Ras NP69 cells and BARF1 NP69 cells) were more resistant to growth factor deprivation, and their proliferation could still be detected at day 12. In contrast, all other cells, especially NP69-LMP1 cells, underwent growth arrest by day 7, with a marked decrease in cell number. Under the culture condition without growth factor and bovine pituitary extract, we noticed that the NP69-LMP1 + Ras cells could proliferate and spread across the surface of the dish until the entire surface was covered with a monolayer of cells, but the NP69-LMP1 cells grew in a scattered pattern with a marked reduction in cell-cell contact. After confluence, all cell populations except NP69-LMP1 cells could be kept for more than 4 weeks by only changing the medium without passage, but the NP69-LMP1 cells would peel off, and the number of attached cells significantly decreased from approximately the 10th day. In addition, the NP69-LMP1 cells seemed to be more sensitive to trypsinization than the others. These observations are consistent with previous reports that high levels of LMP1 expression might be toxic to some cell lines, including several human B-lymphoid cell lines (Raji, GG68, BL60, etc.), BALB/3T3, 143/EBNA1, HEp-2 cells [38,39], and CNE2 cells [40]. Although the precise mechanisms of LMP1-induced inhibition of cell proliferation are not clear, we found that the introduction of H-Ras into LMP1- expressing NP69 cells could partially overcome this inhibition and conferred these cells with an increased resistance to the toxicity.

We then examined the anchorage-independent growth properties of transfected PATAS and NP69 cells in soft agar. For PATAS cells, we only observed colony formation in BARF1 and H-Ras coexpressed PATAS cell clones (P-BA-R; Figure 3D, c). Within NP69 cells, the colonies formed by NP69-BARF1 + Ras (Figure 3E, f1 and f2) cells were larger in size and became microscopically visible 3 days earlier than those formed by NP69-BARF1 (Figure 3E, e1 and e2), NP69-LMP1 (Figure 3E, c1 and c2), and NP69-LMP1 + Ras cells (Figure 3E, d1 and d2). Probably because of the LMP1 expression-induced toxicity that we described previously, the cells in the LMP1-expressing clones underwent apoptosis after approximately 10 days (Figure 3E, c1, c2, d1, and d2). No colonies formed within 3 weeks by the NP69 vector (Figure 3E, a1 and a2) or NP69-Ras (Figure 3E, b1 and b2) cells in three independent experiments (Table 1).

Table 1.

Colony Formation in Soft Agar and Tumor Formation in Nude Mice.

Cell Lines Injected	Genotype	Colony Formation	Tumors/Injection Sites	Tumor Size (cm)
				Day 14	Day 28
HeLa			4/4	1.3 x 1.6	2.7 x 3.4
PATAS		0	0/4	0	0
P-BA-clone 1	BARF1+, hTERT-, Ras-	0	0/4	0	0
P-BA-clone 3	BARF1+, hTERT-, Ras-	0	0/4	0	0
P-BA-R-clone 2	BARF1+, hTERT-, Ras+	42	4/4	0.8 x 1.2	1.4 x 2.3
P-BA-R-clone 3	BARF1+, hTERT-, Ras+	36	4/4	0.7 x 1.0	1.1 x 2.4
P-BA-R-clone 5	BARF1+, hTERT-, Ras+	46	4/4	0.9 x 1.3	1.7 x 2.7
P-TE-clone 1	BARF1-, hTERT+, Ras-	0	0/4	0	0
P-TE-clone 2	BARF1-, hTERT+, Ras-	0	0/4	0	0
P-TE-clone 3	BARF1-, hTERT+, Ras-	0	0/4	0	0
P-TE-R-clone 1	BARF1-, hTERT+, Ras+	0	0/4	0	0
P-TE-R-clone 3	BARF1-, hTERT+, Ras+	0	0/4	0	0
P-TE-R-clone 7	BARF1-, hTERT+, Ras+	0	0/4	0	0
NP69 vector	BARF1-, SV40 LT/ST+, Ras-	0	0/4	0	0
NP69-LMP1	BARF1-, SV40 LT/ST+, Ras-, LMP1+	0	0/4	0	0
NP69-Ras-clone 1	BARF1-, SV40 LT/ST+, Ras+	0	0/4	0	0
NP69-Ras-clone 2	BARF1-, SV40 LT/ST+, Ras+	0	0/4	0	0
NP69-BARF1-clone 2	BARF1+, SV40 LT/ST+, Ras-	0*	0/4	0	0
NP69-BARF1-clone 3	BARF1+, SV40 LT/ST+, Ras-	0*	0/4	0	0
NP69-BARF1-clone 4	BARF1+, SV40 LT/ST+, Ras-	0*	0/4	0	0
NP69-LMP1 + Ras-clone 1	BARF1-, SV40 LT/ST+, Ras+, LMP1+	0	0/4	0	0
NP69-LMP1 + Ras-clone 2	BARF1-, SV40 LT/ST+, Ras+, LMP1+	0	0/4	0	0
NP69-LMP1 + Ras-clone 3	BARF1-, SV40 LT/ST+, Ras+, LMP1+	0	0/4	0	0
NP69-BARF1 + Ras-clone 1	BARF1+, SV40 LT/ST+, Ras+, LMP1-	38	4/4	0.4 x 0.9	0.9 x 1.8
NP69-BARF1 + Ras-clone 2	BARF1+, SV40 LT/ST+, Ras+, LMP1-	41	4/4	0.6 x 0.8	1.0 x 1.7
NP69-BARF1 + Ras-clone 3	BARF1+, SV40 LT/ST+, Ras+, LMP1-	43	4/4	0.9 x 1.1	1.2 x 1.9

For colony formation, data shown here are the mean values of the measurements from triplicate wells. The number of colonies of 100 cells or more formed in each well was counted under a phase-contrast microscope.

For tumor formation, 10₇ cells for each cell line were injected subcutaneously into nude mice with Matrigel. Equal volumes of serum-free DMEM with Matrigel were injected as a negative control. The size of tumor in centimeters is the mean of four tumors for each cell line and clone.

^*Observed some colonies containing 10 to 30 cells per heap in wells.

Cooperation of BARF1 and Other Oncogenes Conferred Tumorigenicity in Nude Mice

Finally, we investigated the cooperative effect of different oncogenes on the tumorigenic properties of various PATAS and NP69 cells in nude mice. HeLa cells, as a positive control, readily formed tumors in this assay, although no tumors were observed in PATAS cells expressing BARF1, hTERT, or hTERT and H-Ras after a 1-month observation. In marked contrast, when PATAS cells expressing BARF1 and H-Ras were introduced, rapidly growing tumors were observed as early as 1 week (Table 1). These data suggest that, together, expression of BARF1 and H-Ras is sufficient to induce the transformation of primary epithelial PATAS cells. No tumor was observed in NP69 vector, NP69-Ras, NP69-LMP1 [34], NP69-BARF1, or NP69-LMP1 + Ras cells after 2 months as expected. However, injection of HeLa cells or NP69 cells coexpressing BARF1 and H-Ras induced progressively enlarged tumors at the sites of inoculation (Table 1).

To ensure that tumors developed in nude mice were transfected cells in origin, the expression of H-Ras, BARF1, SV40T antigens, and keratin was analyzed. As illustrated in PATAS cells in Figure 4A, the expression of BARF1 and H-Ras protein was present in all cases: before injection (P-BA-Ras-2), tumor (P-BA-Ras-2-T, P-BA-Ras-3-T and P-BA-Ras-4-T), and established cell line from tumor biopsy induced by injection of P-BA-Ras (P-BA-R-TL1 and P-BA-R-TL2). To verify whether the established cell lines from the tumor biopsy were of epithelial origin, immunofluorescence with monoclonal anti-AE1/AE3 was performed [22]. In the detection of keratin expression, PATAS primary cells exhibited a low level of expression of AE1/AE3 keratin (Figure 4B, a), and enhanced expression was detected in BARF1-transfected PATAS cells (Figure 4B, b), BARF1/Ras-transfected PATAS cells (data not shown), and tumor cells (Figure 4B, c and d).

As shown in Figure 4C, upregulated expression of c-myc and Bcl-2 was detected in the NP69 cells when BARF1 or LMP1 was introduced. As to tumor formed by transfected NP69 cells, the expressions of BARF1, Ras, and SV40 T antigens (Figure 4D) were confirmed.

Discussion

NPC represents a superb model of gene-environment-virus interaction in the pathogenesis of cancer [25]. Previous studies revealed that, in combination with EBV infection, multiple genetic and epigenetic alterations accumulate, including p53 inactivation by either overexpression of ΔN-p63 or loss of p14/ARF [9,31], mutations in RB2/p130 gene [41] or constitutional RB phosphorylation resulting from loss of p16 [42]. Aberrant expression and point mutation of the Ras gene, up-regulation of Bcl-2 [43] and c-myc [44–46], as well as increased telomerase activity [32] have also been shown to be involved in the development of NPC, thereby indicating that EBV infection and cumulative genetic alterations are critical events for the deregulation of cell proliferation in NPC cells.

Although elucidation of the mechanism of NPC pathogenesis is difficult, such investigations are important not only for understanding the mechanisms of tumorigenesis but also for finding methods for cancer prevention and development of more effective anticancer drugs.

Previous attempts have established several NPC epithelial cell lines, such as HONE1 [47,48], CNE1, CNE2, and C666 [49], from NPC biopsy tissues. Seto et al. [50,51] generated BARF1-rEBV carrying the BARF1 gene under the SV40 promoter using an Akata cell system and established BARF1-rEBV-infected HONE-1 and CNE-1 cell NPC cell clones and then produced tumors in nude mice. However, such cell lines already contained chromosomal abnormalities, and thus, their genetic constitution remains obscure. Therefore, the development of a suitable model system with which to study the viral and genetic events underlying the malignant transformation of human nasopharyngeal epithelial cells is critical.

By introducing the BARF1 gene, we established immortalized primate epithelial PATAS cells that could proliferate more than 100 passages and grow in highly diluted culture condition in vitro, but no tumors were induced after the injection in nude mice. In addition, an SV40 T antigens-immortalized human nasopharyngeal epithelial cell line, NP69, was established recently [23]. Therefore, the establishment of BARF1-immortalized PATAS cells and SV40 T antigens-immortalized NP69 cell line did provide a valuable experimental model to investigate the role of BARF1 in tumorigenesis of NPC.

Previous studies, including our own, demonstrated that immortalization is necessary but not sufficient for malignant transformation. This notion was exemplified by the studies of Hahn et al. [35,52] and Elenbaas et al. [53] who demonstrated that human fibroblasts and epithelial cells could be immortalized by SV40 LT plus hTERT and be transformed to a tumorigenic state by ectopic expression of an activated form of the Ras^V12G oncogene, hTERT, and SV40 T (LT and ST) antigens. Interestingly, here we showed that BARF1-immortalized PATAS cells grew continuously in the same manner as hTERT-immortalized cells during 150 days in vitro, but no tumors formed in nude mice. In contrast, malignant transformation of PATAS cells was induced by the additional transfection of H-Ras gene into BARF1-immortalized PATAS cells but not to hTERT-immortalized cells. The observation suggested that not only telomerase activation but also some other signal pathways, including c-myc, have been altered in the immortalization and transformation of PATAS cells by BARF1. In comparison with the data obtained by Hahn et al. and Elenbaas et al., primary primate epithelial PATAS cells were immortalized by BARF1 and malignantly transformed by introduction of BARF1 and H-Ras. The discrepancy might have resulted from different cell types (different species and tissue origin) used or unknown gene alteration.

In addition, we introduced BARF1 and H-Ras^V12G oncogenes into SV40 T antigens-immortalized NP69 cells and established the cooperation of BARF1, H-Ras, SV40 small and large T antigens in this immortalized cells, thus for the first time driving tumorigenesis in non-malignant human nasopharyngeal epithelial cells. We presume that in this system, SV40 LT simultaneously disables the tumor suppressor p53 and RB proteins to release cells from growth arrest and apoptosis [37,54], whereas SV40 ST inhibits the activity of phosphatase 2A [35,55–57] and H-Ras^V12G activation facilitates cellular proliferation, transformation, angiogenesis, invasion, and metastasis [58,59]. Combined with these effects, the introduction of BARF1 oncogene perturbs several pathways, including c-myc [21] and Bcl-2 [18] pathways, and activates telomerase to permit indefinite replication of cells. Ultimately, the collective and cooperative interaction of these genes might confer the malignant transformation to NP69 cells. Therefore, our results suggested that in NPC carcinogenesis, EBV-encoded BARF1 overexpression cooperates with the disruption of a number of cellular pathways, including inactivation of tumor suppressors p53 and RB, overexpression of oncogene H-Ras^V12G, and activation of hTERT. In addition, all these effects may induce human nasopharyngeal epithelial cell into malignancy. Meanwhile, we found that elevated expression of c-myc and Bcl-2 was detected not only in BARF1-expressing cell lines but also in hTERT- and H-Ras-expressing cell lines, and in LMP1- and H-Ras-expressing cell lines when compared with the vector control; however, increased colony formation was only observed in BARF1- and BARF1 + Ras-expressing cells, whereas only BARF1 + Ras-expressing cells can form tumors in nude mice. Deregulation of c-myc and/or BCL-2 expression, therefore, cannot be the main mechanism of cell transformation by BARF1. Some other signaling pathway might have been disrupted and played an important role in the transformation process. Further investigation with the established models may help to elucidate the underlying mechanism.

This study provides a blueprint of the genetic and viral events that could lead to NPC development and permits identification of other oncogenic signals needed to transform normal nasopharyngeal epithelial cells, particularly these mimicked by the introduction of equivalent to SV40 T antigens and BARF1. In conclusion, this establishes an ideal basis for the testing of novel preventive or therapeutic approaches in the treatment of NPC.

Abbreviations

BARF1

BamH I-A right frame 1

EBNA1

nuclear antigen 1

EBV

Epstein-Barr virus

LMP1 and LMP2

latent membrane proteins 1 and 2

NPC

nasopharyngeal carcinoma

TERT

the telomerase catalytic subunit

TRAP

telomeric repeats amplification protocol