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Keywords:

  • Esptein-Barr virus;
  • premalignant nasopharyngeal epithelium;
  • nasopharyngeal epithelial carcinoma

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

Epstein-Barr virus (EBV) infection has been postulated to be an early event involved in the pathogenesis of nasopharyngeal carcinomas (NPC). The lack of representative premalignant nasopharyngeal epithelial cell system for EBV infection has hampered research investigation into the regulation and involvement of EBV infection in NPC pathogenesis. We have compared the efficiency of EBV infection in nasopharyngeal epithelial cells with different biological properties including immortalized, primary and cancerous nasopharyngeal epithelial cells. EBV infection could be achieved in all the nasopharyngeal epithelial cells examined with variable infection rate. TGF-β effectively enhanced EBV infection into nasopharyngeal epithelial cells both in the immortalized and primary nasopharyngeal epithelial cells. Stable infection of EBV was achieved in a telomerase-immortalized nasopharyngeal epithelial cell line, NP460hTert. The expression pattern of EBV-encoded genes and biological properties of this EBV infected cell line on long-term propagation were monitored. The EBV-infected nasopharyngeal epithelial cells acquired anchorage-independent growth and exhibited invasive growth properties on prolonged propagation. A distinguished feature of this EBV-infected nasopharyngeal epithelial cell model was its enhanced ability to survive under growth factor and nutrient starvation. This was evidenced by the suppressed activation of apoptotic markers and sustained activation of pAkt of EBV-infected cells compared to control cells under nutrient starvation. Examination of cytokine profiles of EBV-infected NP460hTert cells to nutrient and growth factor deprivation revealed upregulation of expression of MCP-1 and GRO-α. The establishment of a stable EBV infection model of premalignant nasopharyngeal epithelial cells will facilitate research investigation into the pathogenic role of EBV in NPC development.

Infection of Epstein-Barr virus (EBV) is ubiquitous and more than 95% of the adult population world-wide is infected with this virus.1 EBV infection is life-long and largely asymptomatic. EBV is implicated as an etiological agent in several human malignancies of either lymphoid or epithelial origin. These include Burkitt's lymphoma, Hodgkin's lymphoma, nasal T/NK lymphomas, undifferentiated nasopharyngeal carcinomas (NPC), gastric adenocarcinomas, smooth muscle tumors and various B-cells lymphomas in AIDS patients or transplant recipients with compromised immune functions.2–4 EBV infection can be detected in most, if not all, undifferentiated NPC, regardless of geographical and ethnical origin of NPC patients.2 Expression of latent EBV genes including EBER, EBNA1, LMP1 and LMP2A as well as lytic EBV genes, such as BZLF1, could be detected in cancer cells inside the nasopharyngeal carcinoma specimens. Clonal infection of EBV has been detected in precancerous lesions of NPC, implicating its involvement at the early stage of NPC pathogenesis.5 EBV infection has long been postulated to play an important role in transformation of premalignant nasopharyngeal epithelial cells into cancer cells. However, the lack of representative EBV infection cell model has hampered the elucidation of its transformation mechanisms in nasopharyngeal epithelial cells. Previous EBV infection studies in epithelial cells were mainly conducted in established cancer cell lines that are not representative of premalignant nasopharyngeal lesions.6 Recently, EBV infection was achieved in primary cultures of epithelial cells derived from nasopharyngeal region7, 8. However, due to the limited lifespan of primary epithelial cells in culture, these EBV-infected cells could not be propagated to examine for long-term effects of EBV infection.7 In our study, we report the stable infection of EBV in a premalignant nasopharyngeal epithelial cell model and its long-term propagation in vitro (over 450 days with more than 140 population doublings). The premalignant nasopharyngeal epithelial cell model, NP460hTert, used for EBV infection in our study was established from primary nasopharyngeal epithelial outgrowth of a tumor-free nasopharyngeal biopsy after transduction of the catalytic component (hTert) of human telomerase.9 The NP460hTert cells are nontumorigenic as indicated by their inability to form tumor in nude mice and their lack of anchorage-independent growth in soft agar. Detail molecular and cytogenetic analysis revealed a discrete deletion at the p16INK4A locus of chromosome 9p and loss of p16 function in the telomerase-immortalized NP460hTert cells. Prolonged propagation of NP460hTert cells resulted in epigenetic suppression of another tumor suppressor gene, RASSF1A, mapped to chromosome 3p21. Inactivation of p16 and RASSF1A are common events in NPC and can be detected in premalignant nasopharyngeal lesions.10 Hence the immortalized NP460hTert cell line represents the closest premalignant nasopharyngeal cell system available for EBV infection study. Elucidation of pathological properties of NP460hTert after EBV infection may reveal early events associated with EBV transformation of premalignant nasopharyngeal epithelial cells.

In the present study, a recombinant EBV tagged with GFP, was used to study the effects of different inflammatory cytokines in regulating EBV infection in this premalignant nasopharynageal epithelial cell model. EBV infection of immortalized NP460hTert cells was achieved via cell–cell contact by coculturing with EBV-infected lymphocytes. NPC is associated with an inflammatory stroma heavily infiltrated with lymphocytes in vivo.11 The model developed here revealed a role of inflammatory cytokines in regulating EBV infection in premalignant nasopharyngeal epithelial cells. Furthermore, we have achieved stable infection of EBV in this cell model and have characterized some of the phenotypic changes on long-term propagation, which may provide insights into the role of EBV infection in NPC pathogenesis.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

Cell lines used for EBV infection

EBV infection was attempted in 3 telomerase-immortalized nasopharyngeal epithelial cell lines (NP460hTert, NP5501hTert and NP3611hTert), 3 primary cultures of nasopharyngeal epithelial cells (NP460, NP461 and NP550) and 2 established nasopharyngeal carcinoma cell lines (HONE1 and HK1) commonly used in nasopharyngeal carcinoma studies. Among the 3 telomerase-immortalized nasopharyngeal epithelial cells, the NP460hTert cells have been extensively characterized.9 The NP5501hTert and NP3611hTert were recently established in one of our laboratories (SWT), and the NP460, NP461 and NP550 were primary cultures of epithelial cells established from nonmalignant nasopharyngeal epithelial tissues. The culture conditions of primary and immortalized nasopharyngeal epithelial cells have been previously published.9 The immortalized nasopharyngeal epithelial cells were grown in 1:1 mixture of Defined Keratinocyte-SFM (Gibco, Grand Island, NY) and EpiLife™ medium with growth supplements (Cascade Biologics, Portland, OR). HONE1 and HK1 were maintained in RPMI 1640 containing 10% FCS. The Akata cells carrying the recombinant EBV was maintained in RPMI 1640 containing 10% FCS with G418 (700 μg/ml; Gibco). The HONE1-EBV cell line was established in our laboratory according to a previously published protocol12 and maintained in RPMI 1640 containing 10% FCS with G418 (500 μg/ml; Gibco). All cells were maintained at 37°C with 5% CO2 in humidified air.

EBV infection

EBV infection of epithelial cells was achieved by cocultivation of epithelial cells with EBV-infected Akata cells using an established protocol.13 Briefly, the epithelial cells were seeded into a 6-well culture plate at 105 cells per well in 1.5 ml medium overnight. Before cocultivation with epithelial cells, EBV-infected Akata cells (2 × 106/ml) were induced for lytic production of infectious EBV particles by crosslinking the surface immunoglobulin G (0.5% vol/vol, Sigma, St Louis, MO) for 24 hr. To examine for the effects of cytokines on EBV infection, epithelial cells were treated with cytokines prior to cocultivation with lytically induced-Akata cells. Cytokines were added individually at the following concentrations: TGF-β1 (CalBiochem, La Jolla, CA; 2 ng/ml); TNF-α (R&D Systems, Minneapolis, MN; 50 ng/ml); IL-6 (R&D Systems; 50 ng/ml) and GM-CSF (R&D Systems; 50 ng/ml). After 24 hr, the medium was removed and replaced with 1 ml of fresh epithelial culture medium plus 1 ml of Akata cell culture previously induced for lytic infection. Coculture of epithelial cells with the lytically induced Akata cells was performed for 24 hr. After that, the epithelial culture was thoroughly washed with PBS to remove the Akata cells. The EBV infection rate in the epithelial cells was determined after 3 days by fluorescence-activated cell sorter (FACS) analysis (FACScalibur, BD Sciences, San Jose, CA) based on GFP expression and the results were analyzed by the Cell Quest program (BD sciences).

Immunofluorescene staining for CR2 receptor

Epithelial cells were detached from the culture flask by treatment with 2 mM EDTA at 37°C for 15 min. The cells were washed in PBS containing 0.5% BSA and incubated with anti-CR2 antibody (Becton Dickinson, Franklin Lakes, NJ). FITC-labeled rabbit antibody to mouse IgG (DakoCytomation, Carpinteria, CA) was used as the secondary antibody. The presence of CR2 positive cells were examined by flow cytometric analysis.

Live cell imaging of NP460hTert-EBV cells

The Perkim Elmer Ultraview Live Cell Imaging system was used to monitor the proliferation of NP460hTert-EBV cells. The cells were maintained in a humidified chamber at 37°C and 5% CO2 in air on a motorized microscopic stage. EBV-infected cells were identified by activation of GFP fluorescence by a krypton–argon laser at 488 nm. Mitosis of EBV-infected cells was captured by time lapse acquisition of images at 8-min interval over a period of 24 hr.

Detection of EBV gene expression in EBV-infected cells by RT-PCR

RNA was isolated with TRIzol (Invitrogen, Carlsbad, CA) and cDNAs were prepared according to previously published protocols14 with random primers using SuperScript II reverse transcriptase (Invitrogen). Detection of EBV-encoded transcripts was performed by semiquantitative RT-PCR using gene specific primers listed in Table 1. The number of PCR cycles was optimized for each primer set and PCR products were visualized at their linear ranges of amplification. GAPDH mRNA was coamplified as control.

Table 1. Oligonucleotide primers used in RT-PCR analysis
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Growth and invasive properties of NP460hTert-EBV cells

For growth rate determination, 105 EBV-infected and uninfected NP460hTert cells were plated onto each well of a 6-well plate. The cultures were trypsinized every day, and the number of viable cells was determined by a haemocytometer with trypan blue exclusion method (Sigma). The growth curves were constructed by plotting the means of 3 independent culture wells with standard errors. The invasive ability of cells was examined by 3D collagen invasion assay as previously described.15 Collagen gel was prepared under 4°C by mixing collagen type 1 (BD biosciences), 0.34 N NaOH, 10× PBS and medium at 7:2:1:10 ratio. Cells (1 × 105) were mixed in 400 μl of collagen gel and allowed to set at 37°C in 24-well plate. After 24 hr, morphology of the cells in the collagen was observed and bright-field images were captured.

Anchorage independent growth assay and tumorigenicity in nude mice

Soft-agar cloning assay was used to determine the anchorage independent growth property of EBV-infected and uninfected cells. Bottom agar (0.66%) was prepared by mixing 6.6% bacto-agar (Sigma) with medium at 1:9 ratio. Two milliliters of upper agar (0.33%) mixed with 1 × 105 cells were then plated on top of the bottom agar. Images of colony formation were taken after 4 weeks. For examination of tumorigenicity of cells in nude mice, EBV-infected and uninfected NP460hTert cells (at 5 × 106 cells) as well as HONE1-EBV cells (1 × 106 cells) were resuspended in 100 μl of PBS mixed with Matrigel (BD Biosciences; 1:1 ratio) and implanted subcutaneously into nude mice and observed for more than 6 months for tumorigenic growth.

Metaphase chromosome preparation and spectral karyotyping

Metaphase cells were obtained by Colcemid (Gibco) treatment at a final concentration of 0.03 mg/ml for 3 h. The harvested cells were fixed in 3:1 methanol/acetic acid. Metaphase chromosome spreading was performed as previously described.16 The slide with metaphase cells was aged at room temperature (RT) for 7 d prior to spectral karyotyping (SKY). The slide was treated with DNase-free RNase solution (0.1 mg/mL) at 37°C for 1 h, washed in 2× SSC for 10 min at RT, treated with proteinase K (0.05 μg/mL) solution for 10 min at 37°C, and washed in 2× SSC for 10 min. The slide was then fixed in 2% paraformaldehyde for 10 min, washed in 2× SSC for 10 min, dehydrated in 70, 85 and 95% ethanol for 2 min each, air-dried using nitrogen gas, denatured in 70% formamide/2× SSC at 75°C for 5 min and dehydrated again in ethanol as above. Other protocols including SKY probe (Applied Spectral Imaging, Migdal Ha'Emek, Israel) denaturation, hybridization and detection followed the manufacturer's instructions. SKY image capturing and karyotyping were performed using the SkyVision Imaging System equipped with a Zeiss Axioplan 2 fluorescence microscope. International System for Human Cytogenetic Nomenclature (ISCN 1995) recommendations was followed in karyotyping descriptions.

Examination of growth properties of EBV-infected and uninfected NP460hTert cells in growth factor deprived conditions

EBV-infected and uninfected NP460hTert cells were seeded in 6-well plate at a density of 2 × 105 cells per well. For growth factor deprivation study, the cells were cultured in plain growth medium without growth factor supplement for 22 days. The cultures were trypsinized at days 2, 7, 13, 18, 22, 24 and 26, and the percentages of viable cells were counted using trypan blue exclusion method. The growth curves were constructed by plotting the means of 3 independent cultures with standard errors. Phase-contrast photographs were also taken at the indicated days to record for any change in cell morphology. To determine the colony formation ability of EBV-infected and uninfected cells under growth factor deprived condition, 1,000 cells were plated in a 25-cm2 flask for 3 weeks without changing medium. Phase contrast and GFP fluorescence photographs were taken for selected clones of NP460hTert-EBV cells after 3 weeks.

Detection of survival markers and cytokine secretion of EBV-infected or uninfected NP460hTert cells under acute amino acid deprivation

(7 × 105) EBV-infected and uninfected NP460hTert cells were seeded onto each well in 6-well plate. After overnight incubation, the medium was changed to Earle's Balanced Salt solution (EBSS) and protein lysates were collected at 0, 0.5, 1.5, 4.5, 7, 12 and 24 hr. The secretory profile of 79 different cytokines in the culture supernatant of EBV-infected and uninfected cells was examined using the antibody-based RayBio Human Cytokine Array V (RayBiotech Inc., Norcross, GA) according to the manufacturer's protocol. Briefly, 2 × 106 cells were allowed to seed in a 25-cm2 flask overnight and then cultured in 2.5-ml EBSS (Sigma). After 48 hr, 1.5 ml of cell-free culture supernatant was collected and incubated for one hour with the membrane previously blocked with blocking buffer. The membrane with the bound cytokines was then washed and incubated with biotin-conjugated anti-cytokines antibodies for another hour. After subsequent washings, the membrane was probed with horseradish peroxidase-conjugated streptavidin for 1 hr and the antibody–cytokine complexes were detected using ECL chemiluminescent detection system (Amersham, Piscataway, NJ). RNA was extracted from EBV-infected and uninfected cells after culturing in EBSS for 6 hr. RT-PCR was performed to examine for the transcripts of MCP-1 and Gro-α. The sequences of primers are listed in Table 1.

Western blotting analysis

Cells were harvested and protein concentrations were determined as previously described.9 Twenty micrograms of extracted protein was resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). Primary antibodies for pAkt (ser 743), cleaved caspase 3 and cleaved poly ADP ribose polymerase (PARP) were obtained from Cell signaling; OT1× and K67-3 antibody for EBNA1 was kindly provided by Prof. Middeldorp, β-actin was from Santa Cruz. β-actin was used as loading control. The blots were visualized using ECL-Plus chemiluminescence kit (Amersham) according to manufacturer's instruction.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

Variable rates of EBV infection in primary, immortalized and cancerous nasopharyngeal epithelial cells

We first examined if the rates of EBV infection rate were different among nasopharyngeal epithelial cells with different biological properties. The rate of EBV infection was determined by flow cytometric analysis based on the percentage of GFP-fluorescent cells after infection. Incubating the nasopharyngeal epithelial cells directly with cell-free supernatant medium collected from EBV-producing Akata cells yielded a very low EBV infection rate (<0.1%; data not shown). A much higher rate of infection was achieved by coculturing nasopharyngeal epithelial cells with EBV-producing Akata cells13 (Fig. 1a). Hence, the cell–cell contact route of EBV infection was adopted in all subsequent experiments. The infection rate of EBV was variable among different cell lines. For telomerase-immortalized nasopharyngeal epithelial cells, the highest infection rate was achieved in NP460hTert cells achieved (around 10%). The infection rate of the other 2 telomerase-immortalized nasopharyngeal epithelial cell lines varied from 1% in NP550hTert cells to less than 0.5% in NP361hTert cells. In primary nasopharyngeal epithelial cells, the EBV infection rate varied from 2.5% infection rate in NP550 cells to 6.5% infection rate in NP461 cells. Established cancerous nasopharyngeal epithelial cell lines also varied in EBV infection rate (Fig. 1a). Infection rate in HONE1 was much higher (approaching 10%) compared to HK1, which had a much lower rate of infection (1.5%). The underlying mechanisms regulating EBV infection rate in nasopharyngeal epithelial cells is likely to be complex and highly dependent on cell context, such as differentiation status of cells.17 We have examined the expression levels of CR2 receptor in nasopharyngeal epithelial cells used in EBV infection by flow cytometry analysis (Fig. 1b). CR2 expression is unlikely to play a major role in influencing EBV infection rate in nasopharyngeal epithelial cells as they were not detectable in all nasopharyngeal epithelial cells examined.

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Figure 1. EBV infection rate and CR2 expression in immortalized, primary and cancerous nasopharyngeal epithelial cells. (a) EBV infection rate in telomerase-immortalized nasopharyngeal epithelial cells (NP460hTert, NP5501hTert and NP3611hTert), primary nasopharyngeal epithelial cells (NP460, NP550 and NP461) and established nasopharyngeal carcinoma cell lines (HONE1 and HK1 cells). Cells were plated at a density of 105 cells/ well in 6-well culture plate and cocultured with the EBV-producing Akata cells. The EBV-producing Akata cells were crosslinked with IgG to induce the production of GFP-tagged EBV. After 24 hr of coculture, the Akata cells were washed away and the epithelial cells were allowed to grow for another 72 hr before the percentage of EBV-infected (GFP positive) cells were estimated by flow cytometry. Mean results ± SD from 3 repeated assays are shown. (b) CR2 receptor staining of epithelial cells. Cells were detached and stained with mAb of anti-CD21 (CR2 receptor). All isotyptic controls of different cell lines were set within the dotted line. The Daudi cells, which express CR2 receptor, were used as positive control. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Enhancement of EBV infection rate in nasopharyngeal cells by TGF-β1

The stromal inflammatory microenvironment is known to play a crucial role in modifying the properties and behaviors of tumor cells. Inflammation is common in the nasopharyngeal mucosa. The various cytokines present in the inflammatory stroma may influence the infection rate of EBV into premalignant nasopharyngeal epithelial cells. We have examined the influence of defined inflammatory cytokines on EBV infection rate in our premalignant immortalized nasopharyngeal epithelial cell model. Pretreatment of NP460hTert cells with TGF-β1 and TNF-α significantly upregulated the infection rate of EBV into cells (Fig. 2a). Among the cytokines examined, the TGF-β1 had the most profound effect in the infection rate of EBV infection in NP460hTert cells with at least 4 folds increase of GFP-positive (EBV-infected) cells compared to untreated cells (Figs. 2a and 2b). Enhancement of EBV infection by TGF-β1 is not limited to immortalized nasopharyngeal epithelial cells and could also be observed in primary nasopharyngeal epithelial cells prior to immortalization (Fig. 2c). These observations support a role of microenvironment in regulating EBV infection in nasopharyngeal epithelial cells. In contrast, pretreatment of cells with IL-6 and GM-CSF did not reveal obvious effects on infection rate of EBV into NP460hTert cells.

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Figure 2. Effects of cytokines on EBV infection rate. (a) TGF-β and TNF-α increased the EBV infection rate on NP460hTert cells. NP460hTert cells were treated with TGF-β, TNF-α, IL-6 and GM-CSF for 24 hr, rinsed with PBS to remove residual cytokines, before coculturing with Akata cells. TGF-β and TNF-α increased the rate of infection by 4 and 2 folds, respectively. Results are expressed as mean + SD from 3 independent experiments. (b) Phase-contrast and fluorescence images of NP460hTert-EBV cells with or without treatment of TGF-β after 3 or 5 days postinfection. (c) TGF-β also increased the EBV infection rate in primary NP cells. Primary NP460 and NP550 cells were subjected to the same infection procedure as the immortalized NP460hTert cells. Comparable increase of infection rate could be found after the treatment of TGF-β. Results are expressed as mean + SD from three independent experiments. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Establishment of stable EBV infection in immortalized NP460hTert cells

An EBV infection model for premalignant nasopharyngeal epithelial cells is not currently available. Our next objective was to establish stable EBV infection in a premalignant nasopharyngeal cell model. As shown previously, NP460hTert was the most susceptible cell line for EBV infection among other immortalized cell lines. It also harbors common genetic alterations (notably p16 and RASSF1A inactivation), which were also detected in premalignant nasopharyngeal epithelium.10 The NP460hTert cells did not exhibit transformed properties when examined by soft-agar growth and were nontumorigenic when injected into nude mice and may represent an appropriate cell model for examination of malignant transformation associated with EBV infection. The EBV used in our study was tagged with G418-resistant gene and GFP expressing gene. We did not use G418 to select EBV-infected NP460hTert cells as the NP460hTert cells have been previously transduced with a telomerase expression vector harboring a G418 resistant gene. Guided by the presence of GFP fluorescence, we cloned the EBV-infected NP460hTert cells and pooled them together for long-term propagation. The pooled EBV-infected NP460hTert cell line was designated as NP460hTert-EBV. The purity of NP460hTert-EBV cells was further confirmed by the high percentage of GFP fluorescent cells using flow cytometric analysis (Fig. 3a). The epithelial origin of NP460hTert-EBV was confirmed by the presence of keratin staining (data not shown). The presence of EBV infection in NP460hTert-EBV was further confirmed by the positive expression of EBV latent gene, EBNA1 (Fig. 3b). The cells were subcultured at a 1:4 splitting ratio and examined for the persistence of EBV infection upon long-term propagation. At the time of submission of this article, the population doubling of NP460hTert-EBV cells reached over 140 with EBV detected in over 90 percent of cells (Fig. 3d).

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Figure 3. Establishment and long-term propagation of EBV-infected NP460hTert cell line. (a) Selectively cloning and expansion of EBV-infected NP460hTert cells. (b) Western blot of EBNA1. Proteins extracted from EBV-infected Akata cell line were served as the positive control for EBNA1 staining. (c) EBV-infected NP460hTert cells could be propagated at early stage of EBV infection. The EBV-infected cells were GFP positive. The green fluorescence color is shown as pseudocolor in white. The arrow indicates one of the EBV-infected cells undergoing mitosis. Fluorescence intensity of GFP was not significantly diminished after mitosis. (d) Flow cytometry analysis of EBV infected NP460hTert cells on prolonged culture from PD (population doubling) 3–98. M1 was marked by detecting the background fluorescence intensity using control uninfected NP460hTert cells. M2 was marked to show the percentage of GFP fluorescent (EBV infected) cells and geometric mean of fluorescence intensity of EBV-infected cells. M3 was marked within the M2 region showing a population of infected cells with particularly high fluorescence intensity. The nature and significance of M3 population is discussed in the main text. (e) RT-PCR analysis of EBV genes in NP460hTert-EBV cells. The positive controls used include LCL B-lymphoblastoid cell line (immortalized by EBV) for detection of LMP1, LMP2A and LMP2B, EBV-infected Akata cell line for detection of EBER and EBNA1 and EBV-infected Akata cell line treated by anti-IgG (to induce lytic cycle of EBV) for detection of BARF1, BZLF1, BALF4, BMRF1 and gp350/220. Independent RT-PCR reactions were performed in triplicates with essentially identical results. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Previous reports have shown that primary nasopharyngeal epithelial cells could be infected by EBV, but the infected cells failed to propagate in culture.17, 18 In our study, we have examined the proliferation potential of NP460hTert-EBV cells at the early stage of EBV infection by live-cell imaging. We were able to confirm that most of the NP460hTert-EBV cells retain their proliferative potential and undergo mitosis with undiminished GFP fluorescence (Fig. 3c). Our findings indicate that immortalized nasopharyngeal epithelial cells can support the persistent infection of EBV.

Long-term propagation of NP460hTert-EBV cells and profile of EBV gene expression

We then examined the status of EBV infection by monitoring the population of GFP-fluorescent cells at different population doublings (Fig. 3d) and by detecting the EBV gene expression profile using semiquantitative RT-PCR (Fig. 3e). The M1 represents the non-GFP fluorescent cells. The M2 represents the total population of GFP fluorescent cells. Interestingly, we observed a subpopulation of cells (M3) in the GFP fluorescent M2 population with exceptionally high GFP fluorescence at early population doublings (PD 3 and 9). We suspected that it may represent a subpopulation of EBV-infected cells undergoing lytic infection. The early population of NP460hTert-EBV cells is likely to comprise EBV-infected cells undergoing latent as well as lytic viral infection. This is supported by the detection of both latent EBV genes (EBER, EBNA1 and LMP1) and lytic EBV genes (BALF4, BMRF1 and BZLF1) (Fig. 3e). in NP460hTert-EBV cells with strong GFP fluorescence. The high GFP fluorescence intensity may also be reflective of higher EBV copy number in those cells. Interestingly, the M3 population was detected only at early population doubling (PD 3) but not at late populations (e.g. PD 35, PD 80 and PD 120), which correlated with the disappearance of lytic gene transcripts (Fig. 3e). Overall, the profiles of GFP fluorescence and EBV gene expression indicate that long-term propagation of NP460hTert-EBV cells is associated with selection of latently EBV-infected cells.

Alteration of growth properties in NP460hTert-EBV cells

We further examined if long-term propagation of NP460hTert-EBV cells may result in the tumorigenic transformation. We had examined the proliferative potential, invasiveness, anchorage-independent growth property of NP460hTert-EBV cells and their tumorigenic property after long-term propagation (9 months after infection; population doubling >70) and compared to their parental NP460hTert cells cultured at equivalent number of population doublings. NP460hTert-EBV cells could undergo anchorage independent growth in soft agar (Fig. 4a) and acquired a more invasive growth morphology in 3D collagen gel (Fig. 4b), which were not observed in the uninfected NP460hTert cells. However, the NP460hTert-EBV cells remained nontumorigenic when injected into athymic nude (Table 2). This shows that EBV infection and long-term propagation of EBV-infected cells could confer transformation properties to the immortalized NP460hTert cells. Additional events are still required to complete the tumorigenic transformation of the EBV-infected cells. Interestingly, the NP460hTert-EBV displayed a slower growth rate compared to control uninfected cells (Fig. 4c) indicating to us that increase in cell proliferation may not be the major role of EBV in the tumorigenic transformation of immortalized nasopharyngeal epithelial cells. The slow down in growth rate was not due to the expression of GFP as another NP460hTert cell line expressing H2B-GFP (established separately) did not reveal growth rate reduction (data not shown). Enhanced proliferation was also not observed in other nasopharyngeal carcinoma cell systems infected with non-GFP-tagged EBV (data not shown).

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Figure 4. Characterization of transformed phenotypes and growth properties in EBV-infected NP460hTert cells upon long term-propagation. (a) Anchorage independent growth could be observed in NP460hTert-EBV cells. Colonies were observed in soft-agar culture of EBV-infected cells after 3 weeks but not in control NP460hTert. Number of colony formation per 1 × 105 cells is shown on the graph (mean ± SD). (b) Invasive phenotype of EBV-infected NP460hTert cells. Cells were embedded inside the collagen gel. The EBV-infected cells exhibited invasive cytoplasmic extension after 24 hr while the uninfected cells remained round in shape. (c) Growth rate of EBV-infected NP460hTert-EBV cells. EBV-infected cells grew at a slower rate compared to the uninfected cells. EBV-infected and uninfected NP460hTert cells were plated at a density of 105 cells per well of a 6-well plate. Cells were trypsinized and counted on consecutive days as indicated (mean ± SD).

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Table 2. Tumorigenicity assay of EBV-infected and noninfected nasopharyngeal (NP) epithelial cells (immortalized by telomerase) in nude mice
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Karyotype of NP460hTert-EBV cells

The NP460hTert cells are well-suited to study the role of EBV infection on chromosome instability with its relatively low chromosomal aberrations compared to established cancer cell lines previously used for EBV infection. The karyotypes of EBV-infected or uninfected NP460hTert cells were analyzed using SKY to examine for both structural and numerical chromosome aberrations. We observed a clonal gain of chromosome 6 NP460hTert-EBV cells which was not observed in uninfected NP460hTert cells (Fig. 5). The significance of this clonal chromosomal aberration is unclear at this stage.

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Figure 5. Karyotype analysis of EBV-infected and uninfected NP460hTert cells. Spectral karyotyping anslysis was used to analyze both numeral and structural chromosomal aberrations in EBV-infected or uninfected NP460hTert cells. Except for the clonal gain of chromosome 6 in EBV-infected cells, no excessive structural chromosomal aberrations were observed. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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NP460hTert-EBV cells were more resistant to growth factor deprivation and nutrient starvation

Despite the fact that EBV infection is commonly present in nasopharyngeal carcinoma cells in vivo, we did not observed enhanced cell growth NP460hTert-EBV cells in vitro. Because ischemia is a major limiting factor on growth of tumor cells in vivo, we postulated that EBV infection may confer growth and survival advantage to NP460hTert cells under stress or starvation conditions. We then examined the survival ability of NP460hTert-EBV cells after growth factor deprivation and nutrient starvation. Both NP460hTert-EBV and uninfected NP460hTert cells were grown in growth-factor–deprived medium over a period of 4 weeks. As shown in Figure 6a, most of the NP460hTert and NP460hTert-EBV cells maintained their viabilities with a decreased growth rate at the initial periods (weeks 1 and 2). After that, the growth rate was halted in both the EBV-infected and uninfected NP460hTert cells with loss of viable cells (Fig. 6a). At the end of week 3, we replenished the medium with growth factors, and interestingly, the NP460hTert-EBV cells were able to recover rapidly and assumed proliferation while the uninfected NP460hTert cells were not able to recover from the growth-factor depletion treatment (Fig. 6a). To examine the ability of NP460hTert-EBV cells to sustain viability under nutrient deficiency, we plated the EBV-infected and uninfected NP460hTert cells at low cell density and did not change the culture medium for 3 weeks (Fig. 6b). Interestingly, viable and proliferative cell clones were only found in NP460hTert-EBV cells but not in control uninfected-NP460hTert cells, which again confirmed the survival ability of NP460hTert-EBV cells to withstand growth factor deprivation and nutrient starvation.

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Figure 6. Survival properties of NP460hTert-EBV cells under growth factor deprivation and nutrient starvation. (a) NP460hTert-EBV cells are more resistant than NP460hTert cells to starving conditions. The cells were grown in growth-supplement–deprived plain RPMI medium for 20 days. The NP460hTert-EBV cells, but not NP460hTert cells, could recover from the growth-supplement–deprived condition. (b) Enhanced ability of EBV-infected cells to form viable clones under nutrient deficiency. Cells were plated in flask at low density with no change in medium for 3 weeks. GFP-positive clones derived from single dispersed cells were found only in EBV-infected NP460hTerts but not in the uninfected NP460hTert cells. (c) EBV-infected NP460hTert cells had enhanced survival advantage over uninfected cells. EBV-infected cells expressed lower levels of apoptotic markers (cleaved PARP and cleaved caspase 3) with substained pAkt activation when cultured in EBSS. (d) Elevated expression of MCP-1 and Gro-α in NP460hTert-EBV cells under acute amino acid deprivation. Cells were cultured in EBSS for 24 hr. Cell free culture supernatants from NP460hTert or NP460hTert-EBV cells were harvested and hybridized with the cytokine array (Raybiotech). Positive and negative controls were designated at (1A, 1B, 1C, 1 D, 8J and 8K) and (1E, 1F and 8I), respectively. The arrows indicate to the positions of upregulated cytokines [MCP-1 (3E), Gro-α (1K)], and downregulated cytokines [angiogenin (4K) and MIF (7H)] in EBV-infected cells. Experiments were performed in duplicates with essential identical results. (e) RT-PCR analysis confirming upregulated transcription of cytokines, MCP-1 and Gro-αin EBV-infected NP460hTert cells. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Activation of cell-survival signaling pathways and altered cytokine secretion in NP460hTert-EBV cells to amino acid withdrawal

We have further explored the potential mechanisms involved in enhanced resistance to nutrient and growth factor deprivation by examining the apoptotic induction in EBV-infected and uninfected cells under acute amino acids withdrawal. The cells were starved in the EBSS for various indicated time periods and then examined for the expression of apoptotic markers and survival marker (Fig. 6c). The EBV-infected cells were able to sustain the activation of pAkt on nutrients deprivation (Fig. 6c). The uninfected cells were more prone to apoptosis with much higher levels of cleaved PARP and cleaved caspase 3 than the EBV-infected cells (Fig. 6c). To further elucidate the underlying mechanism of EBV-infected cells against nutrient starvation, we then examined the alteration of cytokine secretion in EBV-infected and uninfected NP460hTert cells using a protein expression array. The cytokine expression profiles of EBV-infected and uninfected NP460hTert cells are shown in Figure 6e. Among the different cytokines examined, we were able to confirm the upregulation of secretion of MCP-1 and Gro-α in NP460hTert-EBV cells (Fig. 6f). The possible roles of MCP-1 and Gro-α in maintaining growth under starvation or in pathogenesis of NPC will be elaborated in the Discussion section. These results indicate that NP460hTert-EBV cells have the ability to activate multiple survival mechanisms on starvation stress, which may confer survival advantage for EBV-infected cells in vivo.

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References

EBV infection in B cells has been extensively investigated but not in nasopharyngeal epithelial cells. Events relating to EBV infection at early development of NPC are largely unknown. The use of recombinant EBV tagged with GFP has improved the detection of EBV infected cells and facilitated the establishment of stable EBV infection in our immortalized nasopharyngeal epithelial cell system. In concordance with previous studies,7, 13 cell-to-cell contact was shown to be an efficient mode of EBV infection in primary cultures of both primary and immortalized nasopharyngeal epithelial cells as well as established nasopharyngeal carcinoma cells (Fig. 1a). Around 1–10% of EBV infection rate could be achieved by coculture method compares to less that 0.1% of EBV infection rate when cell-free virus preparations were directly used for infection. Hence, close interaction of target epithelial cells with EBV-producing B cells is essential for EBV entry into nasopharyngeal epithelial cells. EBV infects B cells by interaction of the viral glycoprotein gp350/220 with the complement receptor (CR2) present on B cells surface. The route of EBV infection into nasopharyngeal epithelial cells is unclear. Recent studies demonstrated that EBV infection of human epithelial cells could be achieved in vitro by cocultivating the target epithelial cells with an EBV-producing B-cell line.13 Our study showed that the CR2 receptor, which is involved in EBV infection in B cells, is unlikely to be involved in the cell–cell contact route of EBV infection. An alternative mechanism could involve cell–cell contact via EBV encoded BMRF2 membrane protein, containing a RGD-binding motif and expressed in virus producer cells, capable of interacting with specific types of integrins, that may be upregulated during inflammation and oxygen stress.19–21 Hence, inflammation and stromal cytokines may play a role in regulation of EBV infection into nasopharyngeal epithelium in vivo. The great variation of EBV infection rate across the different nasopharyngeal cells used for infection also indicates that the cellular context is crucial in the susceptibility of nasopharyngeal epithelial cells for EBV infection.

To further investigate the regulation of EBV infection in premalignant nasopharyngeal epithelial cells, we have chosen NP460hTert for investigation. It was shown to be the most susceptible cell line for EBV infection. The NP460hTert cells also harbor some of the early genetic alterations present in precancerous nasopharyngeal epithelial cells, notably inactivation of p16 and RASSF1A.10 Inactivation of p16 is known to prevent onset of replicative senescence while inactivation of RASSF1A supports cell proliferation, both may play crucial roles in supporting long-term propagation of EBV-infected cells. NPC is associated with an inflammatory stroma. An array of cytokines was detected in the NPC stroma.22 We have examined the influence of inflammatory cytokines on EBV infection using this premalignant nasopharyngeal cell system. We found that the inflammatory cytokines, TGF-β1 and TNF-α could significantly increase the EBV infection rate in NP460hTert cells suggesting that inflammatory cytokines could play a significant role to facilitate EBV infection in premalignant nasopharyngeal epithelial cells (Fig. 2a). In particular, TGF-β1 was found to have a profound effect to facilitate EBV infection. Pretreating the NP460hTert cells with TGF-β1 could enhance the EBV infection by about 4 folds. The enhancing effect of TGF-β1 in EBV infection could also be observed in primary NP cells (Fig. 2c). Interestingly, higher levels of TGF-β1 were reported in serum samples from nasopharyngeal carcinoma patient with relapsing tumor.23 It has been proposed that this cytokine may have immunosuppressive role in the pathogenesis of NPC.24 Our study indicates that TGF-β1 may also facilitate the development of NPC by facilitating EBV infection in NP cells. This observation is in concordance with the previous study that EBV showed marked tropism to differentiated primary epithelial cells.17, 18 We have observed that TGF-β1 could induce differentiation of primary nasopharyngeal epithelial cells by increasing the levels of involucrin and suppression of cell growth (results not shown). The NP460hTert cells used for EBV infection are also sensitive to the action of TGF-β1. We are currently pursuing the involvement of TGF-β1 signaling and downstream events which may be involved in EBV infection. Our preliminary studies using flow cytometry indicates that upregulation of integrin receptors may be involved (data not shown).

EBV infection was shown to be an early event in NPC development and could be detected in high grade precancerous nasopharyngeal lesions. Many events relating to EBV infection of premalignant nasopharyngeal epithelial cells are largely undefined. In our study, we have established for the first time, long-term propagation of EBV infection in a premalignant nasopharyngeal epithelial cell model. The establishment of this EBV-infected premalignant nasopharyngeal epithelial cell model allows us to examine some of phenotypic changes associated with long-term propagation of EBV infection in this experimental cell model. We have selected the GFP fluorescent NP460hTert-EBV cells and subsequently expanded into a stable EBV-infected line. Flow cytometric analysis of GFP-fluorescent cells and staining of EBNA1 expression confirmed the high infection percentage (> 95%) of NP460hTert-EBV cells. EBV infection of B lymphocytes readily invokes cell proliferation. In contrast, we did not observe stimulation of cell proliferation in EBV-infected primary and immortalized nasopharyngeal epithelial cells. EBV-infected cells often grow at a slower rate compared to uninfected cells at early stage of infection (Fig. 4c), possibly because some cells in culture enter into lytic cycle and die. Long-term propagation of EBV-infected cells was achieved by the selection for pure EBV-infected cell clones guided by GFP fluorescence. Using live cell imaging, we confirmed that EBV-infected NP460hTert cells could undergo mitosis without significant loss of GFP intensity. The number of cells entering into the lytic cycle gradually decreased over time, with cells expressing only latent EBV genes remaining. The discrepancy between the growth rate of EBV infected and uninfected cells become less prominent (data not shown). This may also reflect the natural progression of infected epithelial cells into NPC tumor cells in vivo.

We have examined the contribution of EBV infection to genomic instability of EBV-infected NP460hTert cells using spectral karyotypic analyses. Previous reports have suggested that EBV may promote genomic instability through multiple mechanisms. EBNA1 induced DNA damage and chromosome abnormality by enhancing reactive oxygen species production25; we have previously showed that LMP1 could destabilize mitotic spindles and induce abnormal mitosis.26 Recent studies indicate an important role for EBNA1 in promoting host cell instability, by enhancing P53 degradation and disturbing PML body formation.27, 28 The contribution of EBV to chromosome instability in nasopharyngeal epithelial cells is not clear at this stage. Clonal and nonclonal chromosomal alterations could be detected in NP460hTert-EBV cells. However, we did not detect extensive chromosomal aberration in EBV-infected NP460hTert cells. One explanation is that mitotic deregulation often induces mitotic arrest and apoptosis, thus eliminating cells with extensive chromosome aberrations. It would be interesting to observe if additional genetic alterations including oncogene activation may further drive chromosomal instability in NP460hTert-EBV cells. Further investigations on mitotic checkpoint regulation in NP460hTert-EBV cells are warrant to elucidate the contribution of EBV infection to chromosome instability in nasopharyngeal epithelial cells and their involvement in NPC development.

The establishment of stable EBV infection in NP460hTert cells has enabled us to examine some of the phenotypic alterations of EBV-infected nasopharyngeal epithelial cells on long-term propagation. Long-term propagation of NP460hTert-EBV cells induced a more transformed phenotype compared to control uninfected cells as shown by the formation of invasive cytoplasmic extensions inside 3D collagen gel and anchorage-independent growth in soft-agar (Figs. 4a and 4b). The acquisition of these invasiveness and anchorage independent growth property in EBV-infected cells was a gradual process and not obvious in EBV-infected cells at early stage of infection. The selection of malignant phenotype was not observed in control cells which have undergone equivalent number of population doublings. The acquisition of malignant phenotype in NP460hTert-EBV cells may involve intricate interaction of intracellular factors and EBV gene expression. Long-term propagation of EBV-infected cells apparently facilitates the selection of cells with a more transformed phenotype. Despite the acquisition of these in vitro transformed properties, the NP460hTert-EBV cells remained nontumorigenic when injected into nude mice (Table 2) indicating that additional events are involved in the malignant transformation of EBV-infected cells into cancer cells.

A major obstacle for tumor growth in vivo is ischemia, i.e., inadequate supply of oxygen and nutrients, which associated with insufficient blood supply. Interestingly, the NP460hTert-EBV cells were highly resistant to starvation stress, which may be an important property for tumorigenesis in vivo. We were able to demonstrate that the NP460hTert-EBV cells could survive nutrient starvation or growth factor deprivation much better than uninfected cells. Enhanced survival ability was confirmed by suppression of apoptotic pathways and sustained activation of survival Akt pathway in NP460hTert-EBV cells under amino acid deprivation (Fig. 6c). Examination of secreted cytokines of the EBV-infected and uninfected cells under acute amino acid deprivation revealed elevation of MCP-1 and Gro-α secretion in NP460hTert-EBV cells. MCP-1 could protect prostate cancer cells from autophagic cell death, and allows prolonged survival in serum-free condition by upregulation of survivin.29 Interestingly, production of MCP-1 in epithelial cells could be regulated by the EBV-encoded LMP1.30 On the other hand, the secretion of Gro-α, which is an autostimulatory growth factor,11 may also help to maintain cell proliferation upon nutrient starvation. Gro-α expression by squamous cell carcinoma had been reported to promote tumor growth, metastasis, leukocyte infiltration and angiogenesis.31 All these results strongly indicate that EBV-infected cells are much more resistant against starvation, which may play a crucial role in facilitating tumorigenic transformation of premalignant nasopharyngeal epithelial cells in vivo. This EBV infection cell model of nasopharyngeal cells will support further investigations into elucidation of the multiple pathways involved in survival.

In conclusion, we have established an EBV infection cell system using premalignant nasopharyngeal epithelial cells and demonstrated a role for proinflammatory cytokines in promoting EBV infection of nasopharyngeal epithelial cells. We also established prolonged EBV infection in a well-characterized immortalized nasopharyngeal cell line. Finally, we showed that EBV may contribute to host cell survival under stress conditions. The establishment and characterization of this EBV-infected nasopharyngeal cell system will facilitate systematic investigation into events involved in EBV infection in NP cells and to elucidate the pathogenic roles of EBV in NPC development.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References