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E7 viral oncogenes
Biochimica et Biophysica Acta 1590 (2002) 150 – 158 www.bba-direct.com
Establishment of two immortalized nasopharyngeal epithelial cell lines using SV40 large T and HPV16E6/E7 viral oncogenes Sai Wah Tsao a,*, Xianghong Wang a, Yu Liu a, Yuk Chun Cheung a, Huichen Feng a, Zhong Zheng a, Natalie Wong b, P.W. Yuen c, Angela K.F. Lo a, Y.C. Wong a, D.P. Huang d a
Department of Anatomy, Faculty of Medicine, University of Hong Kong, New Medical Complex, 1st Floor, Laboratory Block, 21 Sassoon Road, Pokfulam, Hong Kong, China b Department of Clinical Oncology at the Sir Y.K. Pao Center for Cancer, Chinese University of Hong Kong, China c Department of Surgery, Faculty of Medicine, University of Hong Kong, Hong Kong, China d Department of Anatomical and Cellular Pathology, Chinese University of Hong Kong, Hong Kong, China Received 5 December 2001; received in revised form 9 April 2002; accepted 11 April 2002
Abstract Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, especially in southern China. One of the most striking features of this disease is its close relationship with Epstein – Barr Virus (EBV). However, to date there is no direct study on the mechanisms involved in the role of EBV in the tumorigenesis of NPC, largely due to lack of an experimental model. Available hypotheses on the association between EBV and NPC are generated from non-nasopharyngeal epithelial cell systems such as human keratinocytes or mouse epithelial cells, which may not truly represent the biological properties of nasopharyngeal epithelial (NP) cells. In this study, we report the establishment of two immortalized NP cell lines, NP69SV40T and NP39E6/E7, using SV40T and HPV16E6/E7 oncogenes. We found that NP60SV40T and NP39E6/E7 cell lines not only maintained many characteristics of normal NP cells (i.e. keratin profile and responsive to TGFh inhibition) but also highly responsive to one of the EBV encoded genes, LMP1. Comparative genome hybridization (CGH) analysis showed that these two cell lines contained multiple genetic alterations, some of which have been described in NPC. The immortalized NP cell lines are nontumorigenic and exhibit anchorage-dependent growth. These cell lines may provide a possible cell model system for studying the mechanisms involved in the tumorigenesis of NPC. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Nasopharyngeal epithelial; HPVE6/E7; SV40T
1. Introduction Nasopharyngeal carcinoma (NPC) is a common cancer in southern China. In Hong Kong, the incidence rate of NPC is 25 times higher than in other countries around the world. As one of the striking features of NPC is the presence of Epstein – Barr Virus (EBV), therefore EBV infection is proposed as one of the significant etiological factors in NPC [1]. A direct role of EBV in tumorigenesis has been established in Burkitt’s lymphoma, however, due to lack of specimens and experimental models, little information is available on the role of EBV in the tumorigenesis process of NPC. Most of the functional studies on the transformation
*
Corresponding author. Tel.: +852-281-99227; fax: +852-281-70857. E-mail address: [email protected] (S.W. Tsao).
ability of EBV were demonstrated in non-nasopharyngeal epithelial systems such as mouse embryonic fibroblasts [2], immortalized human keratinocytes [3] or gastric carcinoma cells [4]. The fact that most of the established NPC cell lines lose EBV after long-term in vitro culture makes it even more difficult to study the involvement of EBV in NPC. To date, although etiological evidence strongly suggests that EBV plays important roles in the tumorigenesis process of NPC, there is no direct evidence to support this hypothesis in nasopharyngeal cell system. In this study, we report the establishment of two immortalized nasopharyngeal epithelial cell lines NP69SV40T and NP39E6/E7, using SV40 large T and HPV16E6/E7 oncogenes, respectively. These two cell lines not only showed keratin profiles of primary nasopharyngeal epithelial cells but also showed response to TGFh1, indicating that they retain some characteristics of non-malignant nasopharyngeal epithelial cells. In addition, expression of one of the EBV encoded genes, LMP1,
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resulted in an increase in cell cycle S phase fraction and cell growth rate in NP69SV40T cells. NP69SV40T and NP39E6/E7 cell lines provide potential nasopharyngeal epithelial cell models for studying mechanisms involved in tumorigenesis of NPC.
up the viral oncogenes would eventually undergo senescence and stop proliferating. Presence of SV40T protein in NP69SV40T was detected using Western blotting analysis as described [7]. The presence of E6/E7 mRNA in NP39E6/ E7 cells was detected using RT-PCR as previously described [8].
2. Materials and methods
2.3. Telomerase assay
2.1. Establishment of in vitro primary nasopharyngeal epithelial cell culture
The presence of telomerase in the immortalized nasopharyngeal epithelial cells was examined using the PCRbased TRAP assay (telomeric repeats amplification protocol) according to a previously published protocol [9].
Primary cultures of nasopharyngeal epithelial cells were established from biopsies from the nasopharynx of patients with symptoms such as nasal obstruction and excessive bleeding. Prior patient’s consent and approval from the research ethics committee were obtained for the use of these surgical materials for research purposes. To initiate primary culture of nasopharyngeal epithelial cells, the biopsies were cut into approximately 1 mm3 sized explants and placed onto 6 cm culture dishes (Falcon) containing 2 ml of culture medium RPMI1640 supplemented with 1% dialyzed fetal bovine serum, 0.25 AM transferrin, 3.32 nM epidermal growth factor, 0.7 AM insulin, 0.5 AM phosphoethanolamine, 0.5 AM ethanolamine, 10 nM triodo-L-thyronine, 2 AM hydrocortisone, 2.7 AM L-epinephrine, 2 mM L-glutamine, 0.03 AM sodium selenium, 1 nM molybdic acid, 0.5 nM stannous chloride and 0.75 nM nickel sulfate. The calcium concentration of the above medium was 0.4 mM, which was designed to facilitate the attachment of explants and initiation of epithelial outgrowth. The monolayer epithelial cells were then cultured in a low calcium (0.1 mM) medium (MCDB151 medium supplemented with 1% dialyzed fetal bovine serum and the same ingredients described above). The low calcium concentration stimulated the epithelial cell proliferation but suppressed the growth of contaminated fibroblasts. Proliferating fibroblasts, which may be present in the culture flask, were removed manually using the tip of a glass Pasteur pipette circled by Bunsen flame. 2.2. Immortalization of nasopharyngeal epithelial cells The nasopharyngeal epithelial cells at passage one were transfected with the PX8 plasmid containing SV40T antigen [5] or infected with a retroviral vector containing the HPV16E6/E7 ORFs [6]. The retroviral vector HPV16E6/ E7 was kindly provided by Dr. D. Galloway at the Fred Hutchison Cancer Center, University of Washington, USA. For transfection of the SV40T expression plasmid (PX8), 1 Ag of the plasmid DNA was added in 100 Al of serum-free culture medium with 6 Al of Fugene 6 solution (Roche). Expression of the HPV16E6/E7 viral genes was achieved by infecting the cells with the HPV16E6/E7 expressing viral vector according to a previously described protocol [6]. No drug selection was required as the cells which failed to take
2.4. Keratin profiles The expression of cytokeratins in primary and immortalized nasopharyngeal epithelial cells was determined using two-dimensional SDS-PAGE electrophoresis as previously described [6]. The solubilized cytoskeletal proteins were separated by non-equilibrium pH gradient electrophoresis (NEPHGE) using a two-dimensional gel system (Hoefer). The presence of cytoskeletal proteins was visualized using a silver staining kit from Amersham Pharmacia. 2.5. Growth inhibition by TGFb1 Cellular responses to TGFh1 were determined by thymidine incorporation after 3 days treatment with various concentrations of TGFh1 (0.1, 1, and 10 ng/ml) (Gibco BRL, Bethesda, MD). Details of the assay protocol were described previously [10]. 2.6. Comparative genome hybridization (CGH) CGH experiment was carried out according to the published method [11]. Briefly, tumor and normal reference DNA labeled with biotin-16-dUTP (Boehringer Mannheim, Mannheim, Germany) and digoxigenin (dig)-11-dUTP (Boehringer Mannheim), respectively by nick translation were competitively co-hybridized onto metaphase preparations. After 2 days of hybridization at 37 jC, biotin labeled signals were detected through avidin conjugated-fluorescein isothiocyanate (FITC) antibodies (Sigma, St. Louis, USA), and dig-labeled probes by antibodies conjugated with tetramethylrhodamine isothiocyanate (TRITC) (Sigma). The preparations were counterstained with 4V,6-diamidino-2phenylindole (DAPI) (Sigma). Hybridized metaphases were captured with a cooled CCD camera mounted on a Leitz DM RB (Leica, Wetzlar, Germany) fluorescence microscope. Three band-pass filter sets (DAPI, FITC and TRITC) arranged in an automated filterwheel were employed for image acquisition. A CGH software version 3.1 on Cytovision (Applied Imaging, Sunderland, UK), was used for digital image analysis of fluorescence intensity. Chromosome identification was per-
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Fig. 1. Keratin profiles of primary nasopharyngeal epithelial cells and immortalized NP69SV40T and NP39E6/E7 cells detected by two-dimensional SDS PAGE electrophoresis. (A) Keratin expression profile of NPE cells is similar to previously described in epithelial cells of the upper respiratory tract. (B and C) Keratin expression pattern is similar in NP69SV40T and NP39E6/E7 cells compared to NPE cells. Inserted photographs show the morphological characteristics of nasopharyngeal epithelial cells.
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formed on the reverse DAPI banding images. The average ratio profiles were calculated based on the analysis of 14 or more selected metaphases. Thresholds for gains and losses were defined as the theoretical value of 1.25 and 0.75, respectively. High level gain of regional or whole chromosome was considered to be present when ratios exceeded 1.5. 2.7. LMP-1 gene transfer mediated by retroviral vector pLNSX The LMP-1 gene was introduced into immortalized NP69SV40T cells by infecting the cells with a retroviral vector pLNSX-LMP1. The construction of the pLNSXLMP1 plasmid was described previously [2]. Briefly, a 1.95-kb fragment containing a NPC derived full-length LMP1 gene was cloned into the retroviral vector pLNSX to generate pLNSX-LMP1. Control cells were infected with the empty retroviral vector pLNSX. All of the transduced cells were selected and maintained in G418 (400 Ag/ml). Immunoblotting was used to confirm the expression of LMP1 in the immortalized nasopharyngeal cells using the specific antibody (S12) against LMP1 [2]. To demonstrate the effect of LMP1 on morphological alteration, a B95-8 LMP1 cloned in pcDNA vector was transfected into the NP69SV40T and selected with G418. Control cells were transfected with an empty pcDNA vector
Fig. 3. Evidence of telomerase activity in NP69SV40T and NP39E6/E7 cells. Telomerase activity was determined at two different concentrations of protein (0.3 and 3 Ag/ml) using TRAP assay. HeLa cells were used as a positive control.
2.8. Cell cycle analysis
2.9. Growth curve
Cells (5 105) were trypsinized and fixed in cold 70% ethanol for at least 1 h and stored at 4 jC. Before testing, the fixed cells were washed with PBS, treated with RNAse (1 mg/ml) and stained with propidium iodide (50 Ag/ml) for 30 min at 37 jC. DNA content analysis was performed on an EPICS ELITE flow cytometer (Beckman Coulter) and cell cycle distribution was analyzed using the ModFit LT2.0 software.
To determine in vitro cell growth rate, control and LMP1 expressing cells were plated in triplicates at 4 104 cells/ well in 24-well plates. The number of viable cells was determined every 48 h using the trypan blue exclusion method. The growth curves were drawn by plotting the means of three independent experiments with the standard deviation as error bars. 2.10. Tumorigenicity study Cells (106 to 107) were injected subcutaneously into two flanks of 6– 8-week-old Balb/c female athymic nude mice and observed for tumorigenic growth every week for a 4month period.
3. Results 3.1. Establishment and keratin profiles of nasopharyngeal epithelial primary culture
Fig. 2. SV40T and HPV16 E6/E7 expression in NP69SV40T and NP39E6/ E7 cells. (A) RT-PCR analysis of E6/E7 mRNA expression in NP39E6/E7 cells. (B) Western blotting analysis of SV40T protein expression in NP69SV40T cells.
To initiate primary culture of nasopharyngeal epithelial growth, biopsies from patients diagnosed with non-malignant nasal diseases were cut into approximately 1 mm3 sized explants and placed onto 6 cm dishes containing 2 ml culture medium. The epithelial-like outgrowth from the explants was observed 3 – 4 days after explantation and the
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Fig. 4. Effect of TGFh1 on NP and NPC cells. Effect of TGFh1 on cell growth was determined by measuring the percentage of thymidine uptake in cells after 3-day treatment. Results were derived from three independent experiments and the error bars indicate standard deviation. NP69SV40T and NP39E6/E7 cells showed decreased sensitivity to TGFh1 treatment compared to primary NPE cells but more sensitive to TGFh1 than NPC cells (CNE1 and CNE2).
Fig. 5. Representative results of CGH analysis on NP69SV40T cells. The main ratio profile of at least 10 chromosomes is shown. The central line represents balanced hybridization (signal ratio = 1). Signal ratios skewed to the left or right represent thresholds for chromosomal losses (signal ratio < 0.75) and gains (signal ratio > 1.25) respectively.
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cells showed morphology of epithelial cells (Fig. 1A). To confirm the epithelial origin of these cells, cytoskeletal proteins were extracted and two-dimensional SDS PAGE electrophoresis was performed. As shown in Fig. 1A, the cultured nasopharyngeal cells showed detectable expression of cytokeratins K5, K6 and K13-19, indicating that they were of respiratory epithelial origin [12].
istics and the keratin profiles of these two cell lines were similar to the primary nasopharyngeal epithelial cells (Fig. 1B and C). The expression of SV40T or HPV16E6/E7 oncogenes was also detected in these two cell lines, respectively (Fig. 2). These results indicate that NP69SV40T and NP39E6/E7 cell lines represent characteristics of nasopharyngeal epithelial origin.
3.2. Immortalization of nasopharyngeal epithelial cells using HPV16E6/E7 and SV40T viral oncogenes
3.3. Evidence of telomerase activation and TGFb1 sensitivity in NP69SV40T and NP39E6/E7 cell lines
The primary nasopharyngeal epithelial cells had limited proliferative potential. They usually underwent terminal differentiation after 2 – 3 weeks in culture and showed senescent-like phenotype including flattened cell morphology and expression of senescent associated-h-galactosidase (data not shown). In order to establish long-term growth of these cells, vectors containing HPV16 E6/E7 or SV40T viral oncogenes were infected or transfected, respectively into the primary cultured cells. After overcoming crisis stage (approximately 3 – 6 months), two immortalized cell lines NP69SV40T and NP39E6/E7 were established, which have undergone over 100 population doublings without showing any evidence of senescence. The morphological character-
Telomerase activation has been shown to be a common characteristic during the cell immortalization process. We next studied if activation of telomerase was apparent in NP69SV40T and NP39E6/E7 cells. As shown in Fig. 3, both immortalized cell lines showed telomerase activation at similar levels to a cancer cell line, HeLa (Fig. 3). However, NP69SV40T and NP39E6/E7 cells were sensitive to TGFh1-induced cell growth inhibition, which has been suggested as one of the characteristics of normal epithelial cells [13], compared to NPC cells. As shown in Fig. 4, both cell lines responded to TGFh1 in a pattern similar to the primary nasopharyngeal epithelial cells (NPE) in a dose-dependent manner as decreased thymidine
Fig. 6. Effect of LMP1 expression on NP69SV40T cells. (A) Presence of LMP1 protein (upper band) in NP60SV40T cells after transfection of pLNSX vector containing a LMP1 gene but absent in the vector control (NP69SV40T-pLNSX) by Western blotting analysis. (B) Morphological changes induced by LMP1 expression. Elongated and fibroblast-like morphology was observed when a B95-8 LMP1 was expressed in NP69SV40T. Photos were taken under 200 magnification. (C) Cell growth curves of NP69SV40T-LMP1 and its control (-pLNSX). LMP1 expression significantly increased the growth rate of NP69SV40T cells ( P < 0.05). (D) Evidence of LMP1-induced cell cycle S phase fraction in NP69SV40T cells by flow cytometric analysis.
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uptake rate was correlated with increased TGFh1 concentrations. However, the sensitivity in these two cell lines to TGFh1 was decreased compared to NPE cells, especially in NP39E6/E7 cells. In contrast, there was no evidence of growth inhibition in two NPC cell lines, CNE1 and CNE2, treated with the same concentrations of TGFh1. In addition, injection of both NP69SV40T and NP39E6/E7 cells into nude mice did not show any evidence of tumor formation after 3 months, indicating that they are not tumorigenic (data not shown). These results indicate that although NP69SV40T and NP39E6/E7 cells showed similar telomerase activity as cancer cells, they still retained characteristics of non-malignant nasopharyngeal epithelial cells. 3.4. Comparative genome hybridization (CGH) analysis of NP69SV40T and NP39E6/E7 cells CGH analysis on NP69SV40T and NP39E6/E7 cells showed that five genetic alterations including loss of material on chromosomes 9p (NP69SV40T) and 3 (NP39E6/E7), and gains of material on chromosomes 17q (NP69SV40T), 12q and 11 (NP39E6/E7), were correlated to the changes commonly observed in NPC (Table 1 and Fig. 5) [14 – 16]. In NP69SV40T cells, losses on chromosome 6, 11 and 16 and gains on chromosomes 8, 9 and 22 were observed as previously described to be associated with SV40T infection [17 –19]. In NP39E6/E7 cells, typical E6/E7 related genetic alterations such as amplification of chromosomes 5 and 20, were observed [20,21]. 3.5. Evidence of LMP1-induced cell proliferation in NP69SV40T cells In order to study whether the immortalized cells responded to EBV infection, one of the EBV encoded genes, LMP1, which has been shown to play important roles in oncogenic transformation in certain types of epithelial cells [2– 4], was expressed into NP69SV40T cells. As shown in Fig. 6A, LMP1 protein was detected in the NP69SV40T-LMP1 cells but absent in the vector control. Expression of LMP1 significantly altered the morphology of NP69SV40T cells to an elongated and fibroblast-like appearance. Interestingly, this morphological transformation is even more remarkable in NP69SV40T cells expressing the prototype B95-8 derived LMP1 (Fig. 6B). Increased growth rate was also observed after LMP1 expression and increased growth rate of NP69SV40T cells compared to the vector control (Fig. 6C). The increased growth rate may be due to the stimulation of DNA synthesis by LMP1 in the transfectants, as the cell cycle S phase fraction was greatly increased in NP69SV40T-LMP1 cells (40.7%) (Fig. 6D) compared to the vector control (22.67%). However, there was no evidence of tumor formation after NP69SV40T cells were injected into nude mice (n = 10) and observed for a 4month period.
4. Discussion In this study, we report the establishment of two immortalized nasopharyngeal epithelial cell lines NP60SV40T and NP39E6/E7 using SV40T and HPV16E6/E7 viral oncogenes. The fact that these cell lines maintained characteristics of primary nasopharyngeal epithelial cells such as keratin profile and are non-tumorigenic (Figs. 1 and 4), indicates that they provide potential in vitro models for studying tumorigenesis of NPC. The nasopharynx is located at the back of the nasal passageway and surgical access to nasopharynx is limited, therefore, nasopharyngeal tissues are not usually available for investigation. Limited numbers of NPC cell lines have been established but due to the fact that the majority of the available cell lines do not contain EBV, studies on the role of EBV in tumorigenesis of NPC have limited resources. In addition, to date, there is not one non-malignant nasopharyngeal epithelial cell system available for such studies. Our report on the establishment of two immortalized nasopharyngeal epithelial (NP) cell lines may provide an experimental cell model to facilitate investigation in mechanisms involved in tumorigenesis in NPC. In this study, results on cellular morphology, keratin profiles and response to TGFh1 treatment have shown that NP69SV40T and NP39E6/E7 cells retain characteristics of primary NP cells (Figs. 1 and 4). Both cell lines showed activation of telomerase activity, indicating that they are immortal (Fig. 2). Genetic studies showed a number of losses or gains of materials on chromosomes commonly associated with both E6/E7 or SV40T infection such as gains on chromosomes 5 and 20 and losses on chromosome 19 (E6/E7) [19,20] or gains on chromosome 8, 9, 22 and losses on 6, 11 and 16 (SV40T) [16 – 18] (Fig. 5, Table 1), indicating that the expression of E6/E7 or SV40T viral
Table 1 Summary of CGH results
NP69SV40T
NP39E6/E7
Losses
Gains
Amplification
4p15.3 – qter, 6p11.2 – qter 9pter – p23 11pter – q11 11q14 – qter 12p11.2 – p13 16pter – q11 19pter – q12 Xp11.2 – qter 3, 4, 10pter – q21 12 15q11.2 – q15 16pter – p11.2 16q11.22 – qter
2 5pter – q21 7 8p11.2 – qter 9p13 – qter 15q14 – q26 17q11.2 – q25 20p12 – q13.3 22cen – q13 1pter – p32 2pter – p24 6p22 – cen 7pter – q35 11 13q22 – qter 17 19q12 – q13.3 X
5q21 – qter 9p13 – qter
5 20
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oncogenes had significant effects on these cells and these genetic changes may be crucial for the immortalization process of these cells. However, we found only five genetic alterations (out of 33) which are commonly observed in NPC (gains on chromosomes 1q, 11q, 12q and 17q and losses on 3p and 9p) [14 –16]. These results indicate that NP69SV40T and NP39E6/E7 cell lines may provide good models to study the multiple-step pathogenesis in NPC. In order to study the effect of EBV on the immortalized NP cells, we transfected one of the EBV encoded genes, LMP1, into NP69SV40T cells and studied its effect on cell growth and tumorigenecity (Fig. 6). LMP1 is one of the EBV encoded genes expressed in NPC and it has been suggested to be involved in the tumorigenic processes of NPC [22]. LMP1 has been shown to be oncogenic in immortalized epithelial cells derived from human skin and canine kidney [3,23 – 25]. A number of studies showed that LMP1 alone was able to induce tumorigenecity in these epithelial systems [3,23,24]. Others have reported that LMP1 alone had significant effect on cell growth morphology and differentiation [25,26]. In this study, we found that expression of LMP1 in NP69SV40T cells resulted in a striking morphological change from relatively flat to fibroblast-like and disorganized bundled morphology (Fig. 6B), which agrees with results found in previous studies on human keratinocytes and canine epithelial cells [23,26]. A significant increase in cell growth rate was also observed in NP69SV40T cells which was associated with increased cell cycle S phase fraction (Fig. 6C and D). This suggests that expression of LMP1 in nasopharyngeal epithelial cells had significant stimulating effects on cell growth. However, in contrast to previous studies [3,24], we did not observe tumor formation when the NP69SV40T-LMP1 cells were injected into nude mice (data not shown). Our results confirmed previous evidence on the cell growth-stimulating role of LMP1 in epithelial cell growth. However, unlike some observations from other epithelial cell systems, LMP1 alone was not sufficient to induce tumorigenesis in immortalized NP cells. Our evidence supports the hypothesis that EBV infection may be an early factor in the pathogenesis of NPC and additional factors such as genetic or etiological factors are needed in the development of NPC. In summary, we provide potential cell model systems for studying the mechanisms involved in tumorigenesis of NPC. The availability of NP69SV40T and NP39E6/E7 cell lines will provide possible tools to advance studies on the association between EBV and NPC.
Acknowledgements This work is supported by the Research Retreat Grant 2001 (Faculty of Medicine, HKU) and research grants from Research Grant Council (Hong Kong) and CRCG grant (HKU) awarded to SWT.
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[23] K.R. Kim, T. Yoshizaki, H. Miyamori, K. Hasegawa, T. Horikawa, M. Furukawa, S. Harada, M. Seiki, H. Sato, Transformation of Madin – Darby canine kidney (MDCK) epithelial cells by Epstein – Barr virus latent membrane protein 1 (LMP1) induces expression of Ets1 and invasive growth, Oncogene 19 (2000) 1764 – 1771. [24] L.F. Hu, F. Chen, X. Zheng, I. Ernberg, S.L. Cao, B. Christensson, G. Klein, G. Winberg, Clonability and tumorigenicity of human epithelial cells expressing the EBV encoded membrane protein LMP1, Oncogene 8 (1993) 1575 – 1583. [25] C.W. Dawson, A.B. Rickinson, L.S. Young, Epstein – Barr virus latent membrane protein inhibits human epithelial cell differentiation, Nature 344 (1990) 777 – 780. [26] R. Fahraeus, L. Rymo, J.S. Rhim, G. Klein, Morphological transformation of human keratinocytes expressing the LMP gene of Epstein – Barr virus, Nature 345 (1990) 447 – 449.
Establishment of two immortalized nasopharyngeal epithelial cell lines using SV40 large T and HPV16E6/E7 viral oncogenes Sai Wah Tsao a,*, Xianghong Wang a, Yu Liu a, Yuk Chun Cheung a, Huichen Feng a, Zhong Zheng a, Natalie Wong b, P.W. Yuen c, Angela K.F. Lo a, Y.C. Wong a, D.P. Huang d a
Department of Anatomy, Faculty of Medicine, University of Hong Kong, New Medical Complex, 1st Floor, Laboratory Block, 21 Sassoon Road, Pokfulam, Hong Kong, China b Department of Clinical Oncology at the Sir Y.K. Pao Center for Cancer, Chinese University of Hong Kong, China c Department of Surgery, Faculty of Medicine, University of Hong Kong, Hong Kong, China d Department of Anatomical and Cellular Pathology, Chinese University of Hong Kong, Hong Kong, China Received 5 December 2001; received in revised form 9 April 2002; accepted 11 April 2002
Abstract Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, especially in southern China. One of the most striking features of this disease is its close relationship with Epstein – Barr Virus (EBV). However, to date there is no direct study on the mechanisms involved in the role of EBV in the tumorigenesis of NPC, largely due to lack of an experimental model. Available hypotheses on the association between EBV and NPC are generated from non-nasopharyngeal epithelial cell systems such as human keratinocytes or mouse epithelial cells, which may not truly represent the biological properties of nasopharyngeal epithelial (NP) cells. In this study, we report the establishment of two immortalized NP cell lines, NP69SV40T and NP39E6/E7, using SV40T and HPV16E6/E7 oncogenes. We found that NP60SV40T and NP39E6/E7 cell lines not only maintained many characteristics of normal NP cells (i.e. keratin profile and responsive to TGFh inhibition) but also highly responsive to one of the EBV encoded genes, LMP1. Comparative genome hybridization (CGH) analysis showed that these two cell lines contained multiple genetic alterations, some of which have been described in NPC. The immortalized NP cell lines are nontumorigenic and exhibit anchorage-dependent growth. These cell lines may provide a possible cell model system for studying the mechanisms involved in the tumorigenesis of NPC. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Nasopharyngeal epithelial; HPVE6/E7; SV40T
1. Introduction Nasopharyngeal carcinoma (NPC) is a common cancer in southern China. In Hong Kong, the incidence rate of NPC is 25 times higher than in other countries around the world. As one of the striking features of NPC is the presence of Epstein – Barr Virus (EBV), therefore EBV infection is proposed as one of the significant etiological factors in NPC [1]. A direct role of EBV in tumorigenesis has been established in Burkitt’s lymphoma, however, due to lack of specimens and experimental models, little information is available on the role of EBV in the tumorigenesis process of NPC. Most of the functional studies on the transformation
*
Corresponding author. Tel.: +852-281-99227; fax: +852-281-70857. E-mail address: [email protected] (S.W. Tsao).
ability of EBV were demonstrated in non-nasopharyngeal epithelial systems such as mouse embryonic fibroblasts [2], immortalized human keratinocytes [3] or gastric carcinoma cells [4]. The fact that most of the established NPC cell lines lose EBV after long-term in vitro culture makes it even more difficult to study the involvement of EBV in NPC. To date, although etiological evidence strongly suggests that EBV plays important roles in the tumorigenesis process of NPC, there is no direct evidence to support this hypothesis in nasopharyngeal cell system. In this study, we report the establishment of two immortalized nasopharyngeal epithelial cell lines NP69SV40T and NP39E6/E7, using SV40 large T and HPV16E6/E7 oncogenes, respectively. These two cell lines not only showed keratin profiles of primary nasopharyngeal epithelial cells but also showed response to TGFh1, indicating that they retain some characteristics of non-malignant nasopharyngeal epithelial cells. In addition, expression of one of the EBV encoded genes, LMP1,
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resulted in an increase in cell cycle S phase fraction and cell growth rate in NP69SV40T cells. NP69SV40T and NP39E6/E7 cell lines provide potential nasopharyngeal epithelial cell models for studying mechanisms involved in tumorigenesis of NPC.
up the viral oncogenes would eventually undergo senescence and stop proliferating. Presence of SV40T protein in NP69SV40T was detected using Western blotting analysis as described [7]. The presence of E6/E7 mRNA in NP39E6/ E7 cells was detected using RT-PCR as previously described [8].
2. Materials and methods
2.3. Telomerase assay
2.1. Establishment of in vitro primary nasopharyngeal epithelial cell culture
The presence of telomerase in the immortalized nasopharyngeal epithelial cells was examined using the PCRbased TRAP assay (telomeric repeats amplification protocol) according to a previously published protocol [9].
Primary cultures of nasopharyngeal epithelial cells were established from biopsies from the nasopharynx of patients with symptoms such as nasal obstruction and excessive bleeding. Prior patient’s consent and approval from the research ethics committee were obtained for the use of these surgical materials for research purposes. To initiate primary culture of nasopharyngeal epithelial cells, the biopsies were cut into approximately 1 mm3 sized explants and placed onto 6 cm culture dishes (Falcon) containing 2 ml of culture medium RPMI1640 supplemented with 1% dialyzed fetal bovine serum, 0.25 AM transferrin, 3.32 nM epidermal growth factor, 0.7 AM insulin, 0.5 AM phosphoethanolamine, 0.5 AM ethanolamine, 10 nM triodo-L-thyronine, 2 AM hydrocortisone, 2.7 AM L-epinephrine, 2 mM L-glutamine, 0.03 AM sodium selenium, 1 nM molybdic acid, 0.5 nM stannous chloride and 0.75 nM nickel sulfate. The calcium concentration of the above medium was 0.4 mM, which was designed to facilitate the attachment of explants and initiation of epithelial outgrowth. The monolayer epithelial cells were then cultured in a low calcium (0.1 mM) medium (MCDB151 medium supplemented with 1% dialyzed fetal bovine serum and the same ingredients described above). The low calcium concentration stimulated the epithelial cell proliferation but suppressed the growth of contaminated fibroblasts. Proliferating fibroblasts, which may be present in the culture flask, were removed manually using the tip of a glass Pasteur pipette circled by Bunsen flame. 2.2. Immortalization of nasopharyngeal epithelial cells The nasopharyngeal epithelial cells at passage one were transfected with the PX8 plasmid containing SV40T antigen [5] or infected with a retroviral vector containing the HPV16E6/E7 ORFs [6]. The retroviral vector HPV16E6/ E7 was kindly provided by Dr. D. Galloway at the Fred Hutchison Cancer Center, University of Washington, USA. For transfection of the SV40T expression plasmid (PX8), 1 Ag of the plasmid DNA was added in 100 Al of serum-free culture medium with 6 Al of Fugene 6 solution (Roche). Expression of the HPV16E6/E7 viral genes was achieved by infecting the cells with the HPV16E6/E7 expressing viral vector according to a previously described protocol [6]. No drug selection was required as the cells which failed to take
2.4. Keratin profiles The expression of cytokeratins in primary and immortalized nasopharyngeal epithelial cells was determined using two-dimensional SDS-PAGE electrophoresis as previously described [6]. The solubilized cytoskeletal proteins were separated by non-equilibrium pH gradient electrophoresis (NEPHGE) using a two-dimensional gel system (Hoefer). The presence of cytoskeletal proteins was visualized using a silver staining kit from Amersham Pharmacia. 2.5. Growth inhibition by TGFb1 Cellular responses to TGFh1 were determined by thymidine incorporation after 3 days treatment with various concentrations of TGFh1 (0.1, 1, and 10 ng/ml) (Gibco BRL, Bethesda, MD). Details of the assay protocol were described previously [10]. 2.6. Comparative genome hybridization (CGH) CGH experiment was carried out according to the published method [11]. Briefly, tumor and normal reference DNA labeled with biotin-16-dUTP (Boehringer Mannheim, Mannheim, Germany) and digoxigenin (dig)-11-dUTP (Boehringer Mannheim), respectively by nick translation were competitively co-hybridized onto metaphase preparations. After 2 days of hybridization at 37 jC, biotin labeled signals were detected through avidin conjugated-fluorescein isothiocyanate (FITC) antibodies (Sigma, St. Louis, USA), and dig-labeled probes by antibodies conjugated with tetramethylrhodamine isothiocyanate (TRITC) (Sigma). The preparations were counterstained with 4V,6-diamidino-2phenylindole (DAPI) (Sigma). Hybridized metaphases were captured with a cooled CCD camera mounted on a Leitz DM RB (Leica, Wetzlar, Germany) fluorescence microscope. Three band-pass filter sets (DAPI, FITC and TRITC) arranged in an automated filterwheel were employed for image acquisition. A CGH software version 3.1 on Cytovision (Applied Imaging, Sunderland, UK), was used for digital image analysis of fluorescence intensity. Chromosome identification was per-
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Fig. 1. Keratin profiles of primary nasopharyngeal epithelial cells and immortalized NP69SV40T and NP39E6/E7 cells detected by two-dimensional SDS PAGE electrophoresis. (A) Keratin expression profile of NPE cells is similar to previously described in epithelial cells of the upper respiratory tract. (B and C) Keratin expression pattern is similar in NP69SV40T and NP39E6/E7 cells compared to NPE cells. Inserted photographs show the morphological characteristics of nasopharyngeal epithelial cells.
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formed on the reverse DAPI banding images. The average ratio profiles were calculated based on the analysis of 14 or more selected metaphases. Thresholds for gains and losses were defined as the theoretical value of 1.25 and 0.75, respectively. High level gain of regional or whole chromosome was considered to be present when ratios exceeded 1.5. 2.7. LMP-1 gene transfer mediated by retroviral vector pLNSX The LMP-1 gene was introduced into immortalized NP69SV40T cells by infecting the cells with a retroviral vector pLNSX-LMP1. The construction of the pLNSXLMP1 plasmid was described previously [2]. Briefly, a 1.95-kb fragment containing a NPC derived full-length LMP1 gene was cloned into the retroviral vector pLNSX to generate pLNSX-LMP1. Control cells were infected with the empty retroviral vector pLNSX. All of the transduced cells were selected and maintained in G418 (400 Ag/ml). Immunoblotting was used to confirm the expression of LMP1 in the immortalized nasopharyngeal cells using the specific antibody (S12) against LMP1 [2]. To demonstrate the effect of LMP1 on morphological alteration, a B95-8 LMP1 cloned in pcDNA vector was transfected into the NP69SV40T and selected with G418. Control cells were transfected with an empty pcDNA vector
Fig. 3. Evidence of telomerase activity in NP69SV40T and NP39E6/E7 cells. Telomerase activity was determined at two different concentrations of protein (0.3 and 3 Ag/ml) using TRAP assay. HeLa cells were used as a positive control.
2.8. Cell cycle analysis
2.9. Growth curve
Cells (5 105) were trypsinized and fixed in cold 70% ethanol for at least 1 h and stored at 4 jC. Before testing, the fixed cells were washed with PBS, treated with RNAse (1 mg/ml) and stained with propidium iodide (50 Ag/ml) for 30 min at 37 jC. DNA content analysis was performed on an EPICS ELITE flow cytometer (Beckman Coulter) and cell cycle distribution was analyzed using the ModFit LT2.0 software.
To determine in vitro cell growth rate, control and LMP1 expressing cells were plated in triplicates at 4 104 cells/ well in 24-well plates. The number of viable cells was determined every 48 h using the trypan blue exclusion method. The growth curves were drawn by plotting the means of three independent experiments with the standard deviation as error bars. 2.10. Tumorigenicity study Cells (106 to 107) were injected subcutaneously into two flanks of 6– 8-week-old Balb/c female athymic nude mice and observed for tumorigenic growth every week for a 4month period.
3. Results 3.1. Establishment and keratin profiles of nasopharyngeal epithelial primary culture
Fig. 2. SV40T and HPV16 E6/E7 expression in NP69SV40T and NP39E6/ E7 cells. (A) RT-PCR analysis of E6/E7 mRNA expression in NP39E6/E7 cells. (B) Western blotting analysis of SV40T protein expression in NP69SV40T cells.
To initiate primary culture of nasopharyngeal epithelial growth, biopsies from patients diagnosed with non-malignant nasal diseases were cut into approximately 1 mm3 sized explants and placed onto 6 cm dishes containing 2 ml culture medium. The epithelial-like outgrowth from the explants was observed 3 – 4 days after explantation and the
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Fig. 4. Effect of TGFh1 on NP and NPC cells. Effect of TGFh1 on cell growth was determined by measuring the percentage of thymidine uptake in cells after 3-day treatment. Results were derived from three independent experiments and the error bars indicate standard deviation. NP69SV40T and NP39E6/E7 cells showed decreased sensitivity to TGFh1 treatment compared to primary NPE cells but more sensitive to TGFh1 than NPC cells (CNE1 and CNE2).
Fig. 5. Representative results of CGH analysis on NP69SV40T cells. The main ratio profile of at least 10 chromosomes is shown. The central line represents balanced hybridization (signal ratio = 1). Signal ratios skewed to the left or right represent thresholds for chromosomal losses (signal ratio < 0.75) and gains (signal ratio > 1.25) respectively.
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cells showed morphology of epithelial cells (Fig. 1A). To confirm the epithelial origin of these cells, cytoskeletal proteins were extracted and two-dimensional SDS PAGE electrophoresis was performed. As shown in Fig. 1A, the cultured nasopharyngeal cells showed detectable expression of cytokeratins K5, K6 and K13-19, indicating that they were of respiratory epithelial origin [12].
istics and the keratin profiles of these two cell lines were similar to the primary nasopharyngeal epithelial cells (Fig. 1B and C). The expression of SV40T or HPV16E6/E7 oncogenes was also detected in these two cell lines, respectively (Fig. 2). These results indicate that NP69SV40T and NP39E6/E7 cell lines represent characteristics of nasopharyngeal epithelial origin.
3.2. Immortalization of nasopharyngeal epithelial cells using HPV16E6/E7 and SV40T viral oncogenes
3.3. Evidence of telomerase activation and TGFb1 sensitivity in NP69SV40T and NP39E6/E7 cell lines
The primary nasopharyngeal epithelial cells had limited proliferative potential. They usually underwent terminal differentiation after 2 – 3 weeks in culture and showed senescent-like phenotype including flattened cell morphology and expression of senescent associated-h-galactosidase (data not shown). In order to establish long-term growth of these cells, vectors containing HPV16 E6/E7 or SV40T viral oncogenes were infected or transfected, respectively into the primary cultured cells. After overcoming crisis stage (approximately 3 – 6 months), two immortalized cell lines NP69SV40T and NP39E6/E7 were established, which have undergone over 100 population doublings without showing any evidence of senescence. The morphological character-
Telomerase activation has been shown to be a common characteristic during the cell immortalization process. We next studied if activation of telomerase was apparent in NP69SV40T and NP39E6/E7 cells. As shown in Fig. 3, both immortalized cell lines showed telomerase activation at similar levels to a cancer cell line, HeLa (Fig. 3). However, NP69SV40T and NP39E6/E7 cells were sensitive to TGFh1-induced cell growth inhibition, which has been suggested as one of the characteristics of normal epithelial cells [13], compared to NPC cells. As shown in Fig. 4, both cell lines responded to TGFh1 in a pattern similar to the primary nasopharyngeal epithelial cells (NPE) in a dose-dependent manner as decreased thymidine
Fig. 6. Effect of LMP1 expression on NP69SV40T cells. (A) Presence of LMP1 protein (upper band) in NP60SV40T cells after transfection of pLNSX vector containing a LMP1 gene but absent in the vector control (NP69SV40T-pLNSX) by Western blotting analysis. (B) Morphological changes induced by LMP1 expression. Elongated and fibroblast-like morphology was observed when a B95-8 LMP1 was expressed in NP69SV40T. Photos were taken under 200 magnification. (C) Cell growth curves of NP69SV40T-LMP1 and its control (-pLNSX). LMP1 expression significantly increased the growth rate of NP69SV40T cells ( P < 0.05). (D) Evidence of LMP1-induced cell cycle S phase fraction in NP69SV40T cells by flow cytometric analysis.
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uptake rate was correlated with increased TGFh1 concentrations. However, the sensitivity in these two cell lines to TGFh1 was decreased compared to NPE cells, especially in NP39E6/E7 cells. In contrast, there was no evidence of growth inhibition in two NPC cell lines, CNE1 and CNE2, treated with the same concentrations of TGFh1. In addition, injection of both NP69SV40T and NP39E6/E7 cells into nude mice did not show any evidence of tumor formation after 3 months, indicating that they are not tumorigenic (data not shown). These results indicate that although NP69SV40T and NP39E6/E7 cells showed similar telomerase activity as cancer cells, they still retained characteristics of non-malignant nasopharyngeal epithelial cells. 3.4. Comparative genome hybridization (CGH) analysis of NP69SV40T and NP39E6/E7 cells CGH analysis on NP69SV40T and NP39E6/E7 cells showed that five genetic alterations including loss of material on chromosomes 9p (NP69SV40T) and 3 (NP39E6/E7), and gains of material on chromosomes 17q (NP69SV40T), 12q and 11 (NP39E6/E7), were correlated to the changes commonly observed in NPC (Table 1 and Fig. 5) [14 – 16]. In NP69SV40T cells, losses on chromosome 6, 11 and 16 and gains on chromosomes 8, 9 and 22 were observed as previously described to be associated with SV40T infection [17 –19]. In NP39E6/E7 cells, typical E6/E7 related genetic alterations such as amplification of chromosomes 5 and 20, were observed [20,21]. 3.5. Evidence of LMP1-induced cell proliferation in NP69SV40T cells In order to study whether the immortalized cells responded to EBV infection, one of the EBV encoded genes, LMP1, which has been shown to play important roles in oncogenic transformation in certain types of epithelial cells [2– 4], was expressed into NP69SV40T cells. As shown in Fig. 6A, LMP1 protein was detected in the NP69SV40T-LMP1 cells but absent in the vector control. Expression of LMP1 significantly altered the morphology of NP69SV40T cells to an elongated and fibroblast-like appearance. Interestingly, this morphological transformation is even more remarkable in NP69SV40T cells expressing the prototype B95-8 derived LMP1 (Fig. 6B). Increased growth rate was also observed after LMP1 expression and increased growth rate of NP69SV40T cells compared to the vector control (Fig. 6C). The increased growth rate may be due to the stimulation of DNA synthesis by LMP1 in the transfectants, as the cell cycle S phase fraction was greatly increased in NP69SV40T-LMP1 cells (40.7%) (Fig. 6D) compared to the vector control (22.67%). However, there was no evidence of tumor formation after NP69SV40T cells were injected into nude mice (n = 10) and observed for a 4month period.
4. Discussion In this study, we report the establishment of two immortalized nasopharyngeal epithelial cell lines NP60SV40T and NP39E6/E7 using SV40T and HPV16E6/E7 viral oncogenes. The fact that these cell lines maintained characteristics of primary nasopharyngeal epithelial cells such as keratin profile and are non-tumorigenic (Figs. 1 and 4), indicates that they provide potential in vitro models for studying tumorigenesis of NPC. The nasopharynx is located at the back of the nasal passageway and surgical access to nasopharynx is limited, therefore, nasopharyngeal tissues are not usually available for investigation. Limited numbers of NPC cell lines have been established but due to the fact that the majority of the available cell lines do not contain EBV, studies on the role of EBV in tumorigenesis of NPC have limited resources. In addition, to date, there is not one non-malignant nasopharyngeal epithelial cell system available for such studies. Our report on the establishment of two immortalized nasopharyngeal epithelial (NP) cell lines may provide an experimental cell model to facilitate investigation in mechanisms involved in tumorigenesis in NPC. In this study, results on cellular morphology, keratin profiles and response to TGFh1 treatment have shown that NP69SV40T and NP39E6/E7 cells retain characteristics of primary NP cells (Figs. 1 and 4). Both cell lines showed activation of telomerase activity, indicating that they are immortal (Fig. 2). Genetic studies showed a number of losses or gains of materials on chromosomes commonly associated with both E6/E7 or SV40T infection such as gains on chromosomes 5 and 20 and losses on chromosome 19 (E6/E7) [19,20] or gains on chromosome 8, 9, 22 and losses on 6, 11 and 16 (SV40T) [16 – 18] (Fig. 5, Table 1), indicating that the expression of E6/E7 or SV40T viral
Table 1 Summary of CGH results
NP69SV40T
NP39E6/E7
Losses
Gains
Amplification
4p15.3 – qter, 6p11.2 – qter 9pter – p23 11pter – q11 11q14 – qter 12p11.2 – p13 16pter – q11 19pter – q12 Xp11.2 – qter 3, 4, 10pter – q21 12 15q11.2 – q15 16pter – p11.2 16q11.22 – qter
2 5pter – q21 7 8p11.2 – qter 9p13 – qter 15q14 – q26 17q11.2 – q25 20p12 – q13.3 22cen – q13 1pter – p32 2pter – p24 6p22 – cen 7pter – q35 11 13q22 – qter 17 19q12 – q13.3 X
5q21 – qter 9p13 – qter
5 20
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oncogenes had significant effects on these cells and these genetic changes may be crucial for the immortalization process of these cells. However, we found only five genetic alterations (out of 33) which are commonly observed in NPC (gains on chromosomes 1q, 11q, 12q and 17q and losses on 3p and 9p) [14 –16]. These results indicate that NP69SV40T and NP39E6/E7 cell lines may provide good models to study the multiple-step pathogenesis in NPC. In order to study the effect of EBV on the immortalized NP cells, we transfected one of the EBV encoded genes, LMP1, into NP69SV40T cells and studied its effect on cell growth and tumorigenecity (Fig. 6). LMP1 is one of the EBV encoded genes expressed in NPC and it has been suggested to be involved in the tumorigenic processes of NPC [22]. LMP1 has been shown to be oncogenic in immortalized epithelial cells derived from human skin and canine kidney [3,23 – 25]. A number of studies showed that LMP1 alone was able to induce tumorigenecity in these epithelial systems [3,23,24]. Others have reported that LMP1 alone had significant effect on cell growth morphology and differentiation [25,26]. In this study, we found that expression of LMP1 in NP69SV40T cells resulted in a striking morphological change from relatively flat to fibroblast-like and disorganized bundled morphology (Fig. 6B), which agrees with results found in previous studies on human keratinocytes and canine epithelial cells [23,26]. A significant increase in cell growth rate was also observed in NP69SV40T cells which was associated with increased cell cycle S phase fraction (Fig. 6C and D). This suggests that expression of LMP1 in nasopharyngeal epithelial cells had significant stimulating effects on cell growth. However, in contrast to previous studies [3,24], we did not observe tumor formation when the NP69SV40T-LMP1 cells were injected into nude mice (data not shown). Our results confirmed previous evidence on the cell growth-stimulating role of LMP1 in epithelial cell growth. However, unlike some observations from other epithelial cell systems, LMP1 alone was not sufficient to induce tumorigenesis in immortalized NP cells. Our evidence supports the hypothesis that EBV infection may be an early factor in the pathogenesis of NPC and additional factors such as genetic or etiological factors are needed in the development of NPC. In summary, we provide potential cell model systems for studying the mechanisms involved in tumorigenesis of NPC. The availability of NP69SV40T and NP39E6/E7 cell lines will provide possible tools to advance studies on the association between EBV and NPC.
Acknowledgements This work is supported by the Research Retreat Grant 2001 (Faculty of Medicine, HKU) and research grants from Research Grant Council (Hong Kong) and CRCG grant (HKU) awarded to SWT.
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