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Upregulated expression of kappa light chain by Epstein–Barr virus encoded latent membrane protein 1 in nasopharyngeal carcinoma cells via NF-κB and AP-1 pathways
Cellular Signalling 19 (2007) 419 – 427 www.elsevier.com/locate/cellsig
Upregulated expression of kappa light chain by Epstein–Barr virus encoded latent membrane protein 1 in nasopharyngeal carcinoma cells via NF-κB and AP-1 pathways Hai-dan Liu, Hui Zheng, Ming Li, Duo-sha Hu, Min Tang, Ya Cao ⁎ Cancer Research Institute, Xiangya School of Medicine, Central South University, Xiangya Road #110, Changsha, Hunan 410078, PR China Received 13 July 2006; accepted 24 July 2006 Available online 18 September 2006
Abstract B lymphocytes are generally considered to be the only source of immunoglobulins. However, increasing evidence revealed that some human epithelial cancer cell lines, including nasopharyngeal carcinoma (NPC) cell lines, expressed immunoglobulins. Moreover, we previously found that expression of kappa light chain in NPC cells could be upregulated by EBV-encoded latent membrane protein 1 (LMP1). Here, Western blot and flow cytometric analysis of intracellular kappa staining indicated that upregulation of the expression of kappa was inhibited by using LMP1targeted DNAzyme and that Bay11-7082 and SP600125, inhibitors of JNK and NF-κB, respectively, inhibited LMP1-augmented kappa light chain expression in NPC cells. LMP1-positive NPC cells expressing the dominant-negative mutant of IκBα (DNMIκBα) or of c-Jun (TAM67) exhibited significantly decreasing kappa production compared with their parental cells. These results suggest that LMP1 elevated kappa light chain through activation of the NF-κB and AP-1 signaling pathways. The present study provided some hints of possible mechanisms by which human cancer cells of epithelial origin produced immunoglobulins. © 2006 Elsevier Inc. All rights reserved. Keywords: Activating protein 1; Immunoglobulin; Kappa light chain; Latent membrane protein 1; Nasopharyngeal carcinoma; Nuclear factor kappa B
1. Introduction B lymphocytes are generally considered to be the only source of immunoglobulins (Igs). However, increasing evidence revealed that epithelial cancer cells express immunoglobulins. Kimoto reported that immunoglobulin heavy chain constant region and TCR were detected in four epithelium derived carcinoma cell lines assayed by nest RT-PCR, which suggested that the corresponding protein might exist [1]. Human Ig constant region could be detected in hepatocellular carcinoma total RNA by using cDNA microarray [2]. It was also reported that both protein and mRNA of Ig could be detected by using 2D electrophoresis and cDNA microarray, respectively, in NPC cell lines [3,4]. Qiu found that human epithelial cancer produced IgG with unidentified specificity and further demonstrated that the light chain of the cancerous Ig is kappa by Western blotting [5]. Recently, by analyzing the protein components in the serum-free ⁎ Corresponding author. Tel.: +86 731 4805448; fax: +86 731 4470589. E-mail address: [email protected] (Y. Cao). 0898-6568/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2006.07.012
conditioned medium of lung cancer primary cell using 1-D PAGE and MS analysis, 299 proteins including Ig alpha C region and Ig kappa V region were identified [6]. Our previous work also indicated kappa gene was expressed in NPC and other epithelial tumor cells [7]. To date, the mechanisms underlying the expression of Igs in nonlymphoid cells remain unidentified. Interestingly, we have found that the levels of kappa light chain were significantly higher in EBV-encoded latent membrane protein (LMP1)-positive cells than in LMP1-negative cells [8,9], thus suggesting that LMP1 might play a role in upregulating the ectopic expression of kappa light chain in NPC cells. LMP1 is an Epstein–Barr virus encoded oncogenic protein composed of a short intracellular N terminus, six hydrophobic transmembrane domains, and an intracellular C terminus including three functional domains, CTAR1, CTAR2, and CTAR3. LMP1 activates its target genes via different signaling pathways that include NF-κB, JNK/c-Jun/AP-1, p38-MAPK/ATF, and JAK/STAT [10–15]. Activation of NF-κB or AP-1 by LMP1 has been linked to the upregulation of some cellular proteins. Although CTAR1 can independently activate the NF-κB
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Fig. 1. LMP1 increased kappa light chain expression in NPC cells. (a) Whole cell lysates of HNE2 and HNE2-LMP1 were measured by Western blot for LMP1, kappa. α-tubulin was used as a loading control. (b) Tet-on-LMP1 HNE2 cells were stimulated with the indicated doses of Dox for 24 h, whole cell lysates were measured by Western blot for LMP1, kappa. α-tubulin was used as a loading control. XG6 and Raji acted as negative and positive controls for kappa chain, respectively.
pathway, it is CTAR2 that is mainly responsible for activating both the NF-κB (accounting for ∼70% of total NF-κB activity induced by LMP1) and JNK (100% of LMP1-mediated JNK activation) pathways [16,17]. NF-κB activation is associated with phosphorylation of the inhibitor of NF-κB, IκBα, at Ser-32 and Ser-36, by IκB kinase (IκK) complexes, degradation of the phosphorylated IκB, and translocation of the freed NF-κB to the nucleus, which triggers its target gene expression [18–22]. By contrast, LMP1 effects on c-Jun/AP-1 mediated through activation of the c-Jun N-terminal kinase (JNK) cascade. The kinase is activated by phosphorylation and subsequently translocates into the cell nucleus. Once into the nucleus, it phosphorylates serine-63 and -73 residues of c-Jun and increases the transcription activity of the AP-1 complex [23,24]. kappa light chain gene expression is under the control of distinct cis-regulatory elements, including the kappa intron enhancer (iEκ) and the kappa 3′enhancer (3′Eκ) [25,26], which is located within the Jκ–Cκ region and downstream of Cκ region, respectively. Both enhancers are inactive in pre-B cell lines in which the κ locus is silent but active at the B cell and plasma stages, indicated they are required for immunoglobulin kappa gene expression [27]. The function of enhancers is mediated by DNA binding proteins that recruit to the enhancer [26]. A κB site within the iEκ and the presence of nuclear NF-κB DNA binding activity in Ig-expressing B lymphocytes, but not at earlier stages of B cell differentiation, suggested that it might play a key role in kappa light chain gene expression [26]. The human kappa gene J–C intron also contains a perfect consensus AP−1 site, which located 320 bases downstream of the κB site. This motif binds a c-Fos/c-Jun protein complex. The κAP-l site in the context of the iEκ can influence kappa expression in B cells, suggesting that it plays a role in kappa gene regulation [28]. According to our previous observations that the level of kappa induction in LMP1-
positive NPC cells was higher than that of LMP1-negative NPC cells, as well as LMP1 can activate NF-κB, AP-1 signal pathways in NPC cell lines [29–31]. We speculated whether the NFκB and AP-1 pathways were involved in LMP1-upregulated kappa chain expression in NPC cells. In the present study, the DNAzymes technology which is one of the commonly used technologies for gene suppression [32] to specifically suppress LMP1 gene expression, the specific NFκB inhibitor Bay11-7082 [33] as well as the selective JNK inhibitor SP600125 [34] were used to investigate the role of NFκB, AP-1 pathways in LMP1-enhanced kappa light chain production in NPC cell lines. Our data demonstrate that upregulation of kappa expression by LMP1 was abolished by treatment with LMP1-specific DNAzymes. LMP1-augmented kappa production was correlated with the elevation of JNK and IκBα phosphorylation, SP600125 and Bay11-7082 inhibited LMP1-induced JNK and IκBα phosphorylation, finally decreased the expression of kappa chain. The NPC cells expressing DNMIκBα which competitively inhibited the activation of NF-κB [29] or TAM67 which competitively quench the transactivation activity of endogenous wild-type Jun [35] exhibited significantly decreasing kappa induction compared with their parental cells. These results suggest that LMP1 induces kappa light chain expression through the activation of the NF-κB and AP-1 signaling pathways. 2. Materials and methods 2.1. Cell lines and cell culture HNE2 is a EBV-LMP1-negative human nasopharyngeal carcinoma (NPC) cell line, HNE2-LMP1 is a cell line constantly expressing LMP1 after the introduction of full-length LMP1 cDNA into HNE2 cells [36], Tet-on-LMP1 HNE2 is a Doxycline-inducible NPC cell line in which the expression of LMP1 would be turned on by Doxycline (Dox, Sigma) [37,38]. HNE2-LMP1DNMIκBα is a cell line constantly expressing dominant-negative mutant of IκBα (DNMIκBα) that had a deletion of 71 amino acids at the N terminus, which competitively inhibited the activation of NF-κB [29]. HNE2-LMP1-TAM67 is a cell line in which the mutant form of c-Jun lacking the NH2-terminal transactivation domain was stably transfected [35]. Human Burkitts Lymphoma cell line Raji was used as kappa chain positive control. Human myeloma cell lines XG6 expressing cytoplasmic λ light chain and XG7 expressing cytoplasmic κ light chain [39] (kindly provided by Prof. X.G. Zhang, Su Zhou Medical
Fig. 2. The effect of inducible LMP1 on the induction of kappa mRNA levels in Tet-on-LMP1 HNE2 cell line. Tet-on-LMP1 HNE2 cells were incubated with medium containing the indicated concentration of Dox for 24 h. Total RNA was isolated from cells and subjected to RT-PCR, using specific primers designed to amplify kappa light chain and actin mRNAs. XG6 and Raji acted as negative and positive controls for kappa chain, respectively. Actin was used as a loading control.
H. Liu et al. / Cellular Signalling 19 (2007) 419–427 University) were used as kappa chain negative and positive controls, respectively. HNE2, HNE2-LMP1, HNE2-LMP1-DNMIκBα, HNE2-LMP1-TAM67 and Raji were grown in RPMI 1640 (GIBCO) supplemented with 10% fetal bovine serum, 1% glutamine, and 1% antibiotics. Tet-on-LMP1 HNE2 was cultured in RPMI 1640 (GIBCO) medium with 10% fetal calf serum, 100 mg/l of G418 and 50 mg/l of hygromycin. XG6 and XG7 were cultured in RPMI 1640 (GIBCO) supplemented with 10% fetal calf serum and 1 ng/ml rIL-6. All cell lines were cultured at 37 °C in a humidified incubator containing 5% CO2. The cells in logarithmic growth phase were used in all experiments.
2.2. Chemicals and cell treatments The selective JNK inhibitor SP600125 and specific NF-κB inhibitor Bay117082 (both from Calbiochem) were prepared as a stock solution of 20 mM in
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dimethylsulfoxide (DMSO, Sigma). Subconfluent cells were treated with the compound at indicated concentrations for an indicated time. Detailed treatment procedures were described in figure legends. The final concentration of DMSO in the culture media was kept less than 0.1% which had no significant effect on the cell growth. Vehicle controls were prepared for all treatments.
2.3. Reverse transcription and polymerase chain reaction Subconfluent Tet-on-LMP1 HNE2 cells were treated with indicated concentrations of Dox for 24 h. Total RNA was isolated from the treated cells, XG6 cells and Raji cells using the TRIzol reagent (GIBCOBRL), according to the instructions of the manufacturer. RNA was dissolved in 20 μl of DEPC-treated water and quantitated at 260 nm. Two microgram of total RNA was reverse transcribed with SuperScript™ IIRT (Invitrogen) at 42 °C for 50 min, and the
Fig. 3. Flow cytometric analysis of induction of LMP1 enhanced kappa light chain expression in Tet-on-LMP1 HNE2 cell line. Tet-on-LMP1 HNE2 cells were stimulated with the indicated doses of Dox for 24 h, expression of kappa chain was detected by flow cytometric analysis with anti-kappa mAb (blue). The background fluorescence was determined using cells incubated without the primary antibody but with the secondary antibody (green). The experiment was performed twice and a representative histogram is shown. The results in ΔMFI were expressed as mean ± SD. XG6 and Raji acted as negative and positive controls for kappa light chain, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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H. Liu et al. / Cellular Signalling 19 (2007) 419–427 ggtgaaagatgag-3′, yield a 231-bp product; or for actin: sense, 5′-ttccagccttcc ttcctggg-3′ and antisense, 5′-ttgcgctcaggaggagcaat-3′, yield a 224-bp product. PCR products were separated on 1.5% agarose gels and visualized with ethidium bromide.
2.4. Transfection of LMP1-targeted DNAzyme
Fig. 4. Inhibition of LMP1-enhanced kappa light chain expression in NPC cells by LMP1-targeted DNAzyme. HNE2 and HNE2-LMP1 cells grown in six-well plates were transfected with DNAzyme (DZ1 at 2 μM), or mock-transfected and incubated in medium containing 10% FBS at 37 °C for 24 h. Total cell lysates were analyzed by Western blot using antibodies targeting LMP1 and kappa light chain, α-tubulin was used as a loading control. XG6 and XG7 cells acted as negative and positive controls for kappa chain, respectively. resulted cDNA was subjected to PCR (94 °C for 5 min followed by 36 cycles of 94 °C for 30 s, 47 °C for 30 s, 72 °C for 30 s, and an extension for 10 min at 72 °C) using specific primers designed for human kappa light chain (GenBank accession no. AJ010442): sense, 5′-tgagcaaagcagactacgaga-3′ and antisense, 5′-ggggtga
Fig. 5. Inhibition of NF-κB and AP-1 pathways prevents LMP1-increased kappa light chain expression. (a, c) HNE2-LMP1 cells were treated with indicated concentrations of Bay11-7082, SP600125 and 0.1% DMSO for 2 h, total cell lysates were gathered. P-IkBα and IkBα, P-JNK and JNK were determined by Western blot. β-actin was used as a loading control. (b, d) HNE2-LMP1 cells were treated with indicated concentrations of Bay11-7082, SP600125 and 0.1% DMSO for 12 h, kappa chain expression in NPC cells were measured with antikappa by Western blot. XG6, XG7 cells were used as kappa light chain negative and positive control, respectively. α-tubulin was used as a loading control.
2 × 105 cells per well of HNE2 and HNE2-LMP1 were seeded in a six-well plate the day before transfection. On the day of transfection, cells were washed once with RPMI 1640. The LMP1-targeted DNAzyme [40]/tetra(4-methylpyridyl) porphyrine (TMP) mixtures were made at a charge ratio of 1 with 2 μm DNAzyme oligonucleotides as described [41]. The mixture was then added to the cells and incubated at 37 °C for 4 h, followed by the addition of complete medium to the wells and further incubation for 20 h.
2.5. Preparation of cell lysates and Western blot analysis Cells were harvested and washed twice with ice-cold phosphate-buffered saline (PBS), and then lysed in the lysis buffer [10 mM Tris–HCl, pH 8.0, 1 mM EDTA, 2% SDS, 5 mM dithiothreitol, 10 mM phenylmethyl sulfonylfluoride, 1 mM Na3VO4, 1 mM NaF, 10% (vol./vol.) glycerol, protease inhibitor cocktail tablet (Roche)] for 30 min on ice and centrifuged at 15,000 ×g for 10 min. The supernatant was collected as the whole cell lysates. Protein concentration was determined by BCA Assay Reagent (Pierce). 100 μg of the total proteins from various cell preparations and rainbow molecular weight markers (Amersham Pharmacia Biotech, Amersham, United Kingdom) were separated on 10% SDSpolyacrylamide gel electrophoresis and then electrotransferred onto the nitrocellulose membrane. The membranes were blocked with buffer containing 5% non-fat milk in PBS with 0.05% Tween-20 for 2 h, and incubated with different primary antibodies overnight at 4 °C. After second wash, the membranes were incubated with anti-rabbit (sc-2004, Santa Cruz) or anti-mouse (sc-2005, Santa Cruz ) horseradish peroxidase-conjugated secondary antibody for 1 h at RT and developed with the enhanced chemiluminescence detection kit (ECL; Pierce). Sometimes, membranes were stripped by incubating at 50 °C for half an hour in stripping buffer [100 mM β-mercaptoethanol, 2% (wt./vol.) sodium dodecyl sulfate and 62.5 mM Tris–HCl (pH 6.8)] and reprobed with primary antibody. The following antibodies were used for Western blotting: mouse LMP1 monoclonal antibody (M0897, DAKO), rabbit anti-human kappa light chains antibody (A0191, DAKO), p-JNK (Thr183/Tyr185) (9251, Cell Signaling Technology) and JNK (9252, Cell Signaling Technology), p-IκBα (sc-8404) against Ser-32 of p-IκBα, IκBα (sc-371) against the C-terminus of IκBα, c-Jun (sc-44) against a highly conserved DNA binding domain of c-Jun, α-tubulin (sc5286), β-actin (sc-8432) (all from Santa Cruz).
Fig. 6. Effects of Bay11-7082 and SP600125 on the transactivation activity of NF-κB and AP-1 induced by LMP1. Transient transfection and luciferase reporter assays were performed as described in Materials and methods. The relative luciferase activity normalized to the value of β-gal activity. Results are expressed as fold induction of vehicle-treated HNE2 cells activity. The data represent the mean ± SD of the three independent experiments performed in triplicate.
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2.6. Intracellular kappa light chain staining for flow cytometric analysis For intracellular staining [42] of the kappa light chain, 1 × 106 cells were harvested and fixed with 4% paraformaldehyde at RT for 40 min. After resuspended and washed with ice-cold PBS, pellets were then permeabilized by resuspending in 1 ml ice-cold PBS containing 0.25% Triton-X100 and 5% BSA and incubated at 4 °C for at least 10 min. Cells were washed twice with PBS
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containing 1% BSA then incubated in 100 μl of PBS containing the anti-human kappa monoclonal antibody (1:100, K-4377, Sigma) and 1% BSA at 4 °C for 40 min. After two washes with PBS containing 1% BSA, the cells were incubated at 4 °C for 40 min in 100 μl of PBS containing FITC-conjugated goat anti-mouse immunoglobulin (1:50, Santa Cruz) and 1% BSA. Subsequently, the cells were washed twice with PBS containing 1% BSA, and the mean fluorescence intensity (MFI) of 10,000 cells was analyzed on a FACSCalibur
Fig. 7. Effects of Bay11-7082 and SP600125 on kappa light chain expression in HNE2 and HNE2-LMP1 cell lines by flow cytometric analysis. HNE2 and HNE2LMP1 cells were treated with 20 μm Bay11-7082, 20 μm SP600125, 0.1% DMSO for 24 h, expression of kappa was detected by flow cytometric analysis with antikappa mAb (blue). The background fluorescence was determined using cells incubated without the primary antibody but with the secondary antibody (green). The experiment was performed three times and a representative histogram is shown. The results in ΔMFI were expressed as mean ± SD. Statistical significance: #P b 0.05 vs. HNE2 vehicle control, ⁎P b 0.05 vs. HNE2-LMP1 vehicle control. XG6 and XG7 cells acted as negative and positive controls for kappa chain, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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using CellQuest software (Becton Dickinson, San Jose, CA.). The background fluorescence was determined using cells incubated without the primary antibody but with the secondary antibody, and kappa-specific mean fluorescence intensity was expressed as ΔMFI = (MFI value of cells incubated with kappa) − (MFI value of cells incubated without kappa) [43]. The experiment was performed in at least two independent experiments.
2.7. Transient transfection and luciferase assay HNE2 and HNE2-LMP1 cells were seeded in 24-well plates before transfection. 0.8 μg pAP-1-Luc or 0.8 μg pNF-κB-Luc (both kindly provided by Dr. Li JJ, National Medical Center and Beckman Research institute, USA) was cotransfected with 0.2 μg pRSV-β-gal constructs using SuperFect Transfection Reagent (Qiagen) in serum-free medium according to the manufacturer's instructions. 24 h after transfection, cells were treated with 20 μm Bay11-7082, SP600125 and 0.1% DMSO as designated for 12 h. Cells were then lysed and reporter gene activity was determined with Luciferase Assay System (Promega) using luminometer (Promega). β-galactosidase activity was measured using βgalactosidase enzyme assay system (Promega) and used for normalization of transfection efficiency. All experiments were performed in triplicate and repeated at least three times.
2.8. Statistical analysis All statistical calculations were performed with the statistical software program SPSS ver.12.0. Differences between various groups were evaluated by the Student's t test. The difference was of statistical significance, when P b 0.05.
flow cytometry correlates well with Western blotting. These results indicate that LMP1 could increase the production of kappa light chain by NPC cells at both mRNA and protein levels. 3.2. Inhibition LMP1 expression by LMP1-specific DNAzyme decreasing κ light chain production Currently, there are several commonly used technologies for gene suppression, which include antisense oligonucleotides, ribozymes, DNAzymes and RNA interference (RNAi). DNAzymes are synthetic, single stranded DNA catalysts that can be engineered to bind to their complementary sequence in a target messenger RNA (mRNA) through Watson–Crick base pairing [32]. We have designed and constructed the specific DNAzymes that were shown to be effective in suppressing expression of the target protein LMP1 [40]. Herein we used the LMP1-specific DNAzyme DZ1 to further investigate the effect of LMP1 on the expression of kappa. Western blotting results indicated that DNAzyme DZ1 obviously inhibited LMP1 protein expression in HNE2-LMP1 cells, compared with TMP transfection reagent and untreated controls. The DNAzyme DZ1 could concomitantly downregulate kappa expression in the LMP1-positive cell line HNE2-LMP1, while has no effect on
3. Results 3.1. LMP1 enhances kappa light chain expression in human nasopharyngeal carcinoma cells that correlates with increased kappa chain mRNA levels To determine whether LMP1 did upregulate kappa expression in NPC cell lines as previously reported [9], we use a constantly expressing full-length LMP1 cell line HNE2-LMP1 and a LMP1-inducible expression NPC cell line, Tet-on-LMP1 HNE2, in which the expression of LMP1 was tightly regulated by doxycycline addition [36–38], as the cell models. Burkitts lymphoma cell line Raji and human myeloma cell line XG6 were used as positive and negative controls for kappa light chain, respectively. We observed kappa expression in HNE2LMP1 cells was higher than that in HNE2 cells (Fig. 1a). When stimulated LMP1 expression by 0, 0.06, 0.6, 6.0 μg/ml of Dox, as evidenced by immunoblotting, Dox induced a marked increase of LMP1 in a dose-dependent manner (Fig. 1b); Raji cells also expressed LMP1. As kappa positive control, Raji cells expressed kappa chain at a very high level. Upon different concentrations of Dox treatment, kappa light chain in Tet-onLMP1 HNE2 cells was also significantly elevated in a dosedependent manner. In order to determine if the observed increase in kappa light chain expression was related to an effect of LMP1 on kappa mRNA levels, Tet-on-LMP1 HNE2 cells under the same treatment with Dox and the levels of kappa light chain mRNA were monitored by RT-PCR. Treatment with Dox induced a marked increase in kappa mRNA, in a dose-dependent manner (Fig. 2). Increased kappa light chain levels by indicated concentrations of Dox in Tet-on-LMP1 HNE2 cells were also confirmed by intracellular flow cytometry analysis (Fig. 3). As can be seen, kappa light chain protein analysis by
Fig. 8. Effect of stable expression TAM67 or DNMIκBα on LMP1-increased kappa light chain production in NPC cells. (a) Expressions of LMP1, endogenous IκBα and dominant-negative IκBα (DNMIκBα), endogenous c-Jun and dominant-negative c-Jun (TAM67) in NPC cell lines were measured by Western blot. Endogenous c-Jun proteins appeared at the upper region of the gel ∼39 kDa and TAM67 at ∼ 29 kDa. Endogenous IκBα appeared at the upper region of the gel ∼ 37 kDa and DNMIκBα at the lower region. (b) Expressions of kappa light chain in NPC cell lines were measured by Western blot. α-tubulin was used as a loading control. XG6 acted as a negative control for kappa. XG7 and Raji were as positive controls for kappa.
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kappa expression in LMP1-negative cell line HNE2 (Fig. 4). These results reinforce the concept that LMP1 upregulates kappa light chain production. 3.3. Inhibition of NF-κB and AP-1 pathways blocks kappa light chain upregulation by LMP1 NF-κB and AP-1 activation mediates upregulation of a number of LMP1-induced gene expression [44]. Our previous studies have shown that both NF-κB and AP-1 could be activated by LMP1 in NPC cell lines [30,36]. To further confirm
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the involvement of the NF-κB and AP-1 pathways in LMP1augmented kappa expression, Bay11-7082, a specific NF-κB inhibitor [33] and SP600125, a JNK inhibitor that inhibits JNK and thus c-Jun phosphorylation [34] were used to investigate JNK and NF-κB activation and kappa chain upregulation induced by LMP1. The levels of IκBα and JNK phosphorylation were higher in HNE2-LMP1 cells than in HNE2 cells (Fig. 5a and c), which demonstrated LMP1 indeed activates NFκB and AP-1 pathways in NPC cells. Treatment of HNE2LMP1 cells with both inhibitors resulted in a dose-dependent suppression of kappa chain induction by the LMP1 (Fig. 5b
Fig. 9. Flow cytometric analysis of the expression of kappa chain in NPC cell lines. Expression of kappa chain was detected by flow cytometric analysis with antikappa mAb (blue). The background fluorescence was determined using cells incubated without the primary antibody but with the secondary antibody (green). The experiment was performed twice and a representative histogram is shown. The results in ΔMFI were expressed as mean ± SD. Statistical significance: #P b 0.05 vs. HNE2 control, ⁎P b 0.05 vs. HNE2-LMP1 control. XG6 acted as a negative control, Raji and XG7 as positive controls for kappa chain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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and d), which is correlated with a dose-dependent attenuation of JNK and IκBα phosphorylation induced by LMP1. The decreasing level of phospho-IκBα was accompanied by accumulation of IκBα in cells due to a block of its degradation (Fig. 5a). Luciferase reporter assays show that Bay11-7082 and SP600125 efficiently inhibited the transactivation activity of NF-κB and AP-1 induced by LMP1 but have no obvious inhibition effect on HNE2 cells (Fig. 6). Moreover, as indicated by flow cytometry analysis, both compounds inhibited kappa expression more efficiently in HNE2-LMP1 cells than in HNE2 cells (Fig. 7). Overall, these results confirm the concept that upregulation of kappa light chain by LMP1 occurs via activation of NF-κB and AP-1 pathways. 3.4. Expression of TAM67 and DNMIκBα inhibits LMP1upregulated κ light chain in NPC cells Stable cell lines expressing TAM67 [35] and DNMIκBα [29] were used to further test the role of AP-1 and NF-κB pathways in regulating kappa light chain expression. DNMIκBα had a deletion of 71 amino acids at the N terminus of IκBα, which competitively inhibited the activation of NF-κB [45]. DNMIκBα expression could be detected by immunoblotting with an antibody against a peptide mapping at the C-terminus of IκBα (Fig. 8a). TAM67 is a mutant form of c-Jun in which the transactivation domain (amino acids 3–122) has been deleted, leaving the C-terminal DNA binding and dimerization domain intact [46]. Such a mutant c-Jun protein is unable to activate its target genes but still possesses the ability to bind to AP-1 site in promoter regions of target genes and competitively quench the transactivation activity of endogenous wild-type Jun and its dimerization partners [47]. Expression of 29 kDa TAM67 could be identified with an antibody against a peptide mapping to DNA binding domain of c-Jun (Fig. 8a). As verified by Western blotting and flow cytometry analysis, the stable expression TAM67 cell lines HNE2-LMP1-TAM67 blocked LMP1-induced increase of kappa chain. The amount of kappa light chain in HNE2-LMP1-TAM67 was significantly lower than that of its parental cell HNE2-LMP1 (Fig. 8b). The result further suggested that LMP1 regulated kappa light chain via JNK/c-Jun cascade. Similarly, the stable expression DNMIκBα cell lines HNE2LMP1-DNMIκBα was efficient in mediating such inhibition (Fig. 8b). FACS results fit the immunoblotting results well (Fig. 9). The data reinforced the concept that the increase of kappa induction by LMP1 occurs via activation of the NF-κB and AP-1 signaling pathways. 4. Discussion Nowadays, several evidences revealed that human epithelial cancer cells express immunoglobulin, though the mechanisms underlying the expression of Igs in nonlymphoid cells have not yet been identified. The present study demonstrated that EBVencoded oncoprotein LMP1 upregulated kappa light chain expression by activating NF-κB and AP-1 signaling pathways, which suggested, at least in part, a mechanism by which LMP1positive human epithelial cancer cells produced immunoglobu-
lins. The activation of enhancer is required for immunoglobulin kappa gene expression. Considerable information has been documented on the function of the two enhancers and the transacting factors that modulate enhancer activity. In addition to NF-κB and AP-1 binding site in human iEκ to modulate enhancer activity, other positive regulatory elements have been identified in the core 3′enhancer, including a consensus binding site for transcription factor PU.1 and the family of Ets-related proteins, a binding motif for factors of the helix-loop-helix (HLH), an 11/12-bp direct repeat (DR) motif, these sequences could potentially regulate kappa gene transcriptional activity [48]. The transcription factors binding to these motif can regulate kappa light chain gene induction. We cannot rule out the participation of other transcription factors via corresponding pathways to regulate kappa gene expression. For instance, the function of Ets-1 and Ets-2 is activated by Ras-MAPK signaling pathways. The MAPKs ERK1 and ERK2 phosphorylate Ets-1 and Ets-2 results in the enhancement of the transactivation activity of these proteins [49]. Kim et al. reported that stable transfection of a LMP1 gene into MDCK cells induces expression of Ets-1, suggesting Ets may be the target genes of LMP1 [50]. Therefore, other signaling cascades such as LMP1/ ERKs/Ets are likely to involve in regulating kappa light chain expression. Data presented here did not clearly show which of two pathways played a leading role in LMP1-augmented kappa light chain expression. However, Schanke et al. [28] reported that the κAP-l site is unable to act independently but can influence kappa expression in the context of the iEκ in B cells, thus suggesting that the factors binding to the κAP-l site are responsible for a portion of the transcriptional activating capacity of the iEκ in B cells. One possible explanation for our observation is other normal or variant AP-1 motifs might exist in the cis-acting elements; these sequences could potentially regulate kappa gene expression. There exists a CRE-sequence in 3′Eκ of murine kappa locus containing two AP-1 half binding sites and c-Fos and c-Jun can bind to this sequence. Mixture of multiple transcription factors can greatly activate the enhancer in NIH 3T3 cell in which the enhancer is normally silent. Removal of cJun expression totally abolished enhancer activity [51], thus raising another possibility that CRE or CRE-like sequence might exist in cis-acting elements of human kappa gene which account for our results. In addition, the manner regulating the expression of kappa chain via AP-1 pathways in tumor cells may be different to that in B cells. Further investigations need to be performed to elucidate the respective roles of NF-κB and AP-1 pathways in upregulating kappa light chain production in LMP1-positive NPC cells. Expression of kappa light chain in cancer cells is complicated. LMP1-negative NPC cell lines also express kappa light chain though at a relatively low level. It was reported that not only NPC cell lines, but many human epithelial cancer cell lines such as MCF-7 (human breast carcinoma), HeLa (human cervical carcinoma), MGC (human gastric carcinoma), SW480 (human colon carcinoma) express kappa light chain [5,7]. Considerable evidence has been found that several infectious agents, particularly viruses, played a role in human cancer.
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Those infectious agents, such as EBV, HBV, HCV, HPVs, have been identified as causes of cancer [52]. In NPC, LMP1 is considered as a major oncogenic protein encoded by EBV and can aberrantly activate many signal pathways. Similarly, over 95% of cervical tumors consistently express E6 and E7 oncoproteins [53]. These suggested that some virus-encoded oncoproteins might induce immunoglobulin gene expression in nonlymphoid cell lineage by abnormal activation corresponding transcription factors and signaling pathways. It seems a candidate to explain the expression of kappa chain in other human epithelial cancer cell lines. In summary, through specifically targeting the molecules on different levels of signaling pathways, we show that LMP1 induces an increasing expression of kappa light chain and that this effect is likely to involve activation of the NF-κB and AP-1 pathways. The present study presented here, using the NPC cell lines as model, might provide some hints of possible mechanisms by which human cancer cells of epithelial origin produced immunoglobulins. Acknowledgement This project was supported by the National Nature Science Foundation of China (No. 30471968). References [1] [2] [3] [4] [5]
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Upregulated expression of kappa light chain by Epstein–Barr virus encoded latent membrane protein 1 in nasopharyngeal carcinoma cells via NF-κB and AP-1 pathways Hai-dan Liu, Hui Zheng, Ming Li, Duo-sha Hu, Min Tang, Ya Cao ⁎ Cancer Research Institute, Xiangya School of Medicine, Central South University, Xiangya Road #110, Changsha, Hunan 410078, PR China Received 13 July 2006; accepted 24 July 2006 Available online 18 September 2006
Abstract B lymphocytes are generally considered to be the only source of immunoglobulins. However, increasing evidence revealed that some human epithelial cancer cell lines, including nasopharyngeal carcinoma (NPC) cell lines, expressed immunoglobulins. Moreover, we previously found that expression of kappa light chain in NPC cells could be upregulated by EBV-encoded latent membrane protein 1 (LMP1). Here, Western blot and flow cytometric analysis of intracellular kappa staining indicated that upregulation of the expression of kappa was inhibited by using LMP1targeted DNAzyme and that Bay11-7082 and SP600125, inhibitors of JNK and NF-κB, respectively, inhibited LMP1-augmented kappa light chain expression in NPC cells. LMP1-positive NPC cells expressing the dominant-negative mutant of IκBα (DNMIκBα) or of c-Jun (TAM67) exhibited significantly decreasing kappa production compared with their parental cells. These results suggest that LMP1 elevated kappa light chain through activation of the NF-κB and AP-1 signaling pathways. The present study provided some hints of possible mechanisms by which human cancer cells of epithelial origin produced immunoglobulins. © 2006 Elsevier Inc. All rights reserved. Keywords: Activating protein 1; Immunoglobulin; Kappa light chain; Latent membrane protein 1; Nasopharyngeal carcinoma; Nuclear factor kappa B
1. Introduction B lymphocytes are generally considered to be the only source of immunoglobulins (Igs). However, increasing evidence revealed that epithelial cancer cells express immunoglobulins. Kimoto reported that immunoglobulin heavy chain constant region and TCR were detected in four epithelium derived carcinoma cell lines assayed by nest RT-PCR, which suggested that the corresponding protein might exist [1]. Human Ig constant region could be detected in hepatocellular carcinoma total RNA by using cDNA microarray [2]. It was also reported that both protein and mRNA of Ig could be detected by using 2D electrophoresis and cDNA microarray, respectively, in NPC cell lines [3,4]. Qiu found that human epithelial cancer produced IgG with unidentified specificity and further demonstrated that the light chain of the cancerous Ig is kappa by Western blotting [5]. Recently, by analyzing the protein components in the serum-free ⁎ Corresponding author. Tel.: +86 731 4805448; fax: +86 731 4470589. E-mail address: [email protected] (Y. Cao). 0898-6568/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2006.07.012
conditioned medium of lung cancer primary cell using 1-D PAGE and MS analysis, 299 proteins including Ig alpha C region and Ig kappa V region were identified [6]. Our previous work also indicated kappa gene was expressed in NPC and other epithelial tumor cells [7]. To date, the mechanisms underlying the expression of Igs in nonlymphoid cells remain unidentified. Interestingly, we have found that the levels of kappa light chain were significantly higher in EBV-encoded latent membrane protein (LMP1)-positive cells than in LMP1-negative cells [8,9], thus suggesting that LMP1 might play a role in upregulating the ectopic expression of kappa light chain in NPC cells. LMP1 is an Epstein–Barr virus encoded oncogenic protein composed of a short intracellular N terminus, six hydrophobic transmembrane domains, and an intracellular C terminus including three functional domains, CTAR1, CTAR2, and CTAR3. LMP1 activates its target genes via different signaling pathways that include NF-κB, JNK/c-Jun/AP-1, p38-MAPK/ATF, and JAK/STAT [10–15]. Activation of NF-κB or AP-1 by LMP1 has been linked to the upregulation of some cellular proteins. Although CTAR1 can independently activate the NF-κB
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Fig. 1. LMP1 increased kappa light chain expression in NPC cells. (a) Whole cell lysates of HNE2 and HNE2-LMP1 were measured by Western blot for LMP1, kappa. α-tubulin was used as a loading control. (b) Tet-on-LMP1 HNE2 cells were stimulated with the indicated doses of Dox for 24 h, whole cell lysates were measured by Western blot for LMP1, kappa. α-tubulin was used as a loading control. XG6 and Raji acted as negative and positive controls for kappa chain, respectively.
pathway, it is CTAR2 that is mainly responsible for activating both the NF-κB (accounting for ∼70% of total NF-κB activity induced by LMP1) and JNK (100% of LMP1-mediated JNK activation) pathways [16,17]. NF-κB activation is associated with phosphorylation of the inhibitor of NF-κB, IκBα, at Ser-32 and Ser-36, by IκB kinase (IκK) complexes, degradation of the phosphorylated IκB, and translocation of the freed NF-κB to the nucleus, which triggers its target gene expression [18–22]. By contrast, LMP1 effects on c-Jun/AP-1 mediated through activation of the c-Jun N-terminal kinase (JNK) cascade. The kinase is activated by phosphorylation and subsequently translocates into the cell nucleus. Once into the nucleus, it phosphorylates serine-63 and -73 residues of c-Jun and increases the transcription activity of the AP-1 complex [23,24]. kappa light chain gene expression is under the control of distinct cis-regulatory elements, including the kappa intron enhancer (iEκ) and the kappa 3′enhancer (3′Eκ) [25,26], which is located within the Jκ–Cκ region and downstream of Cκ region, respectively. Both enhancers are inactive in pre-B cell lines in which the κ locus is silent but active at the B cell and plasma stages, indicated they are required for immunoglobulin kappa gene expression [27]. The function of enhancers is mediated by DNA binding proteins that recruit to the enhancer [26]. A κB site within the iEκ and the presence of nuclear NF-κB DNA binding activity in Ig-expressing B lymphocytes, but not at earlier stages of B cell differentiation, suggested that it might play a key role in kappa light chain gene expression [26]. The human kappa gene J–C intron also contains a perfect consensus AP−1 site, which located 320 bases downstream of the κB site. This motif binds a c-Fos/c-Jun protein complex. The κAP-l site in the context of the iEκ can influence kappa expression in B cells, suggesting that it plays a role in kappa gene regulation [28]. According to our previous observations that the level of kappa induction in LMP1-
positive NPC cells was higher than that of LMP1-negative NPC cells, as well as LMP1 can activate NF-κB, AP-1 signal pathways in NPC cell lines [29–31]. We speculated whether the NFκB and AP-1 pathways were involved in LMP1-upregulated kappa chain expression in NPC cells. In the present study, the DNAzymes technology which is one of the commonly used technologies for gene suppression [32] to specifically suppress LMP1 gene expression, the specific NFκB inhibitor Bay11-7082 [33] as well as the selective JNK inhibitor SP600125 [34] were used to investigate the role of NFκB, AP-1 pathways in LMP1-enhanced kappa light chain production in NPC cell lines. Our data demonstrate that upregulation of kappa expression by LMP1 was abolished by treatment with LMP1-specific DNAzymes. LMP1-augmented kappa production was correlated with the elevation of JNK and IκBα phosphorylation, SP600125 and Bay11-7082 inhibited LMP1-induced JNK and IκBα phosphorylation, finally decreased the expression of kappa chain. The NPC cells expressing DNMIκBα which competitively inhibited the activation of NF-κB [29] or TAM67 which competitively quench the transactivation activity of endogenous wild-type Jun [35] exhibited significantly decreasing kappa induction compared with their parental cells. These results suggest that LMP1 induces kappa light chain expression through the activation of the NF-κB and AP-1 signaling pathways. 2. Materials and methods 2.1. Cell lines and cell culture HNE2 is a EBV-LMP1-negative human nasopharyngeal carcinoma (NPC) cell line, HNE2-LMP1 is a cell line constantly expressing LMP1 after the introduction of full-length LMP1 cDNA into HNE2 cells [36], Tet-on-LMP1 HNE2 is a Doxycline-inducible NPC cell line in which the expression of LMP1 would be turned on by Doxycline (Dox, Sigma) [37,38]. HNE2-LMP1DNMIκBα is a cell line constantly expressing dominant-negative mutant of IκBα (DNMIκBα) that had a deletion of 71 amino acids at the N terminus, which competitively inhibited the activation of NF-κB [29]. HNE2-LMP1-TAM67 is a cell line in which the mutant form of c-Jun lacking the NH2-terminal transactivation domain was stably transfected [35]. Human Burkitts Lymphoma cell line Raji was used as kappa chain positive control. Human myeloma cell lines XG6 expressing cytoplasmic λ light chain and XG7 expressing cytoplasmic κ light chain [39] (kindly provided by Prof. X.G. Zhang, Su Zhou Medical
Fig. 2. The effect of inducible LMP1 on the induction of kappa mRNA levels in Tet-on-LMP1 HNE2 cell line. Tet-on-LMP1 HNE2 cells were incubated with medium containing the indicated concentration of Dox for 24 h. Total RNA was isolated from cells and subjected to RT-PCR, using specific primers designed to amplify kappa light chain and actin mRNAs. XG6 and Raji acted as negative and positive controls for kappa chain, respectively. Actin was used as a loading control.
H. Liu et al. / Cellular Signalling 19 (2007) 419–427 University) were used as kappa chain negative and positive controls, respectively. HNE2, HNE2-LMP1, HNE2-LMP1-DNMIκBα, HNE2-LMP1-TAM67 and Raji were grown in RPMI 1640 (GIBCO) supplemented with 10% fetal bovine serum, 1% glutamine, and 1% antibiotics. Tet-on-LMP1 HNE2 was cultured in RPMI 1640 (GIBCO) medium with 10% fetal calf serum, 100 mg/l of G418 and 50 mg/l of hygromycin. XG6 and XG7 were cultured in RPMI 1640 (GIBCO) supplemented with 10% fetal calf serum and 1 ng/ml rIL-6. All cell lines were cultured at 37 °C in a humidified incubator containing 5% CO2. The cells in logarithmic growth phase were used in all experiments.
2.2. Chemicals and cell treatments The selective JNK inhibitor SP600125 and specific NF-κB inhibitor Bay117082 (both from Calbiochem) were prepared as a stock solution of 20 mM in
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dimethylsulfoxide (DMSO, Sigma). Subconfluent cells were treated with the compound at indicated concentrations for an indicated time. Detailed treatment procedures were described in figure legends. The final concentration of DMSO in the culture media was kept less than 0.1% which had no significant effect on the cell growth. Vehicle controls were prepared for all treatments.
2.3. Reverse transcription and polymerase chain reaction Subconfluent Tet-on-LMP1 HNE2 cells were treated with indicated concentrations of Dox for 24 h. Total RNA was isolated from the treated cells, XG6 cells and Raji cells using the TRIzol reagent (GIBCOBRL), according to the instructions of the manufacturer. RNA was dissolved in 20 μl of DEPC-treated water and quantitated at 260 nm. Two microgram of total RNA was reverse transcribed with SuperScript™ IIRT (Invitrogen) at 42 °C for 50 min, and the
Fig. 3. Flow cytometric analysis of induction of LMP1 enhanced kappa light chain expression in Tet-on-LMP1 HNE2 cell line. Tet-on-LMP1 HNE2 cells were stimulated with the indicated doses of Dox for 24 h, expression of kappa chain was detected by flow cytometric analysis with anti-kappa mAb (blue). The background fluorescence was determined using cells incubated without the primary antibody but with the secondary antibody (green). The experiment was performed twice and a representative histogram is shown. The results in ΔMFI were expressed as mean ± SD. XG6 and Raji acted as negative and positive controls for kappa light chain, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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H. Liu et al. / Cellular Signalling 19 (2007) 419–427 ggtgaaagatgag-3′, yield a 231-bp product; or for actin: sense, 5′-ttccagccttcc ttcctggg-3′ and antisense, 5′-ttgcgctcaggaggagcaat-3′, yield a 224-bp product. PCR products were separated on 1.5% agarose gels and visualized with ethidium bromide.
2.4. Transfection of LMP1-targeted DNAzyme
Fig. 4. Inhibition of LMP1-enhanced kappa light chain expression in NPC cells by LMP1-targeted DNAzyme. HNE2 and HNE2-LMP1 cells grown in six-well plates were transfected with DNAzyme (DZ1 at 2 μM), or mock-transfected and incubated in medium containing 10% FBS at 37 °C for 24 h. Total cell lysates were analyzed by Western blot using antibodies targeting LMP1 and kappa light chain, α-tubulin was used as a loading control. XG6 and XG7 cells acted as negative and positive controls for kappa chain, respectively. resulted cDNA was subjected to PCR (94 °C for 5 min followed by 36 cycles of 94 °C for 30 s, 47 °C for 30 s, 72 °C for 30 s, and an extension for 10 min at 72 °C) using specific primers designed for human kappa light chain (GenBank accession no. AJ010442): sense, 5′-tgagcaaagcagactacgaga-3′ and antisense, 5′-ggggtga
Fig. 5. Inhibition of NF-κB and AP-1 pathways prevents LMP1-increased kappa light chain expression. (a, c) HNE2-LMP1 cells were treated with indicated concentrations of Bay11-7082, SP600125 and 0.1% DMSO for 2 h, total cell lysates were gathered. P-IkBα and IkBα, P-JNK and JNK were determined by Western blot. β-actin was used as a loading control. (b, d) HNE2-LMP1 cells were treated with indicated concentrations of Bay11-7082, SP600125 and 0.1% DMSO for 12 h, kappa chain expression in NPC cells were measured with antikappa by Western blot. XG6, XG7 cells were used as kappa light chain negative and positive control, respectively. α-tubulin was used as a loading control.
2 × 105 cells per well of HNE2 and HNE2-LMP1 were seeded in a six-well plate the day before transfection. On the day of transfection, cells were washed once with RPMI 1640. The LMP1-targeted DNAzyme [40]/tetra(4-methylpyridyl) porphyrine (TMP) mixtures were made at a charge ratio of 1 with 2 μm DNAzyme oligonucleotides as described [41]. The mixture was then added to the cells and incubated at 37 °C for 4 h, followed by the addition of complete medium to the wells and further incubation for 20 h.
2.5. Preparation of cell lysates and Western blot analysis Cells were harvested and washed twice with ice-cold phosphate-buffered saline (PBS), and then lysed in the lysis buffer [10 mM Tris–HCl, pH 8.0, 1 mM EDTA, 2% SDS, 5 mM dithiothreitol, 10 mM phenylmethyl sulfonylfluoride, 1 mM Na3VO4, 1 mM NaF, 10% (vol./vol.) glycerol, protease inhibitor cocktail tablet (Roche)] for 30 min on ice and centrifuged at 15,000 ×g for 10 min. The supernatant was collected as the whole cell lysates. Protein concentration was determined by BCA Assay Reagent (Pierce). 100 μg of the total proteins from various cell preparations and rainbow molecular weight markers (Amersham Pharmacia Biotech, Amersham, United Kingdom) were separated on 10% SDSpolyacrylamide gel electrophoresis and then electrotransferred onto the nitrocellulose membrane. The membranes were blocked with buffer containing 5% non-fat milk in PBS with 0.05% Tween-20 for 2 h, and incubated with different primary antibodies overnight at 4 °C. After second wash, the membranes were incubated with anti-rabbit (sc-2004, Santa Cruz) or anti-mouse (sc-2005, Santa Cruz ) horseradish peroxidase-conjugated secondary antibody for 1 h at RT and developed with the enhanced chemiluminescence detection kit (ECL; Pierce). Sometimes, membranes were stripped by incubating at 50 °C for half an hour in stripping buffer [100 mM β-mercaptoethanol, 2% (wt./vol.) sodium dodecyl sulfate and 62.5 mM Tris–HCl (pH 6.8)] and reprobed with primary antibody. The following antibodies were used for Western blotting: mouse LMP1 monoclonal antibody (M0897, DAKO), rabbit anti-human kappa light chains antibody (A0191, DAKO), p-JNK (Thr183/Tyr185) (9251, Cell Signaling Technology) and JNK (9252, Cell Signaling Technology), p-IκBα (sc-8404) against Ser-32 of p-IκBα, IκBα (sc-371) against the C-terminus of IκBα, c-Jun (sc-44) against a highly conserved DNA binding domain of c-Jun, α-tubulin (sc5286), β-actin (sc-8432) (all from Santa Cruz).
Fig. 6. Effects of Bay11-7082 and SP600125 on the transactivation activity of NF-κB and AP-1 induced by LMP1. Transient transfection and luciferase reporter assays were performed as described in Materials and methods. The relative luciferase activity normalized to the value of β-gal activity. Results are expressed as fold induction of vehicle-treated HNE2 cells activity. The data represent the mean ± SD of the three independent experiments performed in triplicate.
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2.6. Intracellular kappa light chain staining for flow cytometric analysis For intracellular staining [42] of the kappa light chain, 1 × 106 cells were harvested and fixed with 4% paraformaldehyde at RT for 40 min. After resuspended and washed with ice-cold PBS, pellets were then permeabilized by resuspending in 1 ml ice-cold PBS containing 0.25% Triton-X100 and 5% BSA and incubated at 4 °C for at least 10 min. Cells were washed twice with PBS
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containing 1% BSA then incubated in 100 μl of PBS containing the anti-human kappa monoclonal antibody (1:100, K-4377, Sigma) and 1% BSA at 4 °C for 40 min. After two washes with PBS containing 1% BSA, the cells were incubated at 4 °C for 40 min in 100 μl of PBS containing FITC-conjugated goat anti-mouse immunoglobulin (1:50, Santa Cruz) and 1% BSA. Subsequently, the cells were washed twice with PBS containing 1% BSA, and the mean fluorescence intensity (MFI) of 10,000 cells was analyzed on a FACSCalibur
Fig. 7. Effects of Bay11-7082 and SP600125 on kappa light chain expression in HNE2 and HNE2-LMP1 cell lines by flow cytometric analysis. HNE2 and HNE2LMP1 cells were treated with 20 μm Bay11-7082, 20 μm SP600125, 0.1% DMSO for 24 h, expression of kappa was detected by flow cytometric analysis with antikappa mAb (blue). The background fluorescence was determined using cells incubated without the primary antibody but with the secondary antibody (green). The experiment was performed three times and a representative histogram is shown. The results in ΔMFI were expressed as mean ± SD. Statistical significance: #P b 0.05 vs. HNE2 vehicle control, ⁎P b 0.05 vs. HNE2-LMP1 vehicle control. XG6 and XG7 cells acted as negative and positive controls for kappa chain, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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using CellQuest software (Becton Dickinson, San Jose, CA.). The background fluorescence was determined using cells incubated without the primary antibody but with the secondary antibody, and kappa-specific mean fluorescence intensity was expressed as ΔMFI = (MFI value of cells incubated with kappa) − (MFI value of cells incubated without kappa) [43]. The experiment was performed in at least two independent experiments.
2.7. Transient transfection and luciferase assay HNE2 and HNE2-LMP1 cells were seeded in 24-well plates before transfection. 0.8 μg pAP-1-Luc or 0.8 μg pNF-κB-Luc (both kindly provided by Dr. Li JJ, National Medical Center and Beckman Research institute, USA) was cotransfected with 0.2 μg pRSV-β-gal constructs using SuperFect Transfection Reagent (Qiagen) in serum-free medium according to the manufacturer's instructions. 24 h after transfection, cells were treated with 20 μm Bay11-7082, SP600125 and 0.1% DMSO as designated for 12 h. Cells were then lysed and reporter gene activity was determined with Luciferase Assay System (Promega) using luminometer (Promega). β-galactosidase activity was measured using βgalactosidase enzyme assay system (Promega) and used for normalization of transfection efficiency. All experiments were performed in triplicate and repeated at least three times.
2.8. Statistical analysis All statistical calculations were performed with the statistical software program SPSS ver.12.0. Differences between various groups were evaluated by the Student's t test. The difference was of statistical significance, when P b 0.05.
flow cytometry correlates well with Western blotting. These results indicate that LMP1 could increase the production of kappa light chain by NPC cells at both mRNA and protein levels. 3.2. Inhibition LMP1 expression by LMP1-specific DNAzyme decreasing κ light chain production Currently, there are several commonly used technologies for gene suppression, which include antisense oligonucleotides, ribozymes, DNAzymes and RNA interference (RNAi). DNAzymes are synthetic, single stranded DNA catalysts that can be engineered to bind to their complementary sequence in a target messenger RNA (mRNA) through Watson–Crick base pairing [32]. We have designed and constructed the specific DNAzymes that were shown to be effective in suppressing expression of the target protein LMP1 [40]. Herein we used the LMP1-specific DNAzyme DZ1 to further investigate the effect of LMP1 on the expression of kappa. Western blotting results indicated that DNAzyme DZ1 obviously inhibited LMP1 protein expression in HNE2-LMP1 cells, compared with TMP transfection reagent and untreated controls. The DNAzyme DZ1 could concomitantly downregulate kappa expression in the LMP1-positive cell line HNE2-LMP1, while has no effect on
3. Results 3.1. LMP1 enhances kappa light chain expression in human nasopharyngeal carcinoma cells that correlates with increased kappa chain mRNA levels To determine whether LMP1 did upregulate kappa expression in NPC cell lines as previously reported [9], we use a constantly expressing full-length LMP1 cell line HNE2-LMP1 and a LMP1-inducible expression NPC cell line, Tet-on-LMP1 HNE2, in which the expression of LMP1 was tightly regulated by doxycycline addition [36–38], as the cell models. Burkitts lymphoma cell line Raji and human myeloma cell line XG6 were used as positive and negative controls for kappa light chain, respectively. We observed kappa expression in HNE2LMP1 cells was higher than that in HNE2 cells (Fig. 1a). When stimulated LMP1 expression by 0, 0.06, 0.6, 6.0 μg/ml of Dox, as evidenced by immunoblotting, Dox induced a marked increase of LMP1 in a dose-dependent manner (Fig. 1b); Raji cells also expressed LMP1. As kappa positive control, Raji cells expressed kappa chain at a very high level. Upon different concentrations of Dox treatment, kappa light chain in Tet-onLMP1 HNE2 cells was also significantly elevated in a dosedependent manner. In order to determine if the observed increase in kappa light chain expression was related to an effect of LMP1 on kappa mRNA levels, Tet-on-LMP1 HNE2 cells under the same treatment with Dox and the levels of kappa light chain mRNA were monitored by RT-PCR. Treatment with Dox induced a marked increase in kappa mRNA, in a dose-dependent manner (Fig. 2). Increased kappa light chain levels by indicated concentrations of Dox in Tet-on-LMP1 HNE2 cells were also confirmed by intracellular flow cytometry analysis (Fig. 3). As can be seen, kappa light chain protein analysis by
Fig. 8. Effect of stable expression TAM67 or DNMIκBα on LMP1-increased kappa light chain production in NPC cells. (a) Expressions of LMP1, endogenous IκBα and dominant-negative IκBα (DNMIκBα), endogenous c-Jun and dominant-negative c-Jun (TAM67) in NPC cell lines were measured by Western blot. Endogenous c-Jun proteins appeared at the upper region of the gel ∼39 kDa and TAM67 at ∼ 29 kDa. Endogenous IκBα appeared at the upper region of the gel ∼ 37 kDa and DNMIκBα at the lower region. (b) Expressions of kappa light chain in NPC cell lines were measured by Western blot. α-tubulin was used as a loading control. XG6 acted as a negative control for kappa. XG7 and Raji were as positive controls for kappa.
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kappa expression in LMP1-negative cell line HNE2 (Fig. 4). These results reinforce the concept that LMP1 upregulates kappa light chain production. 3.3. Inhibition of NF-κB and AP-1 pathways blocks kappa light chain upregulation by LMP1 NF-κB and AP-1 activation mediates upregulation of a number of LMP1-induced gene expression [44]. Our previous studies have shown that both NF-κB and AP-1 could be activated by LMP1 in NPC cell lines [30,36]. To further confirm
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the involvement of the NF-κB and AP-1 pathways in LMP1augmented kappa expression, Bay11-7082, a specific NF-κB inhibitor [33] and SP600125, a JNK inhibitor that inhibits JNK and thus c-Jun phosphorylation [34] were used to investigate JNK and NF-κB activation and kappa chain upregulation induced by LMP1. The levels of IκBα and JNK phosphorylation were higher in HNE2-LMP1 cells than in HNE2 cells (Fig. 5a and c), which demonstrated LMP1 indeed activates NFκB and AP-1 pathways in NPC cells. Treatment of HNE2LMP1 cells with both inhibitors resulted in a dose-dependent suppression of kappa chain induction by the LMP1 (Fig. 5b
Fig. 9. Flow cytometric analysis of the expression of kappa chain in NPC cell lines. Expression of kappa chain was detected by flow cytometric analysis with antikappa mAb (blue). The background fluorescence was determined using cells incubated without the primary antibody but with the secondary antibody (green). The experiment was performed twice and a representative histogram is shown. The results in ΔMFI were expressed as mean ± SD. Statistical significance: #P b 0.05 vs. HNE2 control, ⁎P b 0.05 vs. HNE2-LMP1 control. XG6 acted as a negative control, Raji and XG7 as positive controls for kappa chain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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and d), which is correlated with a dose-dependent attenuation of JNK and IκBα phosphorylation induced by LMP1. The decreasing level of phospho-IκBα was accompanied by accumulation of IκBα in cells due to a block of its degradation (Fig. 5a). Luciferase reporter assays show that Bay11-7082 and SP600125 efficiently inhibited the transactivation activity of NF-κB and AP-1 induced by LMP1 but have no obvious inhibition effect on HNE2 cells (Fig. 6). Moreover, as indicated by flow cytometry analysis, both compounds inhibited kappa expression more efficiently in HNE2-LMP1 cells than in HNE2 cells (Fig. 7). Overall, these results confirm the concept that upregulation of kappa light chain by LMP1 occurs via activation of NF-κB and AP-1 pathways. 3.4. Expression of TAM67 and DNMIκBα inhibits LMP1upregulated κ light chain in NPC cells Stable cell lines expressing TAM67 [35] and DNMIκBα [29] were used to further test the role of AP-1 and NF-κB pathways in regulating kappa light chain expression. DNMIκBα had a deletion of 71 amino acids at the N terminus of IκBα, which competitively inhibited the activation of NF-κB [45]. DNMIκBα expression could be detected by immunoblotting with an antibody against a peptide mapping at the C-terminus of IκBα (Fig. 8a). TAM67 is a mutant form of c-Jun in which the transactivation domain (amino acids 3–122) has been deleted, leaving the C-terminal DNA binding and dimerization domain intact [46]. Such a mutant c-Jun protein is unable to activate its target genes but still possesses the ability to bind to AP-1 site in promoter regions of target genes and competitively quench the transactivation activity of endogenous wild-type Jun and its dimerization partners [47]. Expression of 29 kDa TAM67 could be identified with an antibody against a peptide mapping to DNA binding domain of c-Jun (Fig. 8a). As verified by Western blotting and flow cytometry analysis, the stable expression TAM67 cell lines HNE2-LMP1-TAM67 blocked LMP1-induced increase of kappa chain. The amount of kappa light chain in HNE2-LMP1-TAM67 was significantly lower than that of its parental cell HNE2-LMP1 (Fig. 8b). The result further suggested that LMP1 regulated kappa light chain via JNK/c-Jun cascade. Similarly, the stable expression DNMIκBα cell lines HNE2LMP1-DNMIκBα was efficient in mediating such inhibition (Fig. 8b). FACS results fit the immunoblotting results well (Fig. 9). The data reinforced the concept that the increase of kappa induction by LMP1 occurs via activation of the NF-κB and AP-1 signaling pathways. 4. Discussion Nowadays, several evidences revealed that human epithelial cancer cells express immunoglobulin, though the mechanisms underlying the expression of Igs in nonlymphoid cells have not yet been identified. The present study demonstrated that EBVencoded oncoprotein LMP1 upregulated kappa light chain expression by activating NF-κB and AP-1 signaling pathways, which suggested, at least in part, a mechanism by which LMP1positive human epithelial cancer cells produced immunoglobu-
lins. The activation of enhancer is required for immunoglobulin kappa gene expression. Considerable information has been documented on the function of the two enhancers and the transacting factors that modulate enhancer activity. In addition to NF-κB and AP-1 binding site in human iEκ to modulate enhancer activity, other positive regulatory elements have been identified in the core 3′enhancer, including a consensus binding site for transcription factor PU.1 and the family of Ets-related proteins, a binding motif for factors of the helix-loop-helix (HLH), an 11/12-bp direct repeat (DR) motif, these sequences could potentially regulate kappa gene transcriptional activity [48]. The transcription factors binding to these motif can regulate kappa light chain gene induction. We cannot rule out the participation of other transcription factors via corresponding pathways to regulate kappa gene expression. For instance, the function of Ets-1 and Ets-2 is activated by Ras-MAPK signaling pathways. The MAPKs ERK1 and ERK2 phosphorylate Ets-1 and Ets-2 results in the enhancement of the transactivation activity of these proteins [49]. Kim et al. reported that stable transfection of a LMP1 gene into MDCK cells induces expression of Ets-1, suggesting Ets may be the target genes of LMP1 [50]. Therefore, other signaling cascades such as LMP1/ ERKs/Ets are likely to involve in regulating kappa light chain expression. Data presented here did not clearly show which of two pathways played a leading role in LMP1-augmented kappa light chain expression. However, Schanke et al. [28] reported that the κAP-l site is unable to act independently but can influence kappa expression in the context of the iEκ in B cells, thus suggesting that the factors binding to the κAP-l site are responsible for a portion of the transcriptional activating capacity of the iEκ in B cells. One possible explanation for our observation is other normal or variant AP-1 motifs might exist in the cis-acting elements; these sequences could potentially regulate kappa gene expression. There exists a CRE-sequence in 3′Eκ of murine kappa locus containing two AP-1 half binding sites and c-Fos and c-Jun can bind to this sequence. Mixture of multiple transcription factors can greatly activate the enhancer in NIH 3T3 cell in which the enhancer is normally silent. Removal of cJun expression totally abolished enhancer activity [51], thus raising another possibility that CRE or CRE-like sequence might exist in cis-acting elements of human kappa gene which account for our results. In addition, the manner regulating the expression of kappa chain via AP-1 pathways in tumor cells may be different to that in B cells. Further investigations need to be performed to elucidate the respective roles of NF-κB and AP-1 pathways in upregulating kappa light chain production in LMP1-positive NPC cells. Expression of kappa light chain in cancer cells is complicated. LMP1-negative NPC cell lines also express kappa light chain though at a relatively low level. It was reported that not only NPC cell lines, but many human epithelial cancer cell lines such as MCF-7 (human breast carcinoma), HeLa (human cervical carcinoma), MGC (human gastric carcinoma), SW480 (human colon carcinoma) express kappa light chain [5,7]. Considerable evidence has been found that several infectious agents, particularly viruses, played a role in human cancer.
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Those infectious agents, such as EBV, HBV, HCV, HPVs, have been identified as causes of cancer [52]. In NPC, LMP1 is considered as a major oncogenic protein encoded by EBV and can aberrantly activate many signal pathways. Similarly, over 95% of cervical tumors consistently express E6 and E7 oncoproteins [53]. These suggested that some virus-encoded oncoproteins might induce immunoglobulin gene expression in nonlymphoid cell lineage by abnormal activation corresponding transcription factors and signaling pathways. It seems a candidate to explain the expression of kappa chain in other human epithelial cancer cell lines. In summary, through specifically targeting the molecules on different levels of signaling pathways, we show that LMP1 induces an increasing expression of kappa light chain and that this effect is likely to involve activation of the NF-κB and AP-1 pathways. The present study presented here, using the NPC cell lines as model, might provide some hints of possible mechanisms by which human cancer cells of epithelial origin produced immunoglobulins. Acknowledgement This project was supported by the National Nature Science Foundation of China (No. 30471968). References [1] [2] [3] [4] [5]
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